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5-2011
Stress Response And Pathogenesis of Salmonella enterica serovar Typhimurium
Jigna D. Shah Utah State University
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STRESS RESPONSE AND PATHOGENESIS OF
SALMONELLA ENTERICA SEROVAR TYPHIMURIUM
by
Jigna D. Shah
A dissertation submitted in partial fulfillment of the requirements for the degree
of
DOCTOR OF PHILOSOPHY
in
Nutrition and Food Sciences
Approved by:
______Bart C. Weimer Marie Walsh Major Advisor Co Advisor
______Dong Chen John Stevens Committee Member Committee Member
______Joanie Hevel Byron Burnham Committee Member Dean of Graduate Studies
UTAH STATE UNIVERSITY Logan, Utah
2011 ii
Copyright © Jigna D. Shah
All Rights Reserved iii
ABSTRACT
Stress Response and Pathogenesis of Salmonella enterica serovar Typhimurium
by
Jigna D. Shah, Doctor of Philosophy
Utah State University, 2011
Major Professor: Dr. Bart Weimer Department: Nutrition, Dietetics, and Food Sciences
Salmonella is a food-borne pathogen that leads to substantial illness worldwide.
The clinical syndromes associated with Salmonella infection are enteric (typhoid) fever
and gastroenteritis, in healthy humans. Typhoid fever is caused by host-adapted S. Typhi
and S. Paratyphi. Gastroenteritis is caused by serovars usually referred to as non typhoidal Salmonellae (NTS). In recent years, an increasing number of outbreaks due to
NTS, despite increased efforts in food safety, were reported because of persistence of
Salmonella in the food chain. Thus I hypothesized that Salmonella is able to withstand stresses in the environment and treatments used during food processing for its elimination and thereby able to develop resistance against subsequent stress encounters. The effect of cold, peroxide, and acid was tested on survival of S. Typhimurium and the survival was persistent under cold stress (5°C) for up to 240 h. Pre-adaptation to cold stress (5°C, 5 h)
also increased survival of S. Typhimurium during subsequent exposure to acid stress (pH
4.0, 90 min) by repressing hydroxyl radical formation. Cold stress (5°C, 48 h) to S.
Typhimurium significantly (p < 0.05) increased its adhesion and invasion in intestinal iv
epithelial cells. This phenotype was attributed to a pair of protein-protein interactors
acting as receptors on microbial (STM2699) and host cell surface (SPTAN1). Cold stress
significantly (q < 0.05) induced STM2699 in S. Typhimurium and SPTAN1 was
significantly (q < 0.05) induced in epithelial cells upon infection with cold-stressed S.
Typhimurium. Cold stress to S. Typhimurium also significantly (q < 0.05) induced genes
related to virulence such as type 3 secretion system apparatus and effectors genes,
prophage genes, and plasmid genes and they remain induced upon infection of epithelial cells with additional induction of spv genes on the plasmid. Infection of epithelial cells with cold-stressed S. Typhimurium significantly (p < 0.05) increased activation of caspase 9 and 3/7. Cold-stressed S. Typhimurium switched metabolism from aerobic respiration to fermentation and it persisted during infection of epithelial cells. As a result, short chain fatty acids formate and acetate, which act as diffusible signal for invasion, were detected in significantly (q < 0.05) high amounts in extracellular media of cells infected with cold-stressed S. Typhimurium supporting the phenotype of high adhesion and invasion of cold-stressed S. Typhimurium in epithelial cells.
(241 pages)
v
ACKNOWLEDGMENTS
With deep gratitude and sincerity, I would like to thank my advisor, Dr. Bart
Weimer, for his guidance, expertise, support, and patience throughout this study. He provided constant encouragement and motivation with great understanding. The scientific discussions with him were very fruitful.
I wish to extend my sincere appreciation to my co-advisor, Dr. Marie Walsh, for her kind support all the way through and my committee members, Dr. Dong Chen for his help with proteomics study, Dr. John Stevens for his help with statistics, and Dr. Joanie
Hevel for her valuable suggestions.
I sincerely thank Dr. Giovanni Rompato and Ninglin Yin from Genomics core at the Center of Integrated Biosystems for their assistance in running GeneChips. I am grateful to Prerak Desai for his extensive discussions around my work, and his interesting explorations in the datasets have been very helpful for this study. I wish to thank Dr.
Balasubramanian Ganesan and Sweta Rajan for their friendly help and scientific debates during my stay at Logan. I am thankful to all my friends and lab mates at Logan for their help, especially Amrita and Ashwini for the care and support they provided.
My deepest gratitude goes to my entire extended family for their unflagging affection and support throughout my life; this study would not have been possible without their continuous encouragement. Most importantly, I am indebted to my mother,
Mrs. Ratan D. Shah, for her unconditional love and constant motivation. I sincerely dedicate this dissertation to my mother who has been instrumental in inspiring me at every step throughout my life on both personal and professional fronts.
Jigna D. Shah vi
CONTENTS
Page
ABSTRACT ...... iii
ACKNOWLEDGMENTS ...... v
LIST OF TABLES ...... xi
LIST OF FIGURES ...... xiii
LIST OF SYMBOLS AND ABBREVIATIONS ...... xvi
CHAPTER
1. INTRODUCTION ...... 1
REFERENCES ...... 4
2. LITERATURE REVIEW ...... 6
Phylogeny and serotypes ...... 6 Genetics and genomics ...... 7 Salmonellosis in animals...... 8 Salmonellosis in humans...... 10 Adhesion...... 11 Invasion...... 13 Prophages...... 16 Stress response of Salmonella ...... 16 Acid stress response ...... 18 Peroxide stress response ...... 20 Cold stress response ...... 22
HYPOTHESIS ...... 24 OBJECTIVES ...... 25 REFERENCES ...... 25
3. COLD STRESS TO S. TYPHIMURIUM INCREASES ITS SURVIVAL DURING SUBSEQUENT ACID STRESS ...... 49 vii
ABSTRACT ...... 49 INTRODUCTION ...... 50 MATERIALS AND METHODS ...... 52
Bacterial strains and culture conditions ...... 52 Stress treatments...... 52 Proteome analysis ...... 53 Gene expression analysis ...... 54 Hybridization and normalization ...... 55 NAD+/NADH quantification ...... 56 Radical measurement ...... 56 Statistical analysis ...... 57
RESULTS ...... 57
Stress survival...... 57 Cold stress proteomics ...... 59 Gene expression during stress ...... 60 Oxidative state and stress ...... 64
DISCUSSION ...... 64 REFERENCES ...... 68
4. COLD STRESS TO S. TYPHIMURIUM INCREASES ADHESION AND INVASION IN INTESTINAL EPITHELIAL CELLS ...... 90
ABSTRACT ...... 90 INTRODUCTION ...... 91 MATERIALS AND METHODS ...... 94
Cell culture ...... 94 Bacterial strains and growth conditions ...... 95 Stress treatments for bacteria ...... 95 Total host association measurements ...... 95 Gentamicin protection assay ...... 96 viii
Infection of epithelial cells for gene expression ...... 97 Bacterial RNA extraction and gene expression ...... 98 Hybridization and normalization ...... 99 Caco-2 RNA extraction and gene expression ...... 100 Hybridization and normalization ...... 100 Antibody blocking assays ...... 100 Construction of isogenic mutants ...... 101 Caspase assay ...... 102 Statistical analysis for gene expression...... 102
RESULTS AND DISCUSSION ...... 103
Effect of stressed bacteria on association to epithelial cells...... 103 Gene expression profile of cold-stressed S. Typhimurium ...... 104 Gene expression profile of cold-stressed S. Typhimurium upon infection of epithelial cells...... 106 Effect of cold-stressed S. Typhimurium on gene expression of epithelial cells...... 108 Induction of apoptosis ...... 111 Receptors involved in association of S. Typhimurium to epithelial cells 111 Phylogenetic distribution of STM2699 and SPTAN1 ...... 114
CONCLUDING REMARKS ...... 115 REFERENCES ...... 116
5. CHANGES IN METABOLISM OF COLD-STRESSED S. TYPHIMURIUM AND EPITHELIAL CELLS DURING INFECTION ...... 158
ABSTRACT ...... 158 INTRODUCTION ...... 159 MATERIALS AND METHODS ...... 160
Cell culture ...... 160 Bacterial strains and growth conditions ...... 161 Stress treatments for bacteria ...... 161 Infection of epithelial cells for gene expression ...... 161 ix
Bacterial RNA extraction and gene expression ...... 162 Hybridization and Normalization ...... 164 Caco-2 RNA extraction and gene expression ...... 164 Hybridization and Normalization ...... 165 Infection of epithelial cells for extracellular metabolite profiling ...... 165 NMR sample preparation ...... 165 NMR spectroscopy...... 166 Data analysis ...... 166 Statistical analysis for gene expression...... 166
RESULTS AND DISCUSSION ...... 167
Gene expression profile and extracellular metabolite profile of cold- stressed S. Typhimurium ...... 167 Gene expression profile and extracellular metabolite profile of cold- stressed S. Typhimurium in the presence of epithelial cells...... 169 Gene expression profile and extracellular metabolite profile of epithelial cells upon infection with cold-stressed S. Typhimurium ...... 172
CONCLUDING REMARKS ...... 173 REFERENCES ...... 174
6. SUMMARY ...... 191 HYPOTHESIS ...... 191 OBJECTIVES ...... 191 FUTURE DIRECTIONS ...... 194 REFERENCES ...... 195
APPENDICES APPENDIX A SUPPLEMENTARY DATA FOR CHAPTER 3 ...... 201 APPENDIX B SUPPLEMENTARY DATA FOR CHAPTER 4 ...... 208 MATERIALS AND METHODS ...... 208 Labeling S. Typhimurium with Sulfo-SBED...... 208 Identification of receptors by cell-cell cross linking...... 208 x
APPENDIX C SUPPLEMENTARY DATA FOR CHAPTER 5 ...... 215
CIRRICULUM VITAE ...... 222
xi
LIST OF TABLES
Table Page
2-1. Examples of host-adapted Salmonella (49)...... 43
2-2. Genome statistics of S. Typhimurium LT2 adapted from (81)...... 44
2-3. Coding sequence homologues shared by S. Typhimurium LT2 and eight other strains of enterobacteria adapted from (81) ...... 45
4-1. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium...... 133
4-2. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium as compared to non-stressed S. Typhimurium, upon infection of epithelial cells...... 134
4-3. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in epithelial cells due to infection with cold-stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S. Typhimurium...... 135
4-4. Salmonella serotypes that contain homologues of STM2699...... 136
5-1. Significantly (q < 0.10) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium as compared to non-stressed S. Typhimurium...... 181
5-2. Significantly (q < 0.10) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium as compared to non-stressed S. Typhimurium, upon infection of epithelial cells. This effect is due to extracellular cold stress to S. Typhimurium, which is beyond the effect of the infection of epithelial cells with non-stressed S. Typhimurium...... 182
A-1. Growth of S. Typhimurium in nutrient broth with hydrogen peroxide (H2O2)...... 201
A-2. Growth of S. Typhimurium in nutrient broth with sodium chloride (NaCl)...... 202
B-1. Composition of tyrodes buffer...... 210
B-2. Growth of S. Typhimurium in cell culture media (excluding FBS) with hydrogen peroxide (H2O2)...... 211
B-3. Host (Caco-2 cells) and microbe (S. Typhimurium) binding protein partners identified as using whole cell cross linking...... 212 xii
C-1. Composition of the chemically defined media...... 215
C-2. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in epithelial cells due to infection with cold-stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S. Typhimurium. This effect is due to extracellular cold stress to S. Typhimurium, which is beyond the effect of the infection of epithelial cells with non-stressed S. Typhimurium...... 217
xiii
LIST OF FIGURES
Figures Page
2-1. Phylogenetic tree of the Salmonella clade adapted from (99)...... 46
2-2. Colonization of murine Peyer’s patches by S. Typhimurium, adapted from (122)....47
2-3. Genetic organization of SPI1 and SPI2 adapted from (51)...... 48
3-1. Survival after shock and stress treatments...... 77
3-2. Heat map representations of the 104 differentially regulated proteins (q < 0.05) due to cold stress (5°C) over time (0.5 h, 5 h, 48 h and 240 h)...... 80
3-3. Distribution in COG categories, of 1809 significantly (q < 0.05) regulated genes due to cold stress (5°C, 5 h) and 357 significantly (q < 0.05) regulated genes due to subsequent acid stress (pH 4.0, 90 min) alone...... 83
3-4. Heat map representations of genes differentially regulated (q < 0.05) due to cold stress (5°C, 5 h), acid stress (pH 4.0, 90 min) and due to pre-adaptation to cold stress (5°C, 5 h) when bacteria were subsequently exposed to acid stress (pH 4.0, 90 min)...... 84
3-5. (A) NAD+/NADH ratio measurements and (B) OH• (hydroxyl radical) measurements using hydroxyphenyl fluorescein (HPF) upon exposure to cold stress (5°C, 5 h), acid stress (pH 4.0, 90 min) and upon pre-adaptation to cold stress (5°C, 5 h) followed by exposure to subsequent acid stress (pH 4.0, 90 min)..88
4-1. Association S. Typhimurium to epithelial (Caco2) cells. (A) Epithelial cells were infected with cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid (pH 4.0, 90 min) stressed S. Typhimurium at MOI of 1:1000 and total association to epithelial cells was measured at 60 min, 90 min and 120 min post infection. (B) Epithelial cells were infected with cold-stressed (5°C, 48 h) S. Typhimurium at MOI of 1:1000 and the number of invaded bacteria was measured using gentamicin protection assay, 60 min post infection...... 137
4-2. GSEA was used to detect coordinate changes in expression of the functionally related genes in S. Typhimurium in response to cold stress (5°C, 48 h)...... 139
4-3. GSEA was used to detect coordinate changes in expression of the functionally related genes in cold-stressed (5°C, 48 h) S. Typhimurium upon infection of epithelial cells (q < 0.05) as compared to infection with non-stressed S. Typhimurium...... 144 xiv
4-4. Expression of significantly regulated (q < 0.20) genes in functional category related to mitochondrial electron transport chain in epithelial cells, upon infection with cold-stressed (5°C, 48 h) S. Typhimurium...... 149
4-5. Caspase activation during infection of epithelial cells with non-stressed and cold-stressed (5°C, 48 h) S. Typhimurium, at MOI of 1:1000...... 150
4-6. Gene expression intensities of the S. Typhimurium protein STM2699 upon no stress and cold stress (5°C, 48 h) alone, and upon infection of epithelial cells with non-stressed and cold-stressed (5°C, 48 h) S. Typhimurium. Gene expression intensities of the epithelial cell protein SPTAN1 in epithelial cells alone, and upon infection of epithelial cells with non-stressed and cold-stressed (5°C, 48 h) S. Typhimurium...... 153
4-7. Role of STM2699 and SPTAN1 in adhesion and invasion of epithelial cells with non-stressed S. Typhimurium (MOI of 1:100)...... 154
4-8. Role of STM2699 and SPTAN1, in adhesion and invasion of epithelial cells with cold-stressed (5°C, 48 h) S. Typhimurium (MOI of 1:100)...... 156
5-1. Heat map showing the expression of coordinately regulating genes within significantly regulated gene sets (q < 0.1) in S. Typhimurium...... 183
5-2. Heat map showing the concentration over time (min), of secreted extracellular metabolites, detected by NMR (q < 0.05)...... 187
5-3. Heat map showing the expression of coordinately regulating genes within significantly regulated gene sets (q < 0.05) in epithelial cells due to infection (60 min post infection) with cold-stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S. Typhimurium...... 188
5-4. Pathways adapted from BioCyc database, in S. Typhimurium for (A) Pyruvate fermentation and (B) Lysine degradation...... 190
6-1. Proposed model for increased survival of S. Typhimurium during exposure to acid stress (pH 4.0, 90 min), upon pre-adaptation to cold stress (5°C, 48 h)...... 197
6-2. Proposed model for increased pathogenesis of cold-stressed (5°C, 48 h) S. Typhimurium...... 198
A-1. Survival of S. Typhimurium during upon pre-adaptation at 10°C for 0.5 h followed by exposure to cold stress at 5°C for 336 h ...... 203
A-2. Heat map representations of the 117 differentially regulated proteins (q < 0.05) due to pre-adaptation at 10°C for 0.5 h followed by exposure to cold stress at 5°C xv
for 240 h...... 204
B-1. Determination of optimum concentration of anti-SPTAN1 to block the exposed alpha spectrin-1 on the cell surface...... 213
C-1. Induction of ‘viable but non-culturable’ (VBNC) state in S. Typhimurium using chemically defined media (for media composition see Table C-1)...... 219
C-2. Association of VBNC and stationary phase S. Typhimurium to epithelial cells (Caco-2 cells) measured using PCR, as described in materials and methods section of Chapter 4...... 221
xvi
LIST OF SYMBOLS AND ABBREVIATIONS
ANOVA Analysis of variance
AS Acid stress
ASP Acid shock proteins
ASR Acid stress response
ATP Adenosine triphospate
CFU Colony forming unit
CMR Comprehensive microbial resource
COG Cluster of orthologus groups
CS Cold stress
CSP Cold shock proteins
DMEM Dubelco modified eagle minimum essential media
DNA Deoxyribonucleic acid
ECM Extracellular matrix
EDTA Ethylene diamine tetra aceticacid
ETC Electron transfer chain
FBS Fetal bovine serum
FDR False discovery rate
GALT Gut-associated lymphoid tissue
GSEA Gene set enrichment analysis
HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethane sulfonic acid
HPF hydroxyphenyl fluorescein xvii
IPA Ingenuity pathway analysis
KEGG Kyoto encyclopedia of genes and genomes
LC Liquid chromatography
LPS Lipopolysaccharide
M Molar
MOI Multiplicity of infection
MOPS 3-morpholinopropane-1-sulfonic acid
MS Mass spectrometry
MS-RMA Multi species-robust multichip average
NAD+ Nicotinamide adenine dinucleotide (Oxidized)
NADH Nicotinamide adenine dinucleotide (Reduced)
NMR Nuclear magnetic resonance
NS No stress
NTS Non-typhoidal Salmonella
OS Osmotic stress
PAGE Polyacrylamide gel electrophoresis
PANP Presence absence calls with negative probesets
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PS Peroxide stress
RFU Relative flourescence unit
RMA Robust multichip average
RNA Ribonucleic acid xviii
SAM Significance analysis of microarrays
SCFA Short chain fatty acids
SCV Salmonella containing vacuole
SDS Sodium dodecyl sulfate
SEM Standard error of mean
SPI Salmonella pathogenicity island
T3SS Type 3 secretion system
T4SS Type 4 secretion system
TCA Tri carboxylic acid
TES N-(Tris(hydroxymethyl)methyl)-2-aminoethanesulfonic acid
VBNC Viable but non culturable
VFDB Virulence factors of pathogenic bacteria database
1
CHAPTER 1
INTRODUCTION
Salmonella is a significant food-borne pathogen throughout the world that leads to substantial illness worldwide (6). In the contemporary classification, the genus
Salmonella contains only two species, Salmonella bongori and Salmonella enterica (S. enterica) but there more than 2,500 serovars of S. enterica. The genus broadly causes food-borne salmonellosis, which is a zoonosis of worldwide economic importance.
Salmonella inhabits the intestinal tracts of vertebrates and excretion results in contamination of environment (13, 15). Although human to human transmission does occur, animals and their products constitute the most important source of the organism for the humans (13). In humans, the clinical syndromes associated with Salmonella infection are enteric (typhoid) fever and gastroenteritis. Typhoid fever is caused by human adapted Salmonella enterica serovars Typhi and Paratyphi and gastroenteritis is caused by other Salmonellae commonly referred as non-typhoidal Salmonella (NTS).
Effective vaccines exist for typhoid (11) but not for gastroenteritis. The incidences of food-borne salmonellosis, every year in the USA are estimated to be between 7.4 x 105
and 5.3 x 10 6 (7). Worldwide there are more than 1.3 billion cases of salmonellosis annually with 3 million deaths (10).
This organism has an unusual ability for adapting in many micro-environments;
as a result the largest reservoir of disease in humans is often domesticated animals. As
Salmonella encounters a variety of drastically different microenvironments and host
defense mechanisms during its course of infection, adaptations to these conditions
involve a large number of functional genes, which may be considered as virulence 2 determinants in a broader sense. Response of Salmonella to various stresses encountered in the environment and in the host has been studied (3). Moreover, exposure of
Salmonella to any stressful conditions results in a protective response and due to overlapping genes, in responses to different stresses, exposure to one stressful condition provides cross protection for any subsequent stressful encounters (4, 8, 12). In the host, the virulence strategy common to Salmonella species after the transit from gastric acidity, is to adhere and invade the intestinal mucosa and multiply in the gut-associated lymphoid tissue (GALT). The pathogens are passed from the infected intestinal tissues to the regional lymph nodes, where macrophages that line the lymphatic sinuses form a first defensive barrier to prevent further spread (14). If this host defense mechanism successfully limits bacterial expansion, the infection remains localized to the intestine and the GALT resulting in gastroenteritis. If, the macrophages located in the draining lymph nodes are ineffective in limiting the spread, Salmonella can cause a systemic disease (14). Systemic disease is generally caused by S. Typhi and S. Paratyphi, while the localized infection or gastroenteritis is caused by NTS. In many cases the host becomes asymptomatic carrier (5) and sheds the organisms for weeks to years after the acute infection is contained.
In recent years multistate outbreaks of NTS from conventional sources such as meat and poultry products and non conventional sources such as peanut butter, dry dog food, dry puffed cereal and fresh produce (9) have been reported. The studies determining the mechanisms used by Salmonella to colonize plants have just begun (1).
We hypothesized that one thing, common to all conventional and non conventional food vectors, food processing methods and existing pathogen elimination methods, was 3 playing a major role in persistent NTS outbreaks. Hence, this study investigated the survival strategies of S. Typhimurium, during cold stress (5°C), which is commonly encountered in the environment and it is also the most common method to increase the shelf life of any food. We further revealed one of the underlying mechanisms, upon pre- adaptation to cold stress (5°C, 5 h), responsible for increased survival under subsequent acid stress (pH 4.0, 90 min) which would be encountered during gastric transit upon ingestion of contaminated food. Further, the effect of cold stress (5°C, 48 h) on pathogenesis of S. Typhimurium is investigated in cell culture model. Significant (q <
0.05) induction of the genes related to the virulence, upon cold stress, is observed and this induction remains persistent in the presence of the intestinal epithelial cells (60 min post infection). The significantly (p < 0.05) high association of cold-stressed S. Typhimurium to the intestinal epithelial cells is attributed to a pair of receptors on host and microbe, which are significantly (q < 0.05) induced due to cold stress to S. Typhimurium. Since metabolic enzymes are attractive targets for antimicrobials due to their central role in physiology and survival, differential regulation in metabolism due to cold stress to S.
Typhimurium, alone and in the presence of epithelial cells, was further investigated. With increasing antibiotic resistance and limitation in developing new antimicrobials due to robust Salmonella metabolism (2), there is strong need to fabricate alternative methods for the control of NTS infection. With this scenario, understanding the interaction of
Salmonella with epithelial cells and elucidating the receptors involved, will lead to new insights for developing strategies, such as competitive exclusion, blocking the receptors with antibodies and vaccine development for NTS, to control the infection.
4
REFERENCES
1. Barak, J. D., L. Gorski, A. S. Liang, and K.-E. Narm. 2009. Previously
uncharacterized Salmonella enterica genes required for swarming play a role in
seedling colonization. Microbiology 155:3701-3709.
2. Becker, D., M. Selbach, C. Rollenhagen, M. Ballmaier, T. F. Meyer, M.
Mann, and D. Bumann. 2006. Robust Salmonella metabolism limits possibilities
for new antimicrobials. Nature 440:303-7.
3. Foster, J. W., and M. P. Spector. 1995. How Salmonella survive against the
odds. Annu Rev Microbiol 49:145-74.
4. Greenacre, E. J., and T. F. Brocklehurst. 2006. The Acetic Acid Tolerance
Response induces cross-protection to salt stress in Salmonella typhimurium. Int J
Food Microbiol 112:62-5.
5. Gyles, C. L., J. F. Prescott, G. Songer, and C. O. Thoen. 1986. Pathogenesis of
bacterial infections in animals.
6. Humphrey, T. 2004. Salmonella, stress responses and food safety. Nat Rev
Microbiol 2:504-9.
7. Jeffreys, A. G., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 1998.
Growth, survival and characterization of cspA in Salmonella enteritidis following
cold shock. Curr Microbiol 36:29-35.
8. Leyer, G. J., and E. A. Johnson. 1993. Acid adaptation induces cross-protection
against environmental stresses in Salmonella typhimurium. Appl Environ
Microbiol 59:1842-7. 5
9. Lynch, M. F., R. V. Tauxe, and C. W. Hedberg. 2009. The growing burden of
foodborne outbreaks due to contaminated fresh produce: risks and opportunities.
Epidemiol Infect 137:307-15.
10. Pang, T., Z. A. Bhutta, B. B. Finlay, and M. Altwegg. 1995. Typhoid fever and
other salmonellosis: a continuing challenge. Trends Microbiol 3:253-5.
11. Pulickal, A. S., and A. J. Pollard. 2007. Vi polysaccharide-protein conjugate
vaccine for the prevention of typhoid fever in children: hope or hype? Expert Rev
Vaccines 6:293-5.
12. Rychlik, I., and P. A. Barrow. 2005. Salmonella stress management and its
relevance to behaviour during intestinal colonisation and infection. FEMS
Microbiol Rev 29:1021-40.
13. Turnbull, P. C. 1979. Food poisoning with special reference to Salmonella -- its
epidemiology, pathogenesis and control. Clin Gastroenterol 8:663-714.
14. Wray, C. 2000. Salmonella in domestic animals.
15. Wray, C., and W. J. Sojka. 1977. Reviews of the progress of dairy science:
bovine salmonellosis. J Dairy Res 44:383-425.
6
CHAPTER 2
LITERATURE REVIEW
Phylogeny and serotypes. Salmonella is a Gram negative bacterium which causes life threatening diseases in humans and animals worldwide. According to contemporary classification, Salmonella has two species, Salmonella bongori and
Salmonella enterica but there are more than 2,500 serovars of S. enterica. Major clinical symptoms associated with humans are enteric (typhoid) fever caused by S. enterica serovar Typhi and Paratyphi; and gastroenteritis caused by non typhoidal Salmonellae
(NTS). Salmonella may be divided in to three groups based on their association with human and animal hosts (104, 115, 123). One group is characterized by specificity for the human host. A second group consists of organisms that are usually adapted to specific animal hosts. A third group consists of un-adapted Salmonellae that cause disease in humans and a variety of animals. Most Salmonellae fall in to the third group with
Salmonella enterica serovar Typhimurium (S. Typhimurium) being the most common species across all hosts. Examples of the host-adapted Salmonellae are listed in Table 2-
1. The presence of homologues of S. Typhimurium LT2 genes was assessed in 22 other
Salmonellae including members of all seven subspecies and Salmonella bongori (99).
Genomes were hybridized to a microarray of over 97% of the 4,596 annotated ORFs in the LT2 genome (99). A phylogenetic tree based on homologue content, relative to LT2, was found to be in agreement with previous studies using sequence information from several loci (99) (Fig 2-1). Overall a high level of gene gain, loss, or rapid divergence was predicted along all lineages (99). 7
Genetics and genomics. S. Typhimurium is a leading cause of gastroenteritis and is used as a model of human typhoid fever. The incidence of non typhoid salmonellosis is increasing worldwide causing millions of infections and many deaths in human population each year. Domestic animals act as a reservoir for the host generalist serovars, which accounts for the high incidence of non typhoid salmonellosis worldwide.
Estimated cost of food-borne diseases (salmonellosis) in the United States range from 4.8 to 23 billion dollars (111).
S. Typhimurium LT2 is the principle strain for both cellular and molecular biology in Salmonella. The general characteristics of S. Typhimurium LT2 genome are summarized in Table 2 and the overall similarity of S. Typhimurium LT2 to eight other enterobacterial genomes is summarized in Table 3, (taken from (81)). The plasmid of S.
Typhimurium LT2 is called pSLT (80) and is about 90 kb (81). Out of 108 annotated
CDS and pseudogenes in this plasmid, only three have been shown to have a close homolog in S. Typhi, S. Paratyphi A or S. Paratyphi B (81). At least 204 pseudogenes have been detected in S. Typhi (97), whereas 39 pseudogenes have been detected in the S.
Typhimurium LT2 chromosome (81). It has been speculated that the restriction of S.
Typhi to growth in humans alone could be due to large numbers of pseudogenes in (97), where as the broad host range of S. Typhimurium LT2 could be due to fewer pseudogenes (81).
The course of S. Typhimurium infection in the mouse is dependent on the dose administered and on the route of inoculation (93). By oral infection approximately 180 genes are required for virulence. In contrast it has been estimated that as few as 60 genes are required for infection by the intraperitoneal (i.p.) route (14). An alternative, more 8 sensitive approach, utilizing tagged transposon, found that roughly 180 genes are required for i.p. infection (57) and 270 genes for oral infection of mice (114). Recent studies with global gene expression profiling of Salmonella during infection of macrophages and epithelial cells give a better idea of gene regulation (29, 55).
Multiple drug resistance is common in Salmonella and there is evidence that the use of antimicrobial drugs in animal feeds has contributed to selection for drug resistant
Salmonella (49). Many Salmonella isolates from farm animals have been shown to be resistant to streptomycin, tetracycline, and sulfonamides (49). Moderately high percentages have been shown to be resistant to ampicillin, kanamycin, neomycin, and chloramphenicol and low percentages have been shown to be resistant to gentamycin and trimethoprim-sulfamethazole (49). Multiple drug resistance is common in S.
Typhimurium and is usually plasmid mediated (49). Additionally, robust metabolism of
Salmonella has been reported as a limiting factor for the development of new antimicrobials (12).
Salmonellosis in animals. Generally cattle, sheep, and horses are more likely to show symptoms of salmonellosis than are pigs, poultry and dogs (123). Salmonellosis in swine usually occurs after weaning and is seen most commonly in pigs 8 -16 weeks old
(49). Pigs are frequently asymptomatic carriers (49). Oral infection of Salmonella in chickens is followed by its passage through crop and stomach, and colonization of small intestine (cecum) and ileocaecal junction (122). The next step is invasion in to intestinal walls followed by phagocytosis of Salmonella by M cells and its drainage in to liver and spleen where it survives and multiplies within the macrophages of liver and spleen (122)
(Fig 2-2). In chicken, drainage of Salmonella to liver and spleen occurs instead of 9 mesenteric lymph node in mice (122). Pullorum disease, caused by S. Pullorum, is a predominantly enteric disease of poultry and fowl typhoid, caused by S. Gallinarum is a septicemic disease of poultry (49). Both are eradicated from flocks in major poultry producing countries and clinical salmonellosis in poultry is not a common problem in most countries (49). Dogs and cats are only infrequent carriers of the organism and even less frequent victims of the disease (49). Tortoises and terrapins are commonly asymptomatic carriers that shed large numbers of Salmonella and may be a source of infection particularly for children (49). Clinical manifestations of septicemia include fever, inappetence and depression in acutely ill animals (49). Abortion often occurs in pregnant animals that develop septicemia, but certain serotypes are more prone to cause abortion (123). For example S. Dublin has been associated with abortion in cattle, and several other serovars adapted to animal hosts have a particular association with abortion
(123). Salmonellosis in horses is often associated with stress such as surgery, parasitism, and hot, humid weather (49). The high frequency of carriage of Salmonella, other than S.
Pullorum and S. Gallinarum, by poultry creates an important source of infection for humans. Transmission of infections through eggs and contamination in the environment and at shipping and slaughter are significant contributors to the problem. Though
Salmonella may survive for long periods in the environment, it is the carrier state that provides the major source of infection for animals and humans (123). Asymptomatic carriers develop as a result of the interaction of several factors including serovar of
Salmonella, the age of the animal and the number of bacteria ingested (49). Certain serovars appear to be more likely to induce the carrier state (49). Even a low dose of
Salmonella, insufficient to cause disease, may result in the carrier state (49). 10
Salmonellosis in humans. Salmonella inhabits the intestinal tracts of vertebrates and excretion results in contamination of environment (115, 123). Although human to human transmission does occur, animals and their products constitute the most important source of the organism for the humans (115). Poultry and cold-blooded animals are frequently incriminated as sources of infection. Fertilizers and feeds containing animal products are sometimes a source of infection. In recent years, unconventional food sources such as peanut butter, breakfast cereals, dry dog food and fresh produce have started to gain recognition as transmission vectors for salmonellosis in humans (6, 78).
Salmonella can enter the body through pharynx, respiratory tract or the conjunctiva but they usually gain entrance to the host by the oral route and are deposited in the intestine where they invade the enterocytes (115, 123). Large number of organisms is required to initiate disease because non specific host defense mechanisms such as gastric acidity, peristalsis, intestinal mucus, lysozyme in secretions, lactoferrin in gastrointestinal tract and the normal flora of intestine are known to affect the ability of organism to establish (49). Salmonellosis is manifested in two major forms in humans i.e. enteritis and septicemia; and abortion has been observed in animals (123). The ability of
Salmonella to survive and multiply inside phagocytes is critical to the outcome of infection. In the intracellular location the bacterium is protected from harmful substances such as antibiotics, antibody and complement (22). A proportion of bacterial population is likely to survive for a prolonged time leading to chronic infection and the carrier state
(49).
Several bacterial structures and products have been suggested as playing a role in the virulence of Salmonella. The factors that aid in survival of Salmonella in the 11 environment, under different stressful conditions, may also be considered as virulence determinants in broader sense. Fluid response and tissue damage may be the result of the toxins produced by the organisms (115). These types of bacterial virulence factors may include enterotoxins, cytotoxins, endotoxin, lipopolysaccharide, flagella and siderophores
(49). Plasmids associated with virulence have been identified in several serotypes of
Salmonella (56, 67, 108). Specific properties that are mediated by this plasmid are adhesion and invasion in the epithelial cells and serum resistance; however virulence plasmids are not essential for pathogenicity of Salmonella and many strains that are free of plasmids are recovered from outbreaks of salmonellosis (49). In some strains plasmids seem to contribute in serum resistance (56, 109, 117) and the resistance of the bacteria to be killed by macrophages (50). The virulence strategy common to Salmonella genus is adhesion, invasion, intracellular replication and bacterial dissemination from the intestinal cells. Infection with NTS serotypes results in exudative inflammation characterized by acutely increased vascular permeability and cellular infiltrates containing neutrophils. Due to recruitment of neutrophils, the infection remains localized to intestinal mucosa and mesenteric lymph nodes and leads to inflammatory diarrhea (28,
82, 87). Death may result from dehydration, electrolyte loss and acid base imbalance.
Enteric fever on the other hand, results in interstitial inflammation in the intestinal mucosa, characterized by mononuclear infiltrates (54, 72, 86, 90, 106).
Adhesion. Microbial adhesion to host tissue is the initial critical event in the pathogenesis and is an attractive target for the development of new antimicrobial therapeutics. Specific microbial components mediate adherence to host tissues by participating in interactions with host surface molecules. Microbial adhesins recognizing 12 fibronectin-, fibrogen-, collagen-, and heparin-related polysaccharides have been studied in terms of structural organization, ligand binding structure, importance in host tissue colonization, invasion, and role as virulence factor. The adhesins reported so far in the literature are proteins in the category of lectins which bind to specific sugar residues (98).
The majority of the complementary host cell receptors are the components of extra cellular matrix (ECM). The ECM is composed of a complex mixture of macromolecules which serve structural functions and also affect the cellular physiology of an organism. Interactions between eukaryotic cells and ECM components are mediated by specific cellular receptors classified in to four main groups: integrins, cadherins, members of immunoglobulin superfamily of CAMs (IgCAMs), and selectins, of which the integrins are the best characterized (15). Members of the integrin receptor families are composed of two sub units (α and β) and link many ECM proteins to the cellular cytoskeleton (98). Integrin-binding bacterial proteins include pertactin (73, 74) and filamentous hemaglutinin (100) of Bordetella pertussis and invasin of Yersinia pseudotuberculosis (64). The microbial component must also recognize macromolecular ligands such as collagen and laminin that are found exclusively in the ECM and other molecules, such as fibronectin, fibrinogen and vitronectin (adhesin ligands) which in addition to ECM, also occur in soluble forms in body fluids such as blood plasma (98).
Other potential adhesin ligands such as heparin sulfated-proteoglycans, occur both in
ECM forms and as intercalated cell membrane proteoglycans (98). Although numerous bacteria bind a variety of ECM components, the molecules involved in these interactions have not been identified nor characterized at a molecular level. 13
The ECM, in addition to serving as substrate for the adhesion of the host cells, also serves as an attachment for colonizing microorganisms. Microorganisms exploit host cell’s adhesion systems in yet another way. Some bacteria appear to use integrins to invade host cells, and many viruses use proteoglycans associated with the host cell surface for similar purposes (98). Microbial adhesins are likely virulence factors that mediate host tissue colonization, which is a prerequisite for the development of invasive infections (15). Many microorganisms have developed several adhesion mechanisms that may involve microbial lectins or adhesins. These adhesion mechanisms may operate independent of each other and adherence to a particular host ligand may set the stage for subsequent signaling cascades. This scenario appears to be particularly important when microbes invade a host tissue. In an intact healthy organism, most of the ECM is not exposed to the environment and ECM-adhesin ligands may not be accessible for interactions with microbial adhesins. However, these ligands and many other so far unknown receptors are exposed after a tissue trauma or alteration in surface structure caused by mechanical or chemical injury or cytopathic changes associated with the infections (98).
Adhesion to host cells is mediated by the fimbriae on the cell surface of S.
Typhimurium LT2 (112). This organism contains 12 putative operons of the chaperon- usher assembly class: stc, bcf, fim, lpf, saf, stb, std, stf , sth, sti and stj, all of which are located on the chromosome and pef, which is located on the plasmid. S Typhimurium
LT2 also has the cgs operon (originally called agf) for curli fimbriae (81).
Invasion. The invasion process is not merely a passive consequence of bacterial contact with epithelial cells, but instead requires the active participation of the 14 bacterium, with the expression of the numerous bacterial virulence genes (2). The expression of these genes is regulated by an array of transcriptional and post transcriptional regulators that exert intricate control over invasion (2). Penetration of the host epithelium is essential for Salmonella virulence (122). The protection against oral challenge is due to immune exclusion at mucosal surface, which prevents entry of
Salmonella in to epithelial cells (83). This implies that the blockage of mucosal invasion results in avirulence of Salmonella serovars for mice by the oral route of infection (122).
However, mutational inactivation of a single virulence factor involved in mucosal invasion results only in a modest increase of the oral LD 50 value for infection of mice
(10, 11, 43). This suggests that S. Typhimurium possesses multiple pathways for intestinal penetration (122).
The major factor for intestinal penetration is encoded by the genes clustered in area on the chromosome designated Salmonella pathogenicity island (SPI). Many SPIs have been identified so far but SPI1 and SPI2 remain crucial for infection of S.
Typhimurium. SPI1 is required for bacterial entry and SPI2 is required for intracellular survival and replication (reviewed in (51)). The genetic organization of the SPI1 and
SPI2 are shown in Fig 2-4 taken from (51). SPI1 encodes a type 3 secretion system
(T3SS) that exports proteins in response to bacterial contact with epithelial cells (44).
T3SS from SPI1 mediates the translocation of the effector and signaling proteins in the host cytosol (reviewed in (24)). The functional T3SS bypasses the surface receptors and triggers the reorganization of actin cytoskeleton. This results in membrane ruffling (122) which ultimately leads in the uptake of Salmonella and concomitant nuclear responses leading to production of pro-inflammatory cytokines (reviewed in (41, 42)). Salmonella 15 induced macropinocytosis is extremely rapid and so vigorous that inert particles and non invasive bacteria can be co-internalized with adherent bacteria. This mechanism of entry is generally referred to as ‘trigger mode of entry’ (25). Shortly after internalization, membrane ruffling subsides and the cytoskeleton resumes its normal architecture.
Internalized bacteria remain in Salmonella containing vacuole (SCV) modified to prevents its fusion with lysosomal compartment (reviewed in (16)). SPI2 also encodes a type 3 secretion system (T3SS) that functions in survival of Salmonella in SCV. SPI2 mutants have been shown with decreased survival rates in macrophages (58) and in monocyte like cell lines (20, 58, 95). Additionally they have been shown to be severely attenuated in virulence during systemic infection (103) and failed to proliferate in liver and spleen (102).
The structure and assembly of the T3SS has been reviewed in (24). Genes encoding T3SS fall into three categories, those encoding regulatory proteins, those involved in formation of the type III secretion apparatus and those encoding the secreted targets of the type 3 exporter. Additional secreted targets of the invasion associated type
3 secretion system are encoded in areas located outside of SPI1 and SPI2. These include genes located on SPI5 (119) and the spoE gene which is carried by a prophage of S.
