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. Upon ingestion, the bacteria encounter an acidic stomach. Salmonella has an acid tolerance response that appears to promote
12 survival in the stomach (55). The first site of action for the bacteria is the ileum
or the final section of the small intestine; the bacteria use their fimbriae
(proteinaceous appendages) to adhere to the intestinal epithelium (116). Here the bacteria will actively invade epithelial cells (M cells in mice) located near the
Peyer’s patches (31,100). Peyer’s patches are lymphoidal tissues that serve as immune surveillance for the intestinal tract recognizing pathogenic microorganisms and antigens that are ingested (112). M cells continually sample the intestinal lumen for antigens and present them to antigen presenting cells
located near them. Salmonella can invade other cell types in the intestine as well
(68,211).
Inside the eukaryotic cell, the bacteria will isolate themselves into a
vacuole, known as the Salmonella containing vacuole (SCV) (48,54), prevent the
cell from undergoing apoptosis, and will produce filaments from the vacuole
known as Sifs (Salmonella induced filaments) (65,66). Bacteria replicate within
Sifs, thus their production is essential for maintaining the virulence lifecycle.
Through both bacterial and epithelial cell mediated events, the epithelial cells
begin produce interleukin-8, which stimulates the recruitment of neutrophils to the
intestine (129). In addition, the normal movements of electrolytes and water are
disturbed resulting in a fluid secretion into the intestinal lumen (71). This is a part
of the innate immune response geared to flush out the invading pathogen.
However, the bacteria also benefit from neutrophil transmigration as the epithelial
cell barrier is broken down allowing more bacteria gain excess to underlying
tissues (30,100). In human infections, the neutrophils and underlying
13 macrophages will typically clear the bacteria (205). Thus in humans, Salmonella
Typhimurium infections are typically self-limiting, last 4-7 days and do not require treatment. Symptoms include diarrhea, nausea, fever, abdominal cramps, headache, and dehydration.
In mice, however, Salmonella’s life cycle continues (110,140). In the subsequent stages of infection, the bacteria leave the epithelial cells by an unknown mechanism and enter the underlying macrophages. Inside the intestinal macrophage the bacteria will isolate themselves in another SCV and attempt to protect itself from the macrophage’s insults (67). Within the intestine, the bacteria cycle from macrophage to macrophage. The bacteria cause the macrophage to undergo apoptosis in order to exit. Then, the bacteria can infect another cell (79). Eventually, the bacteria enter the systemic macrophages and are distributed via blood and lymph to the reticuloendothelial organs, primarily the liver and spleen (110,140). Inside systemic macrophages, Salmonella delays apoptosis so that it can replicate. This severe infection will eventually kill the mice. Infections can become more severe in humans as well, leading to a diagnosis of enterocolitis which can last 2-3 weeks. However, these severe cases occur more often in the immunocompromised, the elderly, and infants.
People who develop severe cases are also more susceptible to bacteremia and focal systemic infections (as in mice) which can lead to death if left untreated
(136). Approximately 600 people die from acute Salmonella infections each year. In some instances, a Salmonella carrier state can arise. In this state,
14 individuals are asymptomatic but continually shed bacteria in their stool and are thus highly contagious.
Animal models of Salmonella Typhimurium infection
As the name Typhimurium suggests, this serovar of Salmonella causes a disease in mice similar to typhoid fever in humans (205). As stated above S.
Typhimurium causes gastroenteritis in humans; thus the same bacterium elicits different pathologies in humans versus mice. Typhoid fever in humans is caused by Salmonella enterica serovar Typhi (136). In humans, some of typhoid fever’s symptoms are similar to those occurring from gastroenteritis. However, symptoms are more severe. Symptoms include a chronic fever up to 104°F, profuse sweating, headaches, and general malaise (136). Also a rash can develop (rose spots). The illness typically last 2-4 weeks and requires treatment.
Over 200 million cases occur worldwide each year with over 600,000 deaths
(110). In mice, typhoid disease is similar to that seen in humans with very little incidence of intestinal pathology due to an inherent resistance of mice to S.
Typhimurium intestinal colonization. Instead bacteria localize to the liver and spleen. Thus, mice serve as a model for typhoid fever when infected with S.
Typhimurium. Most researchers use the mouse typhoid model to study S.
Typhimurium infections. Also, most of what is known about S. Typhimurium has been discovered in this model. Thus, this is the model used in our lab.
Several other models have been established for S. Typhimurium infection
(158): a calf enterocolitis model (157), a bovine ileal loop model (33), a streptomycin-treated mouse enterocolitis model (9), and others which are
15 predominantly used to screen for Salmonella virulence factors. In calves, S.
Typhimurium causes diarrhea and enterocolitis. Clinical and histological
manifestations in calves are analogous to the human gastroenteritis; as a result
calves have become a preferred animal model. However, it is difficult and
expensive to work with such a large animal. Consequently, some work has used
ligated ileal loops from calves. Nevertheless, there are still too many obvious
pitfalls to using calves as an animal model, such as genetic diversity amongst
animals, cost, specialized facilities etc.
Recent work has established a mouse model for S. Typhimurium gastroenteritis, as the typical mouse model is for typhoid fever (9). The inherent issue with using mice is that the flora in their intestines generally prevents colonization by other bacteria. Thus if mice are pretreated with an antibiotic such as streptomycin, then S. Typhimurium can effectively compete for colonization.
This mouse model is similar to human gastroenteritis. However, these mice will also develop typhoid fever. One concern over the use of this model is that it is artificially induced.
Alternative models such as Caenorhabditis elegans (1), Dictyostelium discoideum (170), and Danio rerio (zebrafish) (197) are being researched to generate cheaper, easier and faster approaches to rapidly screen for virulence factors of Salmonella. Caution should be used with these models. Typically, they are useful for preliminary screens but not as complete disease models. For example, 50% of 1 day old C. elegans die within 5 days of being fed S.
Typhimurium whereas those fed non-pathogenic E. coli die within 10 days (1).
16 Infection with S. Typhimurium carrying mutations in the known virulence factors
phoP or phoQ does not kill C. elegans as well as wild type Salmonella (1).
Thus, C. elegans may be good tool for the preliminary screening of virulence
factors when used with appropriate controls. The amoeba, D. discoideum has
not been fully determined to be effective model for screening Salmonella
mutants; however a few laboratories are currently working to develop this
amoeba into a model for Salmonella infection. Work with D. rerio embryos has
been limited to S. Typhimurium strain LT2, which carries a mutation in rpoS, the
stationary phase sigma factor, rendering it avirulent in humans and mice (198).
LPS mutants of LT2 are attenuated compared to wild type in D. rerio, however fully virulent strains of S. Typhimurium have not been assessed (198). Thus, alternative models for Salmonella infection are still in the early stages of being developed.
There are many advantages and disadvantages to different models discussed above for S. Typhimurium infection. Thus it is important to understand the differences among them and choose the correct model for an experiment.
Different virulence effectors are important in different models of infection. Also, the signaling networks involved in Salmonella pathogenesis are distinctive in different animals.
Pathogenicity islands of S. Typhimurium
Salmonella uses a multitude of regulatory networks to successfully infect a host. Many of its virulence determinants are encoded on pathogenicity islands.
These islands are distinct chromosomal regions that have been acquired through
17 horizontal transfer from other bacterial species and are typically not present in closely related non-pathogenic species. Most Salmonellae have five pathogenicity islands, which are referred to as Salmonella Pathogenicity Islands
(SPI) 1-5. Genomic sequencing and analysis have revealed that S. Typhi harbors five additional pathogenicity islands 6-10 (142). SPI6 encodes saf, an atypical fimbriae operon (6). Also SPI9 is present in S. Typhimurium (6). SPI 1-
5 are the best characterized and are described below. Over the recent years it was become increasingly evident that some of the proteins encoded by these
SPIs interact and form a complex signaling network. For example, SPI1 and
SPI2 were long thought to function independently, however they are now known to be closely integrated (2,89).
SPI1 is a 40 kilobase region near centisome 63 and is required for host cell invasion and macrophage apoptosis (58,76). It encodes a type three secretion system (T3SS), which is a molecular syringe used to inject bacterial proteins into a eukaryotic cell (5,8). Specifically, this T3SS injects effector proteins into the non-phagocytic cells, such as M cells in the intestine. The system is composed of a secretion apparatus, effectors, and chaperones. The effectors rearrange the eukaryotic cell’s actin cytoskeleton, which allows the bacteria to enter the cell and replicate within the cell. The following genes make up SPI1: avrA, sprB, hilC, orgA, prgK, prgJ, prgI, prgH, hilD, hilA, iagB, sptP, sicP, iacP, sipA, sipD, sipC, sipB, sicA, spaS, spaR, spaQ, spaP, spaO, spaN, spaM, invC, invB, invA, invE, invG, invF, and invH (Table 1.1, p.43-44). They can be broadly categorized into 4 groups: secreted effectors, chaperones,
18 structural components and transcriptional activators. Not all components of this
T3SS are encoded within SPI1; some are scattered throughout the bacterial
chromosome. Some proteins encoded by SPI1 have dual roles. Several of
these proteins have not been fully characterized thus their true roles in
Salmonella pathogenesis are currently unknown. SPI1 was once thought to be
expressed only during invasion of the intestinal epithelium; however, data from
several labs now suggests that components of this island are used at other
points during infection (2,18a, 19a,45,100a).
SPI2 is located at centisome 30, is 40 kilobases in length, and is required
for systemic infection, survival and replication within macrophages (93,139,166).
It encodes a second T3SS whose effectors prevent the macrophage from killing
the bacterium. The following genes make up SPI2: ssaU, ssaT, ssaS, ssaR,
ssaQ, ssaP, ssaO, ssaN, ssaV, ssaM, ssaL, ssaK, ssaJ, ssaI, ssaH, ssaG, sseG, sseF, sscB, sseE, sseD, sseC, sscA, sseB, sseA, ssaE, ssaD, ssaC, ssaB, ssrA, and ssrB. Unlike SPI1, very little is known about the proteins encoded on SPI2.
Homology to other T3SS’s suggests some putative roles. The
SsaUTSRQPONVMLKJIH operon with SsaG are most likely a part of the needle complex with SsaN energizing secretion by the needle (94). SseBC and D make up a translocon that is used to carry effectors into the eukaryotic cell (154).
SseEF and G are secreted effectors. SseE’s function is unknown, while SseFG
are involved in positioning the SCV near the Golgi and nucleus within the
macrophage (41). SscB is SseF’s chaperone (37). SscA is another chaperone
(154). SsaC (SpiA), SsaE, and SsaD have unknown functions. SsaB (SpiC)
19 interferes with intracellular trafficking (196). SsrAB is a two component system
that regulates the expression of SPI2 (139). SsrA is the sensor and SsrB is the
transcriptional activator of a two component regulatory system. Like SPI1, SPI2
has many secreted effectors that are not encoded within the island itself.
