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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 ,

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 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 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 (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 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 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 are attractive targets for antimicrobials due to their central role in 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 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 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 (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 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 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 (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 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 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 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 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 (sodA), the DNA

repair 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 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 37C to 5C or 10C, 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 30C to 10C) (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 37C, 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.

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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 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 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 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 ( 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.

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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

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

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

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

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

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), (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.

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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.

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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 , fructose-bisphosphate

98PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3

13 13 ALDOA , 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

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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 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)