Typhimurium (53, 84). The type 3 secretion machinery translocates SopE (Salmonella outer protein) through three biological membranes. SopE is first transported across both the cytoplasmic membrane and the outer membrane of the bacterial cell and subsequently traverses the cytoplasmic membrane of the cultured epithelial cell line (120). Once inside the cytosol, SopE induces macropinocytosis in the host cell by inducing signaling events 16 through direct interaction with small GTP-binding proteins, such as CDC42 and RAC-1
(52).
The Salmonella virulence plasmid together with Salmonella pathogenicity island
2 (SPI2) are required for systemic infection (122). A cluster of five genes (the spv operon) located on a large plasmid is essential for systemic infection in the mouse and is present in strains of Salmonella that are most likely to cause a disseminated infection, expect for S. Typhi (33, 121).
Prophages. Prophages are DNA of temperate bacteriophages residing in silent state in bacterial genome. There are several prophages present in S. Typhimurium LT2.
Among them are Fels-2 a prophage of P2 family and Gifsy-1, Gifsy-2 and Fels-1 prophages of lambda family. The role of Gifsy-1 and Gifsy-2 in Salmonella pathogenicity has been shown (34). Hermans et al. has shown the distribution of these four prophages along with additional non-LT2 prophages ST104, ST104B and ST64B (59). All the four prophages were found in other virulent strains of S. Typhimurium.
Stress response of Salmonella. Salmonella must manage stresses ranging from acidic to basic pH, high to low osmolarity, high to low temperature, various types of oxidative stress and a variety of anti-microbial compounds encountered through its journey from the natural environment to an infected host. The defenses used to survive these encounters can be specific or can provide cross protection to a variety of hostile or stress conditions. Inside the host Salmonella species escape the humoral immunity by invading professional and non professional phagocytes in which a new set of defense factors act (38). 17
The ability of bacteria to sense and respond to unexpected changes in the environment is important for their survival (38). This is true for pathogens that not only encounter potentially lethal environmental extremes in the natural environment but must also resist a battery of host defense factors (38). Under adverse cultural or environmental conditions, such as nutrient limitation, changes in temperatures, pH, etc., bacteria activate
“stress responses” that substantially improve their chances of survival in unfavorable environments (21). These responses are particularly manifested in three main areas specifically:
In response to host defense systems during infection (101).
In response to lethal or sub lethal/ inhibitory effects of chemical/physical
treatments- such as disinfectant or heat treatment (21) and
In response to conditions in preserved and minimally processed food (21).
Thus, even though the foods receive heat treatment or treatment with acids, additives or preservatives to suppress bacterial metabolism, it may be insufficient and would enable bacteria to survive and cause spoilage or food-borne illness in consumers.
Simply regulating the temperature or increasing the concentrations of the acid or preservatives is not an easy solution as consumers demand minimally processed and additive free products (1).
Bacterial response to one stress can increase resistance to other stresses (cross protection) and can change a number of other aspects of bacterial metabolism. One such effect can be increased potential to interact with hosts including the ability to invade and colonize hosts during food-borne disease (21). Thus, stress can produce more resistant, persistent and dangerous pathogens. All pathogenic bacteria possess the ability to evade 18 or surmount body defenses, long enough to cause a sufficient reaction, which is then manifested clinically as the disease or illness, while opportunistic pathogens will cause illness in the event of the predisposal weakness in these defenses. This is particularly true of gastrointestinal pathogens such as Listeria monocytogenes and Salmonella species which must circumvent many different stresses in order to arrive at the site of infection
(40). This includes the acid barrier of the stomach, the physical barrier of the epithelial cells lining the gastrointestinal tract and various immune defenses including the initial onslaught of macrophages (40). These organisms have developed elaborate systems for sensing stress and for responding to those stresses in a protective fashion.
Acid stress response. One well characterized adaptive response is to acid stress.
The acid stress response (ASR) is the complex phenomenon, involving a number of changes in the proteins expression and many associated events at the level of gene regulation. A number of molecular approaches have identified numerous interesting genes involved both in sensing and responding to stress and in virulence. Acidic pH is one of the most frequent stress condition encountered by microbial systems. Acid mine drainage, acid rain, and weak acids produced by microorganisms themselves all contribute to acid stress (38). The ability to survive and even to flourish during these conditions is crucial to survival. Some microorganisms (acidophiles) have evolved to the point where the preferred ecological niche is an extremely acidic environment (38).
S. Typhimurium is a neutrophilic bacterium that can grow over a wide range of pH conditions (pH 5-9) because of physiologically triggered pH homeostasis mechanisms
(35). Mechanisms of pH homeostasis include the use of H+ antiport systems to maintain
the internal pH at a relatively constant level (~7.6) over a wide range of external pH 19 conditions (13, 36). S. Typhimurium is capable of growing in minimal media with a pH as low as 5.0 but below pH 4.0 the cells undergo a rapid acid death apparently due to inability to maintain internal pH suitable for viability (36, 37, 61). However, recently it has been demonstrated that S. Typhimurium can be adapted to survive this harsh environment (35).
This organism is shown to have acid stress management including an inducible pH homeostasis system that functions beyond normal constitutive pH homeostasis (36).
The acidification tolerance response has been reported to occur if cells are pre-incubated
(pre-shock-adapted) at pH levels near 6 (optimum, pH 5.8) for one hour prior to exposure at pH 3.3 (acid shock). Under these conditions, survival of adapted cells is 100- to 1000 fold better than that of unadapted cells (35). In one of the studies, analysis of polypeptide profiles with polyacrylamide gel electrophoresis (PAGE) has revealed that 18 polypeptides change during pH 5.8 pre-shock exposure. Of these, 12 are induced and 6 are reported to be repressed during adaptation. This is controlled by the ferric uptake regulator, Fur (35). Thus it was found that, exposure to stress required protein synthesis and as such represented a newly described genetic response to acidic stress. It has been shown that mutations in the fur locus eliminate induction of several acid inducible genes.
They also prevent synthesis of inducible pH homeostasis system and thus confer an extremely acid sensitive phenotype on the cell. Fur has been reported to sense internal pH directly (35). A similar phenomenon has also been described for E. coli (36).
There have been reports of polyacrylamide gel analyses of acid inducible protein synthesis in both E. coli and S. Typhimurium. Protein synthesis during a shift from pH 7 to 5 was examined and it was found that 19 proteins in S. Typhimurium and 13 proteins 20 in E. coli were increased 2- to 14 fold over the controls (61). Further it was identified that
16 proteins of E. coli are produced during a shift from pH 6.9 to 4.3. These proteins were referred as acid shock proteins (ASPs) (60). Consequently, ASPs in S. Typhimurium were studied and it was found that neither the pre-shock ATR nor ASP system alone provided acid tolerance but both were reported to be induced for survival in severe acid conditions (35).
S. Typhimurium can adapt to various other environmental stresses by inducing specific set of genes (75). S. Typhimurium shows adaptive responses to acid (36), salt
(27), heat (76) and H2O2 and oxygen radicals (19). Adaptation enhances cross protection
against these stresses and may promote survival in adverse environment (21).
Acid adaptation promoted survival of S. Typhimurium in chicken meat and has been shown to enhances its survival in macrophage phagolysosomes (36). Acidification is used extensively in food processing to control growth and survival of spoilage causing microorganisms (75). The ability of food-borne pathogens including S. Typhimurium to adapt to acidic conditions and its ultimate survival is a concern in food safety (36).
Peroxide stress response. The inducible systems in bacteria, that protect against oxidative stresses are either OxyR or SoxRS regulated and are switched on by hydrogen peroxide and superoxide respectively (18). The detoxification of superoxide is a two step process in Salmonella; the first step involves the conversion of superoxide to hydrogen peroxide by superoxide dismutases, and the second step is a catalase mediated destruction of hydrogen peroxide. The consequent accumulation of hydrogen peroxide is known to activate the regulator OxyR which then leads to the transcriptional activation of the genes involved in the thioredoxin and glutaredoxin systems. These systems restore protein 21
functions by reducing oxidized residues (8). After treatment with H2O2, OxyR activates
the expression of many genes which have clear antioxidant roles. The protection against the toxic effects of peroxides is provided by hydroperoxidase I (katG) and alkyl
hydroperoxide reductase (ahpCF), which directly eliminate the oxidants (19). The cellular
thiol-disulfide balance is maintained by the induction of glutathione reductase (gorA),
glutaredoxin I (grxA) and thioredoxin 2 (trxC) (19, 107, 127). OxyR induces the
expression of the non specific DNA binding protein Dps, which protects against DNA
damage and mutation (3, 79). OxyR induction of fur, which encodes a global repressor,
generated by H2O2 reacting of ferric ion uptake, should prevent damage caused by HO٠ with intracellular iron (the Fenton reaction) (128). OxyR activation also leads to high level of small RNA denoted OxyS (4). This protects against mutagenesis, the mechanism of which is unknown. The OxyS RNA also activates and represses the expression of numerous genes in E. coli. Another role of OxyS is to integrate the adaptive response to
H2O2 with other cellular stress responses. For both rpoS and fhlA, OxyS acts to repress
translation (5, 126). Several of the activities whose expression is induced by SoxRS in
- generating compounds have roles in a defense against – response to treatment with O2٠
oxidative stress. These include the manganese superoxide dismutase (sodA), the DNA
repair enzyme endonuclease IV (nfo) and glucose-6-phosphate dehydrogenase (zwf)
which increases the reducing power of the cell (47, 113). The expression of some
activities that clearly protect against H2O2 is not under the control of OxyR. These include hydroperoxidase II (katE) and the DNA repair enzymes exonuclease III (xthA),
DNA polymerase I (polA), and RecA (63, 77). The OxyR and SoxRS significantly overlap with other regulatory networks. The rpoS encoded σS subunit of RNA 22 polymerase is important for the expression of the large number of genes that are induced when cells enter in to stationary phase or encounter a number of different stresses including starvation, osmotic stress and acid stress. Starved and stationary phase cells are
S intrinsically resistant to variety of stress conditions including high levels of H2O2 and σ regulates the expression of several antioxidant genes including katE, xthA, and sodC (23,
48). The bacterial killing by macrophages is mainly due to respiratory burst. This raises the question of whether the oxyR and soxRS regulons play a role in virulence. E. coli soxRS mutants are hyper susceptible to killing by murine macrophages (91, 92). In addition, several of the genes of the oxyR regulon in S. Typhimurium have been found to be induced by the macrophage environment, including ahpCF and dps (39, 116). S.
Typhimurium strains carrying mutations in slyA (a transcriptional regulator), rpoS, and recA (a positive regulator of the SOS DNA repair pathway) showed increased sensitivity to reactive oxygen species as well as attenuated virulence in mice (7, 9).
Cold stress response. Temperature is one of the major stresses faced by all living organisms. As refrigeration is commonly used method for extending the shelf life of food, it is necessary to understand the cold stress response of the food-borne pathogens.
S. Typhimurium is a major food-borne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans (62). Following processing poultry products are typically stored under refrigerated conditions until consumed. Therefore, the ability of this pathogen to survive at cold temperatures is of concern. Also, this pathogen has been reported to survive in food products held at cold storage at 5°C for up to 8 months and can cause diarrhea in humans if contaminated food is consumed raw or undercooked (62). 23
S. Typhimurium cultures, when transferred from 37C to 5C or 10C, showed an initial lag period in growth with an approximate generation time of 10 to 25 h (62). The changes in cellular physiology and changes in protein expression profiles in response to cold temperatures have been demonstrated in a number of microorganisms (68) including
E. coli (45, 89), S. enteritidis (65), and Vibrio vulnificus (17). The ability of these microorganisms to survive in cold temperatures requires proteins termed cold shock proteins (CSPs), which are synthesized at the onset of cold temperature shift (68, 96,
110). The cspA gene that codes for major cold shock protein, CS7.4, has been cloned, sequenced and characterized in E. coli (45, 88) and S. enteritidis (65).
Most bacteria react against a sudden decrease in temperature, a cold shock response (from 30C to 10C) (46). During the first phase called acclimation phase, cold shock proteins are synthesized, and subsequently the cells resume growth (62, 69, 70).
CSP genes from Salmonella species, cspA, cspB, cspC, cspE, and cspH, are sequenced and cold shock inducibility of cspA and cspB has been reported (26, 62, 65). CSPs in E. coli have been extensively studied, and cspA is a major CSP (45). Other CSPs, cspB-I, which are collectively known as the cspA family, are known to be present in E. coli. .
Previously it was reported that the expression of cspA and cspB in Salmonella species was slightly different from that in E. coli (46). All 9 CSPs in E. coli are not inducible by a temperature down shift (124, 125). For cspH, neither its expression nor its regulation in
E. coli and S. Typhimurium has been investigated.
Cold shock genes such as cspA, cspB, cspG and cspI have been shown to have an
unusually long 5’ – untranslated region (5’- UTR) (32, 66, 85, 118). In S. Typhimirium, the 5’- UTR of cspA is 145 nucleotides and that of cspB is 162 nucleotides (71). For E. 24 coli, cspA has 159 nucleotides, cspB has 161 nucleotides, cspG has 156 nucleotides and cspI has 145 bp (71). In E.coli, the 5’-UTR are known to be involved in the regulation of their expression (118). The 5’-UTRs of the cspA and cspI mRNAs have a negative effect on the expression of genes at 37C, while they have a positive effect at cold temperatures
(66, 118). In S. enterica serovar Typhimurium the role of the 5’-UTR has not yet been characterized (71).
The downstream box (DB) located in the downstream of AUG initiation codon enhances translation of several bacterial and phage mRNAs and it is also required for efficient translation of CSPs during cold shock (30, 31, 85). It is stated that the DB enhances translation by base pairing transiently to bases 1469 to 1483 of 16s rRNA, the so called anti-DB, during the initiation phase of translation (105). Contrary to this it was reported that the enhancement of translation by the DB did not involve base pairing of mRNA with the stem sequence of 16s rRNA (94). However, a mechanism of translational activation by the DB is not yet known (71).
Few organisms cope as well as S. Typhimurium with the breadth of stresses offered by both niches i.e. host and environment. The way microorganisms survive during periods of environmental stress and still retain potential to cause huge outbreaks, is an exciting area of modern biology to explore. This will lead into new insights and understanding of the mechanisms which will help develop strategies.
HYPOTHESIS
Cold stress increases the pathogenicity of S. Typhimurium in cell culture model.
25
OBJECTIVES
1. Determine influence of cold stress, on the ability of S. Typhimurium to survive during
subsequent acid stress, which would be encountered during gastric transit.
2. Determine the effect of cold stress on virulence of S. Typhimurium during infection
of intestinal epithelial cells.
3. Determine the effect of cold stress on the metabolism of S. Typhimurium during
infection of intestinal epithelial cells.
REFERENCES
1. Abee, T., and J. A. Wouters. 1999. Microbial stress response in minimal
processing. Int J Food Microbiol 50:65-91.
2. Altier, C. 2005. Genetic and environmental control of Salmonella invasion. J
Microbiol 43 Spec No:85-92.
3. Altuvia, S., M. Almiron, G. Huisman, R. Kolter, and G. Storz. 1994. The dps
promoter is activated by OxyR during growth and by IHF and sigma S in
stationary phase. Mol Microbiol 13:265-72.
4. Altuvia, S., D. Weinstein-Fischer, A. Zhang, L. Postow, and G. Storz. 1997. A
small, stable RNA induced by oxidative stress: role as a pleiotropic regulator and
antimutator. Cell 90:43-53.
5. Altuvia, S., A. Zhang, L. Argaman, A. Tiwari, and G. Storz. 1998. The
Escherichia coli OxyS regulatory RNA represses fhlA translation by blocking
ribosome binding. Embo J 17:6069-75. 26
6. Andrews-Polymenis, H. L., A. J. Baumler, B. A. McCormick, and F. C. Fang.
2010. Taming the elephant: Salmonella biology, pathogenesis, and prevention.
Infect Immun 78:2356-69.
7. Arnqvist, A., A. Olsen, and S. Normark. 1994. Sigma S-dependent growth-
phase induction of the csgBA promoter in Escherichia coli can be achieved in
vivo by sigma 70 in the absence of the nucleoid-associated protein H-NS. Mol
Microbiol 13:1021-32.
8. Aslund, F., and J. Beckwith. 1999. The thioredoxin superfamily: redundancy,
specificity, and gray-area genomics. J Bacteriol 181:1375-9.
9. Atlung, T., and L. Brondsted. 1994. Role of the transcriptional activator AppY
in regulation of the cyx appA operon of Escherichia coli by anaerobiosis,
phosphate starvation, and growth phase. J Bacteriol 176:5414-22.
10. Baumler, A. J., R. M. Tsolis, and F. Heffron. 1996. The lpf fimbrial operon
mediates adhesion of Salmonella typhimurium to murine Peyer's patches. Proc
Natl Acad Sci USA 93:279-83.
11. Baumler, A. J., R. M. Tsolis, P. J. Valentine, T. A. Ficht, and F. Heffron.
1997. Synergistic effect of mutations in invA and lpfC on the ability of
Salmonella typhimurium to cause murine typhoid. Infect Immun 65:2254-9.
12. Becker, D., M. Selbach, C. Rollenhagen, M. Ballmaier, T. F. Meyer, M.
Mann, and D. Bumann. 2006. Robust Salmonella metabolism limits possibilities
for new antimicrobials. Nature 440:303-7.
13. Booth, I. R. 1985. Regulation of cytoplasmic pH in bacteria. Microbiol Rev
49:359-78. 27
14. Bowe, F., C. J. Lipps, R. M. Tsolis, E. Groisman, F. Heffron, and J. G.
Kusters. 1998. At least four percent of the Salmonella typhimurium genome is
required for fatal infection of mice. Infect Immun 66:3372-7.
15. Boyle, E. C., and B. B. Finlay. 2003. Bacterial pathogenesis: exploiting cellular
adherence. Curr Opin Cell Biol 15:633-9.
16. Brumell, J. H., and S. Grinstein. 2004. Salmonella redirects phagosomal
maturation. Curr Opin Microbiol 7:78-84.
17. Bryan, P. J., R. J. Steffan, A. DePaola, J. W. Foster, and A. K. Bej. 1999.
Adaptive response to cold temperatures in Vibrio vulnificus. Curr Microbiol
38:168-75.
18. Carmel-Harel, O., and G. Storz. 2000. Roles of the glutathione- and
thioredoxin-dependent reduction systems in the Escherichia coli and
saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol
54:439-61.
19. Christman, M. F., R. W. Morgan, F. S. Jacobson, and B. N. Ames. 1985.
Positive control of a regulon for defenses against oxidative stress and some heat-
shock proteins in Salmonella typhimurium. Cell 41:753-62.
20. Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998.
Macrophage-dependent induction of the Salmonella pathogenicity island 2 type
III secretion system and its role in intracellular survival. Mol Microbiol 30:175-
88.
21. CJ, D. 1994. Genetics of Bacterial Virulence, vol. Blackwell Scientific. London. 28
22. Collins, F. M., and S. G. Campbell. 1982. Immunity to intracellular bacteria.
Vet Immunol Immunopathol 3:5-66.
23. Conter, A., C. Menchon, and C. Gutierrez. 1997. Role of DNA supercoiling
and rpoS sigma factor in the osmotic and growth phase-dependent induction of
the gene osmE of Escherichia coli K12. J Mol Biol 273:75-83.
24. Cornelis, G. R. 2006. The type III secretion injectisome. Nat Rev Microbiol
4:811-25.
25. Cossart, P. 2004. Bacterial invasion: a new strategy to dominate cytoskeleton
plasticity. Dev Cell 6:314-5.
26. Craig, J. E., D. Boyle, K. P. Francis, and M. P. Gallagher. 1998. Expression of
the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold
temperature. Microbiology 144 ( Pt 3):697-704.
27. Csonka, L. N. 1989. Physiological and genetic responses of bacteria to osmotic
stress. Microbiol Rev 53:121-47.
28. Day, D. W., B. K. Mandal, and B. C. Morson. 1978. The rectal biopsy
appearances in Salmonella colitis. Histopathology 2:117-31.
29. Eriksson, S., S. Lucchini, A. Thompson, M. Rhen, and J. C. Hinton. 2003.
Unravelling the biology of macrophage infection by gene expression profiling of
intracellular Salmonella enterica. Mol Microbiol 47:103-18.
30. Etchegaray, J. P., and M. Inouye. 1999. A sequence downstream of the
initiation codon is essential for cold shock induction of cspB of Escherichia coli. J
Bacteriol 181:5852-4. 29
31. Etchegaray, J. P., and M. Inouye. 1999. Translational enhancement by an
element downstream of the initiation codon in Escherichia coli. J Biol Chem
274:10079-85.
32. Fang, L., Y. Hou, and M. Inouye. 1998. Role of the cold-box region in the 5'
untranslated region of the cspA mRNA in its transient expression at low
temperature in Escherichia coli. J Bacteriol 180:90-5.
33. Fierer, J., M. Krause, R. Tauxe, and D. Guiney. 1992. Salmonella
typhimurium bacteremia: association with the virulence plasmid. J Infect Dis
166:639-42.
34. Figueroa-Bossi, N., and L. Bossi. 1999. Inducible prophages contribute to
Salmonella virulence in mice. Mol Microbiol 33:167-76.
35. Foster, J. W. 1991. Salmonella acid shock proteins are required for the adaptive
acid tolerance response. J Bacteriol 173:6896-902.
36. Foster, J. W., and H. K. Hall. 1990. Adaptive acidification tolerance response of
Salmonella typhimurium. J Bacteriol 172:771-8.
37. Foster, J. W., and H. K. Hall. 1991. Inducible pH homeostasis and the acid
tolerance response of Salmonella typhimurium. J Bacteriol 173:5129-35.
38. Foster, J. W., and M. P. Spector. 1995. How Salmonella survive against the
odds. Annu Rev Microbiol 49:145-74.
39. Francis, K. P., P. D. Taylor, C. J. Inchley, and M. P. Gallagher. 1997.
Identification of the ahp operon of Salmonella typhimurium as a macrophage-
induced locus. J Bacteriol 179:4046-8. 30
40. Gahan, C. G., and C. Hill. 1999. The relationship between acid stress responses
and virulence in Salmonella typhimurium and Listeria monocytogenes. Int J Food
Microbiol 50:93-100.
41. Galan, J. E. 1996. Molecular genetic bases of Salmonella entry into host cells.
Mol Microbiol 20:263-71.
42. Galan, J. E. 1994. Salmonella entry into mammalian cells: different yet
converging signal transduction pathways? Trends Cell Biol 4:196-9.
43. Galan, J. E., and R. Curtiss, 3rd. 1989. Cloning and molecular characterization
of genes whose products allow Salmonella typhimurium to penetrate tissue
culture cells. Proc Natl Acad Sci USA 86:6383-7.
44. Ginocchio, C. C., S. B. Olmsted, C. L. Wells, and J. E. Galan. 1994. Contact
with epithelial cells induces the formation of surface appendages on Salmonella
typhimurium. Cell 76:717-24.
45. Goldstein, J., N. S. Pollitt, and M. Inouye. 1990. Major cold shock protein of
Escherichia coli. Proc Natl Acad Sci USA 87:283-7.
46. Graumann, P., and M. A. Marahiel. 1996. Some like it cold: response of
microorganisms to cold shock. Arch Microbiol 166:293-300.
47. Greenberg, J. T., P. Monach, J. H. Chou, P. D. Josephy, and B. Demple.
1990. Positive control of a global antioxidant defense regulon activated by
superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci USA
87:6181-5. 31
48. Groat, R. G., J. E. Schultz, E. Zychlinsky, A. Bockman, and A. Matin. 1986.
Starvation proteins in Escherichia coli: kinetics of synthesis and role in starvation
survival. J Bacteriol 168:486-93.
49. Gyles, C. T., CO. 1986. Pathogenesis of bacterial infections in animals.
50. Hackett, J., I. Kotlarski, V. Mathan, K. Francki, and D. Rowley. 1986. The
colonization of Peyer's patches by a strain of Salmonella typhimurium cured of
the cryptic plasmid. J Infect Dis 153:1119-25.
51. Hansen-Wester, I., and M. Hensel. 2001. Salmonella pathogenicity islands
encoding type III secretion systems. Microbes Infect 3:549-59.
52. Hardt, W. D., L. M. Chen, K. E. Schuebel, X. R. Bustelo, and J. E. Galan.
1998. S. typhimurium encodes an activator of Rho GTPases that induces
membrane ruffling and nuclear responses in host cells. Cell 93:815-26.
53. Hardt, W. D., H. Urlaub, and J. E. Galan. 1998. A substrate of the centisome
63 type III protein secretion system of Salmonella typhimurium is encoded by a
cryptic bacteriophage. Proc Natl Acad Sci USA 95:2574-9.
54. Harris, J. C., H. L. Dupont, and R. B. Hornick. 1972. Fecal leukocytes in
diarrheal illness. Ann Intern Med 76:697-703.
55. Hautefort, I., A. Thompson, S. Eriksson-Ygberg, M. L. Parker, S. Lucchini,
V. Danino, R. J. Bongaerts, N. Ahmad, M. Rhen, and J. C. Hinton. 2008.
During infection of epithelial cells Salmonella enterica serovar Typhimurium
undergoes a time-dependent transcriptional adaptation that results in simultaneous
expression of three type 3 secretion systems. Cell Microbiol 10:958-84. 32
56. Helmuth, R., R. Stephan, C. Bunge, B. Hoog, A. Steinbeck, and E. Bulling.
1985. Epidemiology of virulence-associated plasmids and outer membrane
protein patterns within seven common Salmonella serotypes. Infect Immun
48:175-82.
57. Hensel, M., J. E. Shea, C. Gleeson, M. D. Jones, E. Dalton, and D. W. Holden.
1995. Simultaneous identification of bacterial virulence genes by negative
selection. Science 269:400-3.
58. Hensel, M., J. E. Shea, S. R. Waterman, R. Mundy, T. Nikolaus, G. Banks, A.
Vazquez-Torres, C. Gleeson, F. C. Fang, and D. W. Holden. 1998. Genes
encoding putative effector proteins of the type III secretion system of Salmonella
pathogenicity island 2 are required for bacterial virulence and proliferation in
macrophages. Mol Microbiol 30:163-74.
59. Hermans, A. P., A. M. Beuling, A. H. van Hoek, H. J. Aarts, T. Abee, and M.
H. Zwietering. 2006. Distribution of prophages and SGI-1 antibiotic-resistance
genes among different Salmonella enterica serovar Typhimurium isolates.
Microbiology 152:2137-47.
60. Heyde, M., and R. Portalier. 1990. Acid shock proteins of Escherichia coli.
FEMS Microbiol Lett 57:19-26.
61. Hickey, E. W., and I. N. Hirshfield. 1990. Low-pH-induced effects on patterns
of protein synthesis and on internal pH in Escherichia coli and Salmonella
typhimurium. Appl Environ Microbiol 56:1038-45. 33
62. Horton, A. J., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 2000.
Adaptive response to cold temperatures and characterization of cspA in
Salmonella typhimurium LT2. Antonie Van Leeuwenhoek 77:13-20.
63. Imlay, J. A., and S. Linn. 1988. DNA damage and oxygen radical toxicity.
Science 240:1302-9.
64. Isberg, R. R., D. L. Voorhis, and S. Falkow. 1987. Identification of invasin: a
protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell
50:769-78.
65. Jeffreys, A. G., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 1998.
Growth, survival and characterization of cspA in Salmonella enteritidis following
cold shock. Curr Microbiol 36:29-35.
66. Jiang, W., L. Fang, and M. Inouye. 1996. The role of the 5'-end untranslated
region of the mRNA for CspA, the major cold-shock protein of Escherichia coli,
in cold-shock adaptation. J Bacteriol 178:4919-25.
67. Jones, G. W., D. K. Rabert, D. M. Svinarich, and H. J. Whitfield. 1982.
Association of adhesive, invasive, and virulent phenotypes of Salmonella
typhimurium with autonomous 60-megadalton plasmids. Infect Immun 38:476-
86.
68. Jones, P. G., and M. Inouye. 1994. The cold-shock response--a hot topic. Mol
Microbiol 11:811-8.
69. Jones, P. G., and M. Inouye. 1996. RbfA, a 30S ribosomal binding factor, is a
cold-shock protein whose absence triggers the cold-shock response. Mol
Microbiol 21:1207-18. 34
70. Jones, P. G., R. A. VanBogelen, and F. C. Neidhardt. 1987. Induction of
proteins in response to low temperature in Escherichia coli. J Bacteriol 169:2092-
5.
71. Kim, B. H., I. S. Bang, S. Y. Lee, S. K. Hong, S. H. Bang, I. S. Lee, and Y. K.
Park. 2001. Expression of cspH, encoding the cold shock protein in Salmonella
enterica serovar Typhimurium UK-1. J Bacteriol 183:5580-8.
72. Kraus, M. D., B. Amatya, and Y. Kimula. 1999. Histopathology of typhoid
enteritis: morphologic and immunophenotypic findings. Mod Pathol 12:949-55.
73. Leininger, E., C. A. Ewanowich, A. Bhargava, M. S. Peppler, J. G. Kenimer,
and M. J. Brennan. 1992. Comparative roles of the Arg-Gly-Asp sequence
present in the Bordetella pertussis adhesins pertactin and filamentous
hemagglutinin. Infect Immun 60:2380-5.
74. Leininger, E., M. Roberts, J. G. Kenimer, I. G. Charles, N. Fairweather, P.
Novotny, and M. J. Brennan. 1991. Pertactin, an Arg-Gly-Asp-containing
Bordetella pertussis surface protein that promotes adherence of mammalian cells.
Proc Natl Acad Sci USA 88:345-9.
75. Leyer, G. J., and E. A. Johnson. 1993. Acid adaptation induces cross-protection
against environmental stresses in Salmonella typhimurium. Appl Environ
Microbiol 59:1842-7.
76. Lindquist, S. 1986. The heat-shock response. Annu Rev Biochem 55:1151-91.
77. Loewen, P. C., B. Hu, J. Strutinsky, and R. Sparling. 1998. Regulation in the
rpoS regulon of Escherichia coli. Can J Microbiol 44:707-17. 35
78. Maki, D. G. 2009. Coming to grips with foodborne infection--peanut butter,
peppers, and nationwide Salmonella outbreaks. N Engl J Med 360:949-53.
79. Martinez, A., and R. Kolter. 1997. Protection of DNA during oxidative stress by
the nonspecific DNA-binding protein Dps. J Bacteriol 179:5188-94.
80. Matsui, H., C. M. Bacot, W. A. Garlington, T. J. Doyle, S. Roberts, and P. A.
Gulig. 2001. Virulence plasmid-borne spvB and spvC genes can replace the 90-
kilobase plasmid in conferring virulence to Salmonella enterica serovar
Typhimurium in subcutaneously inoculated mice. J Bacteriol 183:4652-8.
81. McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L.
Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S.
Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan,
H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R.
K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar
Typhimurium LT2. Nature 413:852-6.
82. McGovern, V. J., and L. J. Slavutin. 1979. Pathology of Salmonella colitis. Am
J Surg Pathol 3:483-90.
83. Michetti, P., N. Porta, M. J. Mahan, J. M. Slauch, J. J. Mekalanos, A. L.
Blum, J. P. Kraehenbuhl, and M. R. Neutra. 1994. Monoclonal
immunoglobulin A prevents adherence and invasion of polarized epithelial cell
monolayers by Salmonella typhimurium. Gastroenterology 107:915-23.
84. Mirold, S., W. Rabsch, M. Rohde, S. Stender, H. Tschape, H. Russmann, E.
Igwe, and W. D. Hardt. 1999. Isolation of a temperate bacteriophage encoding 36
the type III effector protein SopE from an epidemic Salmonella typhimurium
strain. Proc Natl Acad Sci USA 96:9845-50.
85. Mitta, M., L. Fang, and M. Inouye. 1997. Deletion analysis of cspA of
Escherichia coli: requirement of the AT-rich UP element for cspA transcription
and the downstream box in the coding region for its cold shock induction. Mol
Microbiol 26:321-35.
86. Mukawi, T. J. 1978. Histopathological study of typhoid perforation of the small
intestines. Southeast Asian J Trop Med Public Health 9:252-5.
87. Murphy, T. F., and S. L. Gorbach. 1982. Salmonella colitis. N Y State J Med
82:1236-8.
88. Newkirk, K., W. Feng, W. Jiang, R. Tejero, S. D. Emerson, M. Inouye, and
G. T. Montelione. 1994. Solution NMR structure of the major cold shock protein
(CspA) from Escherichia coli: identification of a binding epitope for DNA. Proc
Natl Acad Sci U S A 91:5114-8.
89. Ng, H., J. L. Ingraham, and A. G. Marr. 1962. Damage and derepression in
Escherichia coli resulting from growth at low temperatures. J Bacteriol 84:331-9.
90. Nguyen, Q. C., P. Everest, T. K. Tran, D. House, S. Murch, C. Parry, P.
Connerton, V. B. Phan, S. D. To, P. Mastroeni, N. J. White, T. H. Tran, V. H.
Vo, G. Dougan, J. J. Farrar, and J. Wain. 2004. A clinical, microbiological,
and pathological study of intestinal perforation associated with typhoid fever. Clin
Infect Dis 39:61-7. 37
91. Nunoshiba, T., T. DeRojas-Walker, S. R. Tannenbaum, and B. Demple. 1995.
Roles of nitric oxide in inducible resistance of Escherichia coli to activated
murine macrophages. Infect Immun 63:794-8.
92. Nunoshiba, T., T. deRojas-Walker, J. S. Wishnok, S. R. Tannenbaum, and B.
Demple. 1993. Activation by nitric oxide of an oxidative-stress response that
defends Escherichia coli against activated macrophages. Proc Natl Acad Sci USA
90:9993-7.
93. O'Brien, A. D. 1986. Influence of host genes on resistance of inbred mice to
lethal infection with Salmonella typhimurium. Curr Top Microbiol Immunol
124:37-48.
94. O'Connor, M., T. Asai, C. L. Squires, and A. E. Dahlberg. 1999. Enhancement
of translation by the downstream box does not involve base pairing of mRNA
with the penultimate stem sequence of 16S rRNA. Proc Natl Acad Sci USA
96:8973-8.
95. Ochman, H., F. C. Soncini, F. Solomon, and E. A. Groisman. 1996.
Identification of a pathogenicity island required for Salmonella survival in host
cells. Proc Natl Acad Sci USA 93:7800-4.
96. Panoff, J. M., B. Thammavongs, M. Gueguen, and P. Boutibonnes. 1998.
Cold stress responses in mesophilic bacteria. Cryobiology 36:75-83.
97. Parkhill, J., G. Dougan, K. D. James, N. R. Thomson, D. Pickard, J. Wain, C.
Churcher, K. L. Mungall, S. D. Bentley, M. T. Holden, M. Sebaihia, S. Baker,
D. Basham, K. Brooks, T. Chillingworth, P. Connerton, A. Cronin, P. Davis,
R. M. Davies, L. Dowd, N. White, J. Farrar, T. Feltwell, N. Hamlin, A. 38
Haque, T. T. Hien, S. Holroyd, K. Jagels, A. Krogh, T. S. Larsen, S. Leather,
S. Moule, P. O'Gaora, C. Parry, M. Quail, K. Rutherford, M. Simmonds, J.
Skelton, K. Stevens, S. Whitehead, and B. G. Barrell. 2001. Complete genome
sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18.
Nature 413:848-52.
98. Patti, J. M., B. L. Allen, M. J. McGavin, and M. Hook. 1994. MSCRAMM-
mediated adherence of microorganisms to host tissues. Annu Rev Microbiol
48:585-617.
99. Porwollik, S., R. M. Wong, and M. McClelland. 2002. Evolutionary genomics
of Salmonella: gene acquisitions revealed by microarray analysis. Proc Natl Acad
Sci USA 99:8956-61.
100. Relman, D., E. Tuomanen, S. Falkow, D. T. Golenbock, K. Saukkonen, and
S. D. Wright. 1990. Recognition of a bacterial adhesion by an integrin:
macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous
hemagglutinin of Bordetella pertussis. Cell 61:1375-82.
101. Rouquette, C., C. de Chastellier, S. Nair, and P. Berche. 1998. The ClpC
ATPase of Listeria monocytogenes is a general stress protein required for
virulence and promoting early bacterial escape from the phagosome of
macrophages. Mol Microbiol 27:1235-45.
102. Shea, J. E., C. R. Beuzon, C. Gleeson, R. Mundy, and D. W. Holden. 1999.
Influence of the Salmonella typhimurium pathogenicity island 2 type III secretion
system on bacterial growth in the mouse. Infect Immun 67:213-9. 39
103. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of a
virulence locus encoding a second type III secretion system in Salmonella
typhimurium. Proc Natl Acad Sci USA 93:2593-7.
104. Smith, H. W., and S. Halls. 1968. The simultaneous oral administration of
Salmonella dublin, S. typhimurium and S. choleraesuis to calves and other
animals. J Med Microbiol 1:203-9.
105. Sprengart, M. L., E. Fuchs, and A. G. Porter. 1996. The downstream box: an
efficient and independent translation initiation signal in Escherichia coli. Embo J
15:665-74.
106. Sprinz, H., E. J. Gangarosa, M. Williams, R. B. Hornick, and T. E.
Woodward. 1966. Histopathology of the upper small intestines in typhoid fever.
Biopsy study of experimental disease in man. Am J Dig Dis 11:615-24.
107. Tao, K. 1997. oxyR-dependent induction of Escherichia coli grx gene expression
by peroxide stress. J Bacteriol 179:5967-70.
108. Terakado, N., T. Sekizaki, K. Hashimoto, and S. Naitoh. 1983. Correlation
between the presence of a fifty-megadalton plasmid in Salmonella dublin and
virulence for mice. Infect Immun 41:443-4.
109. Terakado, N., T. Ushijima, T. Samejima, H. Ito, T. Hamaoka, S. Murayama,
K. Kawahara, and H. Danbara. 1990. Transposon insertion mutagenesis of a
genetic region encoding serum resistance in an 80 kb plasmid of Salmonella
dublin. J Gen Microbiol 136:1833-8.
110. Thieringer, H. A., P. G. Jones, and M. Inouye. 1998. Cold shock and
adaptation. Bioessays 20:49-57. 40
111. Todd, E. 1990. Epidemiology of foodborne illness: North America. Lancet
336:788-90.
112. Townsend, S. M., N. E. Kramer, R. Edwards, S. Baker, N. Hamlin, M.
Simmonds, K. Stevens, S. Maloy, J. Parkhill, G. Dougan, and A. J. Baumler.
2001. Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial
gene sequences. Infect Immun 69:2894-901.
113. Tsaneva, I. R., and B. Weiss. 1990. soxR, a locus governing a superoxide
response regulon in Escherichia coli K-12. J Bacteriol 172:4197-205.
114. Tsolis, R. M., S. M. Townsend, E. A. Miao, S. I. Miller, T. A. Ficht, L. G.
Adams, and A. J. Baumler. 1999. Identification of a putative Salmonella
enterica serotype typhimurium host range factor with homology to IpaH and
YopM by signature-tagged mutagenesis. Infect Immun 67:6385-93.
115. Turnbull, P. C. 1979. Food poisoning with special reference to Salmonella -- its
epidemiology, pathogenesis and control. Clin Gastroenterol 8:663-714.
116. Valdivia, R. H., and S. Falkow. 1996. Bacterial genetics by flow cytometry:
rapid isolation of Salmonella typhimurium acid-inducible promoters by
differential fluorescence induction. Mol Microbiol 22:367-78.
117. Vandenbosch, J. L., D. K. Rabert, and G. W. Jones. 1987. Plasmid-associated
resistance of Salmonella typhimurium to complement activated by the classical
pathway. Infect Immun 55:2645-52.
118. Wang, N., K. Yamanaka, and M. Inouye. 1999. CspI, the ninth member of the
CspA family of Escherichia coli, is induced upon cold shock. J Bacteriol
181:1603-9. 41
119. Wood, M. W., M. A. Jones, P. R. Watson, S. Hedges, T. S. Wallis, and E. E.
Galyov. 1998. Identification of a pathogenicity island required for Salmonella
enteropathogenicity. Mol Microbiol 29:883-91.
120. Wood, M. W., R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov.
1996. SopE, a secreted protein of Salmonella dublin, is translocated into the target
eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry. Mol
Microbiol 22:327-38.
121. Woodward, M. J., I. McLaren, and C. Wray. 1989. Distribution of virulence
plasmids within Salmonellae. J Gen Microbiol 135:503-11.
122. Wray, C. 2000. Salmonella in domestic animals.
123. Wray, C., and W. J. Sojka. 1977. Reviews of the progress of dairy science:
bovine salmonellosis. J Dairy Res 44:383-425.
124. Yamanaka, K., and M. Inouye. 1997. Growth-phase-dependent expression of
cspD, encoding a member of the CspA family in Escherichia coli. J Bacteriol
179:5126-30.
125. Yamanaka, K., T. Mitani, T. Ogura, H. Niki, and S. Hiraga. 1994. Cloning,
sequencing, and characterization of multicopy suppressors of a mukB mutation in
Escherichia coli. Mol Microbiol 13:301-12.