SPI3 is located at centisome 82, is 17 kilobases in length, and contains 10
genes: sugR, rhuM, rmbA, misL, fidL, marT, slsA, cigR, mgtB, and mgtC (17,18).
In contrast to the genes comprising SPI1 and SPI2, these genes do not appear to be functionally related. MgtB is the best characterized protein expressed from
SPI3. It is a Mg2+ transporter, is expressed in response to low extracellular Mg2+,
and is transcriptionally regulated by the two component system PhoPQ (95,182)
(see below). MgtC is also a membrane protein and appears to form an operon with MgtB (see below). MgtC’s function is largely unknown; it may activate the eukaryotic cell’s Na+/K+ ATPase through an unknown mechanism (83). Also,
MgtC is essential for intramacrophage survival (17). MisL is an autotransporter
(195). It also functions as an extracellular matrix adhesion protein by binding
fibronectin and thus promoting intestinal colonization (44). MarT transcriptionally activates misL expression (195). RhuM’s function is unknown. However when
RhuM is mutated, Salmonella is 45% defective for killing C. elegans (189). Also, a rhuM mutant elicits 20% less PMN migration in a transmigration assay (189).
SugR, RmbA, FidL, SlsA, and CigR have unknown functions in Salmonella.
Homology searches reveal some information about the potential roles of some of these proteins. SugR is a putative ATP binding protein. SlsA is similar to amidohydrolases. CigR is a putative inner membrane protein. SPI3 stands out
20 from the other Salmonella pathogenicity islands because its gene products do
not appear to have any cohesive function.
SPI4 is located at centisome 92, is 27 kilobases in length and contains an
operon with 6 genes: siiABCDEF (69). It encodes a type one secretion system, which secretes SiiE, a very large non-fimbrial adhesin of 600 kDa consisting of
53 repeats of Ig domains. SiiC, SiiD, and SiiF make up the secretion apparatus.
SiiA and SiiB are inner membrane proteins but are not required for the secretion of SiiE. This regulon appears to be regulated transcriptionally by HilA and SirA
(70). SPI4 is required for adhesion to epithelial cells.
SPI5 is located at centisome 20, is about 6 kilobases in length, and has
been best characterized in Salmonella Dublin (208). It contains six genes: sopB,
pipA, pipB, pipC, pipD, and STM1092, a putative cytoplasmic protein. In S.
Typhimurium a seventh gene, sigE is also a part of SPI-5. SopB (also known as
SigD), PipA, PipB, PipC, and PipD are required for enteritis as shown in a bovine
ileal loop model for infection; however, for the most part they are not required for
systemic infection in mice, with SopB and PipB as the exceptions (208).
Much is known about the physiological function of SopB
(46,51,61,107,128,150,213). SopB is an effector protein secreted by the T3SS
encoded on SPI1 (106). SigE is its cognate chaperone (105). sopB is regulated
transcriptionally by SirA (73). Expression of sopB is upregulated in both
phagocytic and non-phagocytic cells (144). It is an inositol phosphate
phosphatase that disrupts the eukaryotic phosphatidylinositol signaling pathway
(107). By altering the balance of inositol phosphates in the eukaryotic cell, SopB
21 indirectly activates eukaryotic proteins such as Akt and Cdc42. As a result, chloride secretion into the intestinal lumen increases resulting in increased fluid secretion (diarrhea). SopB activation of Cdc42 is also involved in the rearrangement of the actin cytoskeleton during invasion (213). Other data indicate it has anti-apoptotic activity (107). SopB is also involved in endosome to lysosome trafficking (46), in disrupting tight junctions (19), and in mRNA export from the nucleus (51). Moreover it can alter nitric oxide activity (45). Despite all these putative functions, sopB mutants are fully virulent, even though they are less invasive to epithelial cells (147).
Less is know about the Pip proteins encoded by SPI5. Expression of pipB is upregulated in both phagocytic and non-phagocytic cells (144). PipB is an effector protein secreted by the SPI2 encoded T3SS and most likely plays a role in glycolipid biogenesis (106). A pipB mutant is attenuated upon infection of a mouse. pipA mutants are 50% defective for killing C. elegans (189). Also, a pipB mutant is 90% defective for migration as measured by a PMN transmigration assay (189). PipC’s physiological function is unknown; however it is homologous to IpgE from Shigella, which chaperones effectors into eukaryotic cells. PipD is a putative secreted peptidase, however currently there is no supportive evidence for this activity. Overall, SPI5 is primarily associated with enteritis caused by S. Typhimurium.
Other virulence determinants of S. Typhimurium
Other virulence determinants for S. Typhimurium are encoded on or include: the Salmonella virulence plasmid, prophages found within the
22 Salmonella chromosome, fimbriae, flagella, and lipopolysaccharide (LPS). pSLT is a 94 kb virulence plasmid. Most of the proteins encoded on this plasmid have unknown functions. Putative fimbrial (pefBACD), conjugation, and virulence proteins (SpvD, SpvC, SpvB, SpvA, and SpvR) are encoded on pSLT (10,80).
SpvD, SpvC, and SpvA have unknown functions. SpvB is an ADP- ribosyltransferase, a bacterial toxin similar to cholera and diphtheria toxins used to modify eukaryotic proteins such as actin (115). SpvB may also promote apoptosis of eukaryotic cells (111). SpvR is the transcriptional regulator for spvDCBA operon (21). It induces expression of the operon during stationary phase and when cells are in carbon-poor media. Optimal expression of spvDCBA is dependent on RpoS, a stationary phase sigma factor (91).
Bacteriophages are viruses that infect bacteria (22). Some bacteriophages work much like viruses that infect eukaryotic cells, in that they inject their DNA into the host cell and hijack the cell’s machinery to generate more viruses. Some bacteriophages have both a lytic and lysogenic life cycle.
During the lytic life cycle, they inject their DNA, replicate and immediately lyse the cell releasing the new viruses, infecting another cell to continue the cycle.
During the lysogenic life cycle, the bacteriophage injects its DNA, but instead of generating more virus, the phage DNA becomes incorporated into the bacterial chromosome (or a bacterial plasmid) generating a prophage or can circularize to form a plasmid. These lysogenic phages are relatively harmless for the host cell and usually cause harm only when stress on host cell activates their replication
23 and causes cell lysis. Some prophages provide benefits to the bacteria by giving
the host cell new functions, such as enhanced virulence.
Salmonella has several prophages. P2 class phages include SopEФ,
Fels1, and Fels2 (20). The lambda phages include Gifsy-1, Gifsy-2, Gifsy-3,
P22, E34, ST64T, and ST64B (20). Several essential virulence effectors are encoded on these prophages. SopE, encoded on the SopEФ prophage, is a
SPI-1 secreted effector that functions as a guanine nucleotide exchange factor for Rho GTPases (87). SodCIII encoded on Fels-1 and SodCI encoded on Gifsy-
2 are Mn2+ dependent superoxide dismutases that protect Salmonella from
superoxide generated by macrophages (49,53). Several other proteins encoded
on Gifsy-1 and Gifsy-2 are known to be important for Salmonella virulence, but
their functions are currently unknown (20). PagJ and SspH1 encoded on Gifsy-3
are also TTSS virulence related effectors (53). Finally, there are also many
stretches of DNA in the Salmonella chromosome known as phage remnants (20).
Two of those stretches carry T3SS effectors, SopE2 and SspH2. Thus, bacteriophages are an evolutionarily important tool for transferring virulence determinants between bacterial species.
Fimbriae are proteinaceous cell surface appendages that help the bacteria stick to each other and to eukaryotic cells (117). Each bacterium can express up to 1000 fimbriae at one time. Whole-genome sequencing has identified 13 putative fimbrial operons in the S. Typhimurium genome: agf (csg), fim, lpf, pef,
bcf, stb, stc, std, stf, sth, sti, saf, and stj (207). These can be subdivided into four
types: Type 1 fimbriae (e.g. fimAICDHF), plasmid-encoded fimbriae (e.g.
24 pefBACD), long polar fimbriae (e.g. lpfABCDE), and thin aggregative fimbriae or
curli (e.g. agfBAC & agfDEFG) (11). Some fimbrial mutants, such as agf, lpf, pef,
and fim, are less virulent than wild type bacteria upon oral infection because they
are needed for attachment to and invasion of intestinal epithelial cells (199).
Moreover, there appears to be functional redundancy with fimbriae, in that in
some cases, multiple fimbrial genes must be mutated to obtain an attenuation
phenotype.
Flagella are long helical projections of protein that extend from the surface
of the bacterium and allow the bacterium to swim (119). Each bacterium
possesses 5 to 10 flagella, which are found peritrichously (covered all over with
uniformly distributed flagella) on the bacterial surface. Flagella are composed of
three subunits: the basal body, a transmembrane motor and the filament. The
filament is made up of subunits of flagellin protein, either FliC or FljB. Salmonella
express either fliC or fljB at different times and switch between the two subunits.
This switching is referred to as phase variation.
Controversy exists in the literature whether or not flagella are important for
virulence as not all flagellar mutants have a virulence defect (23,97,99,159-
161,185). One can speculate the flagella may aid with adherence, aid the bacteria with swimming away from immune cells or towards beneficial surroundings, or in invasion. Flagellar phase variation has also been speculated to be important for virulence (97). The flagellin protein has been shown to stimulate neutrophil transmigration by causing the release of IL-8 via direct
interactions with Toll-like receptor 5 (90). Neutrophil transmigration into the
25 intestine breaks down the intestinal barrier, thus allowing a rapid infection of
underlying macrophages by Salmonella (30,100).
LPS or lipopolysaccharide is the main component of Salmonella’s outer
membrane (146). It is an endotoxin that elicits a strong immune response in the
host. LPS is composed of three subunits: lipid A, a core oligosaccharide region,
and O-antigen, which is a polysaccharide (Figure 1.1 p. 48). Salmonella can
modify the subunits that make up LPS to protect itself from the host. For
example, the two component regulatory systems PhoPQ and PmrAB are
involved in modifying lipid A which results in resistance to host antimicrobial
peptides (82,84).
