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Studies on the Virulence Properties and Regulation of the Cora Magnesium Channel

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Studies on the Virulence Properties and Regulation of the Cora Magnesium Channel STUDIES ON THE VIRULENCE PROPERTIES AND REGULATION OF THE CORA MAGNESIUM CHANNEL By KRISZTINA M. PAPP-WALLACE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Michael E. Maguire Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May 2008 1 Table of Contents List of Tables 4 List of Figures 5 List of Abbreviations 8 Abstract 10 Chapter 1: Introduction 12 Salmonella subtyping 12 Infection process of Salmonella enterica serovar Typhimurium 12 Animal models of Salmonella Typhimurium infection 15 Pathogenicity islands of S. Typhimurium 17 Other virulence determinants of S. Typhimurium 22 PhoPQ and virulence 26 The Two Component System: PhoP/PhoQ 27 The PhoPQ regulon 30 PhoPQ and regulation of Mg2+ transport 30 MgtA 31 MgtB and MgtC 33 CorA 35 CorA structure 35 Inhibition of CorA 38 Mg2+ efflux 39 Other features of CorA expression 40 Role of Mg2+ in the cell 41 2 Table of Contents (continued) Summary 41 Chapter 2: Methods 51 Chapter 3: Fe2+ toxicity and CorA studies 77 Introduction 77 Results 80 Discussion 85 Chapter 4: The role of CorA in Salmonella pathogenesis 95 Introduction 95 Results 97 Discussion 102 Chapter 5: Regulation of CorA 136 Introduction 136 Results 137 Discussion 145 Chapter 6: Conclusion and Future Directions 168 Summary 168 Regulation of corA transcription 169 Regulation of corA mRNA and/or CorA protein stability 175 Regulation of CorA function 176 Regulation of Mg2+ and virulence 183 Conclusion and unanswered questions for the field 185 Reference List 190 3 List of Tables Table 1.1: S. enterica serovar Typhimurium pathogenicity island 1 43 Table 1.2: The PhoP/Q regulon 45 Table 1.3: Genes directly regulated by PhoP/PhoQ 47 Table 3.1: S. enterica serovar Typhimurium strains used in Chapter 3 89 Table 3.2: Effect of Fe2+ on viability 90 Table 4.1: S. enterica serovar Typhimurium strains used in Chapter 4 106 Table 4.2: Genes up/downregulated in a corA mutant in log phase 107 Table 4.3: Genes up/downregulated in a corA mutant in stat phase 112 Table 5.1: S. enterica serovar Typhimurium strains used in Chapter 5 150 Table 6.1: Summary of data from Chapter 5 187 4 List of Figures Figure 1.1: Schematic representation of lipopolysaccharide 48 Figure 1.2: Crystal structure of S. enterica serovar Typhimurium PhoQ periplasmic sensor domain 49 Figure 1.3: Crystal structure of T. maritima CorA 50 Figure 3.1: Fe2+ toxicity in S. enterica serovar Typhimurium 91 Figure 3.2: Fe2+ uptake in S. enterica serovar Typhimurium 92 Figure 3.3: Effect of iron on CorA-mediated transport 93 Figure 3.4: Effect of Mg2+ on 55Fe2+ uptake 94 Figure 4.1: Mouse survival upon oral administration of S. enterica serovar Typhimurium 120 Figure 4.2: Mouse survival upon i.p. administration of S. enterica serovar Typhimurium 121 Figure 4.3: RTqPCR of selected genes from microarray 122 Figure 4.4: SPI1 Western blot for cells grown in LB to log phase 123 Figure 4.5: SPI1 Western blot for cells grown in LB pH 6.0 to 8.0 shift 124 Figure 4.6: Motility assay 125 Figure 4.7: CAS assay 126 Figure 4.8: Congo red binding assay 127 Figure 4.9: ELISA for TGFβ1 128 Figure 4.10: ELISA for IL1β 129 Figure 4.11: ELISA for TNFα 130 Figure 4.12: LacZ reporter assay 131 5 List of Figures (continued) Figure 4.13: Epithelial cell invasion (wild type and corA) 132 Figure 4.14: Replication within epithelial cells (wild type and corA) 133 Figure 4.15: Immunohistochemistry for LAMP1 134 Figure 4.16: Macrophage survival (wild type and corA) 135 Figure 5.1: Total intracellular Mg2+ content 151 Figure 5.2: Replication within epithelial cells (wild type, corA, corA pBSmgtE, and corA pECcorA) 152 Figure 5.3: Epithelial cell invasion (wild type and corA pMJcorA) 153 Figure 5.4: 57Co2+ uptake (wild type, corA, and corA pBSmgtE) 154 Figure 5.5: Replication within epithelial cells (wild type, corA, corA pF266A, and corA pP269A) 155 Figure 5.6: Replication within epithelial cells with chronic or acute cobalt hexaammine (wild type and corA) 156 Figure 5.7: Replication within epithelial cells (wild type, corA, corB, corC, and corD) 157 Figure 5.8: corA transcription (wild type and corA) 158 Figure 5.9: corA transcription (wild type, corA, phoP, and phoQ) 159 Figure 5.10: CorA protein content (wild type and corA) 160 Figure 5.11: CorA protein content (wild type, corA, phoP, and phoQ) 161 Figure 5.12: Total uptake of 63Ni2+ (wild type and corA) 162 Figure 5.13: Total uptake of 63Ni2+ (wild type, corA, phoP, and phoQ) 163 6 List of Figures (continued) Figure 5.14: 54Mn2+ uptake for cells grown in high Mg 164 Figure 5.15: pH dependence on 63Ni2+ uptake for cells grown in high Mg 165 Figure 5.16: Total intracellular Mg2+ content (wild type and corA) 166 Figure 5.17: Total intracellular Mg2+ content (wild type, corA, phoP, and phoQ) 167 Figure 6.1: Regulation of corA transcription 188 Figure 6.2: Epithelial cell invasion and replication within epithelial cells by bacteria grown