126. Zhang, A., S. Altuvia, A. Tiwari, L. Argaman, R. Hengge-Aronis, and G.
Storz. 1998. The OxyS regulatory RNA represses rpoS translation and binds the
Hfq (HF-I) protein. Embo J 17:6061-8.
127. Zheng, M., F. Aslund, and G. Storz. 1998. Activation of the OxyR transcription
factor by reversible disulfide bond formation. Science 279:1718-21. 42
128. Zheng, M., B. Doan, T. D. Schneider, and G. Storz. 1999. OxyR and SoxRS
regulation of fur. J Bacteriol 181:4639-43.
43
TABLE 2-1. Examples of host-adapted Salmonella (49).
Host Serovar Human S. typhi, S. paratyphi, S.schottmuelleri, S. hirschfeldii, S. sendai Cattle S. dublin Swine S. cholerae-suis, S. typhisuis Poultry S. pullorum, S. gallinarum Sheep S. abortus-ovis Horse S. abortus-equi
44
TABLE 2-2. Genome statistics of S. Typhimurium LT2 adapted from (81).
Parameter Chromosome Plasmid pSLT
Size (bp) 4,857,432 93,939 G + C content 53% 53% rRNA clusters 7 0 tRNAs 85 0 tRNA pseudogene 1 0 Structural RNAs 11 1 CDS (including pseudogenes) 4,489 108 CDS pseudogenes 39 6
45
TABLE 2-3. Coding sequence homologues shared by S. Typhimurium LT2 and eight other strains of enterobacteria adapted from (81).
Median homology of reciprocal best hit CDS (%) Homologues Amino Organism Data source of DNA acid STM CDS (%) Salmonella Typhimurium LT2 Complete sequence 100 100 100 Salmonella Typhi CT18 Complete sequence 89 98 99% ~97% sequence Salmonella Paratyphi A Sample - 87/89 98 99 Microarray Salmonella Paratyphi B Microarray 92 - - Salmonella Arizonae Microarray 83 - - Salmonella bongori Microarray 85 - - Escherichia coli K12 Complete sequence 71 80 90 Escherichia coli O157:H7 Complete sequence 73 80 89 ~97% sequence Klebsiella pneumoniae Sample 73 76 88
46
FIG 2-1. Phylogenetic tree of the Salmonella clade adapted from (99). Five crucial stages in Salmonella evolution are indicated: 1. Divergence of Salmonella from E. coli; 2.
Separation of S. enterica from S. bongori; 3. Evolution of the diphasic S. enterica strains;
4. Partition of S. enterica ssp. I; 5. Development of Salmonella Typhimurium (99).
47
FIG 2-2. Colonization of murine Peyer’s patches by S. Typhimurium, adapted from
(122). Cross section of the intestinal wall at the area of the Peyer’s patch showing a lymph follicle (center) and part of an intestinal villus (left). Consecutive stages of Peyer’s patch colonization and S. enterica virulence genes required during infection are described in boxes 1-6.
48
FIG 2-3. Genetic organization of SPI1 and SPI2 adapted from (51). Several genes in SPI1 and SPI2 have different designations: sprA = hilC = sirC; sipBCDA = sspBCDA; ssaB = spiC; ssaC = spiA; ssaD = sipB; ssrA = spiR. The functional classes of SPI1 and SPI2 genes are represented by different colors.
49
CHAPTER 3
COLD STRESS TO S. TYPHIMURIUM INCREASES ITS SURVIVAL DURING
SUBSEQUENT ACID STRESS
ABSTRACT
Salmonella is an important food-borne pathogen that causes gastroenteritis. The mechanisms that enable Salmonella to persist in food and the environment to cause disease in humans are not clearly established. Salmonella encounters multiple abiotic stresses during zoonotic transmission that influence survival. This study tested the hypothesis that abiotic stresses regulate mechanisms that enable survival and persistence.
The response of Salmonella enterica serovar Typhimurium (S. Typhimurium) was determined using cold (5°C), peroxide (1 mM H2O2), osmotic (5% NaCl), and acid (pH
5.3) shock (30 min exposure) or stress (5 h exposure) alone and followed by acid (pH 4.0,
90 min) to mimic stomach transit. Peroxide shock significantly (p < 0.05) reduced the
survival. Treatment with peroxide shock, osmotic shock, and osmotic stress followed by
acid stress (pH 4.0, 90 min) significantly (p < 0.05) reduced survival. Unexpectedly,
exposure to cold stress followed by acid stress (pH 4.0, 90 min) significantly (p < 0.05)
enhanced survival. Proteomic profiles revealed substantial protein repression during cold shock; however, extended exposure to cold induced many proteins for cytoskeleton production, DNA repair, nitrogen metabolism, acid response, and oxidative stress.
Importantly, subsequent acid stress further induced genes involved in aerobic respiration.
Cold stress significantly (p < 0.05) reduced the oxidative potential by modifying the
NAD+/NADH ratio and hydroxyl (OH•) radical formation. The oxidative potential was 50 further reduced with the double stress of cold followed by acid at pH 4.0. Treating the cell with cold before acid stress rescued the cell from the effect of acid via modification of nitrogen metabolism, redox regulation, and aerobic respiration.
INTRODUCTION
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a gram-negative enteric food borne pathogen that causes gastroenteritis in humans and a typhoid-like disease in rodents. Population-based surveillance for bacterial food borne infections with laboratory confirmed cases in 2005 found that Salmonella dominated with ~39 % cases.
Of the 5,869 Salmonella isolates serotyped, S. Typhimurium accounted for 19 % of infections with very high number of infections in children under 4 years (40). Two nation-wide outbreaks caused by S. Typhimurium in 2006 and 2008 linked to contaminated tomatoes and peanut butter, respectively, led to enormous economic loss and 714 confirmed cases of salmonellosis as of April 2009 according to CDC (38). These outbreaks along with the outbreak associated with eggs in 2010, prompted the US government to re-examine the entire food inspection service and protocols in the USA.
Common treatments used to reduce food and animal pathogens include acid, osmotic (salt), oxidative, and temperature. Additionally, refrigeration (5-14°C) is most commonly used method to increase the shelf life of food. Salmonella has efficient mechanisms to survive in the stressful environment and upon consumption of contaminated food it retains the potential to cause an infection in humans. Mechanisms to resist host defense are included in the stress response mechanisms of Salmonella for acid
(2, 31), peroxide (8, 43), osmotic (3, 10-12) and heat stress (37, 52). Acid resistance 51 includes glutamate/arginine/lysine dependent metabolism changes (29, 44, 49).
Salmonella are also capable of resisting oxidative stress (7) using soxSR, oxyR and kat, as well as resisting heat stress using hsp (35). The genes involved in the cold stress response in E. coli (13, 28, 45-48, 57) and Bacillus (18-20, 60) are characterized to include nine csp genes (21) but only some of the csp genes (cspA, cspB, cspC, cspE, and cspH) genes
are found in Salmonella (9, 23, 27).
Pre-adaptation to one stress sometimes increases tolerance and survival during
subsequent stress exposures (32). For example, the acid tolerance response describes
exposure to slightly acidic conditions protects against subsequent severe acidic conditions
in many organisms (15, 16). Similarly, pre-exposure to acetic acid increased survival
during salt stress (22). In addition to stressful conditions during transit in food and the
environment (24, 39, 50) Salmonella must overcome extreme stresses and defense
mechanisms encountered in the host during digestion and cellular invasion (50). Upon
ingestion of contaminated food, Salmonella encounters stomach acidity as host’s first line
of defense. There is initial gut transit time of 90 min (4, 5), and pH of the stomach after
consumption of food is between pH 3.0 – 5.0 in adults is while the pH a child’s stomach
is between pH 4.0 – 5.0 (1, 36). Consequently, to cause food borne illness, Salmonella
experiences abiotic stresses followed by acid stress during ingestion. The effect of abiotic
and biotic stress prior to gastroenteritis in Salmonella is not clearly understood. Hence,
we hypothesized that abiotic stresses regulate mechanisms that enable survival and
persistence that will lead to viable cells.
In this study, S. Typhimurium persisted during cold stress for at least 336 h and
was the only stress that rescued the culture from subsequent acid stress (pH 4.0). 52
Proteome profiles during cold stress revealed induction of proteins during 240 h. Cold stress significantly (p < 0.05) reduced the oxidative potential and hydroxyl radical formation. Cold stress also significantly (q < 0.05) induced genes involved in nitrogen metabolism, acid stress, oxidative stress and DNA repair, of which very few were significantly (q < 0.05) induced due to subsequent acid stress alone (pH 4.0). The oxidative potential was additionally reduced when S. Typhimurium was treated with cold stress followed by exposure to acid stress (pH 4.0), which rescued the cell from the inhibition by acid alone.
MATERIALS AND METHODS
Bacterial strains and culture conditions. Salmonella enterica serovar
Typhimurium LT2 ATCC 700720 was used in this study. Prior to the each experiment a new vial of stock culture was thawed from -70˚C storage, transferred twice in nutrient broth (Difco, Detroit, MI) grown at 37°C with shaking at 220 rpm for 12-16 h. Just prior to each experiment log phase cells were collected (~5 h of growth) and used for all stress treatments.
Stress treatments. Stress treatments were divided into shock (30 min) and stress
(5 h). In one case an extended stress was given (336 h). In each treatment log phase cells were centrifuged at 7200 × g for 5 min and re-suspended in an equal volume of nutrient broth and treated with cold (5˚C), oxidative (1 mM H202), acid (pH 5.3), and osmotic (5%
NaCl) stress. All treatments, except cold, were incubated at 37˚C in a volume of 25 ml.
During the stress treatment each condition was sampled to determine the viable cell count
with pour plate method using nutrient agar (Difco) and for gene expression. A control 53 culture was included with incubation at 37˚C for the same time intervals as the stress treatment. Sequential double stress was done by pre adapting the bacteria to each of the above treatments followed by exposure to stronger acid stress (pH 4.0) for 90 min. The viable cell count was determined using pour plates of nutrient agar (Difco).
Proteome analysis. Samples were collected for intracellular proteins by centrifugation at 7200 × g for 5 min, washed twice with 0.9% sterile saline, and re- suspended in 1 ml of distilled water.
Cell free extracts. Cells were lysed using 334 μg of micro beads of size 106 microns and finer (Sigma, St. Louis, MO), in MinibeadbeaterTM (Biospec Products,
Bartlesville, OK). The samples were centrifuged at 16000 × g for 3 min in micro
centrifuge (Eppendorf, Hauppauge, NY). The supernatant (cell free extract) was collected
and stored in aliquots at -70°C freezer for total protein estimation and the proteome
profile by LC/MS/MS.
Total protein determination. Protein estimation for the above cell free extracts
was done by BCA Protein Assay Kit (Pierce) according to manufacturer’s instruction,
before submitting the samples for proteome profile.
Proteome determination. Proteome profile was determined using a
NanoACQUITY UPLC/MS/MS (Waters, Manchester, UK) as described by Liang et al.
(33), and Tang et al. (56). Briefly, each sample was digested with trypsin and introduced
into a Symmetry C18 trapping column for analysis. The peptides were eluted from
trapping column before introduction into the mass spectrometer that was set for parallel fragmentation (MSE) with the scan times of 1.0 second with low fragmentation energy of
5 volts and a high fragmentation of 17 to 35 volts. Fibrinopeptide B (Glu1).was used as 54 the internal standard with LockSpray. Enolase was used as a spiked control. Waters
Protein Lynx Global SERVER Version 2.2.5 was used to analyze the mass spectrometry dataset.
Gene expression analysis. Sample preparation for gene expression profiling was done as described by Xie et al. (62) and Ganesan et al. (17), except RNA was isolated from TRIzol LS (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions.
Briefly, at various time points the samples were collected as described in the stress treatments and centrifuged at 7200 × g for 5 min. The pellet was re-suspended in TRIzol
LS and gently mixed followed by centrifugation at 7200 × g for 5 min. Trizol supernatant was stored in a clean tube. To the pellet, 1 ml of lysis enzyme cocktail containing 50 mg/ml of lysozyme (Sigma) and 200 U/ml mutanolysin (Sigma) in TE buffer (10 mM
Tris containing 1 mM EDTA, pH 8) was added. The solution was mixed gently and incubated at 37°C for 1 h followed by centrifugation at 7200 × g for 5 min. The supernatant was discarded and pellet was re-suspended in 250 μl of proteinase K buffer
(100 mM Tris-HCl, 5 mM EDTA, 200 mM NaCl, and 0.2 % SDS, pH 8) containing 8
U/ml of proteinase K (Fermentas, Glen Burnie, MD). This was incubated at 55°C for 1 h with intermittent mixing.
RNA was obtained as described by manufacturer’s protocol (TRIzol LS,
Invitrogen). RNA concentration and quality (A260/280≥1.8 and A260/230 ≥1.8) were
measured using a NanoDrop (Thermo scientific, Waltham, MA). The RNA samples were
analyzed for integrity using a 2100 BioAnalyzer (Agilent Technologies, Santa Clara,
CA). 55
Prior to expression analysis total RNA (0.5 g/μl) was reverse transcribed into cDNA with random hexamers and Superscriptase II (Invitrogen) as described by Xie et al. (62) and Ganesan et al. (17). Briefly, after cDNA synthesis, the enzyme was heat inactivated and the RNA templates were degraded with RNaseH (Epicentre, Madison,
WI)). The reaction mixture was cleaned by using the Qiaquick-PCR purification kit
(Qiagen, Valencia, CA) according to the manufacturer’s instructions, except that the Tris- containing buffer was replaced with 75% ethanol for washing the column. The purified single-strand cDNA was eluted from the columns twice with a total of 100 μl of nuclease free water (Ambion, Austin, TX).
cDNA fragmentation was done using DNaseI (Promega, Madison, WI) according to the manufacturer’s instructions. The fragmented cDNA was labeled using GeneChip
DNA Labeling reagent (Affymetrix, Santa Clara, CA) and Terminal Transferase enzyme
(New England Biolabs, Ipswich, MA). The samples were denatured prior to hybridization, at 98°C for 10 min followed by snap cooling at 4°C for 5 min.
Hybridization and normalization. Labeled cDNA was hybridized onto a custom made Affymetrix GeneChip designed against all the annotated coding sequences of S.
Typhimurium LT2 ATCC 700720. Briefly, the array contained 9852 probe sets, of which
4735 probe sets were designed against S. Typhimurium. Each probe set contained 11 probes of 25 nucleotides long. Labeled cDNA (500 ng) extracted from a pure culture of
S. Typhimurium was hybridized to the chip as a positive control, which demonstrated that each probe set was specific for the intended ORF. The samples were hybridized and scanned at the Center for Integrated BioSystems (Utah State University, Logan, UT) as per manufacturer’s protocols for E. coli. Raw data (.cel files) was back ground corrected, 56
quantile normalized and summarized using ms-RMA (53).The resultant normalized log2 transformed intensity matrix was used for further statistical analysis.
NAD+/NADH quantification. The nicotinamide nucleotides quantification was done using NAD+/NADH quantification kit (BioVision Research Products, Mountain
View, CA) according to manufacturer’s instructions. Briefly, cells were centrifuged at
7200 × g for 5 min and washed once with cold, sterile PBS (pH 7.2). Cells were lysed in
PBS, using 334 μg of micro beads of size 106 microns (Sigma), in Mini beadbeater. The
samples were centrifuged (16000 × g for 3 min) to collect the supernatant. The resulting
cell free extracts were used instead of whole cells for this assay and the measurements
were normalized with respect to protein quantity. Protein estimation for cell free extracts
was done as described above followed by nicotinamide nucleotides quantification using
NAD+/NADH quantification kit, according to the instructions.
Radical measurement. Hydroxyl radical (OH•) measurement was done using
hydroxyphenyl fluorescein (HPF) (51) as described by Mols et al. (41). Briefly, the cells
were centrifuged (7200 × g, 5 min, 4˚C) and washed once with an equal volume of sterile
PBS. For the positive control of the assay, 20 mM H202 was used to treat bacteria and for
the negative control of the assay, only bacterial suspension was used. The samples and
assay controls were further diluted to obtain 106 cells/ml followed by addition of HPF at
5 μM final concentration per reaction (30). The reactions were incubated for 60 min at
25°C, in dark. Fluorescence was measured in a fluorescence plate reader DTX 880
Multimode Detector (Beckman Coulter, Brea, CA) using excitation 488 nm and emission
515 nm. 57
Statistical analysis. Growth and survival experiments used an unpaired one-tailed
T-test to compare the means between the treatment and control. The difference was considered significant if the p ≤ 0.05.
Data were collected at 0.5 h, 5 h, 48 h and 240 h for treatment (cold stress at 5°C) and control (no stress at 37°C) during the proteomic analysis. Since many proteins were not found in every sample leading to a large number of zeros that impacted the statistical assessment, random noise was inserted using Bioconductor (R version 2.4.0). Two class unpaired time course (repeated measures) with a corrected T-statistic was used in significance analysis of microarrays (SAM) (58) to calculate the q-value. The difference was considered significant if the q ≤ 0.05.
Upon respective stress treatments, samples were collected and processed for gene expression. Two class (treatment and control) unpaired with T statistic was used in significance analysis of microarrays (SAM) to calculate q-values. Genes with q ≤ 0.05 were considered as significantly different.
NAD+/NADH quantification and OH• measurement analysis used an unpaired T- statistic to compare the difference between the means of treatment and control. The difference was considered significant if the p < 0.05.
RESULTS
Stress survival. To determine the survival of S. Typhimurium during stress we
examined a series of commonly encountered stressed during environmental transit and
food processing. Initially, experiments examined the ability of S. Typhimurium to survive cold stress for at least 336 h to test the hypothesis that Salmonella can withstand long 58 periods of exposure to low temperatures during the transition between animal hosts in the environment. This revealed that S. Typhimurium survived cold stress at a cell density of
~108 cfu/ml (Fig 3-1A). During this time, survival at 37°C declined after 48 h, while at
5°C, a significantly (p < 0.05) high number of viable bacteria were observed at 240 h.
This observation led to the additional hypothesis that S. Typhimurium may respond
differently to shock (i.e. 30 min) and stress (i.e. 5 h) conditions. This was tested using
peroxide, osmotic, and acid (pH 5.3) for 0.5 h (shock) and 5 h (stress) to find that only peroxide shock significantly reduced survival (p < 0.05) while none of the stress treatments reduced survival (Fig 3-1B). These observations indicate the S. Typhimurium has the ability withstand multiple hours of stress during transit in the environment which raises questions about how stress impacts the microbe to survive host defenses. The hypothesis of this work specifically questions the ability of Salmonella to survive stress.
Consequently the effect of the above treatments was further examined in combination to mimic environmental transit followed by consumption of Salmonella
where it would encounter a secondary acid stress in the stomach. Gastric emptying begins
~90 min (4, 5) after consumption of a meal, which exposes organisms to a pH of 3-4
initially upon consumption of food. Consequently, this study investigated survival after
pre-exposure to abiotic stress followed by exposure to acid (pH 4.0) for 90 min. Pre-
exposure to peroxide shock, osmotic shock, and osmotic stress followed by exposure to
acid stress (pH 4.0, 90 min) significantly (p < 0.05) inhibited survival by ~2 decades with
increasing exposure time. Conversely, pre-exposure to cold stress (5°C, 5 h) with
subsequent acid stress (pH 4.0, 90 min) significantly (p < 0.05) induced survival (Fig 3-
1C). This surprising result was examined further to find that pre-exposure to cold stress 59 rescued the cells from effect of acid stress and led to no reduction in cell density during treatment with acid (Fig 3-1D). These observations confirm that the stress conditions and exposure time have complex interactions that impact survival that may impact the exposure dose to levels above that expected considering the treatment. Presumably, a definition of the complex network of gene regulation and protein production used for survival during treatment with cold and acid stress will enable one to design reduction strategies to inhibit Salmonella.
Cold stress proteomics. Since S. Typhimurium survived cold stress for at least
336 h and was induced survival during subsequent acid stress, the proteome was determined to define the specific proteins associated with survival and growth during cold stress. This analysis found that cold stress significantly (q < 0.05) regulated 104 proteins. The proteins were grouped into three categories and 16 COGs: a) information storage and processing (COG J, K, L), b) cellular processes (COG D, O, M, N, P, T), and c) metabolism (COG C, G, E, F, H, I, Q) for subsequent analysis. Protein expression significantly (q < 0.05) declined at 48 h in the control group, while it remained consistently induced in cold-stressed bacteria during the entire treatment period. Of the ribosomal proteins detected in the proteome, 77% (44/57) remained significantly (q <
0.05) induced during the entire cold stress exposure. Cold shock proteins CspA, CspE and CspC were also significantly (q < 0.05) induced and remained constant during the cold stress treatment, but declined after 48 h during incubation at 37˚C. Two DNA recombination and repair proteins (RecA and Ssb) were significantly (q < 0.05) induced with cold stress (Fig 3-2A). Importantly, the cell division proteins, FtsZ and MinD remained induced at 5°C, but declined after 48 h in the control (Fig 3-2B). The proteins 60 associated with metabolism also followed the trend of the other proteins, which were significantly (q < 0.05) induced by cold stress and remained induced for 336 h in cold stress. Carnitine metabolism (Cai) was induced during cold stress and remained expressed after 240 h, while in control cells this metabolism was repressed. Interestingly,
PurU (formyltetrahydrofolate deformylase), which declined due to cold stress, was induced at 37°C after 48 h (Fig 3-2C). This expression trend was pervasive among the proteins found between cold stress and the control, except for CheY (chemotaxis regulation) and PurU (formyltetrhydrofolate deformylase), which were repressed during stress.
While these data are interesting and explain a portion of the survival mechanism, the relatively short list of proteins involved in cellular processes and metabolism did not fully explain the ability to survive and grow to higher cell densities. Proteomic analysis commonly finds only the highly abundant proteins, but overlooks proteins at lower concentrations and the more subtle changes that occur during regulation of the stress response and enzymes that likely enable energy production for growth and persistence as well as membrane proteins. Consequently, we examined response to cold and acid stress using gene expression to define the specific regulatory and membrane transporter changes that led to the induction of survival.
Gene expression during stress. Gene expression analysis resulted in 1,809 significantly (q < 0.05) differentially expressed genes during cold stress. This is well beyond the 104 proteins found previously and enabled analysis of regulation and metabolism changes during each stress condition. COG analysis of cold stress revealed high number of genes being regulated for transcription (COG K), membrane biogenesis 61
(COG M) and amino acid metabolism (COG E) (Fig 3-3). To overcome the effect of cold stress, the cold shock (csp) genes are reported to be induced in the transient growth lag phase, which function in regulating membrane fluidity and resumption of protein synthesis (13, 45, 48). In this study, the cold stress proteins (CspA, CspE and CspC) were found, using proteomics, to be significantly (q < 0.05) induced through 240 h of exposure. Additionally, gene expression analysis found significant (q < 0.05) induction of genes cspB and cspD due to cold stress. The effect of the cold stress proteins was reflected in the gene expression profile where high number of genes associated with membrane biogenesis (COG M) and transcription (COG K) were regulated (Fig 3-3). The oxidative stress genes katG, oxyR, soxS, sodA and sodC were also significantly (q < 0.05) induced in response to cold stress (Fig 3-4C). Among the genes associated with amino acid metabolism (COG E), many genes related to glutamate, glutamine, arginine and lysine transport and metabolism were significantly (q < 0.05) induced due to cold stress
(Fig 3-4B).
Gene expression analysis resulted in 357 significantly (q < 0.05) differentially expressed genes during subsequent acid stress. Subsequent acid stress regulated high number of genes related to energy metabolism (COG C), cell motility (COG N) and ion transport (COG P) (Fig 3-3). To overcome the effect of acid stress, acid induced glutamate, arginine and lysine system functions in maintaining internal pH homeostasis.
Exposure to acid also induces oxidative system which provides protection against hydrogen peroxide. Acid stress induced few metabolic genes for glutamate, arginine and lysine that regulate pH via production of ammonia and carbon dioxide. Significant (q <
0.05) induction of glnA (glutamine synthetase), glnQ (glutamine ABC transporter) and 62 gltI (glutamate/aspartate transporter), along with guaA (bidirection GMP amidotrasnferase) that is involved in glutamate metabolism was observed (Fig 3-4B). The putative Arg repressor (STM4463) was repressed with the double stress, suggesting that
Arg catabolism also increased via the Arg deiminase pathway that was induced during the stress treatments and will lead to ammonia, energy, and precursors for nucleic acid production. Acid stress had little effect on decarboxylase regulation, except for repression of speD (S-adenosylmethionine decarboxylase proenzyme) and speF (ornithine decarboxylase isozyme), suggesting that metabolism was being directed to the arginine deiminase pathway, whereas cold stress alone induced STM2360 (putative decarboxylase) and cbiT (precorrin decarboxylase) (Fig 3-4B). Conversely, cold stress induced many genes responsible for synthesis and transport of glutamine, glutamate, arginine and lysine (Fig 3-4B). Also lysine decarboxylases (cadA and ldcC) and lysine/cadaverine transport (cadB) were significantly (q < 0.05) induced with cold stress, but not with acid stress. The double stress of cold and acid further repressed the oxaloacetate decarboxylase (dcoBC), suggesting decarboxylation was not a method used by the cell to deal with acid stress upon pre adaptation to cold treatment and redirected the TCA intermediates into other metabolites that did not directly modulate the pH. With the acid induced oxidative system, only sodA (superoxide dismutase) and trxC
(thioredoxin 2) were induced. On the other hand, cold stress significantly (q < 0.05) induced many genes involved in oxidative stress such as ahpC (alkyl hydroperoxide reductase), katG (hydroperoxidase), oxyR (oxidative stress regulatory protein), soxS
(transcriptional activator), sodA (superoxide dismutase), sodC (superoxide dismutase precursor), grxA (glutaredoxin 1) and fdx (electron carrier protein). Taken together, the 63 metabolic genes induced during cold stress and the genes induced with subsequent acid stress provide the cell with method to quickly regulate pH, utilize nitrogen and carbon, produce energy, and modulate redox. This was further reflected by the high number of genes regulating in category associated with energy metabolism (COG C) due to subsequent acid stress exposure (Fig 3-3).
Cold stress induced genes involved in TCA cycle include acnA, icdA and sucAD
(Fig 3-4A). All these genes were also induced due to acid stress, except acnA; and additionally acnB and mdh were induced suggesting no significant difference in NADH production due to cold and subsequent acid stress. NADH and succinate generated in
TCA cycle are oxidized in electron transfer chain (ETC), with regeneration of NAD+ and
fumarate, thus providing energy to power ATP synthase. Genes encoding complexes of
ETC were regulated very differently upon cold stress as compared to subsequent acid stress. Genes involved in Complex I - NADH dehydrogenase of ETC, were induced due to cold stress whereas only two of them, nuoG and nuoH were induced due to acid stress
(Fig 3-4A). Genes involved in Complex II – succinate dehydrogenase of the ETC, were
induced due to acid stress whereas cold stress did not regulate this complex. Genes
encoding fumarate reductase subunits (frdA – frdD) and cytochrome o ubiquinol oxidase
subunits (cyoA – cyoB) were repressed due to cold stress except for two genes, cyoD and
cydB. On the other hand, acid stress induced cytochrome o ubiquinol oxidase subunits
(cyoA – cyoD) and ATP synthase subunits (atpA, atpD and atpF – atpH). These
observations indicate differences in NADH consumption and conversion to NAD+ in
ETC due to cold and acid stress and are likely to play a role in survival. To test this 64 observation further the oxidative state was determined upon exposure to cold and acid stress.
Oxidative state and stress. Conversion of NADH to NAD+ in the ETC leads to
superoxide generation, thus higher the NAD+/NADH ratio, the more oxidative damage
(30). The single stress of cold stress decreased the ratio, whereas acid stress increased the ratio. When cold stressed S. Typhimurium was exposed to subsequent acid stress, it resulted in a significant (p < 0.05) reduction of the NAD+/NADH ratio as compared to
acid stress alone and unstressed cells (Fig 3-5A). This ratio directly contributes to the oxidative state of the cell via nitrogen metabolism, Fe-S cluster proteins, and the ETC system that is linked to the TCA cycle.
When the capacity to dismutate superoxide is not sufficient, free superoxide radicals damage the Fe-S clusters releasing Fe2+ ions. The free Fe2+ ions react with hydrogen peroxide to produce OH• radicals that damage the cell and cause death and
DNA damage (26, 30, 42). During the stress treatments OH• radical formation was
measured to determine the oxidative mechanism involved in survival. The OH• radical
formation decreased with cold stress and acid stress did not change the amount of OH• radicals formed. However, when cold stressed bacteria were subsequently acid stressed,
OH• radical formation further declined significantly (p < 0.05) (Fig 3-5B). These results
combined with gene expression of the oxy, sox, and glutamate metabolism systems suggest that the double stress induced multiple metabolic cascades that relieved redox damage via nitrogen metabolism.
DISCUSSION
65
Response to cold stress is extensively studied in E. coli (13, 28, 45-48, 57) and B. subtilis (18-20, 60) to demonstrate complex networks of stress response regulation and adaptation that are induced. Similarly, the relatively few studies using S. Typhimurium demonstrate that this organism also adapts to various stress environments by inducing specific stress responses. Salmonella pre-treated with mild acidic conditions shows induced survival upon exposure to severe acidic conditions (15); however, pre-treatment with cold stress has not been demonstrated to enable adaptation to subsequent acid stress.
Surprisingly, we observed that among the four stresses in this study, cold stress alone significantly induced survival (Fig 3-1). Additionally, cold stress alone rescued the cell from a subsequent acid stress (pH 4) effect and enabled the cell to grow to cell densities beyond that of the control (Fig 3-1). To address this issue proteomic and gene expression profiles were used to determine regulatory networks used for survival.
In this study, the response can be divided into regulation of pathways used for regulation of pH, energy, and oxidative state during shock and stress treatment with cold and acid. Proteomics found ~100 proteins to be induced during cold stress but only three cold stress proteins (CSPs) were found. In E. coli CspA, CspB, CspG, and CspI are cold shock inducible (14, 59), CspD is induced upon nutrition starvation (63), and CspC and
CspE are constitutively expressed (64). Upon cold stress to S. Typhimurium, significant
(q < 0.05) induction of CspA, CspC and CspE proteins was observed and they remained induced over 240 h. Gene expression analysis found additional genes, cspB and cspD being induced upon exposure to cold stress. Additional regulatory mechanisms are needed to explain the response to subsequent acid stress when the cells were pre adapted to cold stress. 66
In contrast to heat shock response, the response to cold stress does not require any sigma factor. The molecular systems to protect against acid stress require sigma factor
RpoS (29, 34, 44, 49). The lack of rpoS expression due to a mutation in the start codon for the strain used in this study (55, 61), further confirmed by no expression of rpoS in gene expression analysis, suggested that operons and genes regulated with this sigma factor must have additional operators and mechanisms. For example, E. coli uses rpoS to regulate expression of cspC and cspE; along with osmY, dps, proP and katG all of which are involved in survival to cold, osmotic, oxidative, and stationary phase stress (48).
While the specific identification of these mechanism are beyond the scope of this paper, the resulting induction adds further information about how the cell regulates the networks used in pH regulation, growth, and oxidative state. Consequently, further examination of other molecular mechanisms to explain survival was done using COG analysis (Fig 3-3).
A large number of metabolic genes regulated were centered around those amino acids that provide carbon and nitrogen. Acid stress induced glutamate, arginine and lysine
– dependent system functions in maintaining pH homeostasis (34). The role of inducible amino acid decarboxylases has been shown in surviving acid stress (44), despite the lack of induction in this study. In E. coli two acid resistance systems AR2 and AR3 have been reported to utilize extracellular glutamate and arginine and the decarboxylation products are exchanged, through antiport systems, for new amino acid substrates (49). The induction of only two decarboxylases was observed in S. Typhimurium LT2 during cold stress. Rather, cold shock and stress induced transporters for glutamate, glutamine, lysine, and arginine, as well as metabolism of these amino acids that results in production of ammonia and energy, was observed. In Lactococcus lactis, which lacks rpoS and most 67 other sigma factors, carbon starvation and acid stress induces the arginine deiminase pathway to enable survival via pH regulation, energy production, and nucleic acid production (6, 54). In addition, these amino acids provide a metabolic link to the TCA cycle which feeds substrates in the ETC for energy production.
Cold and acid stress both induced genes involved in the TCA cycle, suggesting additional ATP and reducing equivalence are induced during stress. This mechanism will produce additional NADH production that will oxidize the cell unless removed. This striking change leads to questions about how the NAD+/NADH balance was regulated since portions of the ETC were regulated differently upon exposure to cold and acid stress (Fig 3-4A), presumably leading to difference in NADH utilization and conversion to NAD+. The NAD+/NADH ratio significantly (p < 0.05) increased with acid stress, but
significantly (p < 0.05) decreased upon cold stress and further decreased when cold
stressed bacteria were exposed to subsequent acid stress. Increase in the NAD+/NADH
ratio during acid stress will also lead to more superoxide generation, which in turn
2+ damages the Fe-S clusters causing free Fe ions to react with H2O2 to form highly toxic
OH• radicals as described in the Fenton reaction (2525, 26). During acid stress no significant change in OH• radical formation was observed, but during sequential treatment with cold and acid the OH• radical formation significantly decreased. The lack
of change in OH• despite an increase in the NAD+/NADH ratio could in part be explained
by the induction of sodA to dismutate superoxide or induction of the TCA cycle to
rebalance the NAD+/NADH ratio, or induction of amino acid utilization that also will
modify the NAD+/NADH ratio while producing energy. 68
This study demonstrated that S. Typhimurium is induced to survive and even grow after exposure to abiotic stresses alone and in combination. Use of cold stress followed by acid stress enabled the cell to grow to significantly higher cell densities as compared to the control, despite having defective expression of rpoS. Induction of the csp genes only partly explained this response. Induction of amino acid metabolism, genes associated with the superoxide metabolism, and redox control provide evidence that cold and acid stress together induce gene sets that provide the cell with mechanisms to grow and survive. The results confirm the hypothesis that cold stress induced metabolic capabilities rescue the cell from inhibition during subsequent acid exposure. The molecular mechanisms for survival center around amino acid utilization for pH regulation and energy production along with oxidative state regulation via induced pathways to decrease NAD+/NADH ratio and the OH• radical formation.
REFERENCES
1. Agunod, M., N. Yamaguchi, R. Lopez, A. Luhby, and G. Glass. 1969.
Correlative study of hydrochloric acid, pepsin, and intrinsic factor secretion in
newborns and infants. Digestive Diseases and Sciences 14:400-414.
2. Bearson, S., B. Bearson, and J. W. Foster. 1997. Acid stress responses in
enterobacteria. FEMS Microbiology Letters 147:173-180.
3. Booth, I. R., and C. F. Higgins. 1990. Enteric bacteria and osmotic stress:
intracellular potassium glutamate as a secondary signal of osmotic stress? FEMS
Microbiology Letters 75:239-246. 69
4. Camilleri, M., L. J. Colemont, S. F. Phillips, M. L. Brown, G. M. Thomforde,
N. Chapman, and A. R. Zinsmeister. 1989. Human gastric emptying and
colonic filling of solids characterized by a new method. Am J Physiol Gastrointest
Liver Physiol 257:G284-290.
5. Cann, P. A., N. W. Read, J. Cammack, H. Childs, S. Holden, R. Kashman, J.
Longmore, S. Nix, N. Simms, K. Swallow, and J. Weller. 1983. Psychological
stress and the passage of a standard meal through the stomach and small intestine
in man. Gut 24:236-40.
6. Chou, L. S., B. C. Weimer, and R. Cutler. 2001. Relationship of arginine and
lactose utilization by Lactococcus lactis ssp. lactis ML3. Intl Dairy J 11:253-258.
7. Christman, M. F., R. W. Morgan, F. S. Jacobson, and B. N. Ames. 1985.
Positive control of a regulon for defenses against oxidative stress and some heat-
shock proteins in Salmonella typhimurium. Cell 41:753-762.
8. Christman, M. F., R. W. Morgan, F. S. Jacobson, and B. N. Ames. 1985.
Positive control of a regulon for defenses against oxidative stress and some heat-
shock proteins in Salmonella typhimurium. Cell 41:753-62.
9. Craig, J. E., D. Boyle, K. P. Francis, and M. P. Gallagher. 1998. Expression of
the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold
temperature. Microbiology 144 ( Pt 3):697-704.
10. Csonka, L. N. 1988. Regulation of cytoplasmic proline levels in Salmonella
typhimurium: effect of osmotic stress on synthesis, degradation, and cellular
retention of proline. J Bacteriol 170:2374-2378. 70
11. Csonka, L. N. 1982. A third L-proline permease in Salmonella typhimurium
which functions in media of elevated osmotic strength. J Bacteriol 151:1433-
1443.
12. Dunlap, V. J., and L. N. Csonka. 1985. Osmotic regulation of L-proline
transport in Salmonella typhimurium. J Bacteriol 163:296-304.
13. Ermolenko, D. N., and G. I. Makhatadze. 2002. Bacterial cold-shock proteins.
Cell Mol Life Sci 59:1902-13.
14. Etchegaray, J. P., P. G. Jones, and M. Inouye. 1996. Differential
thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of
Escherichia coli. Genes Cells 1:171-8.
15. Foster, J. W. 1991. Salmonella acid shock proteins are required for the adaptive
acid tolerance response. J Bacteriol 173:6896-902.
16. Foster, J. W., and H. K. Hall. 1990. Adaptive acidification tolerance response of
Salmonella typhimurium. J Bacteriol 172:771-8.
17. Ganesan, B., P. Dobrowolski, and B. C. Weimer. 2006. Identification of the
leucine-to-2-methylbutyric acid catabolic pathway of Lactococcus lactis. Appl
Environ Microbiol 72:4264-73.
18. Graumann, P., K. Schroder, R. Schmid, and M. Marahiel. 1996. Cold shock
stress-induced proteins in Bacillus subtilis. J Bacteriol 178:4611-4619.
19. Graumann, P., T. M. Wendrich, M. H. W. Weber, K. Schröder, and M. A.
Marahiel. 1997. A family of cold shock proteins in Bacillus subtilis is essential
for cellular growth and for efficient protein synthesis at optimal and low
temperatures. Mol Microbiol 25:741-756. 71
20. Graumann, P. L., and M. A. Marahiel. 1999. Cold shock response in Bacillus
subtilis. J Mol Microbiol Biotechnol 1:203-9.
21. Graumann, P. L., and M. A. Marahiel. 1998. A superfamily of proteins that
contain the cold-shock domain. Trends Biochem Sci 23:286-90.
22. Greenacre, E. J., and T. F. Brocklehurst. 2006. The Acetic Acid Tolerance
Response induces cross-protection to salt stress in Salmonella typhimurium. Int J
Food Microbiol 112:62-5.
23. Horton, A. J., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 2000.
Adaptive response to cold temperatures and characterization of cspA in
Salmonella typhimurium LT2. Antonie Van Leeuwenhoek 77:13-20.
24. Humphrey, T. 2004. Salmonella, stress responses and food safety. Nat Rev
Microbiol 2:504-9.
25. Imlay, J. A. 2006. Iron-sulphur clusters and the problem with oxygen. Mol
Microbiol 59:1073-1082.
26. Imlay, J. A., and S. Linn. 1988. DNA damage and oxygen radical toxicity.
Science 240:1302-9.
27. Jeffreys, A. G., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 1998.
Growth, survival and characterization of cspA in Salmonella enteritidis following
cold shock. Curr Microbiol 36:29-35.
28. Jones, P. G., and M. Inouye. 1994. The cold-shock response--a hot topic. Mol
Microbiol 11:811-8.
29. Kieboom, J., and T. Abee. 2006. Arginine-dependent acid resistance in
Salmonella enterica serovar Typhimurium. J Bacteriol 188:5650-3. 72
30. Kohanski, M. A., D. J. Dwyer, B. Hayete, C. A. Lawrence, and J. J. Collins.
2007. A common mechanism of cellular death induced by bactericidal antibiotics.
Cell 130:797-810.
31. Koutsoumanis, K. P., and J. N. Sofos. 2004. Comparative acid stress response
of Listeria monocytogenes, Escherichia coli O157:H7 and
SalmonellaTyphimurium after habituation at different pH conditions. Letters in
Appl Microbiol 38:321-326.
32. Leyer, G. J., and E. A. Johnson. 1993. Acid adaptation induces cross-protection
against environmental stresses in Salmonella typhimurium. Appl Environ
Microbiol 59:1842-7.
33. Liang, Y., D. R. Gardner, C. D. Miller, D. Chen, A. J. Anderson, B. C.
Weimer, and R. C. Sims. 2006. Study of biochemical pathways and enzymes
involved in pyrene degradation by Mycobacterium sp. strain KMS. Appl Environ
Microbiol 72:7821-8.
34. Lin, J., M. Smith, K. Chapin, H. Baik, G. Bennett, and J. Foster. 1996.
Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl
Environ Microbiol 62:3094-3100.