PhoPQ and virulence
PhoP/PhoQ is a two component system consisting of PhoQ, the
sensor/receptor histidine kinase, and PhoP, the response regulator and
transcriptional activator. Activation of PhoQ is controlled by Mg2+, Ca2+,
antimicrobial peptides, and acid (7,12,62,131,182). PhoPQ was first discovered
in S. Typhimurium because of its role in virulence; phoP and phoQ mutant strains have survival deficits in the macrophage and are less virulent in the mouse
(52,60,131). PhoPQ negatively regulates genes in SPI1, thus repressing epithelial cell invasion (8,13). However, recently, orgBC of SPI1 was found to be positively activated by PhoP under PhoPQ inducing conditions (2), as well as invasion inducing conditions. This regulation occurs through a PhoP box lying just upstream of orgBC; these same genes are also regulated by HilA.
26 PhoPQ positively regulates genes in SPI2 by activating SsrB/SsrA(SpiR),
a two component system that increases SPI2 expression (15,39,40). PhoPQ
directly promotes transcription of ssrB and positively regulates ssrA at the
posttranscriptional level through binding to the 5’-UTR. However, mouse
competition experiments using mutations in SPI2 genes or in phoP suggested
that the PhoPQ and SsrB/SsrA regulatory networks work independently (14).
PhoP is also involved in macrophage cell death (42) in part by promoting proper
trafficking of the Salmonella containing vacuole (67). PhoPQ regulates other
virulence related properties, such as resistance to host antimicrobial peptides,
resistance to bile, biofilm formation, and stabilization of RpoS levels
(132,145,193,200). Moreover, a putative surge in transcription created by
phospho-PhoP may be necessary for virulence because a mutant constitutively
expressing PhoP and which therefore lacks the initial surge in transcription is
completely attenuated in the mouse (168). Thus, PhoPQ is a key player in S.
Typhimurium pathogenesis.
The Two Component System: PhoP/PhoQ
PhoPQ controls over 100 genes in S. Typhimurium. Two general classes
exist: pags or PhoPQ activated genes and prgs or PhoPQ repressed genes.
The mechanisms of gene regulation by PhoPQ and the molecular details of metal regulation of PhoQ and PhoP are complex. Disagreement exists over several aspects of regulation and biochemistry.
phoP and phoQ form an operon with two promoters (184). Expression of phoP and phoQ from promoter two is constitutive at a basal level and
27 independent of Mg2+ concentration. Environmental Mg2+ controls expression
through promoter one, by stimulating PhoQ to phosphorylate PhoP.
Phosphorylated PhoP binds promoter one and increase expression of PhoP and
PhoQ dramatically; thus phoPQ transcription is autoregulated (62,184). PhoP’s
autoregulation of transcription results in a surge of transcription of PhoPQ
regulated genes. This surge is followed by a decrease in transcription that
eventually reaches a steady state level of transcription. The pattern of
transcription is mirrored by levels of phosphorylated PhoP, indicating that
phospho-PhoP is responsible for the surge (168).
Upon encountering low environmental Mg2+ concentrations, Mg2+ dissociates from PhoQ which then undergoes a conformation change resulting in a typical autophosphorylation of the PhoQ sensor-kinase on a histidine residue
(H277) (25,29,62,135). PhoQ transfers this phosphate to an aspartate residue on PhoP through direct interactions with PhoP (24,25,135). The mechanism by which PhoP regulates gene transcription is complex and appears to differ for different PhoPQ-regulated genes. PhoP typically binds to a consensus sequence of (T)G(T)TT(AA) (PhoP box) (102,210). PhoP can also bind another class of promoters which have a reverse PhoP box (214). At some but not all
PhoP-regulated sites, an additional regulatory protein is required.
Most unclear at this time is the control and function of PhoP phosphorylation. All investigators agree that increasing Mg2+ concentration
activates a Mg2+-dependent phosphatase activity of PhoQ that readily
dephosphorylates either PhoQ or PhoP. There is disagreement however on
28 whether the autokinase activity of PhoQ is Mg2+-activated and on whether
phosphorylation of PhoP is required for binding to its promoters. These
discrepancies are most likely due to differences in assay conditions such as
temperature, high versus low salt, pH and the presence or absence of a His-tag
on PhoP. Moreover, given the complexity of PhoP-regulated promoters, a
requirement for PhoP phosphorylation may differ at different promoters.
The 2.4 Å resolution crystal structure of the PhoQ periplasmic sensor domain reveals a dimer with a flat negatively charged region parallel to the membrane surface (Figure 1.2, p.49) (29). Bound metal ions bridge this highly negatively charged region and the bacterial inner membrane. Upon loss of metal, these negatively charged regions should repel each other; this repulsion is thought to be responsible for initiating signal transduction. Several acidic residues including D149 through E154 of the PhoQ sensor domain are important for metal binding (29,204). Reporter assays monitoring the repression of expression of a psiD:lacZ transcriptional fusion in wild type or phoQ mutant strains when exposed to different concentrations of Mg2+ and Ca2+ suggest that
PhoQ has distinct binding sites for Mg2+ and Ca2+. The levels of repression by
Mg2+ and Ca2+ alone are distinct and full repression of psiD:lac expression
requires the presence of both metal ions (62). Moreover, a phoQ mutant (pho-
24) had differential responses to Mg2+ and Ca2+ (202). However, NMR analyses do not support these conclusions since identical conformational changes occur in the extracellular PhoQ sensor domain upon binding either Ca2+ or Mg2+ (29).
29 Several mutants of PhoP and PhoQ have been characterized. A T48I mutant of PhoQ (pho-24) is constitutively active possibly due to either decreased phosphatase activity of PhoQ or an increased net phosphotransfer onto PhoP
(62,82). The T48 residue may be required for PhoQ to switch between acting as a PhoP kinase or a PhoP phosphatase (156). S93N and Q203R PhoP mutants are constitutively active independent of the presence of PhoQ . The combination of these two mutations is synergistic (27,81). The S93N but not the Q203R PhoP mutant can still interact with PhoQ and thus its activity can still be influenced by
Mg2+ concentration as long as PhoQ is present.
The PhoPQ regulon
Studies suggest that the PhoPQ regulon contains over 100 genes, however thus far PhoP has been shown to directly induce transcription of only ten genes or gene sets. Most of the 100 genes in the PhoPQ regulon have been suggested to be PhoPQ regulated because transcription of those genes is affected by mutation of PhoPQ (Table 1.2, p. 45-46). A microarray study indicated that the regulon is even larger, with 214 genes within 189 operons regulated by PhoPQ (134). (The microarray data is not presented in Table 1.2 due to its complexity.) About ten promoters have been shown to directly be bound by PhoP through either an electrophoretic mobility shift assay (EMSA), a chromatin immunoprecipitation (ChIP) assay, or DnaseI footprinting (Table 1.3, p.47). Other genes among the 100 genes in the PhoPQ regulon may also be directly regulated by PhoPQ; however, most of those genes are more likely
30 regulated indirectly by PhoPQ through crosstalk with other signaling networks
(HilA, SsrA/B, SlyA, and PmrAB).
PhoPQ and regulation of Mg2+ transport
S. Typhimurium has three Mg2+ transporters: MgtA, MgtB, and CorA.
Expression of both mgtA and the mgtCB operon is markedly induced by low concentrations of Mg2+ in the medium in a PhoPQ-dependent manner (63). CorA
expression is independent of PhoPQ under most growth conditions. corA and
mgtA are found as single open reading frames. In S. Typhimurium, mgtB is found with mgtC in a two gene operon at the 3’ end of SPI3. PhoPQ directly interacts with the mgtA and mgtCB promoters (168). In S. Typhimurium, mgtA
expression is also regulated at the mRNA level by intracellular Mg2+ (36). mgtA
has a 264 nucleotide 5'-UTR between its coding region and promoter. Low Mg2+ concentrations in the growth medium promote the transcription of mgtA, even if the mgtA promoter is replaced with the lac promoter. In silico analysis predicts extensive secondary structure within the mgtA 5' UTR. The 5' UTR appears capable of forming three stem loops: A, B, and C. Loop C can only form when loop A and loop B do not form because of overlap. Loop C forms when intracellular Mg2+ is low, resulting in transcription of mgtA. When intracellular
Mg2+ rises, loops A and B form, and transcriptional elongation of mgtA is
repressed. Low concentrations of extracellular Mg2+ increase PhoPQ-mediated
transcription of mgtA; however MgtA protein will only be expressed if intracellular
Mg2+ concentrations are also low.
MgtA
31 MgtA is a 95 kDa protein that belongs to the P-type ATPase superfamily
(188). MgtA is as similar (≈ 50% a.a. identity) to mammalian Ca2+ ATPases of
the sarcoplasmic reticulum and fungal P-type H+-ATPases as it is to MgtB.
Unlike most prokaryotic P-type ATPases which contain 6 TM segments, MgtA
(and MgtB) have 10 TM segments like most eukaryotic P-type ATPases (171).
P-type ATPases require the ATP dependent phosphorylation of a conserved
aspartyl residue for cation binding and transport (181). The affinity of MgtA for
2+ Mg is 20-30 μM (179). A Vmax value is not relevant because of the regulation,
but maximal uptake via MgtA can reach 20-30% that of CorA (179). It transports
only Mg2+ and Ni2+. Uptake by MgtA is inhibited by Zn2+, Co2+ and Ca2+ but not
by Fe2+ or Mn2+ with a rank order of potency of Zn2+ ≥ Mg2+ > Ni2+ ≈ Co2+ > Ca2+.
These differences in inhibition are probably due to either the chemicals
properties of the metals and the MgtA protein.
When cells are grown in minimal media containing millimolar concentrations of Mg2+, a decrease in external [Mg2+] elicits a biphasic
transcriptional response (187). A small increase in transcription occurs with a
2+ K0.5 for Mg of 0.5 -1.0 mM while a second, far larger increase in transcription has a K0.5 of 0.01 mM (36,187). mgtA regulation is complex. The two
component PhoP/PhoQ system controls transcription of mgtA via activation of
PhoQ by low extracellular concentrations of Mg2+. Intracellular Mg2+ appears to
regulate mgtA mRNA level posttranscriptionally (36,62). Acid shock abolishes
expression of mgtA when grown in low Mg2+. However, chronic exposure to acid
in low Mg2+ conditions induces mgtA expression but only if CorA is present (174).
32 mgtA expression is induced upon invasion of J774 macrophage-like cells,
RAW 264.7 macrophage-like cells, CMT93 epithelial cells, Hep-2 epithelial cells
and Henle-407 epithelial cells (92,174). mgtA expression is also induced in
Balb/C mice. However, a mgtA mutant does not have an invasion defect in any
of the cell lines tested (174). Mutation of mgtA alone in the presence of wild type
alleles of corA and mgtB causes no apparent virulence defect.