in low Mg, LB, or high Mg to stat phase 189 7 List of Abbreviations SCV - Salmonella containing vacuole Sif - Salmonella induced filament SPI – Salmonella pathogenicity island T3SS – type three secretion system PMN – polymorphonuclear leukocytes LPS – lipopolysaccharide TM1 – transmembrane segment 1 TM2 – transmembrane segment 2 PBS – phosphate buffered saline LB – Luria Bertani broth TY – tryptone/yeast extract medium dH20 - deionized water SSC – sodium chloride/sodium citrate buffer SDS – sodium dodecyl sulfate MOI – multiplicity of infection BSA – bovine serum albumin DEPC – diethylpyrocarbonate TAE – Tris/acetate/EDTA DTT – dithiothreitol CAS – chrome azurol S HDTMA - Hexadecyltrimethylammonium ELISA - Enzyme-Linked ImmunoSorbent Assay 8 List of Abbreviations (continued) qRTPCR – quantitative real time polymerase chain reaction qPCR - quantitative polymerase chain reaction PCR - polymerase chain reaction low Mg – N minimal medium with 0.1% casamino acids, 0.4% glucose, and 10 μM MgSO4 high Mg – N minimal medium with 0.1% casamino acids, 0.4% glucose, and 10 mM MgSO4 RLU – relative light units DAPI - 4',6-diamidino-2-phenylindole X-gal - 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside ONPG - ortho-Nitrophenyl-β-galactoside 9 Studies on the Virulence Properties and Regulation of the CorA Magnesium Transporter Abstract by KRISZTINA M. PAPP-WALLACE CorA is the primary Mg2+ channel in Salmonella enterica serovar Typhimurium. A strain lacking corA is attenuated in mice after infection either by oral gavage or intraperitoneal injection. Microarray studies show that several virulence effectors in Salmonella pathogenicity island 1 and Salmonella pathogenicity island 2 are repressed in the corA strain compared to wild type. While these results could be sufficient to explain the virulence deficit, the microarray data suggest additional defects that could also contribute. Motility is significantly reduced in a corA strain whereas enterochelin-dependent iron uptake and curli are upregulated. A corA strain is defective for invasion of and replication within Caco-2 epithelial cells. However, a corA strain does not have a significant survival defect in J774A.1 macrophages. Thus, despite the presence of two other Mg2+ transporters, loss of CorA affects multiple systems which manifests ultimately as a decrease in virulence. We further examined Salmonella interaction with Caco-2 epithelial cells. Inhibiting CorA acutely or chronically with a high concentration of a selective inhibitor, cobalt (III) hexaammine, had no effect on S. Typhimurium invasion of and replication within Caco-2 epithelial cells. Complementing the corA mutation with a corA from various species rescued the invasion defect only if the complementing allele was functional and if it was evolutionarily similar to S. Typhimurium CorA. One explanation for these results could be that regulation of 10 CorA function is needed for optimal virulence. Further experiments examining corA transcription, CorA protein content, CorA transport, and Mg2+ content indicated that both CorA expression and CorA function are differentially regulated. Moreover the rates of Mg2+ influx via CorA are not strictly correlated with either protein levels or Mg2+ content. We conclude that loss of the CorA protein disrupts a regulatory network(s) with the ultimate phenotype of decreased virulence. This conclusion is compatible with the microarray results which showed that loss of corA resulted in changes in transcription in multiple metabolic pathways. Further study of the regulation of CorA expression and function provides an opportunity to dissect the complexity of Mg2+ homeostasis and its ties to virulence within the bacterium. 11 Chapter 1 Introduction Salmonella subtyping Salmonella was named after a United States Department of Agriculture veterinarian, Daniel Salmon. Salmonella is a gram negative rod. Only two species of Salmonella exist: Salmonella enterica and Salmonella bongori. S. bongori only has a single subspecies (190). S. enterica has six subspecies, one of which infects humans and other warm-blooded animals, S. enterica subspecies enterica. Both species have been further separated into 50 serogroups and over 2000 serotypes or serovars. Using the Kauffman-White classification system, serogroups are determined based on cell surface “O” antigens (lipopolysaccharide protein chains), and serovars are determined based on cell surface “H” antigens (flagellar) (163). Infection process of Salmonella enterica serovar Typhimurium In humans, Salmonella enterica serovar Typhimurium causes a gastrointestinal ailment affecting over 1.4 million Americans per year although only 40,000 cases are serious enough to get reported (203). Infection typically begins with oral consumption of live bacteria in contaminated consumables (140,205). Salmonella live in the intestines of animals and on the skin of reptiles and thus food/drink can become contaminated through contact with fecal matter or reptiles. Humans typically suffer from enteritis whereas mice develop enteric fever, much like typhoid fever.
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