35. Lindquist, S. 1986. The heat-shock response. Annu Rev Biochem 55:1151-91.
36. Lonnerdal, B. 2002. Expression of human milk proteins in plants. J Am Coll
Nutr 21:218S-221.
37. Mackey, B. M., and C. M. Derrick. 1986. Elevation of the heat resistance of
Salmonella typhimurium by sublethal heat shock. J Appl Bacteriol 61:389-93. 73
38. Maki, D. G. 2009. Coming to grips with foodborne infection--peanut butter,
peppers, and nationwide salmonella outbreaks. N Engl J Med 360:949-53.
39. McMeechan, A., M. Roberts, T. A. Cogan, F. Jorgensen, A. Stevenson, C.
Lewis, G. Rowley, and T. J. Humphrey. 2007. Role of the alternative sigma
factors sigmaE and sigmaS in survival of Salmonella enterica serovar
Typhimurium during starvation, refrigeration and osmotic shock. Microbiology
153:263-9.
40. MMWR Weekly. 2006. Preliminary FoodNet Data on the incidence of infection
with pathogens transmitted commonly through food – 10 states, United States,
2005. Centers for Disease Control and Prevention, Atlanta, GA.
41. Mols, M., R. van Kranenburg, M. H. Tempelaars, W. van Schaik, R.
Moezelaar, and T. Abee. 2010. Comparative analysis of transcriptional and
physiological responses of Bacillus cereus to organic and inorganic acid shocks.
Int J Food Microbiol 137:13-21.
42. Mols, M., R. van Kranenburg, C. C. van Melis, R. Moezelaar, and T. Abee.
2010. Analysis of acid-stressed Bacillus cereus reveals a major oxidative response
and inactivation-associated radical formation. Environ Microbiol 12:873-85.
43. Morgan, R. W., M. F. Christman, F. S. Jacobson, G. Storz, and B. N. Ames.
1986. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap
with heat shock and other stress proteins. Proc Natl Acad Sci USA 83:8059-63.
44. Park, Y. K., B. Bearson, S. H. Bang, I. S. Bang, and J. W. Foster. 1996.
Internal pH crisis, lysine decarboxylase and the acid tolerance response of
Salmonella typhimurium. Mol Microbiol 20:605-11. 74
45. Phadtare, S. 2004. Recent developments in bacterial cold-shock response. Curr
Issues Mol Biol 6:125-36.
46. Phadtare, S., J. Alsina, and M. Inouye. 1999. Cold-shock response and cold-
shock proteins. Curr Opin Microbiol 2:175-80.
47. Phadtare, S., and M. Inouye. 2001. Role of CspC and CspE in regulation of
expression of RpoS and UspA, the stress response proteins in Escherichia coli. J
Bacteriol 183:1205-14.
48. Phadtare, S., V. Tadigotla, W. H. Shin, A. Sengupta, and K. Severinov. 2006.
Analysis of Escherichia coli global gene expression profiles in response to
overexpression and deletion of CspC and CspE. J Bacteriol 188:2521-7.
49. Richard, H., and J. W. Foster. 2004. Escherichia coli glutamate- and arginine-
dependent acid resistance systems increase internal pH and reverse
transmembrane potential. J Bacteriol 186:6032-41.
50. Rychlik, I., and P. A. Barrow. 2005. Salmonella stress management and its
relevance to behaviour during intestinal colonisation and infection. FEMS
Microbiol Rev 29:1021-40.
51. Setsukinai, K., Y. Urano, K. Kakinuma, H. J. Majima, and T. Nagano. 2003.
Development of novel fluorescence probes that can reliably detect reactive
oxygen species and distinguish specific species. J Biol Chem 278:3170-5.
52. Spector, M. P., Z. Aliabadi, T. Gonzalez, and J. W. Foster. 1986. Global
control in Salmonella typhimurium: two-dimensional electrophoretic analysis of
starvation-, anaerobiosis-, and heat shock-inducible proteins. J Bacteriol 168:420-
424. 75
53. Stevens J.R., G. B., Desai P., Rajan S., and Weimer B.C. 2008. Statistical
issues in the normalization of multi-species microarray data. Proc Conf Appl
Statistics in Agricul. 47-62.
54. Stuart, M. R., L. S. Chou, and B. C. Weimer. 1999. Influence of carbohydrate
starvation and arginine on culturability and amino acid utilization of lactococcus
lactis subsp. lactis. Appl Environ Microbiol 65:665-73.
55. Swords, W. E., B. M. Cannon, and W. H. Benjamin, Jr. 1997. Avirulence of
LT2 strains of Salmonella typhimurium results from a defective rpoS gene. Infect
Immun 65:2451-3.
56. Tang, C. T., Y. Guo, M. Lu, Y. N. Wang, J. Y. Peng, W. B. Wu, W. H. Li, B.
C. Weimer, and D. Chen. 2009. Development of larval Schistosoma Japonicum
was blocked in Oncomelania Hupensis by pre-infection with larval Exorchis sp. J
Parasitol:1.
57. Thieringer, H. A., P. G. Jones, and M. Inouye. 1998. Cold shock and
adaptation. Bioessays 20:49-57.
58. Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of
microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA
98:5116-21.
59. Wang, N., K. Yamanaka, and M. Inouye. 1999. CspI, the ninth member of the
CspA family of Escherichia coli, is induced upon cold shock. J Bacteriol
181:1603-9. 76
60. Weber, M. H., and M. A. Marahiel. 2002. Coping with the cold: the cold shock
response in the Gram-positive soil bacterium Bacillus subtilis. Philos Trans R Soc
Lond B Biol Sci 357:895-907.
61. Wilmes-Riesenberg, M. R., J. W. Foster, and R. Curtiss, 3rd. 1997. An altered
rpoS allele contributes to the avirulence of Salmonella typhimurium LT2. Infect
Immun 65:203-10.
62. Xie, Y., L. S. Chou, A. Cutler, and B. Weimer. 2004. DNA Macroarray
profiling of Lactococcus lactis subsp. lactis IL1403 gene expression during
environmental stresses. Appl Environ Microbiol 70:6738-47.
63. Yamanaka, K., and M. Inouye. 1997. Growth-phase-dependent expression of
cspD, encoding a member of the CspA family in Escherichia coli. J Bacteriol
179:5126-30.
64. Yamanaka, K., T. Mitani, T. Ogura, H. Niki, and S. Hiraga. 1994. Cloning,
sequencing, and characterization of multicopy suppressors of a mukB mutation in
Escherichia coli. Mol Microbiol 13:301-12.
77
FIG 3-1. Survival after shock and stress treatments. (A) Survival of S. Typhimurium during cold stress (5°C) and no stress (37°C) for 336 h. Inset: Enlarged for growth between 0 h – 8 h. The asterisk (*) represents the significant difference (p < 0.05) as compared to no stress. (B) Survival of S. Typhimurium upon cold (5°C), peroxide (1 mM
H2O2), osmotic (5% NaCl) and acid (pH 5.3) shock (0.5 h) and stress (5 h) treatments.
The asterisk (*) represents the significant difference (p < 0.05) as compared to no shock and no stress respectively. (C) Survival of S. Typhimurium upon pre-adaptation to cold
(5°C), peroxide (1 mM H2O2), osmotic (5% NaCl) and acid (pH 5.3) shock (0.5 h) and
stress (5 h) treatments, followed by exposure to acid stress (pH 4.0, 90 min). The asterisk
(*) represents the significant difference (p < 0.05) as compared to no shock/acid (pH 4.0,
90 min) and no stress/acid (pH 4.0, 90 min) respectively. (D) Survival upon cold stress
(5°C, 5 h) and upon pre-adaptation to cold stress (5°C, 5 h) followed by exposure to acid
stress (pH 4.0, 90 min). The asterisk (*) represents the significant difference (p < 0.05) as
compared to no stress.
78
79
80
FIG 3-2. Heat map representations of the 104 differentially regulated proteins (q < 0.05) due to cold stress (5°C) over time (0.5 h, 5 h, 48 h and 240 h). The proteins are grouped based on the COGs functional annotation. (A) Proteins related to information storage and processing (COGs J, K &L). (B) Proteins related to cellular processes (COGs D, O, M,
N, P & T). (C) Proteins related to metabolism (COGs C, G, E, F, H, I & Q). Legends of
COG categories: J-Translation; K-Transcription; L-DNA replication, recombination and repair; D-Cell division and chromosome partitioning; O-Posttranslational modification, protein turnover, chaperones; M-Cell envelope biogenesis, outer membrane; N-Cell motility and secretion; P-Inorganic ion transport and metabolism; T-Signal transduction mechanisms; C-Energy production and conversion; G-Carbohydrate transport and metabolism; E-Amino acid transport and metabolism; F-Nucleotide transport and metabolism; H-Coenzyme metabolism; I-Lipid metabolism; Q-Secondary metabolites biosynthesis, transport and catabolism. Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity.
81
Cold None 0.5 5 48 240 0.5 5 48 240
99989975rpsJ J 30S ribosomal protein S10
A 88988761rpsK J 30S ribosomal protein S11
88888851rpsL J 30S ribosomal protein S12
991099971rpsM J 30S ribosomal protein S13
88988761rpsO J 30S ribosomal protein S15
88987761rpsP J 30S ribosomal protein S16
98888861rpsR J 30S ribosomal protein S18
8888775 0rpsS J 30S ribosomal protein S19
89988875rpsB J 30S ribosomal protein S2
8887665 0rpsT J 30S ribosomal protein S20
88877751rpsU J 30S ribosomal protein S21
99999986rpsC J 30S ribosomal protein S3
99999973rpsD J 30S ribosomal protein S4
99999985rpsE J 30S ribosomal protein S5
88888876rpsF J 30S ribosomal protein S6
991099974rpsG J 30S ribosomal protein S7
8888876 0rpsH J 30S ribosomal protein S8
99998874rpsI J 30S ribosomal protein S9
99999986rplA J 50S ribosomal protein L1
99999986rplJ J 50S ribosomal protein L10
99999886rplK J 50S ribosomal protein L11
99988872rplM J 50S ribosomal protein L13
78878762rplN J 50S ribosomal protein L14
99988874rplO J 50S ribosomal protein L15
88877761rplP J 50S ribosomal protein L16
99998861rplQ J 50S ribosomal protein L17
99999871rplS J 50S ribosomal protein L19
99998874rplB J 50S ribosomal protein L2
99998861rplT J 50S ribosomal protein L20
88888873rplU J 50S ribosomal protein L21
9998887 0rplV J 50S ribosomal protein L22
9998886 0rplX J 50S ribosomal protein L24
78876751rplY J 50S ribosomal protein L25
75566611rpmA J 50S ribosomal protein L27
88988861rpmB J 50S ribosomal protein L28
88887761rpmC J 50S ribosomal protein L29
99999975rplC J 50S ribosomal protein L3
78876621rpmD J 50S ribosomal protein L30
77776631rpmE J 50S ribosomal protein L31
777666 01rpmF J 50S ribosomal protein L32
99989874rplD J 50S ribosomal protein L4
89988875rplE J 50S ribosomal protein L5
99999986rplF J 50S ribosomal protein L6
99989976rplI J 50S ribosomal protein L9
44446631yfiA J ribosome stabilization factor
76777761frr J ribosome releasing factor
77776662infC J translation initiation factor IF-3
11 12 12 11 12 11 11 9 tuf J elongation factor Tu
4665454 0rne J RNase E
66666651gltX J glutamyl-tRNA synthetase
46654531tyrS J tyrosyl-tRNA synthetase
88877775lysS J lysyl-tRNA synthetase
03433 000ychF J putative GTP-binding protein
101111119861cspA K major cold shock protein
99998872cspE K cold shock protein E
99998882cspC K cold shock protein
77766651nusA K transcription elongation factor NusA
77766651rho K transcription termination factor Rho
1123323 0rpoZ K DNA-directed RNA polymerase omega subunit
6776653 0recA L recombinase A
66665542ssb L single-strand DNA-binding protein
12223111polA L DNA polymerase I
8888611 0deaD LKJ cysteine sulfinate desulfinase 82
Cold None 0.5 5 48 240 0.5 5 48 240
677665 02ftsZ D cell division protein FtsZ
B 78877743minD D cell division inhibitor protein
4655452 0galU M glucose-1-phosphate uridylyltransferase
77877754kdsA M 2-dehydro-3-deoxyphosphooctonate aldolase
15544421gcpE M 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase
112422 01murA M UDP-N-acetylglucosamine 1-carboxyvinyltransferase
66666662rfaD MG ADP-L-glycero-D-mannoheptose-6-epimerase
56655551rfbG MG CDP glucose 4,6-dehydratase
6466541 0fklB O FKBP-type 22KD peptidyl-prolyl cis-trans isomerase
66666651ydhD O putative glutaredoxin protein
88988876tig O trigger factor
001 0531 0cheY T chemotaxis regulator
78877643phoP TK response regulator
77776611typA T GTP-binding elongation factor family protein
4676535 0proQ T putative solute/DNA competence effector
01131 01 0aer TN aerotaxis sensor receptor
7887777 0yiiU S putative cytoplasmic protein
35555421STM0327 putative cytoplasmic protein
Cold None 0.5 5 48 240 0.5 5 48 240
67767751caiB C crotonobetainyl-CoA:carnitineCoA-transferase
C 66765641nemA C N-ethylmaleimide reductase
88877762ackA C acetate/propionate kinase
7787887 0fixA C putative electron transfer flavoprotein subunit beta
78877874fixB C putative electron transfer flavoprotein subunit alpha
66655521ppc C phosphoenolpyruvate carboxylase
67766662pta C phosphate acetyltransferase
88877764glpK C glycerol kinase
7787752 0asnA E asparagine synthetase AsnA
6666662 0speF E ornithine decarboxylase isozyme
66656531STM4466 E putative carbamate kinase
88888964STM4467 E putative arginine deiminase
6665551 0thrA E bifunctional aspartokinase I/homeserine dehydrogenase I
7776662 0thrC E threonine synthase
77766643dapA EM dihydrodipicolinate synthase
6675563 0glpB E anaerobic glycerol-3-phosphate dehydrogenase subunit B
311 00146purU F formyltetrahydrofolate deformylase
66655511pyrG F CTP synthetase
66666663ndk F nucleoside diphosphate kinase
6676662 0kbl H 2-amino-3-ketobutyrate coenzyme A ligase
77767751caiD I carnitinyl-CoA dehydratase
77766612fabB IQ 3-oxoacyl-(acyl carrier protein) synthase
67766761caiA I crotonobetainyl-CoA dehydrogenase
83
FIG 3-3. Distribution in COG categories, of 1809 significantly (q < 0.05) regulated genes due to cold stress (5°C, 5 h) and 357 significantly (q < 0.05) regulated genes due to subsequent acid stress (pH 4.0, 90 min) alone. Legends of COG categories: J-Translation;
K-Transcription; L-DNA replication, recombination and repair; D-Cell division and chromosome partitioning; O-Posttranslational modification, protein turnover, chaperones;
M-Cell envelope biogenesis, outer membrane; N-Cell motility and secretion; P-Inorganic ion transport and metabolism; T-Signal transduction mechanisms; C-Energy production and conversion; G-Carbohydrate transport and metabolism; E-Amino acid transport and metabolism; F-Nucleotide transport and metabolism; H-Coenzyme metabolism; I-Lipid metabolism; Q-Secondary metabolites biosynthesis, transport and catabolism. 84
FIG 3-4. Heat map representations of genes differentially regulated (q < 0.05) due to cold stress (5°C, 5 h), acid stress (pH 4.0, 90 min) and due to pre-adaptation to cold stress
(5°C, 5 h) when bacteria were subsequently exposed to acid stress (pH 4.0, 90 min).
Legends: NS No stress, CS Cold stress, AS Acid stress. In each heat map, the first two columns represent the significant (q < 0.05) differences due to cold stress (5°C, 5 h) alone; the third and fourth columns represent significant (q < 0.05) differences due to acid stress (pH 4.0, 90 min) alone; the fourth and fifth columns represent the significant
(q < 0.05) differences due to pre-adaptation to cold stress when bacteria were subsequently exposed to acid stress (pH 4.0, 90 min). Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity. White block or no color means that particular gene for that respective comparison was not significantly (q <
0.05) changing.
85
NS CS NS/NS NS/AS CS/AS
A 67 acnA C aconitate hydratase
78 acnB C aconitate hydratase
11 12 11 12 icdA C isocitrate dehydrogenase
8989 sucA C 2-oxoglutarate dehydrogenase
89810 sucB C dihydrolipoamide acetyltransferase
81089 sucC C succinyl-CoA synthetase subunit beta TCA cycle TCA 7979 sucD C succinyl-CoA synthetase alpha subunit
10 11 mdh C malate dehydrogenase
810 ndh C respiratory NADH dehydrogenase 2
7778 nuoG C NADH dehydrogenase gamma subunit
7878 nuoH C NADH dehydrogenase subunit H
67 nuoI C NADH dehydrogenase subunit I
67 nuoJ C NADH dehydrogenase subunit J
57 nuoK C NADH dehydrogenase kappa subunit
67 nuoL CP NADH dehydrogenase subunit L
67 nuoM C NADH dehydrogenase subunit M
77 nuoN C NADH dehydrogenase subunit N
78 sdhA C succinate dehydrogenase catalytic subunit
89 sdhB C succinate dehydrogenase catalytic subunit
910 sdhC C succinate dehydrogenase cytochrome b556 subunit
78 sdhD C succinate dehydrogenase cytochrome b556 subunit
10 8 frdA C fumarate reductase
98 frdB C succinate dehydrogenase
87 frdC C fumarate reductase subunit C
87 frdD C fumarate reductase subunit D
11 8 11 11 9 cyoA C cytochrome o ubiquinol oxidase subunit II
97897cyoB C cytochrome o ubiquinol oxidase subunit I
89 cyoC C cytochrome o ubiquinol oxidase subunit III Electron transfer chain
7889 cyoD C cytochrome o ubiquinol oxidase subunit IV
67 cyoE O protoheme IX farnesyltransferase
910 cydB C cytochrome d terminal oxidase polypeptide subunit II
8109atpA C ATP synthase subunit A
79 atpC C ATP synthase subunit epsilon
7778 atpD C ATP synthase subunit B
89 atpF C ATP synthase subunit B
8989 atpG C ATP synthase subunit C
89 atpH C ATP synthase subunit D
98 bcp O bacterioferritin comigratory protein
8127 9 bfd P bacterioferritin-associated ferredoxin
911 bfr P bacterioferrin
76 76corA P Mg2+/Ni2+/Co2+ transport protein
810 feoA P ferrous iron transport protein A
81079 feoB P ferrous iron transport protein B
7968 fhuA P outer membrane ferrichrome receptor protein precursor
46 fhuB P hydroxamate-dependent iron uptake protein
46 fhuC PH hydroxymate-dependent iron transport protein
4744 fhuD P hydroxamate-dependent iron uptake protein
5657 fhuE P outer membrane receptor protein precursor
4846 fhuF R ferric hydrozamate transport protein
5857 iroB GC putative glycosyl transferase
5856 iroC V putative ABC transporter protein
4845 iroD P enterochelin esterase=-like protein
4745 iroE R putative hydrolase
7969 iroN P TonB-dependent siderophore receptor protein
10 11 iscA S iron-sulfur cluster assembly protein
65 mgtA P Mg2+ ATPase transporter
95 85mgtB P Mg2+ transporter Fe and Mg transport
11 6 96mgtC S Mg2+ transport protein
67 phsB C thiosulfate reductase electron transport protein
66 phsC C thiosulfate reductase cytochrome B subunit
71069 sitA P putative periplasmic binding protein
61057 sitB P putative ATP-binding protein
61057 sitC P putative permease
5956 sitD P putative permease
6767 sufB O putative ABC transporter protein
6867 sufC O putative transport protein
68 sufD O cysteine desulfurase modulator
57 sufS E selenocysteine lyase
10 11 yfhP K putative iron-sulfur cluster regulatory protein 86
NS CS NS/NS NS/AS CS/AS
B 13 10 12 11 groEL O chaperonin GroEL
14 11 groES O co-chaperonin GroES
10 8 97hslO O Hsp33-like chaperonin
11 9 11 9 hslU O ATP-dependent protease ATP-binding subunit
12912119hslV O ATP-dependent protease peptidase subunit
11 10 12 11 10 dnaJ O heat shock protein
13 10 12 11 dnaK O molecular chaperone DnaK
89 clpA O ATP-binding subunit of serine protease Chaperones 129131110clpB O ATP-dependent protease
10 9 11 10 clpP OU ATP-dependent Clp protease proteolytic subunit
910 clpS S ATP-dependent Clp protease adaptor protein ClpS
777nhaB P Na+/H+ antiporter
47 amtB P putative ammonium transport protein
88 panD H aspartate 1-decarboxylase precursor
78 speD E S-adenosylmethionine decarboxylase proenzyme
7487 speF E ornithine decarboxylase isozyme
76 ases lysA E diaminopimelate decarboxylase
710 STM2360 E putative diaminopimelate decarboxylase
58 cbiT H precorrin-8w decarboxylase
65 cobD E threonine-phosphate decarboxylase
54dcoB C oxaloacetate decarboxylase beta chain
54dcoC C putative oxaloacetate decarboxylase subunit gamma Decarboxyl 87 pyrF F orotidine 5 -phosphate decarboxylase
86 menD H 2-oxoglutarate decarboxylase
57 cheR NT glutamate methyltransferase
7118 9 glnA E glutamine synthetase
67 glnE OT adenylyl transferase for glutamine synthetase
811 glnH ET glutamine ABC transporter periplasmic-binding protein
58 glnP E glutamine ABC transporter permease component
6977 glnQ E glutamine ABC transporter ATP-binding component
56 gltD ER glutamate synthase small subunit
8118 9 gltI ET glutamate/aspartate transporter
69 gltJ E glutamate/aspartate transporter
58 gltK E glutamate/aspartate transporter
68 gltL E glutamate/aspartate transporter
88 gshA H glutamate--cysteine ligase
67 argB E acetylglutamate kinase
45 yfeJ F glutamine amidotransferase
7697 STM2186 ER putative NADPH-dependent glutamate synthase beta chain
97 97guaA F bifunctional GMP synthase/glutamine amidotransferase protein
710 artJ ET arginine transport system component
78 artM E arginine transport system component
79 STM4351 ET putative arginine-binding periplasmic protein
76 96STM4463 K putative arginine repressor
75 86STM4464 S putative arginine repressor
96 11 8 STM4467 E arginine deiminase
96 yggA R arginine exporter protein Glu / Gln / Arg / Lys transportLys /Arg / Gln /Glu
79 cadA E lysine decarboxylase 1
711 cadB E lysine/cadaverine transport protein
67 ldcC E lysine decarboxylase 2
67 argT ET lysine/arginine/ornithine transport protein
56 hisM E histidine/lysine/arginine/ornithine transport protein
57 fliB lysine-N-methylase
87
NS CS NS/NS NS/AS CS/AS
C 610 hmpA C dihydropteridine reductase 2/nitric oxide dioxygenase
97 narP TK response regulator
77 nirD PR nitrite reductase small subunit
55 nrfB P formate-dependent nitrite reductase
65 65STM2840 C anaerobic nitric oxide reductase flavorubredoxin
74 74ygaA KT anaerobic nitric oxide reductase transcription regulator
10 12 N metabolism N ahpC O alkyl hydroperoxide reductase C22 subunit
68 katG P hydroperoxidase
78 oxyR K oxidative stress regulatory protein
10 5 96soxR K redox-sensing transcriptional activator
78 soxS K transcriptional activator
89810 sodA P superoxide dismutase
11 8 sodB P superoxide dismutase
99 sodC P superoxide dismutase precursor
10 9 trxB O thioredoxin reductase
910 trxC OC thioredoxin 2
10 7 11 9 ybbN O putative thioredoxin protein
896fixX C putative ferredoxin
57 yfaE C putative ferredoxin
87 STM1733 R putative ferredoxin Redox responseRedox
87 yffB P putative glutaredoxin
11 10 ydhD O putative glutaredoxin protein
5657 nrdH O glutaredoxin-like protein
11 12 grxA O glutaredoxin 1
76 fldB C flavodoxin
78 fdx C electron carrer protein
45 alkA L 3-methyl-adenine DNA glycosylase II
79 dam L DNA adenine methylase
69 dinG KL SOS repair enzyme
78 dnaE L DNA polymerase III subunit alpha
79 dnaN L DNA polymerase III subunit beta
87 dnaQ L DNA polymerase III subunit epsilon
78 dnaX L DNA polymerase III subunits gamma and tau
76 exoX L exodeoxyribonuclease X
68 holA L DNA polymerase III subunit delta
78 holB L DNA polymerase III subunit delta
810 lexA KT LexA repressor
67 lig L DNA ligase
79 mutL L DNA mismatch repair protein
78 67nei L endonuclease VIII
78 nfi L endonuclease V
67 recB L exonuclease V beta chain
46 recD L exonuclease V alpha chain DNA repairDNA
55 recG LK DNA helicase
67 recJ L single-stranded-DNA-specific exonuclease
89 uvrA L excinuclease ABC subunit A
78 uvrD L DNA-dependent ATPase I/helicase II
67 yejH KL putative ATP-dependent helicase
11 9 STM3517 L putative DNA-damage-inducibile protein
911 ssb L single-strand DNA-binding protein
89 phrB L deoxyribodipyrimidine photolyase
79 ydhM K putative transcriptional repressor
65685yjiY T putative carbon starvation protein
88
FIG 3-5. (A) NAD+/NADH ratio measurements and (B) OH• (hydroxyl radical)
measurements using hydroxyphenyl fluorescein (HPF) upon exposure to cold stress (5°C,
5 h), acid stress (pH 4.0, 90 min) and upon pre-adaptation to cold stress (5°C, 5 h)
followed by exposure to subsequent acid stress (pH 4.0, 90 min). The asterisk (*)
indicates significant difference (p < 0.05) as compared to respective no stress control.
The dot (•) indicates significant difference as compared to no stress followed by acid
stress (pH 4.0, 90 min) treatment.
89
90
CHAPTER 4
COLD STRESS TO S. TYPHIMURIUM INCREASES ADHESION AND
INVASION IN INTESTINAL EPITHELIAL CELLS
ABSTRACT
Salmonella encounters multiple stresses during environmental transit and the infection process that should reduce infection. However, this group of organisms remains the leading cause of food-borne illness, indicating that it routinely resists stress conditions to cause gastroenteritis. This work investigated the hypothesis that Salmonella has multiple mechanisms to resist stress and induce new traits to enable host association.
The effect of cold (5°C, 48 h), peroxide (5 mM H202, 5 h) and simulated gastric transit
(pH 4.0, 90 min) was tested using Salmonella enterica serovar Typhimurium (S.
Typhimurium) to determine their impact on host association and induction of new
capabilities for host association. Cold stress significantly (p < 0.05) increased adhesion
and invasion of colonic epithelial cells in vitro that was coupled with the significant (q <
0.05) induction of virulence genes associated with the type three secretion system, pilus
biosynthesis, and prophages. All of these genes remained induced 60 min post infection
using a colonic epithelial model, implying that abiotic stress potentiates S. Typhimurium for host association and virulence. Significant (p < 0.05) increase of caspase 9 and caspase 3/7 activity confirmed increased rate of cell death when epithelial cells were infected with cold-stressed S. Typhimurium. Infection of epithelial cells with cold- stressed S. Typhimurium resulted in significant (q < 0.05) induction of genes involved in
electron transfer chain, but no simultaneous significant increase in expression of anti- 91 oxidant genes neutralizing the effect of superoxide radicals or reactive oxygen species in the epithelial cell. Further, a prophage gene STM2699, which was induced due to cold stress and a spectrin encoding gene SPTAN1, which was induced due to infection with cold-stressed S. Typhimurium were found to play a role as receptor/ligand partners during host association. Interaction of these two proteins significantly (p < 0.05) contributed to the phenotype of increased adhesion and invasion by cold-stressed S.
Typhimurium in vitro.
INTRODUCTION
Salmonella is an important food-borne pathogen throughout the world. It is transmitted through the fecal-oral route and most infections occur due to ingestion of contaminated food. Salmonella encounters and survives various stresses such as temperature (heat), acid, and oxidation during its journey from the environment to food to infection of the animal host (17, 31, 38, 63, 72, 83). Salmonella is capable of surviving stresses such as nutritional starvation, osmotic, iron and cationic antimicrobial peptides reviewed in (39, 85). In a previous study, it was observed that Salmonella enterica serovar Typhimurium (S. Typhimurium) survived during cold shock (30 min) and stress
(336 h), whereas at 37°C the growth declined after 48 h (Fig 3-1). Survival during cold stress was concomitant with induction of new proteins and genes involved in transcription, translation, energy production, and protein metabolism that also enabled the cell to be rescued from subsequent acid stress (Fig 3-1). The fact that pre-exposure to one stress can increase tolerance or survival during exposure to subsequent stress (49, 61, 70), poses increased risk of infection as Salmonella encounters a range of stresses during its 92 journey from environment to human host (39, 94). Presumably, stress also regulates expression of virulence factors and the adhesion molecules that will produce and expose new ligands on the microbe for interaction with receptors on the host that change adherence specificity and likely the extent of invasion as well as the route of entry.
A necessary step in the successful infection is the ability of Salmonella to adhere to host surfaces. Consequently, identification of adhesins (ligands) and host receptors, that are targets for bacterial association, is important. Bacterial adhesins can be divided in to two major groups: pili (fimbriae) and non-pilus (afimbrial adhesins). The examples of many fimbrial adhesion factors includes genes csg, bcf, fim, lpf, pef, saf, stb, stc, std, stf, sth, sti, and stj, which commonly associate with mucin or the glycocaylix (14, 112).
Salmonella also binds to host cell surface via TLRs or to the components associated with extracellular matrix (ECM) that include laminin, plasminogen, integrins, vitronectin, and fibronectin (88, 86, 98). However, only a small number of afimbrial adhesin factors including misL, ratB, shdA, sinH (14, 112) and siiE (48) have been functionally characterized, and in most cases the specific binding partners on host cells are not known.
Identification of specific host receptor and bacterial ligand partnerships will lead to new association and infection mechanisms which will help in designing strategies to reduce infection.
Upon adhesion, Salmonella induces a network of cytoplasmic and nuclear responses in epithelial cells leading to cytoskeletal rearrangement, membrane ruffling and macropinocytosis, induction of transmembrane fluids and electrolyte fluxes, and synthesis of cytokines and mediators of inflammation that are largely induced by injection of effector toxins from the type three secretion systems (T3SS) (27, 42, 60, 87, 93
108). T3SS are encoded on two Salmonella pathogenicity islands (SPIs), SPI1 and SPI2 and each T3SS has a distinct set of secreted effector proteins. Expression of SPI1 is required for invasion and SPI2 is required for intracellular survival and replication (42,
58, 73). Infection of macrophages induces antibacterial peptides and an oxidative burst to defend against pathogen invasion (30, 75). In spite of these defense mechanisms,
Salmonella proliferate in macrophages within the Salmonella containing vacuole (SCV) that is in part derived from the clathrin-mediated endocytosis (46, 79). This protection in the macrophage survival is again offered by genes on SPIs and a number of virulence plasmids (3, 11, 45, 51, 73). Alterations of the host cell surface due to inflammation and membrane changes can provide pathogens with alternate adhesin receptors in addition to new ligand presentation on the microbe associated with host adhesion that are not well defined. Investigating interactions of afimbrial adhesin factors with non-ECM components, when intestinal epithelial cells are infected with stressed Salmonella, is equally important to discover new mechanisms of infection that can be used to reduce
Salmonella host association.
The hypothesis tested in this study was that abiotic stresses encountered in the environment and biotic stresses encountered in the host induce specific receptor/ligand partnerships and metabolism genes in the host and microbe that result in an increase in adhesion and infection of intestinal epithelial cells. This study investigated the effect of cold stress (5°C, 48 h), peroxide stress (5 mM H2O2, 5 h) and acid stress (pH 4.0, 90 min)
on the adhesion and invasion on intestinal epithelial cells. Cold stress to S. Typhimurium
significantly (p < 0.05) increased adhesion and invasion in epithelial cells and this was
concomitant with significant (q < 0.05) induction of virulence genes associated with 94
T3SS, pili biosynthesis, and prophage genes in cold-stressed S. Typhimurium.
Concomitantly, significant (p < 0.05) increases in caspase 9 and caspase 3/7 activity in the epithelial cells infected with cold-stressed S. Typhimurium was observed. Further, a prophage gene STM2699, significantly (q < 0.05) induced due to cold stress and a spectrin gene SPTAN1, significantly (q < 0.05) induced due to infection with cold- stressed S. Typhimurium, were identified to be receptor/ligand partners that significantly contributed (p < 0.05) to an increase in adhesion and invasion of cold-stressed S.
Typhimurium in epithelial cells.
MATERIALS AND METHODS
Cell culture. Intestinal epithelial cells (Caco-2; ATCC HTB-37) were obtained from American Type Culture Collection (Manassas, VA) and were grown as per the manufacturer’s instructions. Briefly, cells were seeded to a density of 105 cells/cm2 using cell culture media comprising of DMEM/High Modified (Thermo Scientific, Rockford,
IL), non-essential amino acids (Thermo Scientific), 10mM MOPS (Sigma, St. Louis,
MO), 10 mM TES (Sigma), 15 mM HEPES (Sigma) and 2 mM NaH2PO4 (Sigma).
Additionally, 16.6% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) was added to the cell culture media only for feeding cells. Cells were incubated at 37˚C with
5% CO2 for 14-days post confluence so as to let them differentiate. The cells were serum starved for 24 h, prior to sample collection for gene expression profiles, by feeding the cells with only cell culture media (excluding FBS). 95
Bacterial strains and growth conditions. Salmonella Typhimurium LT2 ATCC
700720 (S. Typhimurium) was used in this study. Cell culture media was used to grow the bacteria at 37°C with shaking at 220 rpm.
Stress treatments for bacteria. A log phase culture was centrifuged at 7200 × g for 5 min and re-suspended at equal density in complete cell culture media, maintained at
5°C for cold stress; in cell culture media with 5 mM H202 maintained at 37°C for
peroxide stress; in cell culture media with pH 4.0 maintained at 37°C for acid stress.
These stress treatments were given for 48 h, 5h and 90 min respectively in two biological
replicates. The control for these treatments was stationary phase (~15 h) culture re-
suspended in cell culture media maintained at 37°C.
Total host association measurements. Caco-2 cells were infected with
respective bacterial treatments at multiplicity of infection (MOI) of 1:1000, in a 96 well
plate, in three biological replicates. The infected cells were incubated for 60 min, 90 min
and 120 min at 37°C with 5% CO2. Upon incubation, media was aspirated and cells were
washed three times with 200 μl tyrodes buffer (for composition, see supplementary data
Table B-1). The cells and associated bacteria were lysed using 50 μl commercial lysis
buffer (AEX Chemunex, France) as described by Desai et al. (25) and cell lysate was used to quantify the number of Caco2 cells and associated bacteria. Quantitative bacterial analysis was done using qPCR with a CFX 96 Real Time System (BioRad, Hercules,
CA). Reactions were performed with iQ SYBR Green Supermix (BioRad) as per manufacturer’s instructions. Briefly, a 25 μl reaction contained 1 μl of cell lysate and 100 nM of forward (F) and reverse (R) PCR primers for 16s rDNA gene (F: 5`-TGT TGT
GGT TAA TAA CCG CA -3`; R: 5`-CAC AAA TCC ATC TCT GGA -3`) (114) to 96 quantify S. Typhimurium or G3PDH gene (F: 5`- ACC ACA GTC CAT GCC ATC AC -
3`; R: 5`-TCC ACC ACC CTG TTG CTG TA -3`) to quantify Caco2 cells (Integrated
DNA technologies, Coralville, IA). The thermocycling parameters for both primers consisted of denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation, annealing and extension at 95˚C for 15 s, 56˚C for 30 s, 72˚C for 30 s, respectively, and a final extension at 72°C for 1 min. The amplified product was verified using melt curve analysis from 50˚C to 95˚C with a transition rate of 0.2˚C/s. At each time point, one-way analysis of variance (ANOVA) with Tukey post test was done to find significant differences across treatment’s and control’s group means.
Gentamicin protection assay. Caco-2 cells were infected with respective bacterial treatments at MOI of 1:1000 in a 96-well plate, in three biological replicates.
The infected cells were incubated for 60 min at 37°C with 5% CO2. Upon incubation,
media was aspirated and cells were washed three times with 200 μl tyrodes buffer. To
enumerate invaded bacteria, cells were incubated with 200 μl of 100 µg/ml gentamicin for 2 h at 37°C with 5% CO2. As a control for invaded bacteria, cells were incubated with
only cell culture media (without gentamicin) to enumerate total host associated bacteria.
Cells were again washed three times with 200 μl tyrodes buffer and lysed with 100 μl of
0.01% triton. The total host associated or invaded bacteria were enumerated by pour plate method using nutrient agar (Difco, Detroit, MI). To calculate number of adhered bacteria, the mean of the number of invaded bacteria was subtracted from the mean of the total number of host associated bacteria. The error for adhered bacteria was propagated using equation (ΔZ)2 = (ΔA)2 + (ΔB)2 where, ΔZ standard error of mean (SEM) for adhered
bacteria, ΔA is SEM for total host associated bacteria and ΔB is SEM for invaded 97 bacteria. Independent two sample T-test was used to find significant differences across treatment’s and control’s group means (Fig 4-1B). When appropriate, one-way ANOVA with Tukey post test was done to find significant differences across treatment’s and control’s group means (Fig 4-6 and Fig 4-8).
Infection of epithelial cells for gene expression. Caco-2 cells were cultured in
T-75 flasks and were serum starved 24 h before infection. Respective bacterial treatments i.e. S. Typhimurium cold-stressed for 48 h and non-stressed S. Typhimurium, at MOI of
1:1000, were used to infect epithelial cells. The control for these infections was addition of cold-stressed and non-stressed S. Typhimurium respectively, in T-75 flasks without the epithelial cells, and non infected epithelial cells. The experiment was done in two biological replicates. The infected cells along with the controls were incubated at 37°C with 5% CO2 for 60 min. For infected cells, media with extracellular bacteria was
aspirated and 10 ml of TRIzol LS reagent (Invitrogen, Carlsbad, CA) was added to the
cells. This was gently mixed with pipette followed by centrifugation at 7200 × g for 5
min to pellet the host associated bacteria. TRIzol LS supernatant was stored in a clean
tube and further processed for RNA extraction from infected Caco-2 cells. The bacterial
pellet was re-suspended in 2 ml of fresh TRIzol LS, gently mixed and further processed for RNA extraction from host associated bacteria. For controls, media with bacteria was
collected and centrifuged at 7200 × g for 5 min. To the pellet, 10 ml of TRIzol LS reagent
was added, gently mixed and was further processed for RNA extraction from bacterial
controls. For the non infected Caco-2 cells, media was aspirated and 10 ml of TRIzol LS
reagent was added to the cells, gently mixed and was further processed for RNA
extraction form non infected Caco-2 cells. 98
Bacterial RNA extraction and gene expression. Sample preparation for gene expression profiling was performed as described by Yi Xie et al (111) with modification in RNA isolation, which was done using TRIzol LS reagent (Invitrogen). The 750 µl of the TRIzol LS suspension containing host associated bacteria or bacterial controls was centrifuged at 7200 × g for 5 min. The TRIzol LS supernatant was stored in a clean tube for later use and to the bacterial pellet, 1 ml of lysis enzyme cocktail containing 50 mg/ml of lysozyme (Sigma) and 200 U/ml mutanolysin (Sigma) in TE buffer (10mM Tris and
1mM EDTA, pH 8) was added. The solution was mixed gently and incubated at 37°C for
1 h followed by centrifugation at 7200 × g for 5 min. The supernatant was discarded and pellet was re-suspended in 250 μl of proteinase K buffer (100 mM Tris-HCl, 5 mM
EDTA, 200 mM NaCl, and 0.2 % SDS, pH 8) containing 8 U/ml of proteinase K
(Fermentas, Glen Burnie, MD). This was incubated at 55°C for 1 h with intermittent mixing. To this, previously stored TRIzol LS was added and gently mixed. After this step manufacturer’s (TRIzol LS, Invitrogen) protocol was followed for RNA extraction. RNA concentration, A260/280 and A260/230 were measured on NanoDrop (Thermo scientific,
Waltham, MA) and samples were processed further only if the RNA concentration was at least 0.5 µg/µl and ratios were ≥ 1.8. The RNA samples were analyzed for integrity on
2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Total RNA (10 g in 20 μl) was reverse transcribed into cDNA with 6 µg of random hexamers and 400 U of Superscriptase II (Invitrogen) according to the manufacturer’s protocol. Upon cDNA synthesis, the enzyme was heat inactivated and the
RNA templates were degraded with 80 U of RNaseH (Epicentre, Madison, WI) at 37°C for 20 min. The reaction mixture was cleaned by using the Qiaquick-PCR purification kit 99
(Qiagen, Valencia, CA) according to the manufacturer’s instructions, except that the Tris- containing buffer was replaced with 75% ethanol for washing the column. The purified single-strand cDNA was eluted from the columns twice with a total of 100 μl of nuclease free water (Ambion, Austin, TX).
cDNA fragmentation was done using 0.6 U of DNaseI (Promega, Madison, WI) per µg of cDNA, according to the instructions. The fragmented 1 µg of cDNA was labeled using 2 µl of GeneChip DNA Labeling reagent (Affymetrix, Santa Clara, CA) and 60 U of Terminal Transferase enzyme (New England Biolabs, Ipswich, MA). The samples were denatured prior to hybridization, at 98°C for 10 min followed by snap cooling at 4°C for 5 min.