MgtB AND MgtC
MgtB is also a P-type ATPase and at 101 kDa is slightly larger than MgtA
(181). It is present at the 3’ end of SPI3 as the second gene of a putative two-
gene operon with mgtC. The affinity of MgtB for Mg2+ is slightly better that that of
MgtA, 5-10 μM. A Vmax value is not relevant because of the regulation, but like
MgtA maximal uptake via MgtB can reach 20-30% that of CorA (179). It
transports only Mg2+ and Ni2+. MgtB is inhibited by Co2+ and Mn2+ but not Zn2+ or
Ca2+ with a rank order of potency of Mg2+ ≈ Co2+ ≈ Ni2+ > Mn2+ >>Ca2+/Zn2+. In
intact cells, MgtB is active at 37 °C but completely inactive at 20 °C.
MgtC is a 22.5 kDa protein encoded by the first gene of the mgtCB
operon. Although homologs are widespread in prokaryotes, it lacks significant
homology with any other known protein with a known function, and its function is
unknown (133). MgtC is not required for MgtB function or its insertion into the
membrane (187). Although initially hypothesized to be a Mg2+ transporter
because it is required for growth in liquid medium at extracellular Mg2+ concentrations <50 μM (17), subsequent data has definitively shown that it does not transport Mg2+ (133). Expression of mgtC in Xenopus laevis oocytes
33 revealed that it can constitutively activate eukaryotic Na+,K+ ATPase (83). The
significance of this activation has not been elucidated.
Although mgtC is the first gene of the operon, and although decreased
extracellular Mg2+ markedly increases transcription of mgtCB via PhoP/PhoQ, only MgtB protein is made; no MgtC protein can be detected for several hours after the increase in transcription (133). Even if MgtB is rendered inactive by mutation of the active site aspartyl residue, no MgtC protein is made. MgtC is readily detected however if most or all of the mgtB gene is deleted.
As with mgtA, expression of the mgtCB operon is biphasic with decreasing
concentrations of Mg2+ in the growth medium (187). It is currently unknown if
mgtB mRNA is regulated by Mg2+ through a riboswitch mechanism as with mgtA.
However, expression of mgtB can be induced by inorganic acid in the presence of high Mg2+ (12). These results indicate that potentially two forms of regulation
of the mgtCB operon are present (187). mgtCB response to acid shock is similar to that of mgtA (174).
SPI3 is necessary for intramacrophage survival, thus components were assessed for their involvement in pathogenesis (17). mgtCB expression is induced markedly upon invasion of mice, J774 macrophage-like cells, RAW
264.7 macrophage-like cells, MDCK epithelial cells, CMT-93 epithelial cells, Hep-
2 epithelial cells and Henle-407 epithelial cells (64,92,125,174). A mgtCB mutant strain exhibits no deficit for entry of J774 macrophage-like cells although it is deficient in long-term survival (17,174). After intraperitoneal injection into mice, a mgtCB mutant strain shows about a 2 log decrease in LD50 (17). This virulence
34 effect was attributed to mgtC, since a strain lacking a functional MgtB was as
virulent as wild type. An mgtA mgtCB double mutant exhibits about a 3 log
decrease in LD50 compared to wild type after intraperitoneal injection and marked attenuation after oral gavage (Kehres and Maguire, unpublished). Interestingly,
the Mg2+ growth requirement and the virulence phenotype of mgtC mutant strains
have recently been dissociated, because Yersinia pestis MgtC fully complements
both phenotypes, while Pseudomonas aeruginosa only complements the Mg2+ growth requirement. How each phenotype relates to putative MgtC functions is unknown (148).
CorA
CorA is a unique class of ion channel and is widespread in the Bacteria and Archaea with rather distant homologs in eukaryotes (95,96,179). corA was initially identified in E. coli by the Silver and Kennedy laboratories as a locus giving moderate resistance to Co2+. Their transport and genetic studies indicated
that the locus was responsible for Mg2+ influx (137,169).
Half maximal uptake by CorA is at 15 μM Mg2+. As measured by bulk cell
2+ -1 8 -1 uptake, the Vmax for CorA is >0.5 nmol of Mg min 10 cells (95), but this may
be misleading since the protein is apparently an ion channel (see below). Cation selectivity for CorA is Mg2+=Co2+>Ni2+. Co2+ and Ni2+ have affinities of 20 and
200 μM, respectively (179). Both cations are toxic to the cell at these
concentrations. Thus, the primary physiological role for CorA is Mg2+ influx.
2+ 2+ Mn inhibits Mg influx with a Ki of 30 μM but is not transported. No other
35 cations, including Fe2+ (discussed in Chapter 3 (141)) and Ca2+, are transported
nor do any inhibit Mg2+ influx with good affinity.
CorA structure
CorA is a 37 kDa protein. Topology studies using blaM and lacZ fusions
initially indicated that the S. Typhimurium CorA contained 3 transmembrane (TM)
segments (172); however, subsequent data using a variety of approaches
showed that this superfamily of proteins has only 2 TMs at the extreme C-
terminus (118,149,209). Thus CorA is a two-domain protein with a small
transmembrane domain and a large soluble N-terminal domain resident in the
cytosol. This latter domain is largely α-helical and can be expressed as a
truncated protein with retention of structure (206).
The crystal structure of the Thermatoga maritima CorA protein was
recently solved at resolution of 3.9 Å for the whole protein and 1.85 Å for the
soluble domain (118). Subsequently, virtually identical crystal structures of the T. maritima CorA were published by Eshaghi et al. (47) at 2.9 Å resolution and by
Payandeh and Pai (143) at 3.6 Å resolution. S. Typhimurium CorA shares about
20% sequence identity with T. maritima CorA but is virtually an exact match in
terms of secondary structure predictions (118). Indeed, secondary structure
predictions for CorA and its homologs including ZntB (Zn2+ transporter) from
Salmonella and human mitochondrial Mrs2 are all very similar, an indication that
they all likely have the same basic structure (123).
CorA is a funnel-shaped homopentamer (Figure 1.3, p.50). In the cytosol,
the N-terminus forms an α3β7α3 sandwich domain that comprises a new protein
36 fold. This domain leads to the almost 100 Å long α7 or “stalk” helix which largely forms the interior face of the funnel and continues through the membrane as transmembrane segment 1 (TM1). TM1 ends at the periplasmic face of the membrane in the signature sequence unique to CorA proteins, YGMNF.
Mutation of any of the residues in this sequence in S. Typhimurium abolishes transport through CorA (186). Within the homopentamer, the 5 TM1 helices form the cation pore. A short 9 a.a. sequence in the periplasm connects TM1 and transmembrane segment 2 (TM2). The structure of this loop is unresolved in all current T. maritima structures. TM2 lies outside the pore formed by TM1 and returns the C-terminus to the cytosol. Virtually all CorA proteins have only a short 6 a.a. C-terminal tail that always contains several Arg and/or Lys residues.
For example, the sequence in S. Typhimurium and E. coli CorA is KRKNWL while that in T. maritima is KKKKWL.
From the crystal structure, amino acids N314 at the periplasmic end of the pore, M302 in the middle of the pore, and L294 and M291 at the cytosolic end of the pore would appear to be the narrowest part of the pore and thus would occlude cation passage indicating that the current structure is that of a closed form (118). Mutation of residues of the S. Typhimurium CorA that are near the equivalent of the L294/M291 site in T. maritima alters transport properties of
CorA consistent with these residues occluding the pore (186).
The ring of extreme positive charge at the cytosolic membrane interface created by the C-terminus (20 Lys plus 10 more Lys from the extremely long
“stalk” α-helix) creates a large positive potential field at the same level as the
37 L294/M291 occlusion in the pore that presumably further inhibits passage of
Mg2+ through the pore (118). Thus, the combination of the positive field and the
L294/M291 amino acid pair has been postulated to be the primary “gate” for Mg2+ passage through the channel. A hypothetical control of this gate is provided by the so-called “willow helices” (α5 and α6) that are part of the soluble domain and
lie external to the funnel formed by the stalk helix and extend down towards the
ring of positive charge. The ends of the willow helices contain a plethora of Asp
and Glu residues that could reasonably provide a negative counterpart to the ring
of positive charge. Interactions between these highly charged regions is
potentially critical to the mechanism of transport of CorA (118) and is likely
controlled by the binding of a Mg2+ ion seen in the crystal structure between
D253 of stalk helix in one monomer and D89 of the N-terminal α3-helix in an
adjacent monomer. Two of the crystal structures have an additional adjacent
cation bound partially by E88 (47,143). This is likely an artifact of crystallization
since the other ligands are water molecules, and therefore the affinity would be several mM, well above physiological free Mg2+ concentrations. The
homopentamer thus contains (at least) 5 bound Mg2+ ions, which are poised to
control the association between the monomers (Figure 1.3, p.50) (123). Further
work will be necessary to determine the role of these various parts of the CorA
structure in cation movement.
Inhibition of CorA
Cation hexaammines are selective inhibitors of CorA transport (109).
They can also inhibit TolC, a multidrug efflux protein with even higher affinity.
38 Cobalt (III) hexaammine trichloride (Co(NH3)6Cl3) and the ruthenium (II) and
ruthenium (III) hexaammine analogs all inhibit 63Ni2+ uptake through S.
Typhimurium and Methanococcus jannaschii CorA. These cations are the same
diameter (5.0 Å) as a hydrated Mg2+ cation. Ni (II) hexaammine, which is 6.0 Å in
diameter, does not inhibit. Neither the MgtA or MgtB Mg2+ transporters nor the
PhoQ Mg2+ sensor are inhibited by these cation hexaammines. Co (III)
hexaammine does not inhibit growth of wild type S. Typhimurium but is
bacteriostatic towards a mgtA mgtCB mutant strain that is dependent on corA for
Mg2+ influx. The cation hexaammines do not enter the cell and thus are
presumed to bind to CorA via the short periplasmic loop between TM1 and TM2
(123) although Payandeh and Pai suggest that Co (III) hexaammine can enter
the external portion of the pore (143). These data suggest the hypothesis that
CorA initially binds a fully hydrated Mg2+ and that the primary basis for cation
selectivity is size not charge.
Mg2+ efflux
CorA is also essential for Mg2+ efflux (72,179). Under typical laboratory
growth conditions, the Mg2+ content of the medium is about 10-15 μM in minimal media and 30-40 μM in LB. When cells loaded with 28Mg2+ are diluted or are
washed and resuspended in media without added Mg2+, no significant Mg2+ efflux can be detected even after 2 h incubation. In contrast, if the concentration of extracellular Mg2+ is markedly increased, to the mM range, efflux of intracellular
28Mg2+ can be seen (179). Three additional Co2+ resistant genes, corB, corC,
and corD influence efflux (72). Mutation of corB/corC/corD elicits a progressive
39 right shift of the extracellular Mg2+ concentration required to elicit 28Mg2+ efflux.