Hybridization and normalization. Labeled cDNA was hybridized onto a custom made Affymetrix GeneChip designed against all the annotated coding sequences of S.
Typhimurium LT2 ATCC 700720. Briefly, the array contained 9852 probe sets, of which
4735 probe sets were designed against S. Typhimurium. Each probe set contained 11 probes, each 25 nucleotides long. Labeled cDNA (500 ng) for samples extracted from pure culture of S. Typhimurium and 2000 ng of labeled cDNA for samples extracted from co-culture of S. Typhimurium and Caco-2, was hybridized onto the chips. The chips were hybridized and scanned at the Center for Integrated BioSystems (Utah State University,
Logan, UT) as per manufacturer’s protocols for E. coli. Raw data (.cel files) was back ground corrected, quantile normalized and summarized using MS-RMA (101).The resultant normalized log2 transformed intensity matrix was used for further statistical analysis. 100
Caco-2 RNA extraction and gene expression. The TRIzol LS samples containing infected or non infected Caco-2 cells were frozen (Liquid N2) and thawed
(70°C) twice. To 750 μl of TRIzol LS sample, 250 μl of water was added. This was
further processed for RNA extraction using manufacturer’s (TRIzol LS, Invitrogen)
instructions. RNA concentration, A260/280 and A260/230 were measured on NanoDrop
(Thermo scientific, Waltham, MA) and samples were processed further only if the RNA
concentration was at least 0.5 µg/µl and ratios were ≥ 1.8. The RNA samples were
analyzed for integrity on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Synthesis of cDNA, biotin labeled cRNA, fragmentation and purification of cRNA were
carried out using one-cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA).
Hybridization and normalization. Labeled and fragmented cRNA (10 g) was
hybridized onto the Affymetrix U133Plus2 GeneChips as per manufacturer's
recommendations at the Center for Integrated BioSystems (Utah State University, Logan,
UT). Raw data (.cel files) was back ground corrected, quantile normalized and
summarized using RMA (62). RMA normalized data was then filtered through the PANP
algorithm (107) to make presence-absence calls for each probe set. Probe sets that were
called present in at least one of the samples were included in further statistical analysis.
Antibody blocking assays. Anti-SPTAN1 (Novus Biologicals, Littleton, CO)
was used at four concentrations in to determine the optimum concentration at which
blocking was observed for cold-stressed S. Typhimurium followed by testing of two
concentrations for blocking of non-stressed S. Typhimurium (supplementary data Fig B-
1). This experiment was done in three biological replicates. Optimal concentration
(1:2000) in 25 µl was added to the cells to block exposed alpha spectrin on cell surface. 101
Cells were incubated for 60 min at 37°C with 5% CO2 before adding respective bacterial treatments in 25 µl volume, in three biological replicates. Upon adding bacteria (MOI of
1:100), invasion assays were performed as described earlier. One-way ANOVA with
Tukey post test was done to find significant differences across treatment’s and control’s group means.
Construction of isogenic mutants. Bacterial gene knockouts were performed as described by Datsenko and Wanner (22). Briefly, mini-prep kit (Qiagen, Valencia, CA) was used to isolate plasmid pKD46 carrying ampicillin resistance and λ Red recombinase genes, from E. coli BW25141 (CGSC 7634), plasmid pKD3 carrying chloramphenicol resistance gene, from E. coli BW25141 (CGSC 7631) and plasmid pKD4 carrying kanamycin resistance gene, from E. coli BW25141 (CGSC 7632). This plasmid pKD46 was electroporated in to S. Typhimurium and transformants were selected on LB agar with 100 µg/ml ampicillin (Sigma). S. Typhimurium carrying pKD46 was grown in LB broth in the presence of 100 µg/ml ampicillin and 100 mM L-arabinose to induce λ Red recombinase production. The chloramphenicol or kanamycin resistance genes were amplified using plasmid pKD3 or pKD4 templates respectively. The primers used for amplification were STM2699 P1 (5-CCTTAACGGCGCGGGCAGCCGCGCCAGTAT
TTCATTAACA GGATACGAACGTGTAGGCTGGAGCTGCTTC-3), STM2699 P2 (5-
TGGCGACCAGTGAAAGATGG TGGCGATATCCGCCACAAGATCATCAATCG
ATGGGAATTAGCCATGGTCC3’), invA P1 (5-
GTCGTACTATTGAAAAGCTGTCTTAATTTAATATTAACAGGATACCTATAGTG
TAGGCTGGAGCTGCTTC-3) and invA P2 (5-
TAATTCAGCGATATCCAAATGTTGCATAGA 102
TCTTTTCCTTAATTAAGCCCATGGGAATTAGCCATGGTCC-3). The purified PCR products were electroporated in to S. Typhimurium with induced λ Red recombinase. The transformants were selected on LB agar either with 10 µg/ml chloramphenicol or 40
µg/ml kanamycin. The gene knockout or absence of gene was confirmed by PCR using primers STM2699 J1 (5-CAGGATACGAACGTGTAGGC-3), STM2699 J2 (5-
AAGATCATCAATCGATGGGAAT-3), STM2699 F (5-
ATGAGCGACAAGCTGACTGA-3), STM2699 R (5-GCCCACGTCCATATCCATAA-
3), invA J1 (5-AGGATACCTATAGTGTAGGCTGGA-3), invA J2 (5-
TAAGCCCATGGGAATTAGC-3), invA F (5-AGTGCTCGTTTACGACCTGAA-3), and invA R (5-GCTATCTGCTATCTCACCGAAA-3).
Caspase assay. Caco-2 cells were washed with phosphate buffered saline (PBS) before use. Upon adding bacterial treatments (MOI of 1:1000) in 50 µl volume in a 96 well plate, in three biological replicates, cells were incubated at 37°C with 5% CO2 for 4
h, 6 h and 8 h. At different timepoints, caspase activity was measured using Caspase-Glo
8, 9 and 3/7 assay kits (Promega, Madison, WI) according to manufacturer’s instructions.
Bioluminescence was measured in a plate reader DTX 880 Multimode Detector
(Beckman Coulter, Brea, CA). Two-way ANOVA with Bonferroni post tests was used to
find significant differences across treatment’s and control’s group means, over time.
Statistical analysis for gene expression. Gene expression profile for cold-
stressed (5°C, 48 h) and non-stressed S. Typhimurium alone and in the presence of the
epithelial cells was obtained 60 min post infection. The data was analyzed as two class unpaired (cold stress vs. no stress), with T statistic, using Significance Analysis of
Microarrays (SAM) (105). All the genes were ranked based on the score (d) from SAM 103 output. This pre ordered ranked gene list was then used in Gene Set Enrichment Analysis software (GSEA) (82, 102) to detect the coordinate changes in the expression of groups of functionally related genes, upon respective treatments. The gene sets were defined based on the annotations from Comprehensive Microbial Resource (CMR) (89), Cluster of Orthologous Groups of proteins (COGs) (104), and Virulence Factors of pathogenic bacteria DataBase (VFDB) (15, 112).
Gene expression profile was obtained for epithelial cells alone and upon infection with cold-stressed (5°C, 48 h) and non-stressed S. Typhimurium, 60 min post infection.
The data was analyzed as two class unpaired (infection with cold-stressed S.
Typhimurium vs. infection with non-stressed S. Typhimurium), with T statistic, using
SAM (105). All the genes were ranked based on the score (d) from SAM output. This pre ordered ranked gene list was then used in GSEA (82, 102) to detect the coordinate changes in the expression of groups of functionally related genes, upon infection with cold-stressed S. Typhimurium. The gene sets were defined based on the annotations in
GO gene sets from Molecular Signatures Database (MSigDB) (102).
RESULTS AND DISCUSSION
Effect of stressed bacteria on association to epithelial cells. Cold, oxidation, and acid stress are commonly encountered by pathogens in the environment, during food processing treatments, and inside the host during infection, respectively. This study examined the effect of these stresses to modify host association and infection of S.
Typhimurium. Oxidative stress (5 mM H202, 5 h) to S. Typhimurium significantly (p <
0.05) increased association to epithelial cells at 60 min and 90 min post infection. Cold 104 stress (5°C, 48 h) to S. Typhimurium as well, significantly (p < 0.05) increased association to epithelial cells at all time points (Fig 4-1A) and it remained significantly (p
< 0.05) increased over time (120 min) as compared with effect of oxidative stress.
Consequently, invasion was measured at 60 min post infection to avoid subsequent lysis and reinfection due to an undefined multiplicity of infection (MOI). Cold stress treatment to S. Typhimurium significantly (p < 0.05) increased adhesion and invasion in the epithelial cells as compared to infection with non-stressed S. Typhimurium (Fig 4-1B).
This observation is consistent with reports of the effect of other abiotic conditions encountered by Salmonella during environmental transit such as media ion composition
(109), oxygen availability (29), osmolarity (103), bacterial growth phase (29, 68, 96, 99,
103) on the regulation of the outcome and severity of infection.
Gene expression profile of cold-stressed S. Typhimurium. Cold stress (5°C, 48 h) to S. Typhimurium significantly (q < 0.05) regulated gene categories with virulence associated functions such as T3SS & its effectors, cellular pathogenesis, plasmid genes and prophage genes, in addition to gene categories associated with protein secretion & trafficking, DNA metabolism & repair, and degradation of RNA (Table 4-1). In this study, we focus on molecules directly involved with host association and virulence since cold stress significantly (p < 0.05) increased host association.
Cold stress coordinately induced expression of genes related to T3SS from SPI2, which are required for the later stages of infection and intracellular replication (55). Two component system ssrAB controls the expression of genes encoding the structural
components of the T3SS and the effectors located both in SPI2 and outside of SPI2 (47)
out of which one of the local regulators, ssrA responsible for induction of genes on SPI2 105
(24), was also significantly (q < 0.05) induced. The genes encoding effectors mainly from ssa operons, sse operons, and genes such as sscA, sscB, sifA, sifB, pipA, pipB, and pipB2 were also significantly (q < 0.05) induced. sifA and sifB are located outside SPI2 but these effectors are required for intracellular bacterial replication. PipABD genes, located on SPI5, play an important role in enteric infections (110). Additionally from SPI1, genes invJ and sipC responsible for translocation of the effectors and sipC (55) further involved in actin bundling (57) were also significantly (q < 0.05) induced (Fig 4-1B). The role of
T3SS in allowing bacteria to remain docked at the cellular membrane to deliver effectors in the host cell cytosol (19) has been demonstrated. Consequently, the significant induction of the genes on SPIs encoding T3SS, due to cold stress, implicates enhanced potential to adhere, invade and multiply intracellularly. Genes of the tra and trb clusters forming subset of type four secretion system (T4SS) (67) were also induced (Fig 4-2C).
These genes, encoded on the plasmid, are the elements of pilus required for mating and conjugal DNA transfer. Transcription of the tra operon is activated by TraJ (10), whose synthesis is controlled by FinP (negative regulation) and FinO (positive regulation) (10) and significant (q < 0.05) induction of both traJ and finO was observed (Fig 4-2C). In addition to genes involved in T3SS and T4SS with functions of high association to host, invasion and intracellular replication, genes on prophages Fels-1 (22/35), Gifsy-2
(12/52), Gifsy-1 (24/53) and Fels-2 (9/47) were also significantly (q < 0.05) induced (Fig
4-2A). The prophages are often identified adjacent to virulence genes (23, 32, 53, 56, 91,
106) and tRNA genes (76) due to which they contribute to pathogenicity, insertion of transferable elements (13, 93) and lateral spreading of the pathogenicity determinants.
Moreover, many virulence factors have been reported to be present on prophages (2, 34, 106
36, 59, 78, 80, 100). The role of Gifsy-2 and Gifsy-1 prophages has been studied in great detail and their contribution in virulence is shown (35) but there is no direct evidence showing direct contribution of Fels-1 and Fels-2 in virulence other than role of sodC, nanH and grvA from Fels-1 (7) and a suspected role of abiU from Fels-2 (5, 7). Other phage genes such as pspA and pspD were also significantly (q < 0.05) induced due to cold stress (Fig 4-2A).
Gene expression profile of cold-stressed S. Typhimurium upon infection of epithelial cells. Infection of epithelial cells with cold-stressed (5°C, 48 h) S.
Typhimurium significantly (q < 0.05) regulated gene categories associated with virulence such as prophage genes, plasmid genes including spv genes, transposon genes and stress response in addition to gene categories with functions in DNA transformation, energy metabolism, protein synthesis, polysaccharides metabolism and chemotaxis (Table 4-2) as compared to infection with non-stressed S. Typhimurium. This implies that due to extracellular cold stress to S. Typhimurium, these above described categories are additionally regulated beyond the regulation of the genes or gene categories in non- stressed S. Typhimurium in the presence of the host.
Genes of the tra and trb clusters, forming subset of T4SS coordinately induced due to cold stress alone, remained significantly (q < 0.05) induced in the presence of the host. Additionally, traL required for pilus tip formation on cell surface (1) and traS involved in entry exclusion during mating pair stabilization (67) were also significantly
(q < 0.05) induced (Fig 4-3D). The significant (q < 0.05) induction of genes responsible for conjugal transfer upon cold stress and their persistent induction in the presence of epithelial cells upon infection, could result in high probability of DNA transfer between 107 bacteria upon cold stress or between bacteria and epithelial cells upon infection with cold-stressed S. Typhimurium. Increased frequency of gene transfer between S.
Typhimurium inside the epithelial cells has been reported (33). In either case transfer of genetic material could lead to acquisition of new traits giving rise to more virulent organisms.
Gene category related to genes on prophages Fels-1 (23/35), Gifsy-2 (12/52),
Gifsy-1 (15/53) and Fels-2 (11/47) was also significantly (q < 0.05) induced (Fig 4-3A).
Other phage genes such as pspABCDE were all significantly (q < 0.05) induced in cold- stressed S. Typhimurium, in the presence of the host cells (Fig 4-3A). Since the psp genes are highly conserved in some pathogens and are also involved in infection processes (6,
21, 28), their significant (q < 0.05) induction could contribute to increased pathogenicity.
Genes involved in DNA transposition such as genes encoding resolvase, transposase and integrase on the chromosome as well as on the plasmid were also significantly (q < 0.05) induced (Fig 4-3C). Many of these genes were located near the prophage regions. The spv genes on the plasmid, required for intracellular proliferation and to cause a systemic disease (52), were significantly (q < 0.05) induced in the cold-stressed S. Typhimurium, only in presence of the host cells. spvR is the transcriptional regulator required for activation of spvABCD. Significant (q < 0.05) induction of spvR and spvABC was observed (Fig 4-3B). The other genes being significantly (q < 0.05) induced were cspABE, uspA, relA, htpX and osmB, involved in stress response, (Fig 4-3B) and remained significantly (q < 0.05) induced in cold-stressed S. Typhimurium, in the presence of the host cells. 108
The flagella are usually required during the extracellular phase in the life cycle of
Salmonella to assist with motility and chemotaxis. During the infection, flagellar proteins are recognized by TLR 5, NLR and Ipaf on the host cell, leading to induction of pro- inflammatory cascade, caspase 1 dependent cell death and T-cell mediated immune response (16, 18, 40, 77, 81, 92). However, down-regulation of these genes is considered to be a means by which cold-stressed S. Typhimurium evades its detection (20). The genes related to flagellar functions from fli, flh and flg operons were found to be significantly (q < 0.05) repressed in cold-stressed S. Typhimurium in the presence of the epithelial cells. Lipopolysaccharide (LPS), a component of the outer membrane of
Salmonella has been shown to interact with TLR4 (26) and initiate a pro-inflammatory cascade. The genes encoding surface polysaccharides including LPS were also repressed and repression can contribute in evasion of Salmonella’s detection from host’s immune system.
To summarize, significantly (p < 0.05) increased adhesion and invasion in epithelial cells of cold-stressed S. Typhimurium (Fig 4-1) was concomitant with significantly (q < 0.05) induced expression of genes related to T3SS (Fig 4-2B), T4SS
(Fig 4-2C) and prophage (Fig 4-2A) due to cold stress alone, and persistent induction of many of these genes with induction of additional spv genes (Fig 4-3B) upon infection of epithelial cells. This further implied that cold-stressed S. Typhimurium were more pathogenic as compared to non-stressed S. Typhimurium, which can result in increased epithelial cell damage and death.
Effect of cold-stressed S. Typhimurium on gene expression of epithelial cells.
Infection of epithelial cells with cold-stressed (5°C, 48 h) S. Typhimurium significantly 109
(q < 0.05) regulated 18 gene categories related to mitochondrial function in addition to gene categories associated with ribosomal genes, ribonucleoprotein complex, kinesin complex, RNA splicing and coenzyme metabolism (Table 4-3), when compared to infection with non-stressed S. Typhimurium. A dominant role of mitochondria is to produce ATP through electron transfer chain (ETC) and regulate cellular metabolism.
Due to their central role in cell metabolism, dysfunction due to any external or internal stimuli will have a wide impact on human health and diseases. Mitochondrial dysfunction has been associated with many neurodegenerative diseases (71), oxidative stress (8, 12), ageing (9) and apoptosis (64). Mitochondria have a central role in many forms of cell death. All the genes significantly (q < 0.20) regulated in the gene categories functionally associated with mitochondria were overlaid on the electron transport chain for visualization using Ingenuity Pathway Analysis software (IPA) (Ingenuity Systems,
Redwood City, CA) (Fig 4-4). The genes encoding the subunits of the four complexes of
ETC, Complex I (NADH dehydrogenase/reductase), Complex II (Succinate dehydrogenase), Complex III (Cytochrome bc1 complex) and Complex IV (Cytochrome c oxidase) were significantly (q < 0.20) induced (Fig 4-4). Additionally genes, glycerol-
3-phosphate dehydrogenase 2 (GPD2) and monoamine oxidase A (MAOA), were also significantly (q < 0.20) induced. Although ETC is a very efficient system, it is a major site to produce superoxide radicals and reactive oxygen species as there is a high probability of electron being passed to oxygen directly, instead of next electron carrier in the chain. Appropriate balance of oxidants and antioxidants is required for cell survival; even the slight perturbation can lead to damage of biological macromolecules and hence cell death. Consequently, we further examined the expression intensities of the 110 components of antioxidant defense system (71). No significant changes in gene expression were observed for any of the genes except SOD2 (superoxide dismutase 2),
CAT (catalase), GPX7 (glutathione peroxidase 7) and PRX3 (peroxiredoxin 3) (q < 0.20).
However, the fold induction for these genes was SOD2 – 1.2, CAT – 1.2, GPX7 – 1.3 and PRX3 – 1.5. In functionally intact mitochondria, a large number of antioxidant gene products neutralize the effect of superoxide radicals and reactive oxygen species (71).
These observations suggested induced oxidative stress in epithelial cells infected with cold-stressed S. Typhimurium.
As seen earlier in gene expression profile of S. Typhimurium, the genes related to
SPI2 were significantly (q < 0.05) induced due to cold stress alone and even though they were not induced any further in the presence of epithelial cells, the basal expression and residual protein product can still have the effects. The genes in SPI2, along with various other functions, also cause accumulation of microtubules around the Salmonella containing vacuole (SCV) (66), recruitment of kinesin and dynein to regulate vacuolar membrane dynamics (4, 50, 74), and interference of the activity of ubiquitination pathway (95). This, together with the significantly (q < 0.05) increased adhesion and invasion, explains the induction of genes related to microtubule activity and kinesin complex in epithelial cells, upon infection with cold-stressed S. Typhimurium.
To summarize, induced oxidative stress to mitochondria was implied from the gene expression profile of epithelial cells infected with cold stress S. Typhimurium as compared to infection with non-stressed S. Typhimurium. Oxidative stress has been shown to cause damage leading to apoptosis via caspase activation (54, 84, 113). As a 111 measure of cytotoxicity upon infection of epithelial cells with cold-stressed S.
Typhimurium, activation of caspases 8, 9, and 3/7 was measured.
Induction of apoptosis. Caspases are intracellular cysteine containing, aspartic acid specific proteases implicated in programmed cell death. Caspases are classified in to
"initiators" and "executioners" (12, 90). The activation of the "executioner" caspase i.e. caspase 3/7, is a committed step in apoptosis and can come via extrinsic and/or intrinsic pathway involving "initiator" caspase 8 and/or caspase 9, respectively (12, 90). We measured activation of caspase 3/7 upon infection of epithelial cells with non-stressed and cold-stressed (5°C, 48 h) S. Typhimurium. The activation of caspase 3/7, significantly (p < 0.05) increased at 8 h post infection with cold-stressed S. Typhimurium as compared to infection with non-stressed S. Typhimurium (Fig 4-5C). The activation of caspase 8 did not significantly change upon infection with cold-stressed S. Typhimurium as compared to non-stressed S. Typhimurium, at any time points tested during infection
(Fig 4-5A). On the other hand, the activation of caspase 9 was significantly (p < 0.05) higher at 8 h post infection with cold-stressed S. Typhimurium as compared to infection with non-stressed S. Typhimurium (Fig 4-5B). Moreover, activity of caspase 9 increased with time, further implying the contribution of caspase 9 in activation of caspase 3/7 through intrinsic pathway. To summarize, significant (p < 0.05) activation of caspase 9 and caspase 3/7 was concomitant with the gene regulation observations suggesting induced apoptosis through intrinsic (mitochondrial) route, in epithelial cells infected with cold-stressed S. Typhimurium.
Receptors involved in association of S. Typhimurium to epithelial cells. Since, significant increase in adhesion and invasion of cold-stressed S. Typhimurium to 112 epithelial cells, leading to induction in caspase 9 and 3/7 activity was observed, the receptor/ligand partnership that led to this induction came in question (Fig 4-1 and Fig 4-
4). Examination of protein-protein interactions used a whole cell cross linking method
(Desai et al., unpublished data). Using this method, five proteins from S. Typhimurium were found cross linked to 10 proteins from the colonic epithelial cells in five different locations on the gel (Appendix B Methods and Table B-3). The proteins found included a
Fels-2 prophage gene STM2699 from S. Typhimurium cross linked to SPTAN1 (spectrin) from Caco-2 cells. The expression for STM2699 in S. Typhimurium was significantly (q
< 0.05) induced due to cold stress and it remained induced in the presence of Caco-2 cells during infection (Fig 4-6). Expression of SPTAN1 in Caco-2 cells was further induced during infection with cold-stressed S. Typhimurium as compared to infection with non- stressed S. Typhimurium (Fig 4-6). Consequently, further characterization to define the role of STM2699 in adhesion and invasion of Caco-2 cells was done using gene knock- out in wild type and invasion deficient strain of S. Typhimurium (S. Typhimurium ΔinvA)
(43); and to define the role of SPTAN1 in adhesion and invasion of S. Typhimurium was done by blocking the surface of Caco-2 cells with an anti-SPTAN1 antibody before addition of Salmonella.
Infection of Caco-2 cells with non-stressed S. Typhimurium ΔSTM2699 did not change adhesion, but significantly (p < 0.05) reduced invasion (Fig 4-7A). The invasion deficient strain (ΔinvA), displayed the expected decrease in invasion and also displayed an unexpected significant (p < 0.05) decrease in adhesion. Infection of Caco-2 cells with
S. Typhimurium ΔSTM2699:ΔinvA did not further change adhesion or invasion as compared with S. Typhimurium ΔinvA (Fig 4-7A). Infection of Caco-2 cells blocked with 113 anti-SPTAN1 antibody and infected with non-stressed S. Typhimurium significantly reduced (p < 0.05) adhesion, but did not change invasion (Fig 4-7B). No further reduction in adhesion of Caco-2 cells, blocked with anti-SPTAN1 antibody, was observed during infection with S. Typhimurium ΔSTM2699 but significant (p < 0.05) reduction in invasion was observed. S. Typhimurium ΔinvA and S. Typhimurium ΔSTM2699:ΔinvA, both significantly reduced (p < 0.05) adhesion and invasion in Caco-2 cells blocked with anti-SPTAN1 antibody (Fig 4-7B). No further reduction in adhesion and invasion of
Caco-2 cells, blocked with anti-SPTAN1 antibody, was observed when infected with non-stressed S. Typhimurium ΔSTM2699:ΔinvA as compared to S. Typhimurium ΔinvA
(Fig 4-7B). These observations established that this previously unknown receptor/ligand partnership had a direct role in Salmonella cellular association.
Subsequent examination of cold stress (5°C, 48 h) on the role of the STM2699 and SPTAN1 partnership was done to determine the regulation due to stress and a possible explanation of increased Salmonella association with stress. Infection of Caco-2 cells with cold-stressed S. Typhimurium ΔSTM2699 significantly (p < 0.05) reduced adhesion and invasion, but did not abolish host association (Fig 4-8A). Cold-stressed S.
Typhimurium ΔSTM2699:ΔinvA displayed a significant (p < 0.05) reduction in adhesion
(Fig 4-8A). Infection of Caco-2 cells, blocked with anti-SPTAN1 antibody, with cold- stressed S. Typhimurium did not change adhesion or invasion; however, infection with cold-stressed S. Typhimurium ΔSTM2699 significantly (p < 0.05) reduced both adhesion and invasion (Fig 4-8B). Further reduction in adhesion of Caco-2 cells, blocked with anti-
SPTAN1 antibody, was observed when infected with cold-stressed S. Typhimurium
ΔSTM2699:ΔinvA as compared to S. Typhimurium ΔinvA (Fig 4-8B). 114
To summarize, a decrease in adhesion followed by a decrease in invasion of
Caco-2 cells upon infection with cold-stressed (5°C, 48 h) S. Typhimurium ΔSTM2699 proves the role of STM2699 in adhesion when the bacteria are cold-stressed.
Furthermore, decrease in adhesion to Caco-2 cells due to STM2699 deletion was also observed in invasion deficient strain background with cold stress. Blocking with anti-
SPTAN1 antibody did not change adhesion or invasion of cold-stressed S. Typhimurium which may be due to induction of vast array of genes upon cold stress to S. Typhimurium
(Fig 4-2 and 4-3) with contribution in binding to other receptors on host cells. However, decrease in adhesion and invasion was observed with cold-stressed S. Typhimurium
ΔSTM2699 upon blocking the cell surface with anti-SPTAN1 antibody, again proving the interaction of STM2699 with SPTAN1.
Phylogenetic distribution of STM2699 and SPTAN1. Alpha II spectrin is one of the ubiquitous major proteins responsible for maintaining cytoskeletal integrity of the host cells (69). STM2699 is one of the Fels-2 prophage genes in S. Typhimurium. Table
4-4 shows the % identity of all the homologues of STM2699 from S. Typhimurium.
STM2699 is present in virulent strains of Salmonella such as S. Agona SL483, S.
Typhimurium SL1344, S. Typhi CT18, S. Javiana GAMM04042433 with greater than
90% homology. To conclude, significant (q < 0.05) induction of STM2699 due to cold stress (5°C, 48 h) to S. Typhimurium and significant (q < 0.05) induction of SPTAN1 in
Caco-2 cells due to infection with cold-stressed (5°C, 48 h) S. Typhimurium, along with their interaction and significant (p < 0.05) role in adhesion and invasion will lead to high association of any cold-stressed Salmonella to the host intestinal epithelial cells.
115
CONCLUDING REMARKS
A wide variety of the bacterial pathogens have evolved mechanisms to adhere and invade in to host cells. Infection of epithelial cells with Salmonella requires virulence factors that are encoded on SPIs. In general SPI1 is required for bacterial entry (42) and
SPI2 is required for intracellular survival (97). Both SPI1 and SPI2 encode T3SS and effectors (44). Contact with host cell surface triggers rapid formation of T3SS and delivery of effectors in to host cell cytosol (115) leading to membrane ruffling and internalization of bacteria (37, 41). Salmonella invasion is rapid and bacteria appear in vacuoles within few minutes upon interacting with host cell. The death of cultured intestinal epithelial cells by apoptosis upon invasion of Salmonella has been shown (65).
In this study, we found that cold stress (5°C, 48 h) to S. Typhimurium significantly (p <
0.05) increased adhesion and invasion in epithelial cells as compared to non-stressed S.
Typhimurium. Significant induction (p < 0.05) of genes encoding T3SS and effectors, genes encoding subset of T4SS and prophages due to cold stress exacerbates the damaging effect. Upon interaction with the host cells, several signaling events are also activated in the host cells. We observed significant (p < 0.05) induction of caspase 9 and caspase 3/7 activity upon infection with cold-stressed S. Typhimurium. This increased activity of caspase was attributed to perturbation of the balance of oxidants and antioxidants as evident by gene expression profile of epithelial cells upon infection with cold-stressed S. Typhimurium. Further we discovered pair of receptors i.e. STM2699 on
Salmonella and SPTAN1 on Caco-2 cells, which play a role in adhesion and invasion, and their interaction is significantly (p < 0.05) greater when epithelial cells are infected 116 with cold-stressed S. Typhimurium. These observations together conclude that exposure to cold stress (5°C, 48 h) increases pathogenicity of S. Typhimurium.
REFERENCES
1. Anthony, K. G., W. A. Klimke, J. Manchak, and L. S. Frost. 1999.
Comparison of proteins involved in pilus synthesis and mating pair stabilization
from the related plasmids F and R100-1: insights into the mechanism of
conjugation. J Bacteriol 181:5149-59.
2. Bakshi, C. S., V. P. Singh, M. W. Wood, P. W. Jones, T. S. Wallis, and E. E.
Galyov. 2000. Identification of SopE2, a Salmonella secreted protein which is
highly homologous to SopE and involved in bacterial invasion of epithelial cells.
J Bacteriol 182:2341-4.
3. Beuzon, C. R., S. Meresse, K. E. Unsworth, J. Ruiz-Albert, S. Garvis, S. R.
Waterman, T. A. Ryder, E. Boucrot, and D. W. Holden. 2000. Salmonella
maintains the integrity of its intracellular vacuole through the action of SifA.
EMBO J 19:3235-49.
4. Boucrot, E., T. Henry, J. P. Borg, J. P. Gorvel, and S. Meresse. 2005. The
intracellular fate of Salmonella depends on the recruitment of kinesin. Science
308:1174-8.
5. Boyd, E. F., and H. Brussow. 2002. Common themes among bacteriophage-
encoded virulence factors and diversity among the bacteriophages involved.
Trends Microbiol 10:521-9. 117
6. Brissette, J. L., M. Russel, L. Weiner, and P. Model. 1990. Phage shock
protein, a stress protein of Escherichia coli. Proc Natl Acad Sci USA 87:862-866.
7. Brussow, H., C. Canchaya, and W. D. Hardt. 2004. Phages and the evolution of
bacterial pathogens: from genomic rearrangements to lysogenic conversion.
Microbiol Mol Biol Rev 68:560-602.
8. Buttke, T. M., and P. A. Sandstrom. 1994. Oxidative stress as a mediator of
apoptosis. Immunology Today 15:7-10.
9. Cadenas, E., and K. J. A. Davies. 2000. Mitochondrial free radical generation,
oxidative stress, and aging. Free Radical Biol and Medicine 29:222-230.
10. Camacho, E. M., and J. Casadesus. 2002. Conjugal transfer of the virulence
plasmid of Salmonella enterica is regulated by the leucine-responsive regulatory
protein and DNA adenine methylation. Mol Microbiol 44:1589-98.
11. Chakravortty, D., I. Hansen-Wester, and M. Hensel. 2002. Salmonella
pathogenicity island 2 mediates protection of intracellular Salmonella from
reactive nitrogen intermediates. J Exp Med 195:1155-66.
12. Chandra, J., A. Samali, and S. Orrenius. 2000. Triggering and modulation of
apoptosis by oxidative stress. Free Radical Biol and Medicine 29:323-333.
13. Cheetham, B. F., and M. E. Katz. 1995. A role for bacteriophages in the
evolution and transfer of bacterial virulence determinants. Mol Microbiol 18:201-
8.
14. Chen, L., J. Yang, J. Yu, Z. Yao, L. Sun, Y. Shen, and Q. Jin. 2005. VFDB: a
reference database for bacterial virulence factors. Nucleic Acids Res 33:D325-
D328. 118
15. Chen, S. L., L. Jian, and H. Q. Lang. 2003. Optimization of peroxynitrite-
luminol chemiluminescence system for detecting peroxynitrite in cell culture
solution exposed to carbon disulphide. Luminescence 18:249-53.
16. Choudhary, A., R. P. Tiwari, A. Koul, V. Chanana, S. Gupta, and P. Rishi.
2005. Role of Salmonella surface components in immunomodulation of
inflammatory mediators. Mol Cell Biochem 270:167-75.
17. Christman, M. F., R. W. Morgan, F. S. Jacobson, and B. N. Ames. 1985.
Positive control of a regulon for defenses against oxidative stress and some heat-
shock proteins in Salmonella typhimurium. Cell 41:753-762.
18. Coers, J., R. E. Vance, M. F. Fontana, and W. F. Dietrich. 2007. Restriction of
Legionella pneumophila growth in macrophages requires the concerted action of
cytokine and Naip5/Ipaf signalling pathways. Cell Microbiol 9:2344-57.
19. Cornelis, G. R. 2006. The type III secretion injectisome. Nat Rev Microbiol
4:811-25.
20. Cummings, L. A., W. D. Wilkerson, T. Bergsbaken, and B. T. Cookson. 2006.
In vivo, fliC expression by Salmonella enterica serovar Typhimurium is
heterogeneous, regulated by ClpX, and anatomically restricted. Mol Microbiol
61:795-809.
21. Darwin, A. J., and V. L. Miller. 1999. Identification of Yersinia enterocolitica
genes affecting survival in an animal host using signature-tagged transposon
mutagenesis. Mol Microbiol 32:51-62. 119
22. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of
chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad
Sci USA 97:6640-6645.
23. De Groote, M. A., U. A. Ochsner, M. U. Shiloh, C. Nathan, J. M. McCord, M.
C. Dinauer, S. J. Libby, A. Vazquez-Torres, Y. Xu, and F. C. Fang. 1997.
Periplasmic superoxide dismutase protects Salmonella from products of
phagocyte NADPH-oxidase and nitric oxide synthase. Proc Natl Acad Sci USA
94:13997-4001.
24. Deiwick, J., T. Nikolaus, S. Erdogan, and M. Hensel. 1999. Environmental
regulation of Salmonella pathogenicity island 2 gene expression. Mol Microbiol
31:1759-1773.
25. Desai, P. T., M. K. Walsh, and B. C. Weimer. 2008. Solid-phase capture of
pathogenic bacteria by using gangliosides and detection with real-time PCR. Appl
Environ Microbiol 74:2254-8.
26. Du, X., A. Poltorak, M. Silva, and B. Beutler. 1999. Analysis of Tlr4-mediated
LPS signal transduction in macrophages by mutational modification of the
receptor. Blood Cells Mol Dis 25:328-38.
27. Eckmann, L., M. T. Rudolf, A. Ptasznik, C. Schultz, T. Jiang, N. Wolfson, R.
Tsien, J. Fierer, S. B. Shears, M. F. Kagnoff, and A. E. Traynor-Kaplan.
1997. D-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal
epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-
kinase signaling pathways. Proc Natl Acad Sci USA 94:14456-60. 120
28. Eriksson, S., S. Lucchini, A. Thompson, M. Rhen, and J. C. Hinton. 2003.
Unravelling the biology of macrophage infection by gene expression profiling of
intracellular Salmonella enterica. Mol Microbiol 47:103-18.
29. Ernst, R. K., D. M. Dombroski, and J. M. Merrick. 1990. Anaerobiosis, type 1
fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by
Salmonella typhimurium. Infect Immun 58:2014-2016.
30. Ernst, R. K., T. Guina, and S. I. Miller. 1999. How intracellular bacteria
survive: surface modifications that promote resistance to host innate immune
responses. J Infect Dis 179 Suppl 2:S326-30.
31. Farr, S. B., and T. Kogoma. 1991. Oxidative stress responses in Escherichia coli
and Salmonella typhimurium. Microbiol Mol Biol Rev 55:561-585.
32. Farrant, J. L., A. Sansone, J. R. Canvin, M. J. Pallen, P. R. Langford, T. S.
Wallis, G. Dougan, and J. S. Kroll. 1997. Bacterial copper- and zinc-cofactored
superoxide dismutase contributes to the pathogenesis of systemic salmonellosis.
Mol Microbiol 25:785-96.
33. Ferguson, G. C., J. A. Heinemann, and M. A. Kennedy. 2002. Gene Transfer
between Salmonella enterica Serovar Typhimurium inside Epithelial Cells. J
Bacteriol 184:2235-2242.
34. Figueroa-Bossi, N., and L. Bossi. 1999. Inducible prophages contribute to
Salmonella virulence in mice. Mol Microbiol 33:167-76.
35. Figueroa-Bossi, N., and L. Bossi. 1999. Inducible prophages contribute to
Salmonella virulence in mice. Mol Microbiol 33:167-176. 121
36. Figueroa-Bossi, N., S. Uzzau, D. Maloriol, and L. Bossi. 2001. Variable
assortment of prophages provides a transferable repertoire of pathogenic
determinants in Salmonella. Mol Microbiol 39:260-71.
37. Finlay, B. B., and S. Falkow. 1990. Salmonella interactions with polarized
human intestinal Caco-2 epithelial cells. J Infect Dis 162:1096-106.
38. Foster, J. W., and Z. Aliabadi. 1989. pH-regulated gene expression in
Salmonella: genetic analysis of aniG and cloning of the earA regulator. Mol
Microbiol 3:1605-15.
39. Foster, J. W., and M. P. Spector. 1995. How Salmonella survive against the
odds. Annu Rev Microbiol 49:145-74.
40. Franchi, L., A. Amer, M. Body-Malapel, T. D. Kanneganti, N. Ozoren, R.
Jagirdar, N. Inohara, P. Vandenabeele, J. Bertin, A. Coyle, E. P. Grant, and
G. Nunez. 2006. Cytosolic flagellin requires Ipaf for activation of caspase-1 and
interleukin 1beta in salmonella-infected macrophages. Nat Immunol 7:576-82.
41. Francis, C. L., T. A. Ryan, B. D. Jones, S. J. Smith, and S. Falkow. 1993.
Ruffles induced by Salmonella and other stimuli direct macropinocytosis of
bacteria. Nature 364:639-42.
42. Galan, J. E. 1996. Molecular genetic bases of Salmonella entry into host cells.
Mol Microbiol 20:263-71.
43. Galan, J. E., and R. Curtiss, 3rd. 1989. Cloning and molecular characterization
of genes whose products allow Salmonella typhimurium to penetrate tissue
culture cells. Proc Natl Acad Sci USA 86:6383-7. 122
44. Galan, J. E., and H. Wolf-Watz. 2006. Protein delivery into eukaryotic cells by
type III secretion machines. Nature 444:567-73.
45. Gallois, A., J. R. Klein, L. A. Allen, B. D. Jones, and W. M. Nauseef. 2001.
Salmonella pathogenicity island 2-encoded type III secretion system mediates
exclusion of NADPH oxidase assembly from the phagosomal membrane. J
Immunol 166:5741-8.
46. García-del Portillo, F. Salmonella intracellular proliferation: where, when and
how? Microbes and Infection 3:1305-1311.
47. Garmendia, J., C. R. Beuzon, J. Ruiz-Albert, and D. W. Holden. 2003. The
roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the
Salmonella typhimurium SPI-2 type III secretion system. Microbiology
149:2385-2396.
48. Gerlach, R. G., D. Jäckel, B. Stecher, C. Wagner, A. Lupas, W. D. Hardt,
and M. Hensel. 2007. Salmonella Pathogenicity Island 4 encodes a giant non-
fimbrial adhesin and the cognate type 1 secretion system. Cellular Microbiology
9:1834-1850.
49. Greenacre, E. J., S. Lucchini, J. C. Hinton, and T. F. Brocklehurst. 2006. The
lactic acid-induced acid tolerance response in Salmonella enterica serovar
Typhimurium induces sensitivity to hydrogen peroxide. Appl Environ Microbiol
72:5623-5.
50. Guignot, J., E. Caron, C. Beuzon, C. Bucci, J. Kagan, C. Roy, and D. W.
Holden. 2004. Microtubule motors control membrane dynamics of Salmonella-
containing vacuoles. J Cell Sci 117:1033-45. 123
51. Gulig, P. A., H. Danbara, D. G. Guiney, A. J. Lax, F. Norel, and M. Rhen.
1993. Molecular analysis of spv virulence genes of the Salmonella virulence
plasmids. Mol Microbiol 7:825-30.
52. Gulig, P. A., H. Danbara, D. G. Guiney, A. J. Lax, F. Norel, and M. Rhen.
1993. Molecular analysis of spv virulence genes of the salmonella virulence
plasmids. Mol Microbiol 7:825-830.
53. Gunn, J. S., W. J. Belden, and S. I. Miller. 1998. Identification of PhoP-PhoQ
activated genes within a duplicated region of the Salmonella typhimurium
chromosome. Microb Pathog 25:77-90.
54. Hampton, M. B., and S. Orrenius. 1997. Dual regulation of caspase activity by
hydrogen peroxide: implications for apoptosis. FEBS Lett 414:552-6.
55. Hansen-Wester, I., and M. Hensel. 2001. Salmonella pathogenicity islands
encoding type III secretion systems. Microbes Infect 3:549-59.
56. Hardt, W. D., H. Urlaub, and J. E. Galan. 1998. A substrate of the centisome
63 type III protein secretion system of Salmonella typhimurium is encoded by a
cryptic bacteriophage. Proc Natl Acad Sci USA 95:2574-9.
57. Hayward, R. D., and V. Koronakis. 1999. Direct nucleation and bundling of
actin by the SipC protein of invasive Salmonella. Embo J 18:4926-4934.
58. Hensel, M. 2000. Salmonella pathogenicity island 2. Mol Microbiol 36:1015-23.
59. Ho, T. D., N. Figueroa-Bossi, M. Wang, S. Uzzau, L. Bossi, and J. M. Slauch.
2002. Identification of GtgE, a novel virulence factor encoded on the Gifsy-2
bacteriophage of Salmonella enterica serovar Typhimurium. J Bacteriol
184:5234-9. 124
60. Hobbie, S., L. M. Chen, R. J. Davis, and J. E. Galan. 1997. Involvement of
mitogen-activated protein kinase pathways in the nuclear responses and cytokine
production induced by Salmonella typhimurium in cultured intestinal epithelial
cells. J Immunol 159:5550-9.
61. Husain, M., T. J. Bourret, B. D. McCollister, J. Jones-Carson, J. Laughlin,
and A. Vazquez-Torres. 2008. Nitric oxide evokes an adaptive response to
oxidative stress by arresting respiration. J Biol Chem 283:7682-9.
62. Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U.
Scherf, and T. P. Speed. 2003. Exploration, normalization, and summaries of
high density oligonucleotide array probe level data. Biostatistics 4:249-64.
63. Jeffreys, A. G., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 1998.
Growth, survival and characterization of cspA in Salmonella enteritidis following
cold shock. Curr Microbiol 36:29-35.
64. Joza, N., S. A. Susin, E. Daugas, W. L. Stanford, S. K. Cho, C. Y. J. Li, T.
Sasaki, A. J. Elia, H. Y. M. Cheng, L. Ravagnan, K. F. Ferri, N. Zamzami, A.
Wakeham, R. Hakem, H. Yoshida, Y.-Y. Kong, T. W. Mak, J. C. Zuniga-
Pflucker, G. Kroemer, and J. M. Penninger. 2001. Essential role of the
mitochondrial apoptosis-inducing factor in programmed cell death. Nature
410:549-554.
65. Kim, J. M., L. Eckmann, T. C. Savidge, D. C. Lowe, T. Witthoft, and M. F.
Kagnoff. 1998. Apoptosis of human intestinal epithelial cells after bacterial
invasion. J Clin Invest 102:1815-23. 125
66. Kuhle, V., D. Jackel, and M. Hensel. 2004. Effector proteins encoded by
Salmonella pathogenicity island 2 interfere with the microtubule cytoskeleton
after translocation into host cells. Traffic 5:356-70.
67. Lawley, T. D., W. A. Klimke, M. J. Gubbins, and L. S. Frost. 2003. F factor
conjugation is a true type IV secretion system. FEMS Microbiol Lett 224:1-15.
68. Lee, C. A., and S. Falkow. 1990. The ability of Salmonella to enter mammalian
cells is affected by bacterial growth state. Proc Natl Acad Sci USA 87:4304-4308.
69. Leto, T. L., D. Fortugno-Erikson, D. Barton, T. L. Yang-Feng, U. Francke,
A. S. Harris, J. S. Morrow, V. T. Marchesi, and E. J. Benz, Jr. 1988.
Comparison of nonerythroid alpha-spectrin genes reveals strict homology among
diverse species. Mol Cell Biol 8:1-9.
70. Leyer, G. J., and E. A. Johnson. 1993. Acid adaptation induces cross-protection
against environmental stresses in Salmonella typhimurium. Appl Environ
Microbiol 59:1842-7.
71. Lin, M. T., and M. F. Beal. 2006. Mitochondrial dysfunction and oxidative stress
in neurodegenerative diseases. Nature 443:787-795.
72. Lindquist, S. 1986. The heat-shock response. Annu Rev Biochem 55:1151-91.
73. Marcus, S. L., J. H. Brumell, C. G. Pfeifer, and B. B. Finlay. 2000. Salmonella
pathogenicity islands: big virulence in small packages. Microbes Infect 2:145-56.
74. Marsman, M., I. Jordens, C. Kuijl, L. Janssen, and J. Neefjes. 2004. Dynein-
mediated vesicle transport controls intracellular Salmonella replication. Mol Biol
Cell 15:2954-64. 126
75. Mastroeni, P., A. Vazquez-Torres, F. C. Fang, Y. Xu, S. Khan, C. E.
Hormaeche, and G. Dougan. 2000. Antimicrobial actions of the NADPH
phagocyte oxidase and inducible nitric oxide synthase in experimental
salmonellosis. II. Effects on microbial proliferation and host survival in vivo. J
Exp Med 192:237-48.
76. McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L.
Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S.
Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan,
H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R.
K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar
Typhimurium LT2. Nature 413:852-6.
77. Miao, E. A., E. Andersen-Nissen, S. E. Warren, and A. Aderem. 2007. TLR5
and Ipaf: dual sensors of bacterial flagellin in the innate immune system. Semin
Immunopathol 29:275-88.
78. Miao, E. A., C. A. Scherer, R. M. Tsolis, R. A. Kingsley, L. G. Adams, A. J.
Baumler, and S. I. Miller. 1999. Salmonella typhimurium leucine-rich repeat
proteins are targeted to the SPI1 and SPI2 type III secretion systems. Mol
Microbiol 34:850-64.
79. Mills, S. D., and B. B. Finlay. 1998. Isolation and characterization of Salmonella
typhimurium and Yersinia pseudotuberculosis-containing phagosomes from
infected mouse macrophages: Y. pseudotuberculosis traffics to terminal
lysosomes where they are degraded. Eur J Cell Biol 77:35-47. 127
80. Mirold, S., W. Rabsch, M. Rohde, S. Stender, H. Tschape, H. Russmann, E.
Igwe, and W. D. Hardt. 1999. Isolation of a temperate bacteriophage encoding
the type III effector protein SopE from an epidemic Salmonella typhimurium
strain. Proc Natl Acad Sci USA 96:9845-50.
81. Molofsky, A. B., B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse, K.
Tateda, and M. S. Swanson. 2006. Cytosolic recognition of flagellin by mouse
macrophages restricts Legionella pneumophila infection. J Exp Med 203:1093-
104.
82. Mootha, V. K., C. M. Lindgren, K.-F. Eriksson, A. Subramanian, S. Sihag, J.
Lehar, P. Puigserver, E. Carlsson, M. Ridderstrale, E. Laurila, N. Houstis,
M. J. Daly, N. Patterson, J. P. Mesirov, T. R. Golub, P. Tamayo, B.
Spiegelman, E. S. Lander, J. N. Hirschhorn, D. Altshuler, and L. C. Groop.
2003. PGC-1[alpha]-responsive genes involved in oxidative phosphorylation are
coordinately downregulated in human diabetes. Nat Genet 34:267-273.
83. Morgan, R. W., M. F. Christman, F. S. Jacobson, G. Storz, and B. N. Ames.
1986. Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap
with heat shock and other stress proteins. Proc Natl Acad Sci USA 83:8059-63.
84. Nicholson, D. W., and N. A. Thornberry. 1997. Caspases: killer proteases.
Trends in Biochemical Sciences 22:299-306.
85. Ohl, M. E., and S. I. Miller. 2001. Salmonella: a model for bacterial
pathogenesis. Annu Rev Med 52:259-74.
86. Olsen, A., A. Jonsson, and S. Normark. 1989. Fibronectin binding mediated by
a novel class of surface organelles on Escherichia coli. Nature 338:652-5. 128
87. Pace, J., M. J. Hayman, and J. E. Galan. 1993. Signal transduction and
invasion of epithelial cells by S. typhimurium. Cell 72:505-14.
88. Patti, J. M., B. L. Allen, M. J. McGavin, and M. Hook. 1994. MSCRAMM-
mediated adherence of microorganisms to host tissues. Annu Rev Microbiol
48:585-617.
89. Peterson, J. D., L. A. Umayam, T. Dickinson, E. K. Hickey, and O. White.
2001. The Comprehensive Microbial Resource. Nucleic Acids Res 29:123-125.
90. Pop, C., and G. S. Salvesen. 2009. Human Caspases: Activation, Specificity, and
Regulation. J Biolog Chem 284:21777-21781.
91. Pulkkinen, W. S., and S. I. Miller. 1991. A Salmonella typhimurium virulence
protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage
lambda outer membrane protein. J Bacteriol 173:86-93.
92. Reed, K. A., M. E. Hobert, C. E. Kolenda, K. A. Sands, M. Rathman, M.
O'Connor, S. Lyons, A. T. Gewirtz, P. J. Sansonetti, and J. L. Madara. 2002.
The Salmonella typhimurium flagellar basal body protein FliE is required for
flagellin production and to induce a proinflammatory response in epithelial cells. J
Biol Chem 277:13346-53.
93. Reiter, W. D., P. Palm, and S. Yeats. 1989. Transfer RNA genes frequently
serve as integration sites for prokaryotic genetic elements. Nucleic Acids Res
17:1907-14.
94. Rychlik, I., and P. A. Barrow. 2005. Salmonella stress management and its
relevance to behaviour during intestinal colonisation and infection. FEMS
Microbiol Rev 29:1021-40. 129
95. Rytkonen, A., J. Poh, J. Garmendia, C. Boyle, A. Thompson, M. Liu, P.
Freemont, J. C. Hinton, and D. W. Holden. 2007. SseL, a Salmonella
deubiquitinase required for macrophage killing and virulence. Proc Natl Acad Sci
USA 104:3502-7.
96. Schmidt, L. D., L. J. Kohrt, and D. R. Brown. 2008. Comparison of growth
phase on Salmonella enterica serovar Typhimurium invasion in an epithelial cell
line (IPEC J2) and mucosal explants from porcine small intestine. Comparative
Immunol, Microbiol Infect Dis 31:63-69.
97. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of a
virulence locus encoding a second type III secretion system in Salmonella
typhimurium. Proc Natl Acad Sci USA 93:2593-7.
98. Sjobring, U., G. Pohl, and A. Olsen. 1994. Plasminogen, absorbed by
Escherichia coli expressing curli or by Salmonella enteritidis expressing thin
aggregative fimbriae, can be activated by simultaneously captured tissue-type
plasminogen activator (t-PA). Mol Microbiol 14:443-52.
99. Song, M., H.-J. Kim, E. Y. Kim, M. Shin, H. C. Lee, Y. Hong, J. H. Rhee, H.
Yoon, S. Ryu, S. Lim, and H. E. Choy. 2004. ppGpp-dependent Stationary
Phase Induction of Genes on Salmonella Pathogenicity Island 1. J Biolog Chem
279:34183-34190.
100. Stanley, T. L., C. D. Ellermeier, and J. M. Slauch. 2000. Tissue-specific gene
expression identifies a gene in the lysogenic phage Gifsy-1 that affects
Salmonella enterica serovar typhimurium survival in Peyer's patches. J Bacteriol
182:4406-13. 130
101. Stevens J.R., G. B., Desai P., Rajan S., and Weimer B.C. 2008. Statistical
Issues in the Normalization of Multi-Species Microarray Data. Proc Conf Appl
Statistics in Agricul 47-62.
102. Subramanian, A., P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, M.
A. Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, and J. P.
Mesirov. 2005. Gene set enrichment analysis: A knowledge-based approach for
interpreting genome-wide expression profiles. Proc Natl Acad Sci USA
102:15545-15550.
103. Tartera, C., and E. S. Metcalf. 1993. Osmolarity and growth phase overlap in
regulation of Salmonella typhi adherence to and invasion of human intestinal
cells. Infect Immun 61:3084-3089.
104. Tatusov, R. L., E. V. Koonin, and D. J. Lipman. 1997. A genomic perspective
on protein families. Science 278:631-7.
105. Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of
microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA
98:5116-21.
106. Valdivia, R. H., and S. Falkow. 1997. Fluorescence-based isolation of bacterial
genes expressed within host cells. Science 277:2007-11.
107. Warren, P., D. Taylor, P. G. V. Martini, J. Jackson, and J. Bienkowska.
2007. Presented at the Bioinformatics and Bioengineering, 2007. BIBE 2007.
Proc 7th IEEE Intl Conf. 131
108. Watson, P. R., E. E. Galyov, S. M. Paulin, P. W. Jones, and T. S. Wallis.
1998. Mutation of invH, but not stn, reduces Salmonella-induced enteritis in
cattle. Infect Immun 66:1432-8.
109. Wilson, J. W., C. M. Ott, L. Quick, R. Davis, K. Honer zu Bentrup, A.
Crabbe, E. Richter, S. Sarker, J. Barrila, S. Porwollik, P. Cheng, M.
McClelland, G. Tsaprailis, T. Radabaugh, A. Hunt, M. Shah, M. Nelman-
Gonzalez, S. Hing, M. Parra, P. Dumars, K. Norwood, R. Bober, J. Devich,
A. Ruggles, A. CdeBaca, S. Narayan, J. Benjamin, C. Goulart, M. Rupert, L.
Catella, M. J. Schurr, K. Buchanan, L. Morici, J. McCracken, M. D. Porter,
D. L. Pierson, S. M. Smith, M. Mergeay, N. Leys, H. M. Stefanyshyn-Piper,
D. Gorie, and C. A. Nickerson. 2008. Media ion composition controls regulatory
and virulence response of Salmonella in spaceflight. PLoS One 3:e3923.
110. Wood, M. W., M. A. Jones, P. R. Watson, S. Hedges, T. S. Wallis, and E. E.
Galyov. 1998. Identification of a pathogenicity island required for Salmonella
enteropathogenicity. Mol Microbiol 29:883-891.
111. Xie, Y., L. S. Chou, A. Cutler, and B. Weimer. 2004. DNA Macroarray
profiling of Lactococcus lactis subsp. lactis IL1403 gene expression during
environmental stresses. Appl Environ Microbiol 70:6738-47.
112. Yang, J., L. Chen, L. Sun, J. Yu, and Q. Jin. 2008. VFDB 2008 release: an
enhanced web-based resource for comparative pathogenomics. Nucleic Acids Res
36:D539-D542.
113. Zamzami, N., P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch,
S. A. Susin, P. X. Petit, B. Mignotte, and G. Kroemer. 1995. Sequential 132
reduction of mitochondrial transmembrane potential and generation of reactive
oxygen species in early programmed cell death. J Exp Med 182:367-77.
114. Ziemer, C., and S. Steadham. 2003. Evaluation of the specificity of Salmonella
PCR primers using various intestinal bacterial species*. Letters in Appl Microbiol
37:463-469.
115. Zierler, M. K., and J. E. Galan. 1995. Contact with cultured epithelial cells
stimulates secretion of Salmonella typhimurium invasion protein InvJ. Infect
Immun 63:4024-8.
133
TABLE 4-1. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium.
Enriched gene sets in S. Typhimurium, Genes Size of Regulation FDR due to cold stress regulated the gene set q value SP2-T3SS 26 27 Induced 0 T3SS 27 58 Induced 0 T3SS effectors 10 22 Induced 0 T3SS2 effectors 9 13 Induced 0 Pathogenesis 8 32 Induced 0.03 Prophage functions 70 143 Induced 0.04 Plasmid functions 26 28 Induced 0 Protein and peptide secretion and trafficking 21 54 Induced 0 DNA transformation 28 30 Induced 0 Purine ribonucleotide biosynthesis 9 18 Induced 0.01 Cog L- Replication recombination and repair 79 157 Induced 0.01 Transcription- Degradation of RNA 9 15 Induced 0.05
134
TABLE 4-2. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium as compared to non-stressed S.
Typhimurium, upon infection of epithelial cells.
Enriched gene sets in cold-stressed Genes Size of RegulationFDR S. Typhimurium during to infection Regulated the gene set q value Prophage functions 63 143 Induced 0 Plasmid functions 26 28 Induced 0 Transposon functions 27 44 Induced 0 SPV locus 4 4 Induced 0.04 Adaptations to atypical conditions 10 30 Induced 0.04 Cellular processes- DNA transformation 30 30 Induced 0 Energy metabolism- Anaerobic 32 66 Induced 0.04 Synthesis & modification ribosomal proteins 37 61 Repressed 0 Cog J- Translation 67 170 Repressed 0 Cellular processes- Chemotaxis and motility 20 40 Repressed 0 Energy metabolism- Pyruvate dehydrogenase 3 8 Repressed 0.02 Metabolism of surface polysaccharides & LPS 39 82 Repressed 0.04 Energy metabolism- Aerobic 12 24 Repressed 0.04
135
TABLE 4-3. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in epithelial cells due to infection with cold-stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S. Typhimurium.
Enriched gene sets in Caco-2 cells due to Genes Size of Regulation FDR infection with cold-stressed S. Typhimurium Regulated the gene set q value Ribosome 18 37 Induced 0.01 Ribosomal subunit 11 20 Induced 0 Organellar ribosome 12 22 Induced 0 Mitochondrial ribosome 12 22 Induced 0 Mitochondrial small ribosomal subunit 9 11 Induced 0 Organellar small ribosomal subunit 9 11 Induced 0.01 Small ribosomal subunit 9 11 Induced 0.01 Mitochondrial part 47 128 Induced 0.01 Mitochondrial envelope 28 85 Induced 0.03 Mitochondrial membrane 26 77 Induced 0.03 Mitochondrial inner membrane 24 60 Induced 0.01 Mitochondrial membrane part 20 50 Induced 0 Mitochondrial lumen 18 44 Induced 0.03 Mitochondrial matrix 18 44 Induced 0.03 Cellular respiration 9 18 Induced 0.03 NADH dehydrogenase complex 7 14 Induced 0.03 Mitochondrial respiratory chain complex I 7 14 Induced 0.03 Respiratory chain complex I 7 14 Induced 0.03 Structural constituent of ribosome 43 73 Induced 0.00 Ribosome biogenesis and assembly 6 12 Induced 0.04 Ribonucleoprotein complex 41 111 Induced 0.01 Microtubule motor activity 9 14 Induced 0.01 Kinesin complex 8 11 Induced 0.03 RNA splicing via transesterification reactions 10 30 Induced 0.03 Coenzyme metabolic process 12 31 Induced 0.04
136
TABLE 4-4. Salmonella serotypes that contain homologues of STM2699.
Salmonella Strain E value Amino acids matched % Identity S. Agona SL483 5.00E-50 99/100 99 S. Typhimurium SL1344 2.00E-49 98/100 98 S. Typhi CT18 2.00E-47 94/99 94 S. Javiana GAMM04042433 4.00E-47 94/99 94 S. Typhi CT18 5.00E-46 90/100 90 S. Typhi E01 6750 2.00E-37 76/77 98 S. Agona SL483 3.00E-32 69/103 66 S. Typhi E98 2068 7.00E-17 42/47 89 S. Typhi 404ty 1.00E-14 38/87 43 S. Typhimurium D23580 1.00E-11 32/86 37 S. Typhi E98 3139 2.00E-10 31/36 86 S. Typhi E98 2068 1.00E-06 29/88 32
137
FIG 4-1. Association S. Typhimurium to epithelial (Caco2) cells. (A) Epithelial cells were infected with cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid (pH 4.0, 90
min) stressed S. Typhimurium at MOI of 1:1000 and total association to epithelial cells
was measured at 60 min, 90 min and 120 min post infection. The measurements for
number of epithelial cell associated bacteria and number of epithelial cells were done
with PCR. (B) Epithelial cells were infected with cold-stressed (5°C, 48 h) S.
Typhimurium at MOI of 1:1000 and the number of invaded bacteria was measured using
gentamicin protection assay, 60 min post infection. Results are mean ± SEM of three
biological replicates. The number of adhered bacteria was calculated as described in
materials and methods. The asterisk (*) indicates significant difference (p < 0.05) as
compared to no stress control. The dot (•) indicates significant difference (p < 0.05) due
to cold stress (5°C, 48 h) as compared to peroxide stress (5 mM H2O2, 5 h). 138
139
FIG 4-2. GSEA was used to detect coordinate changes in expression of the functionally related genes in S. Typhimurium in response to cold stress (5°C, 48 h). (A) Significantly
(q < 0.05) regulated gene set with functions associated with prophages. (B) Significantly
(q < 0.05) regulated gene set with functions associated with T3SS. (C) Significantly (q <
0.05) regulated gene set with functions associated with plasmid (pSLT). Color scale:
Light pink - brown corresponds to low expression intensity - high expression intensity. 140
None Cold
A 79PSLT042 putative integrase protein
78PSLT069 PsiB
78PSLT070 PsiA
56STM0893 putative integrase
45STM0895 hypothetical protein
35STM0896 hypothetical protein
35STM0897 hypothetical protein
34STM0899 hypothetical protein
35STM0902 hypothetical protein
45STM0904 hypothetical protein
35STM0908 hypothetical protein
35STM0909 hypothetical protein
45STM0910 hypothetical protein
45STM0911 hypothetical protein
35STM0912 ATPdependent protease
35STM0914 putative phage tail component
46STM0915 hypothetical protein
24STM0916 putative major tail protein
35STM0918 putative minor tail protein
45STM0919 putative minor tail protein
45STM0920 Ail/OmpXlike protein
35STM0921 putative minor tail protein
45STM0922 putative phage tail assembly protein
35STM0926 putative minor tail protein
34STM0927 putative phage tail assembly protein
45STM1005 integrase
36STM1007 hypothetical protein
46STM1009 exodeoxyribonuclease
35STM1010 hypothetical protein
46STM1011 hypothetical protein
34STM1020 hypothetical protein
57STM1025 hypothetical protein
34STM1034 putative RecA/RadA recombinase
34STM1042 probable minor tail protein
34STM1043 attachment/invasion protein
512STM1051 secreted effector protein
45STM1055 hypothetical protein
34STM1092 putative cytoplasmic protein
58STM1242 putative envelope protein
Fig 4-2A continued on next page 141
Continued from above
None Cold
A 35STM1638 putative SAMdependent methyltransferase
46STM1687 phage shock protein
79STM1690 phage shock protein
38STM2233 putative cytoplasmic protein
48STM2235 hypothetical protein
46STM2236 hypothetical protein
45STM2585 transposaselike protein
34STM2592 phage tail component Llike protein
34STM2593 phage tail component Mlike protein
34STM2594 phage tail component Hlike protein
34STM2596 minor taillike protein
35STM2598 hypothetical protein
35STM2600 minor tail protein Zlike
35STM2601 minor capsid protein FII
34STM2602 DNA packaginglike protein
34STM2604 phage headlike protein
34STM2606 headtail preconnectorlike protein
35STM2608 terminaselike large protein
34STM2609 DNA packaginglike protein
35STM2612 morphogenesislike protein
34STM2617 antiterminatorlike protein
44STM2619 hypothetical protein
35STM2620 hypothetical protein
46STM2623 hypothetical protein
46STM2624 hypothetical protein
36STM2625 DNA replication protein DnaC
35STM2626 replication protein 15like
36STM2627 cIlike protein
35STM2631 hypothetical protein
44STM2635 excisionaselike protein
59STM2694 late controllike protein
56STM2697 phage taillike protein
47STM2699 putative phage taillike protein
47STM2700 phage tail fiberlike protein
67STM2701 phage tail sheathlike protein
56STM2705 hypothetical protein
46STM2716 phageholinlike protein
57STM2720 major capsidlike protein
56STM2739 phage taillike protein
36STM2776 putative hydrolase
142
None Cold
B 512STM1051 secreted effector protein
49STM1087 pathogenicity islandencoded protein A
310STM1088 secreted effector protein
69STM1224 secreted effector protein
48STM1392 sensor kinase
49STM1393 secreted effector protein
510STM1394 outer membrane secretin precursor
410STM1395 virulence protein
410STM1395 virulence protein
611STM1396 secretion system effector
10 13 STM1397 secretion system chaperone protein
10 13 STM1398 translocation machinery component
812STM1399 secretion system chaparone
912STM1400 translocation machinery component
410STM1401 translocation machinery component
510STM1402 secreted effector protein
49STM1403 secretion system chaparone
37STM1404 secreted effector protein
47STM1405 secreted effector protein
69STM1406 type III secretion system apparatus protein
69STM1407 type III secretion system apparatus protein
59STM1408 type III secretion system apparatus protein
69STM1409 needle complex inner membrane lipoprotein
510STM1411 type III secretion system apparatus protein
49STM1412 type III secretion system apparatus protein
38STM1413 type III secretion system apparatus protein
49STM1414 type III secretion system apparatus protein
59STM1415 type III secretion system ATPase
610STM1416 type III secretion system apparatus protein
37STM1417 type III secretion system apparatus protein
59STM1418 type III secretion protein
610STM1419 needle complex export protein
511STM1420 type III secretion system apparatus protein
49STM1421 type III secretion system apparatus protein
37STM1422 type III secretion system apparatus protein
510STM1602 secreted effector protein
46STM1687 phage shock protein
48STM2235 hypothetical protein
49STM2241 leucinerich repeat protein
712STM2287 putative cytoplasmic protein
35STM2679 hypothetical protein
10 13 STM2780 secreted effector protein
35STM2884 translocation machinery component
34STM2892 needle length control protein 143
None Cold
C 78PSLT073 mating signal protein
68PSLT075 regulatory protein
68PSLT077 pilus subunit
78PSLT079 pilus assembly protein
78PSLT081 pilus assembly protein
68PSLT082 conjugative transfer protein
67PSLT083 conjugative transfer protein
78PSLT084 pilus assembly protein
78PSLT087 OrfG2
78PSLT088 ATPbinding protein
68PSLT089 pilus assembly protein
68PSLT091 pilus assembly protein
79PSLT092 pilus assembly protein
79PSLT093 OrfF
79PSLT094 pilus assembly protein
78PSLT095 mating pair stabilization protein
68PSLT096 conjugative transfer protein
68PSLT097 pilus assembly protein
78PSLT098 pilin chaperone
67PSLT099 conjugative transfer protein
68PSLT100 pilus assembly protein
89PSLT101 mating pair stabilization and pilus assembly protein
78PSLT105 conjugative transfer protein
78PSLT110 pilin subunit acetylation
78PSLT111 finP bindingprotein
45STM4093 essential cell division protein
144
FIG 4-3. GSEA was used to detect coordinate changes in expression of the functionally related genes in cold-stressed (5°C, 48 h) S. Typhimurium upon infection of epithelial cells (q < 0.05) as compared to infection with non-stressed S. Typhimurium. This effect is due to extracellular cold stress to S. Typhimurium, which is beyond the effect of the infection of epithelial cells with non-stressed S. Typhimurium. (A) Significantly (q <
0.05) regulated gene set with functions associated with prophages. (B) Significantly (q <
0.05) regulated gene set with functions associated with spv genes on the plasmid (pSLT) and stress response. (C) Significantly (q < 0.05) regulated gene set with functions associated with DNA transposition. (D) Significantly (q < 0.05) regulated gene set with functions associated with plasmid (pSLT) excluding spv genes. Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity.
145
None Cold
A 58PSLT042 putative integrase protein
78PSLT044 putative integrase protein
67PSLT069 PsiB
57PSLT070 PsiA
34STM0894 putative excisionase
44STM0895 hypothetical protein
45STM0896 hypothetical protein
34STM0897 hypothetical protein
44STM0899 hypothetical protein
45STM0902 hypothetical protein
34STM0903 putative chaparone
44STM0904 hypothetical protein
44STM0907 putative chitinase
25STM0908 hypothetical protein
44STM0909 hypothetical protein
35STM0910 hypothetical protein
45STM0911 hypothetical protein
45STM0912 ATPdependent protease
34STM0913 hypothetical protein
45STM0914 putative phage tail component
46STM0915 hypothetical protein
34STM0918 putative minor tail protein
45STM0919 putative minor tail protein
45STM0920 Ail/OmpXlike protein
46STM0922 putative phage tail assembly protein
35STM0926 putative minor tail protein
35STM0927 putative phage tail assembly protein
45STM1007 hypothetical protein
45STM1009 exodeoxyribonuclease
24STM1010 hypothetical protein
46STM1011 hypothetical protein
67STM1012 probable regulatory protein
34STM1024 hypothetical protein
46STM1026 hypothetical protein
34STM1033 Clp proteaselike protein
34STM1041 probable minor tail protein
46STM1044 superoxide dismutase precursor
34STM1046 probable tail assembly protein
33STM1053 hypothetical protein
Fig 4-3A continued on next page
146
Continued from above
None Cold
A 23STM1092 putative cytoplasmic protein
56STM1242 putative envelope protein
46STM1687 phage shock protein
611STM1690 phage shock protein
34STM1853 serine/threonine protein phosphatase
33STM2066 secreted effector protein
23STM2590 tail assembly protein Ilike
34STM2593 phage tail component Mlike protein
44STM2598 hypothetical protein
45STM2599 putative virulence protein
34STM2600 minor tail protein Zlike
67STM2610 hypothetical protein
34STM2612 morphogenesislike protein
34STM2613 hypothetical protein
34STM2617 antiterminatorlike protein
35STM2619 hypothetical protein
67STM2621 hypothetical protein
45STM2624 hypothetical protein
44STM2625 DNA replication protein DnaC
35STM2627 cIlike protein
34STM2631 hypothetical protein
47STM2694 late controllike protein
46STM2696 putative phage tail protein
57STM2697 phage taillike protein
46STM2699 putative phage taillike protein
47STM2700 phage tail fiberlike protein
47STM2701 phage tail sheathlike protein
46STM2705 hypothetical protein
67STM2711 phage taillike protein
46STM2716 phageholinlike protein
47STM2720 major capsidlike protein
57STM2739 phage taillike protein
34STM4202 putative phage baseplate protein
34STM4211 putative phage tail protein
34STM4213 putative phage tail sheath protein
147
None Cold
B 57PSLT038 hydrophilic protein
57PSLT039 hydrophilic protein
68PSLT040 outer membrane protein
57PSLT041 regulator of spv operon
55STM0629 cold shock protein E
35STM1686 phage shock protein
48STM1688 phage shock protein
48STM1689 phage shock protein B
66STM1705 lipoprotein B
45STM1844 heat shock protein HtpX
34STM1996 putative coldshock protein
45STM2956 (p)ppGpp synthetase I
89STM3591 universal stress protein A
910STM3649 major cold shock protein
None Cold C 68PSLT008 putative regulatory protein 57PSLT031 resolvase
46PSLT036 putative transposase
67PSLT076 DNAbinding protein
34STM0759 hypothetical protein
67STM0946 transposase
34STM0947 putative integrase
33STM2509 putative transposase
55STM2749 putative cytoplasmic protein
34STM2768 putative transposase
35STM2769 putative transposase
23STM3306 transcriptional regulator
45STM4315 putative DNAbinding protein
148
None Cold
D 47PSLT073 mating signal protein
47PSLT075 regulatory protein
57PSLT077 pilus subunit
57PSLT078 pilus assembly protein
68PSLT079 pilus assembly protein
58PSLT081 pilus assembly protein
57PSLT082 conjugative transfer protein
57PSLT084 pilus assembly protein
57PSLT087 OrfG2
68PSLT088 ATPbinding protein
57PSLT089 pilus assembly protein
67PSLT091 pilus assembly protein
58PSLT092 pilus assembly protein
57PSLT093 OrfF
68PSLT094 pilus assembly protein
57PSLT095 mating pair stabilization protein
57PSLT096 conjugative transfer protein
57PSLT097 pilus assembly protein
67PSLT098 pilin chaperone
56PSLT099 conjugative transfer protein
57PSLT100 pilus assembly protein
58PSLT101 mating pair stabilization and pilus assembly protein
89PSLT102 entry exclusion protein
68PSLT105 conjugative transfer protein
57PSLT110 pilin subunit acetylation
67PSLT111 finP bindingprotein
149
FIG 4-4. Expression of significantly regulated (q < 0.20) genes in functional category related to mitochondrial electron transport chain in epithelial cells, upon infection with cold-stressed (5°C, 48 h) S. Typhimurium. This effect is due to extracellular cold stress to S.
Typhimurium, which is beyond the effect of the infection of epithelial cells with non-stressed S. Typhimurium. Color legends: Red,
Induction; Green, Repression. 150
FIG 4-5. Caspase activation during infection of epithelial cells with non-stressed and cold-stressed (5°C, 48 h) S. Typhimurium, at MOI of 1:1000. (A) Caspase 8 activity, (B)
Caspase 9 activity, and (C) Caspase 3/7 activity. The asterisk (*) indicates significant difference (p < 0.05) due to infection with cold-stressed S. Typhimurium as compared to infection with non-stressed S. Typhimurium.
151
152
153
S. Typhimurium Caco-2
Host Salmonella NS CS NS CS Con NS CS
4746STM2699 SPTAN1 677
Fels-2 prophage gene Alpha II Spectrin
FIG 4-6. Gene expression intensities of the S. Typhimurium protein STM2699 upon no stress and cold stress (5°C, 48 h) alone, and upon infection of epithelial cells with non- stressed and cold-stressed (5°C, 48 h) S. Typhimurium. Gene expression intensities of the epithelial cell protein SPTAN1 in epithelial cells alone, and upon infection of epithelial cells with non-stressed and cold-stressed (5°C, 48 h) S. Typhimurium. Legends: NS, No stress; CS, Cold stress. Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity.
154
FIG 4-7. Role of STM2699 and SPTAN1 in adhesion and invasion of epithelial cells with non-stressed S. Typhimurium (MOI of 1:100). (A) Role of STM2699 in adhesion and invasion of wild type S. Typhimurium and S. Typhimurium ΔinvA, in epithelial cells. (B)
Role of SPTAN1 in adhesion and invasion of wild type S. Typhimurium, S. Typhimurium
ΔSTM2699 and S. Typhimurium ΔinvA ΔSTM2699, in epithelial cells. The asterisk (*) indicates significant difference (p < 0.05) between (A) adhesion and invasion of deletion mutants as compared to adhesion and invasion of wild type S. Typhimurium and (B) adhesion and invasion of wild type S. Typhimurium as compared to adhesion and invasion of wild type S. Typhimurium, S. Typhimurium ΔSTM2699 and S. Typhimurium
ΔinvA ΔSTM2699 upon blocking the surface of epithelial cells with anti-SPTAN1 antibody.
155
156
FIG 4-8. Role of STM2699 and SPTAN1, in adhesion and invasion of epithelial cells with cold-stressed (5°C, 48 h) S. Typhimurium (MOI of 1:100). (A) Role of STM2699 in adhesion and invasion of cold stresses wild type S. Typhimurium and cold-stressed S.
Typhimurium ΔinvA, in epithelial cells. (B) Role of SPTAN1 in adhesion and invasion of cold-stressed wild type S. Typhimurium, cold-stressed S. Typhimurium ΔSTM2699 and cold-stressed S. Typhimurium ΔinvA ΔSTM2699, in epithelial cells. The asterisk (*) indicates significant difference (p < 0.05) between (A) adhesion and invasion of cold- stressed deletion mutants as compared to adhesion and invasion of cold-stressed wild type S. Typhimurium and (B) adhesion and invasion of cold-stressed wild type S.
Typhimurium as compared to adhesion and invasion of cold-stressed wild type S.
Typhimurium, cold-stressed S. Typhimurium ΔSTM2699 and cold-stressed S.
Typhimurium ΔinvA ΔSTM2699 upon blocking the surface of epithelial cells with anti-
SPTAN1 antibody. The dot (•) indicates significant difference (p < 0.05) between (A) adhesion and invasion of cold-stressed S. Typhimurium ΔinvA ΔSTM2699 as compared to adhesion and invasion of cold-stressed S. Typhimurium ΔinvA (B) adhesion and invasion of cold-stressed S. Typhimurium ΔinvA ΔSTM2699 upon blocking the surface of epithelial cells with anti-SPTAN1, as compared to adhesion and invasion of cold- stressed S. Typhimurium ΔinvA upon blocking the surface of epithelial cells with anti-
SPTAN1 antibody. 157
158
CHAPTER 5
CHANGES IN METABOLISM OF COLD-STRESSED S. TYPHIMURIUM AND
EPITHELIAL CELLS DURING INFECTION
ABSTRACT
Salmonella infections represent major threat to human health due to their persistence in the food chain. Salmonella alters its gene regulation in order to survive under different environmental stresses and the mechanism of the regulation of metabolism and its repertoire of virulence determinants, under different conditions, is not yet completely understood. Metabolic enzymes playing a central role in microbial physiology and survival have been promising targets for antimicrobials, but differential regulation of metabolism upon exposure to stress urges development of new targets for antimicrobials. Consequently, in this study, we have investigated the regulation in metabolism of Salmonella Typhimurium (S. Typhimurium) upon exposure to cold stress
(5°C, 48 h) and in cold-stressed S. Typhimurium during subsequent infection, using systems level approach. Exposure to cold stress, which is encountered in the environment and is also the most common method for increasing shelf life of food, indicated switch from aerobic respiration to fermentation in S. Typhimurium. This led to the increased (q
< 0.05) utilization of pyruvate and hence increased (q < 0.05) production of short chain fatty acids formate and acetate in the extracellular medium. This effect was persistent when intestinal epithelial cells were infected with cold-stressed S. Typhimurium leading to increased (q < 0.05) adhesion and invasion. In epithelial cells, increased (q < 0.05) aerobic respiration was indicated upon infection with cold-stressed S. Typhimurium. 159
INTRODUCTION
Salmonellae are gram negative food-borne bacteria causing gastroenteritis and enteric (typhoid) fever. Salmonella must overcome various stresses encountered in the environment, to survive, and in the host, to establish an infection. Response of
Salmonella to various stresses such as starvation, acid, oxidative, temperature, osmotic and cationic antimicrobial peptides has been studied and the gene clusters providing protection against most of these stresses have been identified (reviewed in 7). The response to one stress can be specific, overlapping with another stress response or can provide cross protection against subsequent hostile conditions. Exposure to stress conditions can also affect the expression of virulence genes if they are under the control of similar regulators (5). The function of a large number of virulence genes is required for successful pathogenesis and many underlying mechanisms have been revealed (reviewed in 25). Most of the virulence genes are known to be present on Salmonella pathogenicity islands (SPIs). SPIs confer specific virulence traits and function in different stages of infection process such as entry and replication in the host cells (3, 13, 34), induction of intestinal secretory and inflammatory responses (8), and dissemination from epithelial cells to cause systemic infection (13). Two SPIs, SPI1 and SPI2 encoding for type three secretion system (T3SS) play a central role in pathogenesis of Salmonella (reviewed in
(12)).
While much of the research has focused in determining the virulence determinants of Salmonella and host responses (reviewed in (16)) in an attempt to control and prevent infections, metabolism of both, the stressed pathogen and the host, during the infection has gained little attention. Metabolic enzymes have central role in microbial physiology 160 have been attractive targets for antimicrobials, but exposure to stress regulates metabolism as well in order for the pathogen to survive efficiently. Induction of ‘viable but non-culturable’ (VBNC) state in S. Typhimurium, which are still metabolically active
(9), lead to loss of adhesion and invasion in epithelial cells (see supplementary data Fig
C-1 and C-2) but there have been reports of resuscitation of S. Typhimurium in in-vivo mouse models (1). Hence, to develop novel efficient antimicrobials, there is urgent need to understand the complete metabolic potential and regulation of metabolism in the pathogen, upon exposure to stress and upon subsequent infection.
In this study, we show differential regulation of the metabolism in Salmonella
Typhimurium (S. Typhimurium) due to cold stress (5°C), which can be encountered in the environment and is also the most common method for storage and increasing shelf life of the food. We further observed that the regulation in the metabolism due to stress was persistent upon infection of epithelial cells. Thus, we have attempted to gain insights with systems level approach in to the metabolism of S. Typhimurium upon exposure to cold stress and upon infection of the epithelial cells, in order to develop novel targets for antimicrobials.