Since the apparent half-maximal concentration of Mg2+ required for influx is 15
μM and that required for a half-maximal rate of Mg2+ efflux is about 3-5 mM, such
efflux via CorA cannot be due to a simple Mg2+-Mg2+ exchange process because at the extracellular Mg2+ concentration at which efflux can be detected, influx
would already be saturated (72).
Other features of CorA expression
S. Typhimurium exhibits a Fe2+ hypersensitivity phenotype if either phoP
or both mgtA mgtCB are mutated (28). The increased Fe2+ sensitivity of the
phoP strain is abrogated by mutation of corA. Mutation of mgtA and mgtCB
slightly decreases Fe2+ sensitivity of the phoP strain. 63Ni2+ uptake is reported to
be increased in either mgtA mgtCB or phoP mutants. The increased uptake is
dependent on CorA and is apparently the result of an increased Vmax without
2+ change in the Km for Ni . Neither corA transcription nor CorA protein level are
reported to increase in either the mgtA mgtCB or phoP mutants. These results
imply that CorA transport is regulated (discussed in Chapter 5). Addition of
cation hexaammines, selective CorA antagonists, also decreases the Fe2+ hypersensitivity phenotype of the phoP mutant. However, since the CorA protein does not transport Fe2+ (see Chapter 3 (141)), this Fe2+ sensitivity phenotype is
apparently not a direct consequence of CorA transport, possibly is the result of
increased uptake of Fe2+ via another transporter.
The enzyme lactoperoxidase is produced as a defensive measure by
immune cells; it oxidizes different substrates, primarily thiocyanate, using
40 hydrogen peroxide. Exposure to lactoperoxidase induces expression of several
genes in E. coli, among them corA (164,165). Both E. coli and S. Typhimurium
corA mutants are hypersensitive to lactoperoxidase but not to direct hydrogen
peroxide challenge or to superoxide generated by addition of plumbagin.
Addition of Mg2+ did not alter the response, but addition of Ni2+ increased lactoperoxidase induced killing in wild type but not an E. coli corA mutant, strongly suggesting that the ability of CorA to transport Ni2+ (or Co2+) mediates or
influences the lactoperoxidase sensitivity. The mechanism by which
lactoperoxidase exposure induces corA expression has not been investigated.
Role of Mg2+ in the cell
Mg2+ homeostasis is poorly understood (47,56,108,118,130,143,151-153).
As indicated by the introduction above, most of what is known is with regards to
transport of Mg2+. This work as well as previous work by others indicates the
Mg2+ transport is highly regulated. These observations imply the Mg2+ homeostasis is crucial for cell function. Mg2+ has long been known to play a role in enzyme catalysis. Mg2+ is important for the virulence and has been linked to
pathogenesis in many bacteria, but the role Mg2+ plays in virulence is unknown.
Overall, there is much to learn about Mg2+ homeostasis, but it is clearly
important.
Summary
S. Typhimurium’s environment fluctuates markedly during its pathogenic lifecycle. Coordination of gene expression and protein function is central in
maintaining this lifecycle. Many of the requisite cellular processes for
41 pathogenicity require Mg2+ as a cofactor or environmental signal. Moreover,
Mg2+ is implicated in several stages of S. Typhimurium infection. For example,
exposure to low extracellular Mg2+ results in activation of the PhoPQ two
component system and consequently markedly alters expression of many genes
necessary for virulence including but not limited to Salmonella pathogenicity island 1, Salmonella pathogenicity island 2, host antimicrobial peptide resistance,
bile resistance, and biofilm formation. The primary source of intracellular Mg2+ in
S. Typhimurium and many other bacteria and archaea is the CorA Mg2+ channel.
Data in Chapter 4 show that mutation of corA results in a decrease in
virulence after either oral or intraperitoneal infection. Microarray data indicated
that multiple metabolic pathways are affected by corA mutation. The simplest
explanation for these defects would be that in the absence of CorA, the organism
cannot obtain sufficient intracellular Mg2+ for optimal virulence despite having two
other Mg2+ transporters. However, data in Chapter 5 show that intracellular Mg2+ is relatively unaffected by loss of corA. The virulence phenotype and changes in gene expression are related to the presence of a functional CorA protein.
Studies by Hantke (85) and Champongpol and Groisman (28) indicated that
CorA can transport Fe2+ due to increase in resistance to Fe2+ toxicity of the corA
mutant. Moreover, Champongpol and Groisman indicated that CorA function
was regulated. Chapter 3 shows that CorA does not transport Fe2+. Chapter 5
shows that regulation of CorA function does occur and it is important for the
virulence phenotype.
42 Table 1.1 S. enterica serovar Typhimurium pathogenicity island 1
SPI1 General Role Putative Function gene avrA Secreted Effector Inhibits activation of NFκB thus augmenting epithelial cell apoptosis. sprB Transcription Factor Acts upstream or at the same level as HilA to regulate SPI1 expression sprA Transcription Factor Acts upstream or at the same level as HilA to regulate SPI1 expression hilC Transcription Factor Derepresses hilA and can activate SPI1 expression independent of HilA orgA Unknown Oxygen regulated gene also regulated by PhoPQ orgB Unknown Oxygen regulated gene also regulated by PhoPQ orgC Unknown Oxygen regulated gene also regulated by PhoPQ prgK Structural Component Part of needle complex prgJ Structural Component Helps with needle complex assembly and may serve as needle cap prgI Structural Component Subunit for filament of the needle complex prgH Structural Component Part of needle complex hilD Transcription Factor Derepresses hilA and can activate SPI1 expression independent of HilA hilA Transcription Factor Activates expression of SPI1 iagB Secreted Effector A lytic transglycolase involved in peptidoglycan degradation sptP Secreted Effector Protein tyrosine phosphatase, GTPase activating protein for Rac-1 and Cdc42, and inhibitor of the ERK MAPK pathway sicP Chaperone Chaperone for SptP iacP Unknown Involved in fatty acid metabolism and may function as a acyl carrier protein sipA Secreted Effector Binds actin, inhibits depolymerization of actin and decreases actins critical concentration thus enhancing membrane ruffles during invasion. It also enhances SipC’s concentration. sipD Secreted A needle tip protein that senses the Effector/Chaperone environment. Also is a protein translocase. sipC Secreted Actin binding protein, and helps bundle Effector/Chaperone actin. Also is a protein translocase. sipB Secreted Induces macrophage apoptosis or Effector/Chaperone autophagy by interacting with caspase 1. Also is a protein translocase. sicA Chaperone and Binds SipC and SipB and prevents their Transcription Factor degradation. Acts with InvF to activate expression of SPI1 genes. spaS Structural Component Part of apparatus spaR Structural Required for protein secretion by SPI1. Component/Chaperone
43 spaQ Structural Required for protein secretion by SPI1. Component/Chaperone spaP Structural Required for protein secretion by SPI1. Component/Chaperone spaO Structural Required for protein secretion by SPI1. Component/Chaperone invI Structural Required for antigen presentation for /spaN Component/Effector mammalian cell invasion. invJ Structural Determines the length of the needle, /spaM component/Effector required for proteins secretion, and required for antigen presentation for mammalian cell invasion. invC Structural ATPase, energize protein export, and component/Effector helps drive the separation of chaperone from effector invB Chaperone Chaperone for sipA, sopE, sopE2, and sopA. invA Structural component Involved in protein secretion/part of secretion apparatus invE Structural Involved in protein translocation component/Chaperone invG Structural component Part of the needle complex invF Transcription factor Acts with SicA to regulate expressions of SPI1. invH Structural component Helps localize InvG in the membrane.
44 Table 1.2: The PhoPQ regulon
Gene Function Positively (+) or negatively (-) regulated ebgR Involved in adaptive mutagenesis + gmm or GDP glucose mannose mannosyl - wcaH hydrolase hilA SPI1 T3SS transcriptional activator - lpxO Dioxygenase + mgtA Mg2+ transporter + mgtB Mg2+ transporter + mgtC Unknown + orgB SPI1 T3SS + orgC SPI1 T3SS + pagA or UDP glucose/GDP mannose + ugd dehydrogenase for AMP resistance pagC Virulence effector/Macrophage survival + factor pagD Unknown + pagH Unknown + pagJ Unknown + pagK Unknown + pagL Deacylates lipid A for AMP resistance + pagM Unknown + pagN Putative invasion + pagO Unknown + pagP Acylates lipid A for AMP resistance + pagT Unknown + pbgB Unknown + pbgC Unknown + pbgD or Porphobilinogen deaminase + hemC pbgE Unknown + pbgF Unknown + pbgM Unknown + pbgP or Involved in aminoarabinose synthesis + pmrF for AMP resistance pbgW Unknown + pbgX Unknown + pbgY Unknown + pcgD Unknown + pcgE Unknown + pcgF Unknown + pcgG Unknown +
45 pcgH or Outer membrane lipoprotein + slyB pcgJ Unknown + pcgL D-alanine-D-alanine dipeptidase + pcgN Unknown + pcgP Unknown + pcgQ Unknown + Pdu Unknown - pgtE Surface protease for AMP resistance + phoN Acid phosphatase + pmrA AMP resistance transcriptional regulator + pmrB AMP resistance sensor kinase + pmrC Phosphoethanolamine transferase for + AMP resistance pmrD Regulator of the PmrA/PmrB two + component system prgA Unknown - prgB Unknown - prgC Unknown - prgE Unknown - prgH SPI1 T3SS - prgI SPI1 T3SS - prgJ SPI1 T3SS - prgK SPI1 T3SS - psgA Unknown - psgB Unknown - psgC Unknown - psgD Unknown - psgE Unknown - psgF Unknown - psgG Unknown - psiD Unknown + slyA Transcriptional regulator + sodCI Superoxide dismutase + Soma Involved in polymyxin B resistance + sopD2 SPI2 T3SS + spvB Unknown - ssrA SPI2 T3SS sensor kinase + ssrB SPI2 T3SS transcriptional activator + tlpA Coiled coil temperature sensor - ugtL Involved in AMP resistance + virK Involved in polymyxin B resistance + yqjA Involved in magainin-2 resistance + yqjB Unknown +
46 Table 1.3: Genes directly regulated by PhoP/PhoQ
Gene Function PhoP Assays to indicate box PhoPQ regulation mgtA Mg2+ transporter Yes EMSA & ChIP (114,214) mgtB Mg2+ transporter No ChIP (168) mgtC Unknown No ChIP (168,214) orgB SPI1 T3SS No EMSA & DnaseI footprint (2) orgA SPI1 T3SS No EMSA & DnaseI footprint (2) pagA UDP glucose & GDP No DnaseI footprint or mannose (controversial) ugd dehydrogenase (214) pcgL Unknown Yes EMSA (114) phoP Transcriptional Yes EMSA & ChIP regulator (168) pmrA AMP resistance No EMSA (114) transcriptional regulator pmrB AMP resistance sensor No EMSA (114) kinase pmrC Phosphoethanolamine No EMSA (114) transferase for AMP resistance pmrD Polymyxin resistance Yes ChIP (214) protein slyA Transcriptional No ChIP (controversial) regulator (167) slyB OMP Yes EMSA (114) ssrB SPI2 transcriptional Yes ChIP (15) regulator ugtL Membrane protein No DnaseI footprint (167)
47
Figure 1.1. Salmonella’s LPS is composed of three segments: Lipid A, core polysaccharide, and O-polysaccharide.