MATERIALS AND METHODS
Cell culture. Intestinal epithelial cells (Caco-2; ATCC HTB-37) were obtained from American Type Culture Collection (Manassas, VA) and were grown as per the manufacturer’s instructions. Briefly, cells were seeded to a density of 105 cells/cm2 using cell culture media comprising of DMEM/High Modified (Thermo Scientific, Rockford,
IL) with non-essential amino acids (Thermo Scientific, Rockford, IL), 10mM MOPS 161
(Sigma, St. Louis, MO), 10 mM TES (Sigma), 15 mM HEPES (Sigma) and 2 mM
NaH2PO4 (Sigma). Additionally, 16.6% fetal bovine serum (FBS) (HyClone Laboratories,
Logan, UT) was added to the media only for feeding cells. Cells were incubated at 37˚C with 5% CO2 for 14 days post confluence so as to let them differentiate. The cells were
serum starved for 24 h, prior to sample collection for gene expression profiles, by feeding
the cells with cell culture media with all the components mentioned above excluding
FBS.
Bacterial strains and growth conditions. Salmonella Typhimurium LT2 ATCC
700720 (S. Typhimurium) was used in this study. Cell culture media, was used to grow
the bacteria at 37°C with shaking at 220 rpm.
Stress treatments for bacteria. A log phase culture of S. Typhimurium was
centrifuged at 7200 × g for 5 min and re-suspended at equal density in complete cell
culture media, maintained at 5°C for cold stress. This stress treatment was given for 48 h
before using the cold-stressed bacteria for infection of epithelial cells. The control for the
cold stress treatment was stationary phase (~15 h) culture re-suspended in cell culture
media maintained at 37°C, which is further referred to as non-stressed S. Typhimurium.
Infection of epithelial cells for gene expression. Caco-2 cells were cultured in
T-75 flasks and were serum starved 24 h before infection. Respective bacterial treatments
i.e. cold-stressed (5°C, 48 h) and non-stressed S. Typhimurium, at MOI of 1:1000, were
used to infect epithelial cells. The control for these infections was addition of cold-
stressed and non-stressed S. Typhimurium respectively, in T-75 flasks without the
epithelial cells, and non infected epithelial cells. The experiment was done in two
biological replicates. The infected cells along with the controls were incubated at 37°C 162
with 5% CO2 for 60 min. For infected cells, media with extracellular bacteria was
aspirated and 10 ml of TRIzol LS reagent (Invitrogen, Carlsbad, CA) was added to the
cells. This was gently mixed with pipette followed by centrifugation at 7200 × g for 5
min to pellet the host associated bacteria. TRIzol LS supernatant was stored in a clean
tube and further processed for RNA extraction from infected Caco-2 cells. The bacterial
pellet was re-suspended in 2 ml of fresh TRIzol LS, gently mixed and further processed for RNA extraction from host associated bacteria. For controls, media with bacteria was
collected and centrifuged at 7200 × g for 5 min. To the pellet, 10 ml of TRIzol LS reagent
was added, gently mixed and was further processed for RNA extraction from bacterial
controls. For the non infected Caco-2 cells, media was aspirated and 10 ml of TRIzol LS
reagent was added to the cells, gently mixed and was further processed for RNA
extraction form non infected Caco-2 cells.
Bacterial RNA extraction and gene expression. Sample preparation for gene
expression profiling was performed as described by Yi Xie et al. (36) with modification
in RNA isolation, which was done using TRIzol LS reagent (Invitrogen, Carlsbad, CA).
The 750 µl of the TRIzol LS suspension containing host associated bacteria or bacterial
controls, was centrifuged at 7200 × g for 5 min. The TRIzol LS supernatant was stored in
a clean tube for later use and to the bacterial pellet, 1 ml of lysis enzyme cocktail
containing 50 mg/ml of lysozyme (Sigma) and 200 U/ml mutanolysin (Sigma) in TE
buffer (10mM Tris and 1mM EDTA, pH 8) was added. The solution was mixed gently
and incubated at 37°C for 1 h followed by centrifugation at 7200 × g for 5 min. The
supernatant was discarded and pellet was re-suspended in 250 μl of proteinase K buffer
(100 mM Tris-HCl, 5 mM EDTA, 200 mM NaCl, and 0.2 % SDS, pH 8) containing 8 163
U/ml of proteinase K (Fermentas, Glen Burnie, MD). This was incubated at 55°C for 1 h with intermittent mixing. To this, previously stored TRIzol LS was added and gently mixed. After this step manufacturer’s (TRIzol LS, Invitrogen) protocol was followed for
RNA extraction. RNA concentration, A260/280 and A260/230 were measured on
NanoDrop (Thermo scientific, Waltham, MA) and samples were processed further only if the RNA concentration was at least 0.5 µg/µl and ratios were ≥ 1.8. The RNA samples were analyzed for integrity on 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
CA).
Total RNA (10 g in 20 μl) was reverse transcribed into cDNA with 6 µg of random hexamers and 400 U of Superscriptase II (Invitrogen) according to the manufacturer’s protocol. Upon cDNA synthesis, the enzyme was heat inactivated and the
RNA templates were degraded with 80 U of RNaseH (Epicentre, Madison, WI) at 37°C for 20 min. The reaction mixture was cleaned by using the Qiaquick-PCR purification kit
(Qiagen, Valencia, CA) according to the manufacturer’s instructions, except that the Tris- containing buffer was replaced with 75% ethanol for washing the column. The purified single-strand cDNA was eluted from the columns twice with a total of 100 μl of nuclease free water (Ambion, Austin, TX).
cDNA fragmentation was done using 0.6 U of DNaseI (Promega, Madison, WI) per µg of cDNA, according to the instructions. The fragmented 1 µg of cDNA was labeled using 2 µl of GeneChip DNA Labeling reagent (Affymetrix, Santa Clara, CA) and 60 U of Terminal Transferase enzyme (New England Biolabs, Ipswich, MA). The samples were denatured prior to hybridization, at 98°C for 10 min followed by snap cooling at 4°C for 5 min. 164
Hybridization and Normalization. Labeled cDNA was hybridized onto a custom made Affymetrix GeneChip designed against all the annotated coding sequences of S. Typhimurium LT2 ATCC 700720. Briefly, the array contained 9852 probe sets, of which 4735 probe sets were designed against S. Typhimurium. Each probe set contained
11 probes, each 25 nucleotides long. 500 ng of labeled cDNA for samples extracted from pure culture of S. Typhimurium and 2000 ng of labeled cDNA for samples extracted from co-culture of S. Typhimurium and Caco-2 was hybridized onto the chips. The chips were hybridized and scanned at the Center for Integrated BioSystems (Utah State University,
Logan, UT) as per manufacturer’s protocols for E. coli. Raw data (.cel files) was back ground corrected, quantile normalized and summarized using MS-RMA (28).The resultant normalized log2 transformed intensity matrix was used for further statistical analysis.
Caco-2 RNA extraction and gene expression. The TRIzol LS samples
containing infected or non infected Caco-2 cells were frozen (Liquid N2) and thawed
(70°C) twice. To 750 μl of TRIzol LS sample, 250 μl of water was added. This was
further processed for RNA extraction using manufacturer’s (TRIzol LS, Invitrogen)
instructions. RNA concentration, A260/280 and A260/230 were measured on NanoDrop
(Thermo scientific, Waltham, MA) and samples were processed further only if the RNA
concentration was at least 0.5 µg/µl and ratios were ≥ 1.8. The RNA samples were
analyzed for integrity on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Synthesis of cDNA, biotin labeled cRNA, fragmentation and purification of cRNA were
carried out using one-cycle cDNA synthesis kit (Affymetrix). 165
Hybridization and Normalization. Labeled and fragmented cRNA (10 µg) was hybridized onto the Affymetrix U133Plus2 GeneChips as per manufacturer's recommendations at the Center for Integrated BioSystems (Utah State University, Logan,
UT). Raw data (.cel files) was back ground corrected, quantile normalized and summarized using RMA (15). RMA normalized data was then filtered through the PANP algorithm (33) to make presence-absence calls for each probe set. Probe sets that were called present in at least one of the samples were included in further statistical analysis.
Infection of epithelial cells for extracellular metabolite profiling. Caco-2 cells were cultured in T-75 flasks and were serum starved 24 h before infection. Respective bacterial treatments i.e. cold-stressed (5°C, 48 h) and non-stressed S. Typhimurium, at
MOI of 1:1000, in two biological replicates were used to infect Caco-2 cells. The control for these infections was addition of cold-stressed and non-stressed S. Typhimurium respectively, in T-75 flasks without the epithelial cells, and non infected epithelial cells.
All the flasks were incubated at 37°C with 5% CO2. Upon incubation for 60 min, 90 min
and 120 min, media with or without the extracellular bacteria was aspirated followed by
centrifugation at 7200 × g for 5 min to pellet the bacteria. The extracellular supernatant
was stored in a clean tube at -70°C and further processed for metabolite profiling by
nuclear magnetic resonance spectroscopy (NMR).
NMR sample preparation. Samples were filtered though a 3000 MW cutoff
filters (Pall Life Sciences, Ann Arbor, MI). A volume of 65 µl containing 5 mM DSS-d6
(3-(trimethylsilyl)-1-propanesulfonic acid-d6) with 0.2% NaN3, in 99.8% D2O (Chenomx
internal standard, Chenomx, Edmondton, Canada) was added to 585 µl of each sample filtrate and the pH was adjusted to 6.8 ± 0.1. A 600 uL aliquot of each sample was 166 transferred in a 5-mm NMR tube (Wilmad, Buena, NJ) and stored at 4 °C until NMR data acquisition.
NMR spectroscopy. All one-dimensional NMR spectra of the samples were acquired using the first increment of the standard NOESY pulse sequence on a Bruker
AVANCE 600 MHz NMR spectrometer equipped with a SampleJet. All spectra were recorded at 25°C with a 12 ppm sweepwidth, 2.5 s recycle delay, 100-ms mix, an
acquisition time of 2.5 s, 8 dummy scans, and 32 transients. 1H decoupling of the water
resonance was applied during the recycle delay and the 100 ms mix. All spectra were
zero-filled to 128k data points and multiplied by an exponential weighting function
corresponding to a line-broadening of 0.5 Hz.
Data analysis. Analysis of the NMR data was accomplished through targeted
profiling using the Chenomx NMRSuite v6.1 (Chenomx Inc., Edmonton, Canada) (35).
Statistical analysis for gene expression. Gene expression profile for cold-
stressed (5°C, 48 h) and non-stressed S. Typhimurium alone and in the presence of the
epithelial cells 60 min post infection was obtained. The data was analyzed as two class
(cold stress vs. no stress) unpaired, with T statistic, using Significance Analysis of
Microarrays (SAM) (31). All the genes were ranked based on the score (d) from SAM
output. This pre ordered ranked gene list was then used in Gene Set Enrichment Analysis
software (GSEA) (24, 29) to detect the coordinate changes in the expression of groups of
functionally related genes, upon respective treatments. The gene sets were defined based
on the annotations from BioCyc Database (18). BioCyc offers a comprehensive review of
metabolites and enzymes detected in high throughput datasets (2, 19). 167
Gene expression profile was obtained for epithelial cells alone and upon infection with cold-stressed (5°C, 48 h) and non-stressed S. Typhimurium 60 min post infection.
The data was analyzed as two class (infection with cold-stressed S. Typhimurium vs. infection with non-stressed S. Typhimurium) unpaired, with T statistic, using SAM (31).
All the genes were ranked based on the score (d) from SAM output. This pre ordered ranked gene list was then used in GSEA (24, 29) to detect the coordinate changes in the expression of groups of functionally related genes, upon infection with non-stressed and cold-stressed S. Typhimurium. The gene sets were defined based on the annotations from
REACTOME Database (17, 22, 32).
RESULTS AND DISCUSSION
Gene expression profile and extracellular metabolite profile of cold-stressed
S. Typhimurium. Extracellular cold stress (5°C, 48 h) to S. Typhimurium significantly
(q < 0.1) regulated gene categories with functions in nitrate reduction, electron transfer, oxidative pentose phosphate pathway and pyruvate fermentation (Table 5-1). Cold stress alone coordinately induced expression of four genes narG, narI, napA and napC encoding the subunits of periplasmic and respiratory nitrate reductase (Fig 5-1A). The genes encoding the subunits of NADH dehydrogenase and ATP synthase, involved in energy production along with conversion of NADH to NAD+, were induced in cold-
stressed S. Typhimurium. However, genes encoding other complexes in electron transfer
chain were not regulated. The genes in the oxidative branch of pentose phosphate
pathway, in which NADPH is generated and further used for reductive biosynthesis
reactions, were also significantly (q < 0.1) induced. Consequently, regeneration of 168
NADPH will prevent oxidative stress or production of 5C or 4C sugars further used in nucleic acid and aromatic amino acid metabolism. The three genes tdcE, pflB and yfiD responsible for pyruvate fermentation to formate were significantly (q < 0.05) repressed
(Fig 5-1A) thereby reducing the amount the formate formed.
Glucose and pyruvate were significantly (q < 0.05) depleted from the extracellular media indicating their increased usage by cold-stressed (5°C, 48 h) S. Typhimurium (Fig
5-2). The significant (q < 0.05) repression of the genes tdcE, pflB and yfiD responsible for fermentation of pyruvate to formate, in cold-stressed S. Typhimurium was concomitant with the significantly (q < 0.05) decreased amount of formate in extracellular media.
However, lactate, ethanol, acetate and acetone, were detected in significantly (q < 0.05) higher concentrations in extracellular media of cold-stressed S. Typhimurium indicating fermentation as the process for generating reducing equivalents and hence deriving energy (Fig 5-2). Increased amount of acetate and lactate in extracellular medium was concomitant with significant (q < 0.05) induction of acetate kinase A (ackA) and lactate dehydrogenase (ldhA) along with induction of non specific transporter (aphA) (18).
Additionally, citrate and succinate were also detected in significantly (q < 0.05) high amount and fumarate in significantly (q < 0.05) low amount in extracellular media.
Increased amount of succinate was concomitant with significant (q < 0.05) induction of genes frdABCD which function during fermentation in conversion of fumarate to succinate (Fig 5-1A) and aphA which functions as a transporter. The gene gltA encoding citrate synthase was also induced, however a transporter for export of citrate in
Salmonella is not yet known. Polyamines such as putrescine and cadaverine were also detected in significantly (q < 0.05) high amounts and arginine and lysine, precursors for 169 formation of polyamines, were depleted (q < 0.05) from extracellular medium. The conversion of lysine to cadaverine was further supported by significant (q < 0.05) induction of genes cadABC encoding lysine decarboxylase and lysine/cadaverine transport protein (Fig 5-1A).
To summarize, upon exposure to cold stress (5°C, 48 h), S. Typhimurium shifted metabolism towards fermentation. Additionally, formation of polyamines, putrescine and cadaverine, was also induced.
Gene expression profile and extracellular metabolite profile of cold-stressed
S. Typhimurium in the presence of epithelial cells. Infection of epithelial cells significantly (q < 0.1) regulated gene categories with functions in TCA Cycle, respiration, amino acid biosynthesis, fatty acid biosynthesis, nucleotide metabolism and enterobacterial common antigen biosynthesis (Table 5-2), in cold-stressed (5°C, 48 h) S.
Typhimurium as compared to infection with non-stressed S. Typhimurium. This implies that due to extracellular cold stress to S. Typhimurium, these above described categories are regulated in the presence of the host. However, the genes associated with the above described gene categories were repressed in cold-stressed S. Typhimurium when compared with non-stressed S. Typhimurium upon infection of epithelial cells (Fig 5-1B).
The genes encoding the subunits of NADH dehydrogenase complex, cytochrome o ubiquinol oxidase, ATP synthase and succinate dehydrogenase, along with genes encoding citrate synthase, isocitrate dehydrogenase and malate dehydrogenase were significantly (q < 0.1) repressed. The genes involved in pentose phosphate pathway were also significantly (q < 0.1) repressed which could lead to decreased formation of 5C and
4C sugars, required for nucleic acid and amino acid metabolism. This was further 170 supported by the significant (q < 0.1) repression of genes involved in fatty acid biosynthesis, nucleotide de novo biosynthesis and amino acid metabolism (Fig 5-1B).
The other genes significantly (q < 0.1) repressed in cold-stressed S. Typhimurium upon infection of epithelial cells, were genes from wec, rff and rfb clusters responsible for biosynthesis of enterobacterial common antigen (ECA) (Fig 5-1B). ECA provides resistance against bile and is important in oral than in intraperitoneal infections (21, 26).
The repression of these genes and hence repression of immune response, upon contact with intestinal epithelial cells can be of an advantage to Salmonella.
The repression of citrate synthase in S. Typhimurium can lead to the decreased amount of citrate in extracellular media during infection of epithelial cells with cold- stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S.
Typhimurium. Succinate was present in significantly (q < 0.05) higher amount and fumarate in significantly (q < 0.05) lower amount in extracellular media, which was concomitant with persistent significant (q < 0.1) induction of the genes frdABCD which function in conversion of fumarate to succinate. The utilization of glucose was delayed and pyruvate was being utilized by S. Typhimurium or epithelial cells as indicated by its rapid depletion (q < 0.05) from extracellular media during infection of epithelial cells with cold-stressed S. Typhimurium. The significantly higher amount of formate, acetate and ethanol detected (q < 0.05) in extracellular media can be attributed to increased fermentation and pyruvate utilization in cold-stressed S. Typhimurium during infection of epithelial cells. Even though the genes responsible for formation of acetate, formate and ethanol from pyruvate were not regulated, the possibility of post translational modification of pyruvate formate lyase (23) and promiscuity of dehydrogenases can be 171 responsible for increased amounts detected. Short chain fatty acids in the intestine are formed due to breakdown of complex sources by the intestinal bacteria and provide diffusible signal and regulate invasion genes of S. Typhimurium. Formate and acetate have been shown to induce invasion and propionate and butyrate have been shown to repress invasion (4, 10, 14, 20). Polyamines have also been implicated in stress response and infection processes (reviewed in (27)). Cadaverine has been showed to reduce adherence of E. coli (30) and it has also been showed to inhibit lysis of phagosomes formed by S. flexneri (6), thereby preventing its dissemination within the intestinal epithelium. Cadaverine was present in significantly lower concentrations (q < 0.05) in extracellular media during infection of epithelial cells with cold-stressed S. Typhimurium as compared to infection with non-stressed S. Typhimurium. The detection of lysine at significantly (q < 0.05) higher concentration in extracellular media during infection of epithelial cells with cold-stressed S. Typhimurium along with no regulation of the lysine transporter can lead to the low amount of cadaverine. This could be one of the factors responsible for high host association of cold-stressed S. Typhimurium to epithelial cells
(Fig 4-1A).
To summarize, genes involved in aerobic respiration were significantly (q < 0.1) repressed in cold-stressed (5°C, 48 h) S. Typhimurium upon infection of epithelial cells, but the fermentation products were persistently being accumulated in extracellular media suggesting fermentation as the preferred source to derive energy during initial stages of infection. The fermentation pathway utilizing pyruvate is present in S. Typhimurium (Fig
5-4B), thus the major production of fermentation products can be attributed to S.
Typhimurium. The switch from aerobic respiration could be due to two reasons, either 172 the epithelial cells are using all the dissolved oxygen at higher rate which can partly be supported by induction of genes involved in aerobic respiration or majority of the S.
Typhimurium are intracellular where they experience low oxygen availability (11).
Repression of genes involved in aerobic respiration, in cold-stressed S. Typhimurium, will repress the formation of the superoxide radicals. This may be a mechanism by which cold-stressed S. Typhimurium down regulates its own superoxide generation leading to decreased oxidative stress from the energy generation processes in itself and using its full potential to overcome only the host defense factors. Additionally, extracellular short chain fatty acids, acetate and formate, known to regulate invasion of S. Typhimurium (14,
20), were also present in significantly high amounts in extracellular media during infection of epithelial cells. Further, during infection the formation of cadaverine was significantly (q < 0.05) repressed which can lead to increased association of cold-stressed
S. Typhimurium to epithelial cells as observed (Fig 4-1A).
Gene expression profile and extracellular metabolite profile of epithelial cells upon infection with cold-stressed S. Typhimurium. Infection of epithelial cells with cold-stressed (5°C, 48 h) S. Typhimurium significantly (q < 0.05) regulated gene categories with functions in various cellular processes including electron transport chain and glycolysis, 60 min post infection (Table C-2) as compared to infection with non- stressed S. Typhimurium. Genes involved in electron transport chain were significantly (q
< 0.05) induced and genes involved in glycolysis were significantly (q < 0.05) repressed
(Fig 5-3). This was concomitant with decreased glucose utilization initially and increased pyruvate utilization over time. By 120 min all the pyruvate from the extracellular medium was used up, partly by the cold-stressed S. Typhimurium and partly by the epithelial 173 cells. In epithelial cells, pyruvate can lead to formation of acetyl-CoA which then enters
TCA cycle or lactate formation during fermentation (18). The gene PDHB (pyruvate dehydrogenase) leading to formation of acetyl-CoA, was significantly induced whereas genes encoding lactate dehydrogenase leading to formation of lactate, were not regulated.
This suggested that aerobic respiration through TCA and electron transport chain was main source of deriving energy in epithelial cells during infection and that it was induced or the demand for energy increased upon infection with cold-stressed (5°C, 48 h) S.
Typhimurium. This induction could be due to various reasons but in light of the increased invasion of cold-stressed S. Typhimurium (Fig 4-1B), production of more energy and regeneration of the reducing equivalents, can be utilized for the increased host defense processes (16).
CONCLUDING REMARKS
Metabolic enzymes have a central role in microbial physiology have been attractive targets for antimicrobials. However, in order to survive the exposure to various environmental stresses, the organism regulates number of genes including regulation of metabolism. Consequently, understanding the metabolism of S. Typhimurium upon exposure to stress conditions and metabolism of stressed S. Typhimurium upon infection of epithelial cells, will lead to efficient targets for development of novel antimicrobials.
Exposure to cold stress (5°C, 48 h) shifted the metabolism to fermentation in S.
Typhimurium. The short chain fatty acids, fermentation products of pyruvate which induce invasion in intestinal epithelial cells, were detected in significantly (q < 0.05) high concentration in extracellular medium upon cold stress to S. Typhimurium alone and 174 were present in further high concentration (q < 0.05) upon infection of epithelial cells. In addition to regulation of known virulence factors, the effect of cold stress to S.
Typhimurium on its metabolism seems to provide an added advantage during infection of intestinal epithelial cells. Hence, based on the observations of effect of cold stress to S.
Typhimurium, it can also be concluded that life cycle of S. Typhimurium outside the host will determine its potential to infect and severity of clinical manifestations. Additionally, the observations in this study provided new directions and targets such as pathways for fermentation and formation of short chain fatty acids, which can be further investigated for development of new antimicrobials. Moreover, confirmation of the observations in this study with in-vivo expression technology will provide definitive targets for novel strategies to control infection.
REFERENCES
1. Amel, D., and B. Amina. 2008. Resuscitation of Seventeen-year Stressed
Salmonella typhimurium. Oceanological and Hydrobiological Studies 37:69-82.
2. Becker, D., M. Selbach, C. Rollenhagen, M. Ballmaier, T. F. Meyer, M.
Mann, and D. Bumann. 2006. Robust Salmonella metabolism limits possibilities
for new antimicrobials. Nature 440:303-7.
3. Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998.
Macrophage-dependent induction of the Salmonella pathogenicity island 2 type
III secretion system and its role in intracellular survival. Mol Microbiol 30:175-
88. 175
4. Durant, J. A., D. E. Corrier, and S. C. Ricke. 2000. Short-chain volatile fatty
acids modulate the expression of the hilA and invF genes of Salmonella
typhimurium. J Food Prot 63:573-8.
5. Fang, F. C., S. J. Libby, N. A. Buchmeier, P. C. Loewen, J. Switala, J.
Harwood, and D. G. Guiney. 1992. The alternative sigma factor katF (rpoS)
regulates Salmonella virulence. Proc Natl Acad Sci USA 89:11978-11982.
6. Fernandez, I. M., M. Silva, R. Schuch, W. A. Walker, A. M. Siber,
A. T. Maurelli, and B. A. McCormick. 2001. Cadaverine prevents the
escape of Shigella flexneri from the phagolysosome: a connection between
bacterial dissemination and neutrophil transepithelial signaling. J Infect Dis
184:743-53.
7. Foster, J. W., and M. P. Spector. 1995. How Salmonella survive against the
odds. Annu Rev Microbiol 49:145-74.
8. Galyov, E. E., M. W. Wood, R. Rosqvist, P. B. Mullan, P. R. Watson, S.
Hedges, and T. S. Wallis. 1997. A secreted effector protein of Salmonella dublin
is translocated into eukaryotic cells and mediates inflammation and fluid secretion
in infected ileal mucosa. Mol Microbiol 25:903-12.
9. Ganesan, B., M. R. Stuart, and B. C. Weimer. 2007. Carbohydrate Starvation
Causes a Metabolically Active but Nonculturable State in Lactococcus lactis.
Appl Environ Microbiol 73:2498-2512.
10. Gantois, I., R. Ducatelle, F. Pasmans, F. Haesebrouck, I. Hautefort, A.
Thompson, J. C. Hinton, and F. Van Immerseel. 2006. Butyrate specifically 176
down-regulates salmonella pathogenicity island 1 gene expression. Appl Environ
Microbiol 72:946-9.
11. García-del Portillo, F., C. Núñez-Hernández, B. Eisman, and J. Ramos-
Vivas. 2008. Growth control in the Salmonella-containing vacuole. Current
Opinion in Microbiol 11:46-52.
12. Hansen-Wester, I., and M. Hensel. 2001. Salmonella pathogenicity islands
encoding type III secretion systems. Microbes Infect 3:549-59.
13. Hensel, M., J. E. Shea, S. R. Waterman, R. Mundy, T. Nikolaus, G. Banks, A.
Vazquez-Torres, C. Gleeson, F. C. Fang, and D. W. Holden. 1998. Genes
encoding putative effector proteins of the type III secretion system of Salmonella
pathogenicity island 2 are required for bacterial virulence and proliferation in
macrophages. Mol Microbiol 30:163-74.
14. Huang, Y., M. Suyemoto, C. D. Garner, K. M. Cicconi, and C. Altier. 2008.
Formate acts as a diffusible signal to induce Salmonella invasion. J Bacteriol
190:4233-41.
15. Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U.
Scherf, and T. P. Speed. 2003. Exploration, normalization, and summaries of
high density oligonucleotide array probe level data. Biostatistics 4:249-64.
16. Jones, B. D. 1997. Host responses to pathogenic Salmonella infection. Genes Dev
11:679-87.
17. Joshi-Tope, G., I. Vastrik, G. R. Gopinath, L. Matthews, E. Schmidt, M.
Gillespie, P. D'Eustachio, B. Jassal, S. Lewis, G. Wu, E. Birney, and L. Stein. 177
2003. The Genome Knowledgebase: a resource for biologists and
bioinformaticists. Cold Spring Harb Symp Quant Biol 68:237-43.
18. Karp, P. D., C. A. Ouzounis, C. Moore-Kochlacs, L. Goldovsky, P. Kaipa, D.
Ahrén, S. Tsoka, N. Darzentas, V. Kunin, and N. López-Bigas. Expansion of
the BioCyc collection of pathway/genome databases to 160 genomes. Nucleic
Acids Res 33:6083-6089.
19. Keseler, I. M., C. Bonavides-Martinez, J. Collado-Vides, S. Gama-Castro, R.
P. Gunsalus, D. A. Johnson, M. Krummenacker, L. M. Nolan, S. Paley, I. T.
Paulsen, M. Peralta-Gil, A. Santos-Zavaleta, A. G. Shearer, and P. D. Karp.
2009. EcoCyc: a comprehensive view of Escherichia coli biology. Nucleic Acids
Res 37:D464-70.
20. Lawhon, S. D., R. Maurer, M. Suyemoto, and C. Altier. 2002. Intestinal short-
chain fatty acids alter Salmonella typhimurium invasion gene expression and
virulence through BarA/SirA. Mol Microbiol 46:1451-64.
21. Lew, H. C., H. Nikaido, and P. H. Makela. 1978. Biosynthesis of uridine
diphosphate N-acetylmannosaminuronic acid in rff mutants of Salmonella
tryphimurium. J Bacteriol 136:227-233.
22. Matthews, L., G. Gopinath, M. Gillespie, M. Caudy, D. Croft, B. de Bono, P.
Garapati, J. Hemish, H. Hermjakob, B. Jassal, A. Kanapin, S. Lewis, S.
Mahajan, B. May, E. Schmidt, I. Vastrik, G. Wu, E. Birney, L. Stein, and P.
D'Eustachio. 2009. Reactome knowledgebase of human biological pathways and
processes. Nucleic Acids Res 37:D619-22.
23. Melchiorsen, C. R., K. V. Jokumsen, J. Villadsen, M. G. Johnsen, 178
H. Israelsen, and J. Arnau. 2000. Synthesis and Posttranslational Regulation
of Pyruvate Formate-Lyase in Lactococcus lactis. J Bacteriol 182:4783-4788.
24. Mootha, V. K., C. M. Lindgren, K.-F. Eriksson, A. Subramanian,
S. Sihag, J. Lehar, P. Puigserver, E. Carlsson, M. Ridderstrale, E. Laurila,
N. Houstis, M. J. Daly, N. Patterson, J. P. Mesirov, T. R. Golub, P. Tamayo,
B. Spiegelman, E. S. Lander, J. N. Hirschhorn, D. Altshuler, and L. C.
Groop. 2003. PGC-1[alpha]-responsive genes involved in oxidative
phosphorylation are coordinately downregulated in human diabetes. Nat Genet
34:267-273.
25. Ohl, M. E., and S. I. Miller. 2001. Salmonella: a model for bacterial
pathogenesis. Annu Rev Med 52:259-74.
26. Ramos-Morales, F., A. I. Prieto, C. R. Beuzon, D. W. Holden, and J.
Casadesus. 2003. Role for Salmonella enterica Enterobacterial Common Antigen
in Bile Resistance and Virulence. J Bacteriol 185:5328-5332.
27. Shah, P., and E. Swiatlo. 2008. A multifaceted role for polyamines in bacterial
pathogens. Mol Microbiol 68:4-16.
28. Stevens J.R., G. B., Desai P., Rajan S., and Weimer B.C. 2008. Statistical
Issues in the Normalization of Multi-Species Microarray Data. Proc Conf Appl
Statistics in Agricul 47-62.
29. Subramanian, A., P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert,
M. A. Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, and J.
P. Mesirov. 2005. Gene set enrichment analysis: A knowledge-based approach 179
for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA
102:15545-15550.
30. Torres, A. G., R. C. Vazquez-Juarez, C. B. Tutt, and J. G. Garcia-Gallegos.
2005. Pathoadaptive mutation that mediates adherence of shiga toxin-producing
Escherichia coli O111. Infect Immun 73:4766-4776.
31. Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of
microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA
98:5116-21.
32. Vastrik, I., P. D'Eustachio, E. Schmidt, G. Gopinath, D. Croft, B. de Bono,
M. Gillespie, B. Jassal, S. Lewis, L. Matthews, G. Wu, E. Birney, and L.
Stein. 2007. Reactome: a knowledge base of biologic pathways and processes.
Genome Biol 8:R39.
33. Warren, P., D. Taylor, P. G. V. Martini, J. Jackson, and J. Bienkowska.
2007. Presented at the Bioinformatics and Bioengineering, 2007. BIBE 2007.
Proc of the 7th IEEE Intl Conf.
34. Watson, P. R., S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis. 1995.
Characterization of intestinal invasion by Salmonella typhimurium and
Salmonella dublin and effect of a mutation in the invH gene. Infect Immun
63:2743-54.
35. Weljie, A. M., J. Newton, P. Mercier, E. Carlson, and C. M. Slupsky. 2006.
Targeted profiling: quantitative analysis of 1H NMR metabolomics data. Anal
Chem 78:4430-4442. 180
36. Xie, Y., L. S. Chou, A. Cutler, and B. Weimer. 2004. DNA Macroarray
profiling of Lactococcus lactis subsp. lactis IL1403 gene expression during
environmental stresses. Appl Environ Microbiol 70:6738-47. 181
TABLE 5-1. Significantly (q < 0.10) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium as compared to non-stressed S.
Typhimurium.
Enriched gene sets in S. Typhimurium, Genes Size of Regulation FDR due to cold stress regulated gene set q value Nitrate reduction III dissimilatory 3 7 Induced 0.08 NADH to nitrate electron transfer 3 7 Induced 0.08 Nitrate reduction I denitrification 3 8 Induced 0.08 Formate to nitrate electron transfer 3 7 Induced 0.08 Aerobic respiration - Electron donor III 11 14 Induced 0.10 Pentose phosphate pathway - Oxidative 5 6 Induced 0.10 Pyruvate fermentation to ethanol I 4 5 Repressed 0.01 Reductive monocarboxylic acid cycle 3 5 Repressed 0.02
182
TABLE 5-2. Significantly (q < 0.10) regulated gene sets with coordinately regulating genes, in cold-stressed (5°C, 48 h) S. Typhimurium as compared to non-stressed S.
Typhimurium, upon infection of epithelial cells. This effect is due to extracellular cold stress to S. Typhimurium, which is beyond the effect of the infection of epithelial cells with non-stressed S. Typhimurium.
Enriched gene sets in S. Typhimurium, Genes Size of Regulation FDR due to cold stress regulated gene set q value NADH to cytochrome bo oxidase ETC 8 14 Repessed 0 Pentose phosphate pathway 12 16 Repessed 0.04 Superpathway of leu, val and ile biosynthesis 7 12 Repessed 0.05 TCA cycle 8 12 Repessed 0.05 Superpathway of fatty acid (FA) biosynthesis 6 10 Repessed 0.05 Guanosine nucleotides de novo biosynthesis 6 7 Repessed 0.05 Aerobic respiration - Electron donor III 7 14 Repessed 0.06 Superpathway of FA biosynthesis initiation 3 6 Repessed 0.06 Valine biosynthesis 3 7 Repessed 0.06 TCA cycle variation IV 8 13 Repessed 0.07 Purine degradation II Anaerobic 4 10 Repessed 0.07 tRNA charging pathway 10 21 Repessed 0.07 Formaldehyde oxidation I 4 8 Repessed 0.08 Pyrimidine de novo biosynthesis 5 5 Repessed 0.08 Pentose phosphate pathway – Non oxidative 8 10 Repessed 0.08 Threonine biosynthesis 3 5 Repessed 0.08 Adenosine nucleotides de novo biosynthesis 5 5 Repessed 0.08 Succinate to cytochrome bo oxidase ETC 5 6 Repessed 0.09 Aerobic respiration - Electron donor II 8 16 Repessed 0.09 Enterobacterial common antigen biosynthesis 7 9 Repessed 0.10
183
FIG 5-1. Heat map showing the expression of coordinately regulating genes within significantly regulated gene sets (q < 0.1) in S. Typhimurium. The enrichment analysis was done using GSEA software (24, 29) and the gene sets were defined based on the annotations from BioCyc Database (18). (A) The expression of genes in significantly (q
< 0.1) regulated gene sets due to cold stress (5°C, 48 h) to S. Typhimurium. (B) The expression of genes in significantly (q < 0.1) regulated gene sets in cold-stressed (5°C, 48 h) S. Typhimurium upon infection of epithelial cells. Legends: NS, No stress; CS, Cold stress. Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity.
184
NS CS
A 37narG C nitrate reductase 1 alpha subunit
36narI C nitrate reductase 1 gamma subunit
36napA C periplasmic nitrate reductase
37napC C periplasmic nitrate reductase
35nuoI C NADH dehydrogenase subunit I
36nuoC C NADH dehydrogenase I chain C/D
35nuoK C NADH dehydrogenase kappa subunit
57nuoA C NADH dehydrogenase alpha subunit
46nuoE C ATP synthase subunit E
46nuoF C NADH dehydrogenase I chain F
45nuoH C NADH dehydrogenase subunit H
56nuoB C NADH dehydrogenase beta subunit
45nuoJ C NADH dehydrogenase subunit J
45nuoM C NADH dehydrogenase subunit M
43tdcE C pyruvate formatelyase 4/2ketobutyrate formatelyase
12 11 pflB C pyruvate formate lyase I
11 9 adhE C irondependent alcohol dehydrogenase
35nuoL CP NADH dehydrogenase subunit L
46rfbK G phosphomannomutase
56zwf G glucose6phosphate 1dehydrogenase
36rfbM GM mannose1phosphate guanylyltransferase
46rfbG MG CDP glucose 4,6dehydratase
68rfbF MJ glucose1phosphate cytidylyltransferase
11 9 yfiD R putative formate acetyltransferase
46frdA C fumarate reductase
46frdB C succinate dehydrogenase
45frdC fumarate reductase subunit C
45frdD fumarate reductase subunit D
57gltA C citrate synthase
34cadA E lysine decarboxylase 1
46cadB E lysine/cadaverine transport protein
34cadC K transcriptional activator
185
NS CS
B 54nuoE C ATP synthase subunit E
54cyoD C cytochrome o ubiquinol oxidase subunit IV
55nuoF C NADH dehydrogenase I chain F
54nuoB C NADH dehydrogenase beta subunit
54nuoC C NADH dehydrogenase I chain C/D
65cyoA C cytochrome o ubiquinol oxidase subunit II
64cyoC C cytochrome o ubiquinol oxidase subunit III
64cyoB C cytochrome o ubiquinol oxidase subunit I
54sdhA C succinate dehydrogenase catalytic subunit
44sdhD C succinate dehydrogenase cytochrome b556 small membrane subunit
65sucD C succinylCoA synthetase alpha subunit
87gltA C citrate synthase
54sucC C succinylCoA synthetase subunit beta
76icdA C isocitrate dehydrogenase
75mdh C malate dehydrogenase
64sucB C dihydrolipoamide acetyltransferase
55ackA C acetate/propionate kinase
66nuoA C NADH dehydrogenase alpha subunit
43STM1792 C putative cytochrome oxidase subunit I
44leuB CE 3isopropylmalate dehydrogenase
64pta CR phosphate acetyltransferase
54leuA E 2isopropylmalate synthase
65glyA E serine hydroxymethyltransferase
33sdaB E Lserine dehydratase/Lthreonine deaminase 2
54aspC E aspartate aminotransferase
65thrC E threonine synthase
65asd E aspartatesemialdehyde dehydrogenase
43leuC E isopropylmalate isomerase large subunit
54ilvD EG dihydroxyacid dehydratase
87ilvC EH ketolacid reductoisomerase
43ilvG EH acetolactate synthase II large subunit
66gmk F guanylate kinase
55nrdE F ribonucleotidediphosphate reductase alpha subunit
43guaA F bifunctional GMP synthase/glutamine amidotransferase protein
65nrdB F ribonucleotidediphosphate reductase beta subunit
76nrdF F ribonucleotidediphosphate reductase beta subunit
54adk F adenylate kinase
44ndk F nucleoside diphosphate kinase
54dut F deoxyuridine 5 triphosphate nucleotidohydrolase
Fig 5-1B continued on next page 186
Continued from above
NS CS B 43STM2341 G putative transketolase 33STM2340 G putative transketolase
43STM4080 G ribulosephosphate 3epimerase
43tktB G transketolase
64rpe G ribulosephosphate 3epimerase
53tktA G transketolase
43rpiA G ribose5phosphate isomerase A
43talA G transaldolase
77talB G transaldolase
64zwf G glucose6phosphate 1dehydrogenase
65fabZ I (3R)hydroxymyristoyl ACP dehydratase
65fabD I acyl carrier protein Smalonyltransferase
54accD I acetylCoA carboxylase beta subunit
64accB I acetylCoA carboxylase
65fabB IQ 3oxoacyl(acyl carrier protein) synthase
65fabG IQR 3ketoacyl(acylcarrierprotein) reductase
54leuS J leucyltRNA synthetase
65aspS J aspartyltRNA synthetase
64gltX J glutamyltRNA synthetase
65glyS J glycyltRNA synthetase beta subunit
64ileS J isoleucyltRNA synthetase
87lysS J lysyltRNA synthetase
65alaS J alanyltRNA synthetase
55glyQ J glycyltRNA synthetase alpha subunit
66pheT JR phenylalanyltRNA synthetase beta subunit
44rffH M glucose1phosphate thymidyl transferase
54wecC M UDPNacetylDmannosaminuronic acid dehydrogenase
44rfbA M dTDPglucose pyrophosphorylase
43rffG M dTDPglucose 4,6dehydratase
55wecB M UDPNacetyl glucosamine2epimerase
54rfbG MG CDP glucose 4,6dehydratase
65rfbF MJ glucose1phosphate cytidylyltransferase
65metG RJ methionyltRNA synthetase
54ilvM S acetolactate synthase II small subunit
43STM1939 putative glucose6phosphate dehydrogenase
44wecD lipopolysaccharide biosynthetic protein
54wecF putative enterobacterial common antigen polymerase
56frdA C fumarate reductase
47frdB C succinate dehydrogenase
35frdC fumarate reductase subunit C
46frdD fumarate reductase subunit D 187
12 12 12 10 8 4 14 14 14 0 0 0 3 3 0 Glucose
11 11 11 9 8 6 10 10 9 11 11 9 11 10 2 Pyruvate
9 9 9 10 10 10 4 3 3 10 10 10 10 10 10 Succinate
666777323565555Citrate
777432-1-1-2443342Fumarate
1414141312127 4 4131313141414Formate
1313131313147 5 5131313131313Acetate
1313131313137 7 7131212131313Ethanol
655666231555444Acetone
14 14 14 14 14 14 11 12 12 15 15 15 14 14 14 Lactate
765544898645665Arginine
677888 000000000Putrescine
8767669109877778Lysine
999999 00010109999Cadaverine 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 NS Sal CS Sal Host NS Sal + Host CS Sal + Host
FIG 5-2. Heat map showing the concentration over time (min), of secreted extracellular metabolites, detected by NMR (q < 0.05). Legends: NS, No stress; CS, Cold Stress (5°C,
48 h); Sal, S. Typhimurium LT2; Host, Caco2 cells. Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity. 188
FIG 5-3. Heat map showing the expression of coordinately regulating genes within significantly regulated gene sets (q < 0.05) in epithelial cells due to infection (60 min post infection) with cold-stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S. Typhimurium. The gene sets were defined based on the annotations from
REACTOME Database (17, 22, 32). The enrichment analysis was done using GSEA software (24, 29). Color scale: Light pink - brown corresponds to low expression intensity - high expression intensity 189
NS CS
11 12 NDUFAB1 NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8kDa
11 11 NDUFB1 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7kDa
10 11 NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa
10 11 NDUFA1 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5kDa
11 11 COX7B cytochrome c oxidase subunit VIIb
12 12 NDUFA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa
89NDUFA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14kDa
11 11 NDUFB9 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9, 22kDa
99SDHC succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa
99NDUFS4 NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18kDa (NADH-coenzyme Q reductase)
10 11 NDUFS5 NADH dehydrogenase (ubiquinone) Fe-S protein 5, 15kDa (NADH-coenzyme Q reductase)
99NDUFB2 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8kDa
11 12 COX5B cytochrome c oxidase subunit Vb
99NDUFV3 NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa
11 11 COX7A2L cytochrome c oxidase subunit VIIa polypeptide 2 like
12 12 COX7C cytochrome c oxidase subunit VIIc
10 10 NDUFB3 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12kDa
11 11 NDUFA2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa
10 11 NDUFA12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12
10 10 SDHD succinate dehydrogenase complex, subunit D, integral membrane protein
11 11 UQCRQ ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5kDa
10 10 NDUFV2 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa
910SDHB succinate dehydrogenase complex, subunit B, iron sulfur (Ip)
99NDUFA7 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7, 14.5kDa
12 12 COX6B1 cytochrome c oxidase subunit Vib polypeptide 1 (ubiquitous)
11 11 NDUFA13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13
12 12 COX6C cytochrome c oxidase subunit VIc
10 11 UQCRC2 ubiquinol-cytochrome c reductase core protein II
12 12 UQCRH ubiquinol-cytochrome c reductase hinge protein
10 11 CYCS cytochrome c, somatic
10 11 NDUFA9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa
10 10 NDUFA8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19kDa
11 11 UQCRB ubiquinol-cytochrome c reductase binding protein
10 10 NDUFC1 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6kDa
12 13 COX6A1 cytochrome c oxidase subunit VIa polypeptide 1
77PC pyruvate carboxylase
14 14 GAPDH glyceraldehyde-3-phosphate dehydrogenase
88PCK2 phosphoenolpyruvate carboxykinase 2 (mitochondrial)
76GAPDHS glyceraldehyde-3-phosphate dehydrogenase, spermatogenic
88SLC25A11 solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11
88FBP1 fructose-1,6-bisphosphatase 1
77PFKFB2 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2
11 11 GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2)
13 13 TPI1 triosephosphate isomerase 1
10 10 MDH2 malate dehydrogenase 2, NAD (mitochondrial)
99PCK1 phosphoenolpyruvate carboxykinase 1 (soluble)
88PFKFB4 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4
12 12 ENO1 enolase 1, (alpha)
11 11 ALDOC aldolase C, fructose-bisphosphate
98PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
13 13 ALDOA aldolase A, fructose-bisphosphate
10 10 GPI glucose phosphate isomerase
11 11 PFKP phosphofructokinase, platelet
10 10 PFKM phosphofructokinase, muscle
10 10 PKM2 pyruvate kinase, muscle 190
A
B
FIG 5-4. Pathways adapted from BioCyc database, in S. Typhimurium for (A) Pyruvate fermentation and (B) Lysine degradation. 191
CHAPTER 6
SUMMARY
S. Typhimurium is a significant food-borne pathogen throughout the world that leads to infectious diarrhoea (3). Effective vaccines exist for typhoid (10) but not for gastroenteritis caused by non typhoidal Salmonellae (NTS). The incidences of food-borne salmonellosis, every year in the USA are estimated to be between 7.4 x 105 and 5.3 x 10 6
(4). Worldwide there are more than 1.3 billion cases of salmonellosis annually with 3
million deaths (8). In recent years multistate outbreaks of NTS from conventional sources
such as meat and poultry products have been reported and additionally non conventional
sources such as peanut butter, dry dog food, dry puffed cereal and fresh produce (6) have
started to contribute in these outbreaks. Thus, one thing common to all conventional and
non conventional food vectors, food processing methods and existing pathogen
elimination methods seems to be playing a major role in persistent NTS outbreaks.
Salmonella survives persistently in the food chain by regulating its gene expression in
response to stressful environmental conditions, in order to survive efficiently. Hence, we
hypothesized that exposure to cold stress, which is commonly encountered environmental
stress, increases the pathogenicity of S. Typhimurium.
HYPOTHESIS
Cold stress increases the pathogenicity of S. Typhimurium in cell culture model.
OBJECTIVES
192
1. Determine influence of cold stress, on the ability of S. Typhimurium to survive
during subsequent acid stress, which would be encountered during gastric transit.
2. Determine the effect of cold stress on pathogenesis of S. Typhimurium during
infection of intestinal epithelial cells.
3. Determine the effect of cold stress to S. Typhimurium on the metabolic changes
during infection of intestinal epithelial cells.
Cold stress indeed increased the pathogenicity of S. Typhimurium as seen in cell culture model. This study first investigated the survival of S. Typhimurium, during cold stress (5°C), the most common method to increase the shelf life of any food, peroxide stress and acid stress (Chapter 3). The survival of S. Typhimurium was found to be persistent under cold conditions, which was further supported by continuous induction of proteins at 5°C for up to 240 h whereas survival started to decline after 48 h at 37°C. In addition to significant (q < 0.05) induction of csp genes, genes involved in protection against acid and oxidative stress were also significantly (q < 0.05) induced during exposure to cold stress. The genes involved in TCA cycle were significantly (q < 0.05) induced and many genes involved in electron transfer chain were significantly (q < 0.05) repressed. Regulation of these genes with significantly (p < 0.05) low NAD+/NADH ratio
led to the significant (p < 0.05) repression of hydroxyl radical formation and it remained repressed during subsequent exposure to acid stress, hence increasing the survival of S.
Typhimurium as compared to its survival upon direct exposure to acid stress. This revealed one of the underlying mechanisms leading to increased survival under subsequent acid stress (Fig 6-1). 193
Among the stresses tested, cold stress (5°C, 48 h) to S. Typhimurium significantly
(p < 0.05) increased its adhesion and invasion in to intestinal epithelial cells (Chapter 4).
Further, the effect of cold stress on pathogenesis of S. Typhimurium was investigated.
During cold stress, significant (q < 0.05) induction of the genes related to the virulence was observed and this induction remained persistent in the presence of the intestinal epithelial cells upon infection. The genes induced due to cold stress were related to type
3secretion system apparatus with effectors, genes in tra and trb clusters on plasmid and prophage genes. In the presence of the epithelial cells, these genes remain induced with additional induction of spv genes on plasmid. So far, fimbrial adhesins on microbial surface have been shown to interact with specific sugar residues or glycan receptors on host cell surface, in the initial stages of infection (7, 9, 11). Since infection causes alteration in the surface of epithelium exposing a variety of molecules, understanding other types of interaction leading to successful invasion, is equally important. High association of cold-stressed S. Typhimurium to epithelial cells was attributed to a pair of novel protein-protein receptors found with whole cell cross-linking approach. The
STM2699 in cold-stressed S. Typhimurium and SPTAN1 in epithelial cells were both found to be significantly (q < 0.05) induced during infection of epithelial cells with cold- stressed S. Typhimurium. The STM2699 significantly (p < 0.05) contributed in adhesion as well as invasion, when epithelial cells were infected with cold-stressed S.
Typhimurium and SPTAN1 also had a significant (p < 0.05) contribution in decreasing adhesion of cold-stressed S. Typhimurium ΔSTM2699 (Fig 6-2).
Differential regulation in metabolism due to cold stress (5°C, 48 h) to S.
Typhimurium, alone and in the presence of epithelial cells, was observed (Chapter 5). 194
During infection of epithelial cells with cold-stressed S. Typhimurium, switch from aerobic respiration to fermentation with pyruvate as a substrate was indicated in cold- stressed S. Typhimurium. This was further supported by significantly (q < 0.05) high amounts of formate and acetate in the extracellular media which are also known to regulate invasion of S. Typhimurium (2, 5). Cadaverine which has been showed to reduce adherence of E. coli (12) was present in significantly (q < 0.05) lower amount in extracellular media during infection with cold-stressed S. Typhimurium as compared to infection with non-stressed S. Typhimurium. These observations explain and provide another layer of mechanism for increased association to host epithelial cells upon cold stress to S. Typhimurium (Fig 6-2). Based on the phenotypic observations and analysis of the gene expression profile, Fig 6-1 and 6-2 provide a proposed model for the increased survival and pathogenesis of cold-stressed (5°C, 48 h) S. Typhimurium.
FUTURE DIRECTIONS
Elucidating metabolism changes in cold-stressed (5°C, 48 h) S. Typhimurium upon infection of animal models will provide definitive targets for novel antimicrobials.
In light of robust Salmonella metabolism reported in vivo (1) there is strong need to fabricate alternative methods for the control of NTS infection. Thus, determining LD50 in animal models with cold-stressed S. Typhimurium wild type and ΔSTM2699 will confirm the increased pathogenicity of cold-stressed S. Typhimurium and the role of
STM2699 in vivo. This study has led to new insights for future work in developing alternative strategies, such as competitive exclusion, blocking the receptors with antibodies and vaccine development for NTS, to control the infection. 195
REFERENCES
1. Becker, D., M. Selbach, C. Rollenhagen, M. Ballmaier, T. F. Meyer, M.
Mann, and D. Bumann. 2006. Robust Salmonella metabolism limits possibilities
for new antimicrobials. Nature 440:303-7.
2. Huang, Y., M. Suyemoto, C. D. Garner, K. M. Cicconi, and C. Altier. 2008.
Formate acts as a diffusible signal to induce Salmonella invasion. J Bacteriol
190:4233-41.
3. Humphrey, T. 2004. Salmonella, stress responses and food safety. Nat Rev
Microbiol 2:504-9.
4. Jeffreys, A. G., K. M. Hak, R. J. Steffan, J. W. Foster, and A. K. Bej. 1998.
Growth, survival and characterization of cspA in Salmonella enteritidis following
cold shock. Curr Microbiol 36:29-35.
5. Lawhon, S. D., R. Maurer, M. Suyemoto, and C. Altier. 2002. Intestinal short-
chain fatty acids alter Salmonella typhimurium invasion gene expression and
virulence through BarA/SirA. Mol Microbiol 46:1451-64.
6. Lynch, M. F., R. V. Tauxe, and C. W. Hedberg. 2009. The growing burden of
foodborne outbreaks due to contaminated fresh produce: risks and opportunities.
Epidemiol Infect 137:307-15.
7. Olsen, A., A. Jonsson, and S. Normark. 1989. Fibronectin binding mediated by
a novel class of surface organelles on Escherichia coli. Nature 338:652-5.
8. Pang, T., Z. A. Bhutta, B. B. Finlay, and M. Altwegg. 1995. Typhoid fever and
other salmonellosis: a continuing challenge. Trends Microbiol 3:253-5. 196
9. Patti, J. M., B. L. Allen, M. J. McGavin, and M. Hook. 1994. MSCRAMM-
mediated adherence of microorganisms to host tissues. Annu Rev Microbiol
48:585-617.
10. Pulickal, A. S., and A. J. Pollard. 2007. Vi polysaccharide-protein conjugate
vaccine for the prevention of typhoid fever in children: hope or hype? Expert Rev
Vaccines 6:293-5.
11. Sjobring, U., G. Pohl, and A. Olsen. 1994. Plasminogen, absorbed by
Escherichia coli expressing curli or by Salmonella enteritidis expressing thin
aggregative fimbriae, can be activated by simultaneously captured tissue-type
plasminogen activator (t-PA). Mol Microbiol 14:443-52.
12. Torres, A. G., R. C. Vazquez-Juarez, C. B. Tutt, and J. G. Garcia-Gallegos.
2005. Pathoadaptive mutation that mediates adherence of Shiga toxin-producing
Escherichia coli O111. Infect Immun 73:4766-4776. 197
Cold stress Glucose
Glycolysis Aerobic respiration
TCA ETC (NAD+ NADH) (NADH NAD +) - O2 radicals H2O2 H2O
NAD+/NADH ratio Damage to Fe-S Fenton Fe clusters reaction
OH• radicals
Cell death
Pre adaptation to cold stress leads to increased survival upon subsequent acid stress
FIG 6-1. Proposed model for increased survival of S. Typhimurium during exposure to acid stress (pH 4.0, 90 min), upon pre-adaptation to cold stress (5°C, 48 h). Color legends: Green, repression; Red, induction. Pre-adaptation to cold stress to S.
Typhimurium significantly (q < 0.05) repressed aerobic respiration causing significant (p
< 0.05) decrease in NAD+/NADH ratio and OH• radicals generation, which remained
significantly (p < 0.05) repressed upon exposure to subsequent acid stress.
198
Exposure to cold stress leads to Formate increased adhesion and invasion in Acetate intestinal epithelial cells Ethanol Pyruvate Lysine
Fermentation Cadaverine Pyruvate invJ, sipC SPI2 T3SS Formate spv cluster Amino acid metabolism Acetate tra, trb clusters Ethanol Fels-1, 2 (STM2699) Lysine Gifsy-1,2 Cold stress Cadaverine
Flagella, LPS, ECA (fli, flh, flg, wec, rff, rfb clusters)
Exposure to cold stress leads to efficient evasion of detection by host immune factors
FIG 6-2. Proposed model for increased pathogenesis of cold-stressed (5°C, 48 h) S.
Typhimurium. Color legends: Green, repression; Red, induction. Cold stress to S.
Typhimurium significantly (q < 0.05) repressed aerobic respiration and it remained repressed upon infection of epithelial cells. Instead, fermentation was the preferred source to derive energy and resulting fermentation products, acetate and formate can lead to significant (p < 0.05) increase in invasion of cold-stressed S. Typhimurium in epithelial cells. The invasion and intracellular survival can be more efficient due to significant (q < 0.05) induction of the SPI2 T3SS genes, prophage genes, spv cluster and other plasmid encoded genes. Significant (q < 0.05) repression of the flagella genes, LPS 199 and ECA genes in cold-stressed S. Typhimurium further adds to its advantage since they function in activating immune responses in the host cells. 200
APPENDICES
201
APPENDIX A
SUPPLEMENTARY DATA FOR CHAPTER 3
TABLE A-1. Growth of S. Typhimurium in nutrient broth with hydrogen peroxide (H2O2).
Concentration of H2O2 Growth 0 mM (Positive control) ++++ 1 mM ++++ 2 mM - 3 mM - 4 mM - 5 mM - 6 mM - 7 mM - 8 mM - 9 mM - 10 mM - 15 mM - 20 mM - 30 mM - 40 mM - 50 mM - Negative control (No inoculation) -
Legends: +, Growth; -, No growth.
202
TABLE A-2. Growth of S. Typhimurium in nutrient broth with sodium chloride (NaCl).
Concentration of NaCl Growth 0% (Positive control) ++++ 1% ++++ 2% ++++ 3% ++++ 4% ++ 5% ++ 6% - 7% - 8% - 9% - 10% - Negative control (No inoculation) -
Legends: +, Growth; -, No growth.
203
FIG A-1. Survival of S. Typhimurium during upon pre-adaptation at 10°C for 0.5 h followed by exposure to cold stress at 5°C for 336 h . Inset: Enlarged for growth between
0 h – 8 h. The asterisk (*) represents the significant difference (p < 0.05) as compared to no stress.
204
FIG A-2. Heat map representations of the 117 differentially regulated proteins (q < 0.05) due to pre-adaptation at 10°C for 0.5 h followed by exposure to cold stress at 5°C for 240 h. The proteins are grouped based on the COGs functional annotation. (A) Proteins related to information storage and processing (COGs J, K &L). (B) Proteins related to cellular processes (COGs D, O, M, N, P & T). (C) Proteins related to metabolism (COGs
C, G, E, F, H, I & Q). Legends of COG categories: J-Translation; K-Transcription; L-
DNA replication, recombination and repair; D-Cell division and chromosome partitioning; O-Posttranslational modification, protein turnover, chaperones; M-Cell envelope biogenesis, outer membrane; N-Cell motility and secretion; P-Inorganic ion transport and metabolism; T-Signal transduction mechanisms; C-Energy production and conversion; G-Carbohydrate transport and metabolism; E-Amino acid transport and metabolism; F-Nucleotide transport and metabolism; H-Coenzyme metabolism; I-Lipid metabolism; Q-Secondary metabolites biosynthesis, transport and catabolism. Color legends: Dark-Light is High-Low expression intensity. 205
Pre adapted None 0.5 5 48 240 0.5 5 48 240 A 98889976rplI J 50S ribosomal protein L9 00116631rpmE J 50S ribosomal protein L31
01 01 6 6 01rpmF J 50S ribosomal protein L32
111 07751rpsU J 30S ribosomal protein S21
87778875rplE J 50S ribosomal protein L5
88888874rplO J 50S ribosomal protein L15
99889986rplA J 50S ribosomal protein L1
11 11 11 11 12 11 11 9 tuf J elongation factor Tu
88889974rpsG J 30S ribosomal protein S7
88889874rplD J 50S ribosomal protein L4
88889975rpsJ J 30S ribosomal protein S10
88889986rplF J 50S ribosomal protein L6
98889975rplC J 50S ribosomal protein L3
88888874rpsI J 30S ribosomal protein S9
88889871rplS J 50S ribosomal protein L19
99989986rpsC J 30S ribosomal protein S3
7777886 0rplX J 50S ribosomal protein L24
88889985rpsE J 30S ribosomal protein S5
87879886rplK J 50S ribosomal protein L11
11 014531tyrS J tyrosyl-tRNA synthetase
77778872rplM J 50S ribosomal protein L13
66668761rpsK J 30S ribosomal protein S11
77778875rpsB J 30S ribosomal protein S2
11 006631yfiA J ribosome stabilization factor
02226751rplY J 50S ribosomal protein L25
87778861rplQ J 50S ribosomal protein L17
011 06652rpsQ J 30S ribosomal protein S17
77778762rplN J 50S ribosomal protein L14
7777876 0rpsH J 30S ribosomal protein S8
01216611rpmA J 50S ribosomal protein L27
66667761rpmC J 50S ribosomal protein L29
88889986rplJ J 50S ribosomal protein L10
77777761rplP J 50S ribosomal protein L16
22536651gltX J glutamyl-tRNA synthetase
8888887 0rplV J 50S ribosomal protein L22
88889973rpsD J 30S ribosomal protein S4
111 06651efp J elongation factor P
77778873rplU J 50S ribosomal protein L21
77777775lysS J lysyl-tRNA synthetase
57578851rpsL J 30S ribosomal protein S12
113 08761rpsO J 30S ribosomal protein S15
3 0538861rplT J 50S ribosomal protein L20
66667764pnp J polynucleotide phosphorylase
66666664proS J prolyl-tRNA synthetase
88889987rpsA J 30S ribosomal protein S1
73778861rpsR J 30S ribosomal protein S18
46666765asnS J asparaginyl-tRNA synthetase
121 0665 0rpsT J 30S ribosomal protein S20
66667761frr J ribosome releasing factor
9101099861cspA K major cold shock protein
3344553 0kdgR K putative transcriptional repressor
66666651nusA K transcription elongation factor NusA
66666651rho K transcription termination factor Rho
68888872cspE K cold shock protein E
88888882cspC K cold shock protein
00115542ssb L single-strand DNA-binding protein
2221653 0recA L recombinase A
7777611 0deaD LKJ cysteine sulfinate desulfinase 206
Pre adapted None 0.5 5 48 240 0.5 5 48 240
55546521mreB D rod shape-determining protein
B 666665 02ftsZ D cell division protein FtsZ
66667743minD D cell division inhibitor protein
11114421gcpE M 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase
67667754kdsA M 2-dehydro-3-deoxyphosphooctonate aldolase
1121452 0galU M glucose-1-phosphate uridylyltransferase
111 022 01murA M UDP-N-acetylglucosamine 1-carboxyvinyltransferase
1 0012111stiC NU putativie fimbrial usher
55556651ydhD O putative glutaredoxin protein
88888876tig O trigger factor
01 1 05 2 01ahpF O alkyl hydroperoxide reductase F52a subunit
01114531grxC O glutaredoxin 3
56466631gst O glutathionine S-transferase
11 035664trxB O thioredoxin reductase
77788887yfiD R putative formate acetyltransferase
0011111 0ydjN R kinase/transporter-like protein
00223311ycfP R hypothetical protein
4466777 0yiiU S putative cytoplasmic protein
66666611typA T GTP-binding elongation factor family protein
011 05552arcA TK response regulator
46777776yfcZ putative cytoplasmic protein
00003311STM2729 hypothetical protein
21 035421STM0327 putative cytoplasmic protein
207
Pre adapted None 0.5 5 48 240 0.5 5 48 240
77777751caiB C crotonobetainyl-CoA:carnitineCoA-transferase
C 011 05521ppc C phosphoenolpyruvate carboxylase
111 053 00glpC C sn-glycerol-3-phosphate dehydrogenase K-small subunit
77777874fixB C putative electron transfer flavoprotein subunit alpha
7777887 0fixA C putative electron transfer flavoprotein subunit beta
1 0 01 2 1 01ydcW C putative aldehyde dehydrogenase
111 06652glpA C sn-glycerol-3-phosphate dehydrogenase large subunit
77777775acnB C aconitate hydratase
46647762ackA C acetate/propionate kinase
56666662pta CR phosphate acetyltransferase
2444752 0asnA E asparagine synthetase AsnA
88888964STM4467 E arginine deiminase
5555662 0thrC E threonine synthase
4455662 0speF E ornithine decarboxylase isozyme
1 0111245pepA E leucyl aminopeptidase
0131563 0glpB E anaerobic glycerol-3-phosphate dehydrogenase subunit B
1 021331 0aroG E 3-deoxy-7-phosphoheptulonate synthase
31325511asnB E asparagine synthetase B
77777776glnH ET glutamine ABC transporter periplasmic-binding protein
01116654deoC F deoxyribose-phosphate aldolase
77767765purA F adenylosuccinate synthetase
67666663ndk F nucleoside diphosphate kinase
11124541pfkA G 6-phosphofructokinase
88889987eno G phosphopyruvate hydratase
88888886pgk G phosphoglycerate kinase
11226655eda G keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase
77777766pykF G pyruvate kinase
35555533yneA G putative sugar transport protein
66666665fbp G fructose-1,6-bisphosphatase
11 0043 01cbiF H vitamin B12 biosynthetic protein
4244662 0kbl H 2-amino-3-ketobutyrate coenzyme A ligase
77777751caiD I carnitinyl-CoA dehydratase
1 0114411fabH I 3-oxoacyl-(acyl carrier protein) synthase
57778887acpP IQ acyl carrier protein
64666612fabB IQ 3-oxoacyl-(acyl carrier protein) synthase
0111411 0srlD IQR sorbitol-6-phosphate 2-dehydrogenase
208
APPENDIX B
SUPPLEMENTARY DATA FOR CHAPTER 4
MATERIALS AND METHODS
Labeling S. Typhimurium with Sulfo-SBED. The bacterial surface was labeled with Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)- hexanoamido) ethy-1,3'-dithioproprionate (Sulfo-SBED) (Thermo Scientific, Rockford,
IL). The total protein content on ~109 bacteria’s surface was measured using the BCA
protein assay (Thermo Scientific, Rockford, IL). Assuming an average molecular weight
of 60,000 Da for proteins,10-fold molar excess Sulfo-SBED (dissolved in DMSO at 50
g/l) of the determined protein concentration, was added to 109 bacteria suspended in 1
ml of tyrodes buffer. The labeling reaction was continued in dark on ice for 45 minutes
with intermittent shaking. After 45 min the reaction was quenched by adding 2-fold
molar excess glycine as compared to Sulfo-SBED. The bacteria were washed twice with
tyrodes buffer by centrifugation at 6000 × g for 2 min and re-suspended in 1 ml tyrodes
buffer.
Identification of receptors by cell-cell cross linking. Sulfo-SBED labeled S.
Typhimurium (109) were interacted with ~106 Caco-2 cells in a T-25 flask, in a final
volume of 3.5 ml tyrodes buffer for 60 min at 37˚C. At the end of 60 min the bacterial suspension was aspirated from the flask. The flask was then placed under a 15 watt UV lamp (302 nm) at a distance of 5 cm for 10 min. The cross linked Caco-2 cells and the bacteria were re-suspended in 500 µl of lysis buffer (8 M urea, 6.0% ampholytes pH range 3-10 (Bio-rad Laboratories, Hercules, CA), 0.4 % CHAPS, 0.25 % Triton 100, 0.15 209
% n-Dodecyl-B-β-D maltoside, 0.002% bromophenol blue) and 250 l of glass beads
(0.1 mm, BioSpec Products, Inc, Bartlesville, OK). The samples were homogenized in a mini-beadbeater (BioSpec Products, Inc, Bartlesville, OK) by giving 3 pluses at full speed for 30 s with intermittent 1 min incubation on ice. The samples were stored at -
70˚C until further use. Cross linked extracted samples were processed with 2D gels and
LC/MS/MS for protein identification. Protein spots were selected from the gels if: 1) the spot disappeared in reduced conditions as compared to gel run under non-reducing conditions; 2) the spot had host and the microbe protein in the same spot; 3) the sum of the molecular weight of the identified proteins matched the observed molecular weight on the gel. Table B-3 below lists all the proteins identified.
210
TABLE B-1. Composition of tyrodes buffer.
Constituent Amount NaCl 8.18 g/L KCl 0.37 g/L CaCl2 0.15 g/L MgCl2 0.20 g/L HEPES 2.38 g/L Glucose 1.80 g/L Na pyruvate 1.10 g/L
211
TABLE B-2. Growth of S. Typhimurium in cell culture media (excluding FBS) with hydrogen peroxide (H2O2).
Concentration of H2O2 Growth 0 mM (Positive control) ++++ 1 mM ++++ 2 mM ++++ 3 mM ++++ 4 mM +++ 5 mM +++ 6 mM - 7 mM - 8 mM - 9 mM - 10 mM - 15 mM - 20 mM - 30 mM - 40 mM - 50 mM - Negative control (No inoculation) -
Legends: +, Growth; -, No growth. 212
TABLE B-3. Host (Caco-2 cells) and microbe (S. Typhimurium) binding protein partners identified as using whole cell cross linking.
MW of S. Typhimurium protein Peptides PLGS Bacterial Caco-2 protein Peptides PLGS Host spot on Score protein Score protein gel (Da) MW MW (Da) (Da) 276408 Putative phage tail like 3 98.2 10804 Spectrin, alpha, non- 7 446.5 284105 protein (STM2699) erythrocytic 1 (SPTAN1) 102673 Flagellar biosynthesis 3 87.4 27456 Heat shock 90kDa protein 1 13 651.7 83212 factor FliA (STM1956) beta (HSP90AB1) 93670 Putative cytoplasmic 2 80.1 9306 Tumor rejection antigen 9 394.6 92411 protein (STM4088) gp96 (HSP90B1) Actinin alpha 4 (ACTN4) 5 133 104788 131066 Elongation factor Ef-Tu 5 368 43256 Potein phosphatase 1 14 842.1 115210 (STM4146, STM3445) regulatory subunit 12A (PPP1R12A) Heat shock 90kDa protein 1 4 160.5 83212 beta (HSP90AB1) Catenin alpha 1 (CTNNA1) 3 100.6 100008 Protein tyrosine phosphatase 2 124.8 80590 receptor type E (PTPRE) 119787 Translation initiation 8 415.8 97342 Potein phosphatase 1 8 611.6 89396 factor IF 2 (STM3286) regulatory subunit 9B (PPP1R9B) Elogation factor Ef-Tu 4 220.8 43256 Matrin 3 (MATR3) 5 195.5 94564 (STM4146, STM3445) Beta actin (ACTB) 4 177.7 41709
213
FIG B-1. Determination of optimum concentration of anti-SPTAN1 to block the exposed alpha spectrin-1 on the cell surface. (A) Anti-SPTAN1 was diluted 1:1000, 1:2000,
1:4000 and 1:8000 and added to epithelial cells to determine the association of cold- stressed (5°C, 48 h) S. Typhimurium (MOI of 1:100). (B) Anti-SPTAN1 was diluted
1:2000 and 1:4000 and added to epithelial cells to determine the association of non- stressed S. Typhimurium (MOI of 1:100). Upon incubation with anti-SPTAN1 followed by incubation with respective bacterial treatments, total host association was measured by
PCR as described in experimental procedures.
214
215
APPENDIX C
SUPPLEMENTARY DATA FOR CHAPTER 5
TABLE C-1. Composition of the chemically defined media
Constituent Amount L–Ala 100 mg/L L–Arg 50 mg/L L–Asn 100 mg/L L–Cys 20 mg/L Gly 100 mg/L L–Gln 100 mg/L L–His 50 mg/L L–Ile 20 mg/L L–Leu 20 mg/L L–Lys 100 mg/L DL–Met 20 mg/L L–Pro 200 mg/L DL–Ser 100 mg/L DL–Thr 100 mg/L DL–Val 100 mg/L L–Asp 100 mg/L L–Glu 400 mg/L L–Trp 25 mg/L L–Phe 20 mg/L L–Tyr 50 mg/L Pyridoxal 5 mg/L Pyridoxamine 5 mg/L Adenine 25 mg/L Guanine 25 mg/L Uracil 25 mg/L Xanthine 25 mg/L Mevalonic acid lactone (10%) 0.1 ml Na–acetate 5000 mg/L Na–citrate 2000 mg/L K–phosphate (monobasic) 1000 mg/L K–phosphate (dibasic) 1000 mg/L NaCl 200 mg/L
CaCl2 200 mg/L 216
MgSO4 200 mg/L
MnSO4 50 mg/L Tween 80 1 ml Tween 20 1 ml Glycerol 1 ml Vitaminsa 20 ml Trace Elementsb 2.5 ml Sugar 5000 mg/L MOPS/MES Final Conc 190 mM aVitamins - Sigma R–7256 vitamin mixture. b Trace elements stock solution: 2.85 g H3BO3, 1.8 g MnCl2.4H2O, 1.36 g FeSO4.7H2O,
1.77 g Na Tartarate, 26.9 mg CuCl2.2H2O, 20.8 mg ZnCl2, 40.4 mg CoCl2.6H2O and 25.2 mg Na2MoO4.2H2O dissolved in 1000 ml dH2O and adjusted to pH 4.0 with H2SO4.
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TABLE C-2. Significantly (q < 0.05) regulated gene sets with coordinately regulating genes, in epithelial cells due to infection with cold-stressed (5°C, 48 h) S. Typhimurium as compared to infection with non-stressed S. Typhimurium. This effect is due to extracellular cold stress to S. Typhimurium, which is beyond the effect of the infection of epithelial cells with non-stressed S. Typhimurium.
Enriched gene sets in Caco-2 cells due to Genes Size of Regulation FDR infection with cold-stressed S. gene Typhimurium Regulated set q value Electron transport chain 35 61 Induced 0 Formation of a pool of free 40s subunits 51 74 Induced 0 Formation of ATP by chemiosmotic coupling 10 13 Induced 0 GTP hydrolysis and joining of the 60s ribosomal subunit 56 84 Induced 0 Peptide chain elongation 51 75 Induced 0 Regulation of gene expression in beta cells 52 79 Induced 0 Viral mRNA translation 51 75 Induced 0 Regulation of beta cell development 52 82 Induced 0 Translation 61 97 Induced 0 Influenza viral RNA transcription and replication 56 90 Induced 0 Glucose regulation of insulin secretion 50 125 Induced 0 Formation of the ternary complex and subsequently the 43s complex 25 34 Induced 0 Insulin synthesis and secretion 66 107 Induced 0 Influenza life cycle 71 126 Induced 0 Translation initiation complex formation 26 40 Induced 0 Regulation of insulin secretion 51 147 Induced 0 Cdc20 phospho Apc mediated degradation of cyclin-a 24 57 Induced 0 Cell cycle checkpoints 38 98 Induced 0.01 Cdt1 association with the cdc6 orc origin complex 22 49 Induced 0.01 Snrnp assembly 22 44 Induced 0.01 Regulation of Apc activators between G1 S and early anaphase 26 64 Induced 0.01 Diabetes pathways 130 283 Induced 0.01 Mitotic M G1 phases 62 132 Induced 0.01 Vif mediated degradation of apobec3g 19 45 Induced 0.01 218
G1 S transition 39 87 Induced 0.01 G2 m checkpoints 21 37 Induced 0.02 Scf skp2 mediated degradation of p27 p21 19 47 Induced 0.02 Cyclin e associated events during G1 S transition 24 53 Induced 0.02 Integration of energy metabolism 50 168 Induced 0.02 Cell cycle mitotic 98 251 Induced 0.02 DNA replication pre initiation 27 69 Induced 0.02 Metabolism of proteins 80 171 Induced 0.03 Autodegradation of cdh1 by cdh1 Apc 20 53 Induced 0.04 S phase 38 94 Induced 0.04 Phase II conjugation 18 32 Induced 0.04 P53 independent DNA damage response 17 41 Induced 0.05 M G1 transition 22 57 Induced 0.05 Amino acid and oligopeptide slc transporters 15 27 Repressed 0.01 Amino acid transport across the plasma membrane 13 21 Repressed 0.05 Gluconeogenesis 17 27 Repressed 0.01 Glucose metabolism 24 48 Repressed 0.07 Glycolysis 13 17 Repressed 0.00 Inorganic cation anion slc transporters 23 45 Repressed 0.08 Regulation of insulin like growth factor activity by insulin like growth factor binding proteins 3 8 Repressed 0.08
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FIG C-1. Induction of ‘viable but non-culturable’ (VBNC) state in S. Typhimurium using chemically defined media (for media composition see Table C-1). (A) Viability of bacteria measured using LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen,
Carlsbad, CA). (B) Culturable bacteria measured using pour plate method with nutrient agar (Difco, Detroit, MI) and live bacteria measured using LIVE/DEAD BacLight
Bacterial Viability Kit . After 100 h, the bacteria were not culturable on nutrient agar, but they were still viable as observed by number of live bacteria.
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A
B
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FIG C-2. Association of VBNC and stationary phase S. Typhimurium to epithelial cells
(Caco-2 cells) measured using PCR, as described in materials and methods section of
Chapter 4.
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CURRICULUM VITAE
JIGNA D. SHAH
EDUCATION
Ph.D. Candidate (Advisor Dr. Bart Weimer) March 2006 - present Dissertation: Stress response and pathogenesis of Salmonella enterica serovar Typhimurium. NDFS, Utah State University, Logan, UT-84322. M.Sc. Microbiology April 2004 Thesis: Ethanol production from corn cobs: An alternative feed stock. North Maharashtra University, Jalgaon, MS, India. B.Sc. Microbiology April 2002 Mumbai University, Mumbai, MS, India.
TECHNICAL SKILLS
Cell based assays (Microbiology and Cell culture) Standard bacteriological and cell culture techniques. Adhesion and invasion (gentamicin protection) assays for pathogens alone and in combination with probiotics, in cell culture model (Caco-2). Determine the efficiency of the compounds to inhibit or promote survival and growth of microbes.
Proteomics and Metabolomics Protein and metabolite profiling using LC-MS/MS and NMR. SDS-PAGE and Western blots.
Genomics and Molecular biology Standard molecular biology techniques. Extract DNA & RNA, synthesize and label cDNA, prepare samples for prokaryotic and eukaryotic microarray gene chips (Affymetrix). Primer design, PCR, qPCR, RT-PCR, cloning and sequence analysis. Validation of the custom made Affymetrix GeneChips. Use of PCR, microarrays, comparative genomic hybridization (CGH) to study microbial populations. Construct stable gene knockouts in bacteria using λ red recombinase system.
Bioinformatics and Statistics Analyze high throughput datasets such as proteome profile from LC-MS/MS, metabolite profile from LC-MS & NMR and gene expression profile from microarrays for prokaryotes and eukaryotes. 223
Employ bioinformatics, data visualization and statistical tools such as Ingenuity Pathway Analysis (IPA), Gene Set Enrichment Analysis (GSEA), BioCyc, Kyoto Encyclopedia of Genes and Genomes (KEGG), PRODORIC, and Bioconductor (R).
WORK HISTORY
Junior Specialist February 2010 to August 2010 Department of Population Health and Reproduction, University of California, Davis, CA- 95616. Investigate the infection mechanisms of E. coli O157:H7 and simultaneous response of epithelial cells, using gene expression and metabolic profiling. Design fermentation strategies to induce production of flavor compounds in probiotic bacteria such as Lactobacilli and Brevibacteria.
Graduate Research Assistant March 2006 to January 2010 Center for Integrated Biosystems, Utah State University, Logan, UT-84322. Investigate the effect of environmental stresses on pathogenicity of Salmonella and simultaneous response of epithelial cells, using systems biology based approaches. Discover antimicrobial compounds from natural products.
Head Microbiologist (Quality Assurance) November 2005 to January 2006 Laxmi Enterprises, Navi Mumbai, MS, India. Microbiological analysis of in-process and finished food samples. Implementation of quality management system and internal audits.
Visiting Faculty September 2004 to October 2005 Dr. K. M. V. Pillai’s Campus, New Panvel, MS, India. Course and laboratory instructor in basic microbiology and biotechnology for under-graduate students.
Food Microbiologist July 2004 to August 2004 Microchem Laboratory, Thane, MS, India. Microbiological analysis of ready-to-eat food samples and animal feeds as per the USFDA-BAM methods
PUBLICATIONS
Shah J. D., Chen D., Stevens J. R. and Weimer B. C. Salmonella enterica serovar Typhimurium survives stress associated with environmental transit and digestion. (Manuscript in preparation) Shah J. D., Desai P., Gann R. and Weimer B.C. Cold stress increases adhesion and invasion of Salmonella Typhimurium in colonic epithelial cells. (Manuscript in preparation) 224
Shah J. D., Desai P., Slupsky C. and Weimer B.C. Metabolomics during in vitro infection of colonic epithelial cells with Salmonella Typhimurium. (Manuscript in preparation) Desai P., Shah J. D., Chen D. and Weimer B.C. Identification of Novel Receptors for Salmonella that mediates host-microbe adhesion and invasion. (Manuscript in preparation) Desai P., Shah J. D., Gann R. and Weimer B.C. A systems level analysis of cross talk between Salmonella and Bifidobacterium in presence of the host. (Manuscript in preparation) Desai P., Gann R., Shah J. D. and Weimer B.C. Bifidobacterium infantis protects intestinal cells from Salmonella induced apoptosis. (Manuscript in preparation) Mishchuk D. O.*, Shah J. D.*, Slupsky C. and Weimer B. C. Gene expression and metabolite profile of E. coli O157:H7 during infection of epithelial cells. (Manuscript in preparation)
CONFERENCE POSTERS
Shah J. D., Chen D., Stevens J. and Weimer B. C. 2008. Proteome of Salmonella Typhimurium LT2 under cold stress. 108th ASM General Meeting, Boston, MA. Shah J. D., Desai P., Gann R. and Weimer B.C. 2010. Abiotic stress increases host association of Salmonella typhimurium LT2. 110th ASM General Meeting, San Diego, CA. Shah J. D., Desai P., Gann R. and Weimer B.C. 2010. Abiotic stress increases host association of Salmonella typhimurium LT2. Bay Area Microbial Pathogenesis (BAMPS XIII), UC–San Francisco, CA.
ORAL PRESENTATIONS
Shah J. D., Desai P., Gann R. and Weimer B. C. 2009. Abiotic stress increases survival and adherence of Salmonella Typhimurium. 4th Annual UC Davis Research Retreat on Host Microbe Interactions, Tahoe, CA. Desai P., Shah J. D. and Weimer. B.C. 2010. Characterization of a novel Salmonella adhesin. UC–San Diego, CA. (Invited talk) Weimer. B. C., Desai P., Shah J. D., Gann R. and Pinzon J. 2010. Host microbe interactions modulated by cellular stress and probiotic microbes. Alltech, Inc., Lexington, KY. (Invited talk)