Abbreviations: Abe (Abequose); Ara (4-Amino-Arabinose); EtN (Ethanolamine);
Gal (D-Galactose); Glc (D-Glucose); GlcNAc (N-acetyl-D-Glucosamine); Hep (L-
Glycero-Mannoheptose); KDO (2-Keto-3-Deoxyoctonic acid); Man (D-Mannose);
OAc (O-acetyl); P (phosphoric acid ester); and Rha (L-Rhamnose).
(www.copewithcytokines.de/cope.cgi?key=Endotoxins)
48
Figure 1.2. Crystal structure of the periplasmic sensor domain of S.
Typhimurium PhoQ (J Mol Biol. 2006 Mar 10;356(5):1193-206).
49
Figure 1.3. Crystal structure of T. maritima CorA (top left). One subunit of the
CorA pentamer (top middle). A top view looking down through the channel from the periplasm (top right) (118). A top view look down through the channel indicating the Mg2+ ions bound between monomers (47).
50 Chapter 2
Methods
Bacterial strains, plasmids, and growth conditions in Chapter 3. Bacterial
strains and plasmids used or constructed for the work described in Chapter 3 are listed in Table 3.1, p.89. Unless indicated otherwise, all bacterial strains are
derived from S. enterica serovar Typhimurium SL1344. The molecular biology
methods used were those of Sambrook et al (155). The high-frequency
generalized transducing bacteriophage P22 mutant HT 105/1, int-201 was used
for all transductional crosses, and phage-free, phage-sensitive transductants
were purified using successive rounds of purifications on EBU agar to obtain
white colonies (126). EBU agar is made up of pH indicators, thus phage infected
colonies will turn blue due to lysing of the bacterial cells because of continued
infection. Uninfected colonies will appear white. The transductions were
confirmed by PCR. The corA mutant was constructed by P22 transduction of a
corA::Tn10 insertion cassette from MM385 into MM2089 as a recipient,
generating MM2242. The insertional mutation of corA in the SL1344 background
was further confirmed by a Co2+ resistance assay (96), Western blot analysis,
and PCR. To create a corA feoB strain, the feoB gene of MM2089 was deleted using the technique of Datsenko and Wanner (38) to give MM2985. The deletion
was confirmed by PCR. The corA::Tn10 insertion allele was subsequently
introduced by P22 transduction, giving MM2986. Bacteria were routinely grown
in Luria-Bertani (LB) broth at 37°C with shaking (95,186).
51 Iron toxicity assays. The liquid iron killing assays were adapted from the
methods of Chamnongpol and Groisman (28) and Hantke (85), and two different
protocols were used based on this adaptation. For protocol 1, strains were grown
overnight at 37°C with shaking in 2 ml of N-minimal medium [13.14 g of Tris HCl,
0.196 g of Tris Base, 0.136 g of KH2PO4, 0.372 g of KCl, 1.0 g of (NH4)2SO4, 1.0
liter of double-distilled water] supplemented with 100 mM MgCl2, 0.1% Casamino
Acids, and 0.4% glucose (pH 7.0). Overnight cultures were washed twice in 1 ml
of N-minimal medium containing 10 µM MgCl2 (pH 7.0), resuspended in 200 µl of
the same medium and used to inoculate 10 ml of N-minimal medium with 10 µM
MgCl2, 0.1% Casamino Acids, and 0.4% glucose (pH 5.8 or 7.0) in 125-ml flasks.
Cells were incubated at 37°C with shaking for 6 to 7 h. For toxicity assays, a 0.1
optical density (OD) at 600 nm (OD600) unit of cells was removed and washed
three times in 1 ml of N-minimal medium containing 10 µM MgCl2 (pH 5.8 or pH
7.0). Cells were resuspended in 1 ml of N-minimal medium with 10 µM MgCl2
(pH 5.8 or 7.0). The OD600 was read, and cell samples were adjusted to the
same OD and diluted, initially at 1:100 (10-2) and then twice at 1:1,000 (10-5 and
10-8 final dilutions). A total of 100 µl of each diluted cell sample was mixed with
100 µl of 0, 10, 100, 250, or 500 µM FeCl2 (freshly dissolved in N-minimal
medium containing 10 µM MgCl2 [pH 5.8] preincubated at 37°C) and incubated at
37°C with shaking for 15 min. Cells were immediately plated on N-minimal plates
containing histidine (strain SL1344 is a histidine auxotroph) and 0.4% glucose or on LB, incubated overnight at 37°C before counting. All experiments were performed in duplicate. In addition to the experiments described, other
52 experiments were conducted in medium lacking carbon and nitrogen sources and in liquid medium containing 1 mM sodium ascorbate (data not shown). In all
cases, although Fe2+ clearly killed cells, no differences were seen between wild- type and corA cells (data not shown).
For protocol 2, strains were grown overnight at 37°C with shaking in
tryptone yeast extract (TY) medium (8.0 g of tryptone, 5.0 g of yeast extract, and
5.0 g of NaCl per liter). Overnight cultures were centrifuged, and the cell pellets
were resuspended in a HEPES-salts buffer (30.0 g of HEPES, 4.65 g of NaCl, 1.5
g of KCl, 1.0 g of NH4Cl, and 0.425 g of Na2SO4 per liter at pH 7.2)
9 supplemented with 0.2% glucose and 10 µM MgSO4. Then 10 cells were
incubated with freshly made 30 µM FeSO4 in the presence of 1 mM sodium
ascorbate for 0, 5, 20, 40, and 60 min. At each time point, cells were diluted and plated on TY plates in duplicate. After incubation overnight at 37°C, colonies were counted.
For plate iron killing assays, a lawn of cells was plated on LB plates. A 6-
mm-diameter filter paper disk in the center of the dish received 15 µl of freshly
made 1 mM FeCl2 with 1 mM sodium ascorbate. After overnight incubation, the diameter of the ring around the disk devoid of cells was evaluated (data not shown).
Transport assays used in Chapter 3. The uptake of 63Ni2+ (NEN, Boston,
Mass.) was assayed instead of Mg2+ uptake, as 28Mg2+ is prohibitively expensive
and not readily available. Methods for transport have been described in detail
previously (78,177,179). Briefly, cells were grown aerobically overnight in LB
53 with appropriate antibiotics, washed twice in cold cell wash buffer (N-minimal
medium with 0.4% glucose and 0.2% Casamino Acids at either pH 5.8 or 7.0),
resuspended to an OD600 between 1.0 and 2.0, and placed on ice. 100 μl of cells were added to tubes containing various concentrations of inhibitor cation plus 200
63 2+ 63 2+ µM NiCl2 and 0.3 to 1 µCi Ni in a final volume of 1 ml. Uptake of Ni is linear for the first 15 min after the metal is added. Thus, the reaction mixtures were incubated for 15 min at 37°C, stopped by the addition of 5 ml of ice-cold N-
minimal medium containing 10 mM MgSO4 and 0.5 mM EDTA, filtered
immediately on nitrocellulose filters (Schleicher & Schuell, Keene, N.H.) and
washed once with 5 ml of the same solution. The filters were placed in 3 ml of
Biosafe II scintillation cocktail (Research Products International Corp, Mount
Prospect, Ill.), and radioactivity was measured by scintillation counting.
All solutions containing iron were made fresh immediately before use.
Fe2+ solutions always contained 1 mM ascorbate unless noted, and 55Fe2+ transport assays were always conducted in solutions containing 1 mM freshly
made sodium ascorbate. The final Fe2+ concentration was 1 µM when 55Fe2+ transport was measured, but the transport assay was otherwise similar to that for
63Ni2+ transport assays. When Fe3+ was tested, ascorbate was not used to make
stock solutions and no ascorbate was added to the incubation or wash buffers. In
all cases, the concentration of sodium ascorbate used was chosen based on
methods previously described; most likely some sodium ascorbate was
consumed during the experiment. However, throughout the duration of all
54 experiments and up to 12 h post experiment, oxidation of ferrous iron or
precipitation of ferric iron in stock solutions was not observed.
While our transport assays were very similar to those used by Hantke (85),
there were some differences. Hantke analyzed S. enterica serovar Typhimurium
LT2, whereas we studied S. enterica serovar Typhimurium SL1344 and 14028s.
However, CorA-mediated transport was initially characterized by this laboratory
using S. enterica serovar Typhimurium LT2 as a strain background (78,95,96).
corA expression and CorA-mediated transport results appear to be identical for
the LT2, 14028s and SL1344 strain backgrounds. corA is a single gene locus,
and both the promoter and coding sequences are identical for the different S. enterica serovar Typhimurium strains. In addition, Hantke grew cells overnight in
TY medium and resuspended in a medium buffered with HEPES and containing
NaCl, KCl, NH4Cl, and Na2SO4 and no added magnesium. In our hands and with
our methods, cells can be grown overnight in LB or N-minimal medium with or without various monovalent cations and resuspended in several buffers
containing any monovalent cation without altering transport properties (78).
Thus, these minor differences should not alter interpretation of the transport data.
Bacterial strains and plasmids used in Chapter 4. Bacterial strains used in
Chapter 4 are listed in Table 4.1, p.106. Unless otherwise indicated, all bacterial
strains are derived from Salmonella enterica serovar Typhimurium SL1344.
Mouse experiments. Groups of 6 BALB/c mice (8 weeks old) were infected by
oral gavage with 7X108 or 7x109 cells of wild type, 7X108 cells of a corA strain
carrying a plasmid with a functional CorA, and 5X108 or 5x109 cells of a corA
55 strain. Additionally, groups of seven C3H/HeN female mice (6-8 weeks old) were each infected intraperitoneally with either 700 cfu of wild type, 850 cfu of a corA strain carrying a plasmid with a functional CorA, 900 cfu of a corA strain, or 900 cfu of a phoP strain (WN152 14028s phoP::Tn10dCm). Nine mice were injected with PBS as a control. Mortality was monitored over a 25-28 day period. Mice were checked two times daily. Moribund mice were euthanized.
Microarrays. Microarray protocols were adapted from those at the Pathogen
Functional Genomic Resource Center at The Institute for Genomic Research
(TIGR) (http://pfgrc.tigr.org/resources.html). Cells were grown overnight in LB with antibiotics at 37°C with shaking, subcultured into LB without antibiotics and grown to log phase at 37°C with shaking. Cells were collected on three separate days from wild type or a corA strain strains giving 6 total samples from three independent cultures for each strain. A single microarray was run for each sample, three for wild type and three for a corA strain. Genomic DNA for microarray can be extracted using any method.
A. Total RNA was obtained via hot phenol chloroform extraction:
1. 25.0 ml of phenol (Sigma, P4682-400mL) was pipetted into a glass bottle. The bottle was capped loosely. Double distilled water (ddH20) should not be used instead deionized water (dH20) should be used for all array protocols. ddH2O may contain free radicals, which can inhibit labeling and hybridization of Cy dyes.
2. The bottle of phenol was placed into boiling water at 100°C.
3. Phenol was heated for approximately 10 minutes prior to the RNA extraction.
4. 25.0 ml of a bacterial culture was pipetted into the hot phenol.
56 5. The bottle was shaken to mix the phases.
6. The cap was loosened every so often to release the gas build up.
7. The mixture was heated for about 15-30 min or until the mixture looked like one layer instead of two layers.
8. The mixtures were transferred into two “RNase-treated” centrifuge tubes.
Prior to use, the plastic tubes were RNase-treated by adding 0.5 M sodium hydroxide to the tubes and incubating them at room temperature for 10 min.
Then the tubes were rinsed three times with DEPC (diethylpyrocarbonate) water
(DEPC 0.5 ml /L of dH20) and autoclaved.
9. The tubes were placed on ice.
10. 5.0 ml of chloroform was added to each of the tubes.
11. The solution was thoroughly mixed and the gas build up was released by loosening the cap.
12. The tubes were centrifuged at 10,000 g at 4°C for 10 min.
13. The aqueous phases (top layer) were transferred to fresh centrifuge tubes.
The bottom layer of debris was avoided; this layer can ruin the quality of the
RNA. So, about 1-3 ml of the aqueous layer was left behind.
14. 6.25 ml of phenol was added to each of the tubes.
15. 6.25 ml of chloroform was added to each of the tubes.
16. The tubes were shaken to mix the layers and the caps were loosened to release the gas build up.
17. The tubes were centrifuged at 10,000 g at 4°C for 10 min.
57 18. The aqueous phases (top layer) were transferred to fresh centrifuge tubes.
The bottom layer of debris was avoided; this layer can ruin the quality of the
RNA. So, about 1-3 ml of the aqueous layer was left behind.
19. 0.1 volume of 3.0 M sodium acetate in DEPC treated water (Ambion) was
added to each of the tubes.
20. An equal volume of isopropanol was added to each of the tubes.
21. The tubes were cooled on ice for 30 min. Longer incubations on ice do not
increase the RNA yield.
22. The tubes were centrifuged at 10,000 g at 4°C for 60 min. Longer spins will
increase the RNA yield.
21. All of the supernatant was removed. The tubes were inverted and patted
onto paper towels to remove excess supernatant. This step was completed
carefully not to contaminate the RNA or lose the RNA pellet. The pellet was air
dried upright in uncapped tubes for 5 min in the fume hood.
23. The pellets were resuspended into RNase free water containing 200 U/ml
RNaseOUT (Invitrogen).
B. Extracted RNA is cleaned up using Qiagen RNA maxi kit (Qiagen #75162):
1. The concentration of RNA was measured on spectrophotometer (OD280 and
OD260) prior to the clean-up process.
2. Samples were adjusted to the appropriate volume according to the Qiagen
RNA Cleanup protocols with RNase-free water.
3. The appropriate volume of Buffer RLT with fresh β mercaptoethanol (10 μl/ml) was added and the tubes were mixed well.
58 4. The appropriate volume of 100% ethanol was added and the tubes were mixed well.
5. The samples were transferred to a RNA maxi column (load up to 15 ml).
Then, the samples were centrifuged at 4000 rpm for 5 min. The flow through was discarded.
6. Step 5 was repeated if there is more than 15 ml of starting material.
7. The samples were treated on the column with an RNase-free DNase (Qiagen
#79254) for 15 min by covering the membrane completely with the solution of
DNase.
8. 5 ml of RW1 buffer was added and the tubes were incubated at room temperature for 5 min.
9. The tubes were centrifuged at 4000 rpm for 5 min and the flow through was discarded.
10. 10 ml of RPE buffer was added. Each sample was centrifuged for 2 min at
4000 rpm, and the flow through was discarded.
11. 10 ml of RPE buffer was added. The samples were centrifuged for 5 min at
4000 rpm.
12. The maxi columns were transferred to new 50 ml RNase-free Falcon tubes.
The RNA was eluted by adding the appropriate volume of RNase-free water to the membrane. The tubes were incubated at room temperature for 1 min, and then centrifuged for 3 min at 4000 rpm.
13. Elution step 12 was repeated.
14. The concentration of the RNA was measured on spectrophotometer.
59 15. Clean RNA (0.5 -1.0 μg) was run on a 4.0% TAE (Tris-acetate-EDTA)
agarose gel w/ ethidium bromide to assess quality. The gel box was cleaned
with soap and water every time prior to use to remove any RNases.
16. 3 to 4 bands will be seen on the gel indicated rRNA. 23S rRNA is 3030
bases, 16S rRNA is 1515 bases, and 5S rRNA is 118 bases. 23S rRNA is
typically cleaved by S. Typhimurium thus it will be observed as two bands around
1500 bases. Typically, three distinct bands around 1500 bases will be observed
if the RNA is of good quality.
C1. RNA aminoallyl labeling for microarrays:
1. The following were mixed in RNase free tubes: Two μg of total RNA, 2 μl
random hexamers (3 mg/ml) (Invitrogen #48190-011), 1 μl RNaseOUT
(Invitrogen #10777-019). The reaction was brought up to 18.5 μl with DEPC
water and mixed well. The McClelland lab protocol calls for 30 to 50 μg of RNA,
but 2 μg works well (http://www.skcc.org/mcclelland_protocols_arrays.html). 30
μg were used for the microarrays in this thesis.
2. The tubes were incubated at 70°C for 10 min. Then mixtures were frozen in a
dry ice/ethanol bath for 30 sec. Tubes were centrifuged briefly, and allowed to
reach room temperature.
3. The following were added to the tubes: 18.5 μl of RNA/primer mix from above
step, 6 μl 5X first strand buffer (included with Invitrogen #18080-044), 3 μl 0.1 M
DTT (dithiothreitol) (included with Invitrogen #18080-044), 0.6 μl 25 mM dNTP’s
(Invitrogen #10297-018)/aa-dUTP (2:3 aa-dUTP (Ambion #8439) to dTTP)
60 labeling mix, and 2.0 μl Superscript III reverse transcriptase (200U/μl) (Invitrogen
#18080-044).
4. The tubes were mixed and incubated in a 42°C water bath overnight.
5. The RNA strand was hydrolyzed by adding 10 μl of 0.5 M EDTA and 10 μl of
1.0 M NaOH
6. The tubes were mixed and incubated at 65°C for 15 min.
7. 25 μl of 1.0 M Tris (pH 7.0) was added to each of the tubes to neutralize the
pH.
C2. Genomic DNA aminoallyl labeling for microarrays:
1. The following were mixed in RNase free tubes: 1.5 μg of genomic DNA, 3.3
μl random hexamers (3 mg/ml) (Invitrogen #48190-011), and brought up to 39 μl
with deionized water.
2. The tubes were mixed well and incubated at 100°C for 12 min.
3. Then mixtures were frozen in a dry ice/ethanol bath for 30 sec. Tubes were centrifuged briefly, and allowed to reach room temperature.
4. The following were added to each of the tubes: 39 μl of the above genomic
DNA/primer mix, 5 μl of 10X EcoPol (Klenow) buffer (included with New England
Biolabs #M0212L), 2 μl of 5 mM dNTP’s (Invitrogen #10297-018)/aa-dUTP (2:3 aa-dUTP (Ambion #8439) to dTTP) labeling mix, and 4 μl DNA Polymerase I
Klenow fragment (New England Biolabs #M0212L).
5. The tubes were mixed and incubated at 37°C overnight.
6. The reactions were stopped by adding 5 μl of 0.5 M EDTA (pH 8.0).
D. Cleaning RNA and genomic DNA probes:
61 1. The reactions were cleaned using Qiagen MinElute columns (Qiagen
#28004).
2. Each reaction was mixed with 400 μl of PB buffer (included in Qiagen #28004)
and transferred to MinElute columns.
3. The tubes were centrifuged at 13,000 rpm for 1 min, and the flow through was
discarded.
4. The columns were washed with 750 μl of phosphate wash buffer (5 mM KPO4 pH 8.0 and 80% ethanol) and centrifuged at 13,000 rpm for 1 minute.
5. The flow through was discarded and the tube was centrifuged an additional minute at 13,000 rpm.
6. The column was transferred to a new 1.5 ml centrifuge tube and 30 μl of phosphate elution buffer (4 mM KPO4, pH 8.5) was added.
7. The tubes were incubated at room temperature for 1 min and centrifuged
column for 1 min at 13,000 rpm.
8. Elution step 6-7.was repeated.
9. The open tubes were transferred to a speed vac and dried on high heat for
about 20-30 min. Samples were continually checked to avoid over-drying. When
a few microliters remained in the bottom of tube, samples were closely monitored
until sample was completely dry.
10. 4.5 μl of freshly made 0.1 M sodium carbonate buffer pH 9.3 was placed
onto the pellet for 10-15 minutes. The pellet was resuspended by pipetting
slowly for 2-3 minutes. The pellet is difficult to resuspend. This is a crucial step.
62 11. 4.5 μl Cy3 or Cy5 dye (Amersham Biosciences #PA23001 and #PA25001) was added.
12. The tubes were incubated at room temperature in the dark for 2 hr.
13. Reactions were cleaned using Qiagen MinElute columns.
14. Samples were mixed with 250 μl of PB buffer and transferred to MinElute columns.
15. The columns are centrifuged at 13,000 rpm for 1 min and the flow through was discarded
16. Columns were washed with 750 μl of PE buffer (included in Qiagen #28004) and centrifuged at 13,000 rpm for 1 min.
17. Flow through was discarded, and columns were transferred to a new 1.5 microfuge tube. 50 μl of deionized water was added.
18. Samples were incubated at room temperature for 1 min and centrifuged at
13,000 rpm for 1 min.
19. Another 50 μl of deionized water was added and elution step 17-18 was repeated.
20. Labeled probes were mixed and dried down in a speed vac on high heat for about 30-40 minutes. Samples were continually checked to avoid over-drying.
When a few microliters remained in the bottom of tube, samples were closely monitored until sample was completely dry.
E. Prepping slides for hybridization:
1. A Coplin jar (VWR# 25457-200) containing pre-hybridization solution (25% formamide, 5X SSC (Ambion #9763), 0.1% SDS, 0.1 mg/ml BSA) was warmed to
63 42°C in a water bath. Important: The hybridization protocols that come with the
specific array being hybridized should be used; they are not interchangeable. Do
not use DTT. Slightly old DTT will cause a green film on slide, thus ruining the
slide.
2. Microarray slides were pre-warmed in pre-hybridization solution for 60 min at
42°C.
3. Arrays were washed 10X with deionized water in glass Wheaton dishes
(Wheaton Science Products #900200) for 2 min per wash. Do NOT use ddH20.
4. Arrays were washed 1X with isopropanol.
5. Arrays were dried by centrifugation at 1000 rpm for 15-20 min. Not drying long enough will result in green film on array, thus ruining the array.
6. Hybridization buffer was made containing: 250 μl of formamide, 250 μl of 20X
SSC and 490 μl of deionized water. 10 μl 10% SDS was added last. The hybridization buffer was filtered with a 0.45 μm filter. Steps 7-9 need to be practiced with 60-90 μl of hybridization buffer using clean “non-microarray” glass slides and clean cover slips. The amount of hot hybridization buffer that is required to completely coat the slide needs to be determined. Hybridization buffer should not leak out. The amount used can vary from experiment to experiment. Once steps 7-9 have been practiced, probes are resuspended in the determined amount of hybridization buffer. Placing the probes onto the slide and putting the cover slip on top is the hardest part of the microarray protocol and requires patience and practice.
64 7. The probes were heated to 95°C in heat block for 5-10 min. The tubes were
vortexed to resuspend the probes.
8. Hybridization chambers (Corning #2551) were warmed with slides inside on
heat block.
9. The hot probes were added to center of hot slide. Cover slips were held at
45° angle close to slide surface and dropped. Air bubbles should leave in 20-30
sec because of the heat, but if they remain press lightly with pipette tip to remove
them. Be careful not to let probe mix leak out. Hybridization buffer was added to
the bottom of the chambers to keep them humidified.
10. Chambers were closed, covered with foil since the dyes are slightly light
sensitive, weighed down in 42°C water bath, and incubated overnight.
F. Post-Hybridization and scanning:
1. Cover slips were removed by holding slides upside down in warm (55°C) low stringency buffer (2X SSC and 0.1% SDS in dH20) in a large glass dish. The
fallen cover slip was removed and the slides were washed vigorously (move back
and forth by hand quickly) in large glass dish for 2-3 min. The most important
wash is the first wash because this is when most of the excess dye comes off.
Slides were washed 2X for 5 min with warm (55°C) low stringency buffer (2X
SSC and 0.1% SDS in dH20) in a Wheaton dish on an orbital shaker or by hand
on the bench top mimicking an orbital shaker. Do not use DTT in wash buffers.
Slightly old DTT will cause green film on slide, thus ruining the slide. Do not use
ddH20 for wash buffers. Do not let slides dry out during washing process.
65 Washing is very important to remove excess dye and decrease background.
Extra washes will help remove excess dye and reduce background.
2. Slides were washed vigorously 2X for 5 min with room temperature medium
stringency buffer (0.1X SSC and 0.1% SDS in dH20).
3. Slides were washed vigorously 2X for 5 min with room temperature high
stringency buffer (0.1X SSC in dH20).
4. Slides were washed in dH20 1X for 3 min and transferred to the centrifuge.
5. Slides were dried by centrifugation for 15-20 min at 1000 rpm. Decreasing
slide dry time will cause a greenish haze to develop. Never use Dyesaver from
Genisphere. The slides do not bleach that quickly as long as you read them
within 1-2 hours.
6. Slides should be kept in the dark until they are scanned.
7. Slides were scanned using a GenePix 4000B microarray scanner at the
Cleveland Clinic Foundation’s Lerner Research Institute’s Genomic Research
Core. The contact person was Pieter Faber. Only use a scanner that scans both channels at one time. Slides should be scanned to obtain a ratio between 0.8-
1.2 for Cy3/Cy5 fluorescence.
Microarray analysis. The TM4 Microarray Suite produced by TIGR was used for data analysis (Saeed AI, 2003, Biotechniques). Spotfinder was used to assign gene identifiers to spots (circle algorithm) and to flag spots. MIDAS was used to normalize (Lowess (Locfit) and Standard Deviation Regularization) the data. The normalized data was imported into Microsoft Excel. Normalized intensity values for RNA were divided by those for the genomic DNA control for
66 each slide to obtain a ratio. Three sets of ratios were obtained for wild type and three sets of ratios were obtained for a corA strain. P values were calculated using a t test. Only data with P values less than 0.05 are represented in Table
4.2, p.107-111. The average of the ratios for wild type was divided by the average of the ratios for a corA strain and vice versa to obtain the fold changes indicated in Table 4.2. Microarray descriptions and data are deposited at www.ncbi.nlm.nih.gov/geo (Series record GSE10242). qRTPCR. Total RNA was isolated and reverse transcribed as described for microarrays. qPCR was conducted according to Qiagen’s QuantiTect SYBR
Green PCR protocol. Briefly, 200 ng of cDNA/reaction was mixed with
QuantiTect SYBR green PCR master mix, water, and primers. A Biorad iCycler was used to detect SYBR green fluorescence. Samples were held in the cycler at 95°C for 15 minutes. This was followed by 40 cycles of 94°C for 15 seconds,
50°C for 30 seconds, and 72°C for 30 seconds. Samples then held at 4°C. Data were imported and plotted using Excel.
Western blots used in Chapter 4. For anti-CorA Western blots, triplicate aliquots were grown as described for luciferase assays below to log or stationary phase in the various media. Cells were pelleted and resuspended in 1.0 ml of N minimal medium. Resuspended cells were sonicated for 30 seconds, and protein concentrations were determined using the Bradford protein assay (BioRad). Ten
μg of total protein was loaded onto 12% SDS polyacrylamide gels in triplicate.
For anti-SPI1 Western blots, cells were grown in LB to log phase; 0.2 OD600nm of cell pellets in sample buffer per lane were loaded directly onto 12% SDS
67 polyacrylamide gels. For pH 6.0-8.0 shifted anti-SPI1 Western blots, cells were
grown in LB plus 100 mM MES pH 6.0 to an OD600nm of 0.5, shifted into LB plus
100 mM TAPS pH 8.0, grown for an additional hour, and 0.1 OD600nm of cells
were loaded per lane) Gels were electrophoresed and transferred onto
nitrocellulose (Schleicher & Schuell, Keene, N.H.). The CorA antibody (Smith et
al, JBC, 1998) was used at a dilution of 1:10,000 and a DnaK antibody (Biorad)
was used a dilution of 1:5000. All SPI1 antibodies were used at a dilution of
1:5000. Secondary horseradish peroxidase linked anti-rabbit and anti-mouse antibodies (Amersham) were used at a dilution of 1:10,000. Proteins were visualized using enhanced chemiluminescence as recommended by the manufacturer (Amersham). Western blots were scanned and quantitated by densitometry using DnaK as loading control.
Swimming assay. To measure motility, a swimming assay was performed by plating about 10 μl of log phase cultures of MM2089 and MM2242, equivalent to
OD600nm of 0.005, onto the center of agar swim plates (10 g tryptone, 5 g yeast
extract, 2.5 g agar, 0.5% glucose, and 1.0 L ddH2O). Plates were incubated at
room temperature for 20 hours and scanned.
CAS assay. An assay solution similar to one used by Alexander and Zuberer
was made to measure siderophore production. Hexadecyltrimethylammonium
(HDTMA) (0.876 g) was dissolved in 1.0 L dH20 stirring constantly over low heat.
1.5 ml of 1.0 mM FeCI3·6H20 in 10.0 mM HCl was mixed with 7.5 ml of 2.0 mM
chrome azurol S (CAS). The iron/CAS solution was slowly added to the HDTMA
solution while stirring. Cells were grown to mid log phase, and spun down. The
68 supernatant was filtered through a 0.45 μm filter. The concentration of
siderophore in the filtrate was measured by making 1:1 solution of CAS solution
with filtered supernatant. Solutions were incubated at room temperature for 2-4
hrs and measured with a spectrophotometer at 540 nm. HDTMA-Fe3+-CAS forms a ternary complex that is blue in color. Siderophores have a better affinity for Fe3+ and if the HDTMA-Fe3+-CAS complex loses its Fe3+ it will turn orange.
Congo red liquid assay. Cells were grown in LB with antibiotic supplementation
overnight at 37°C with shaking, spun down, resuspended in LB, and grown to mid-log phase at 37°C with shaking. Cells were spun down and resuspended to obtain a concentration of 109 cells/ml in PBS containing 0.002% (w/v) Congo red.
Cells were then incubated at 37°C with shaking for 1-2 hours. Cells were spun
out samples were read in triplicate on a spectrophotometer at 500nm. The
Congo red PBS solution was also incubated at 37°C with shaking for 1-2 hours
as a control for total Congo red present.
Cytokine assays. Caco-2 epithelial cells were invaded by wild type and a corA
mutant for 2, 4, and 6 hr. At each time point, the media was removed, filtered,
and levels of cytokines were measured. Case Western Reserve University’s
Division of Pediatric Pulmonology’s Inflammatory Mediator Core measured
cytokine levels using Fluorokine MAP assay kits (R&D Systems).
β-galactosidase assay. Cells were grown in LB or N-minimal medium with
0.1% casamino acids, 0.4% glucose, and 10 μM MgSO4 at 37°C with shaking. At
2, 4, and 22 hr time points, 0.25 OD600nm units of cells were pelleted. Pellets
were resuspended in 960 μl of β-galactosidase mix (760 μl of Z-buffer with β-
69 mercaptoethanol (35 μl /100mL), 40 μl of 0.1% SDS, and 160 μl of 4mg/ml o- nitrophenyl-β-galactopyranoside (ONPG) in 100 mM potassium phosphate (pH
8.0)). Then 60 μl of chloroform was added and samples were vortexed for 10 sec. Samples were incubated at 37°C. When samples began to turn yellow, 500
μl of 1.0M NaCO3 was added to stop the reactions. Samples were spun down for
5 min at 14,000 rpm. Full β-galactosidase mix and chloroform were used as a blank. Samples were read at 420 nm and 550 nm (to remove scattering) on a spectrophotometer. If samples read above 1.0 OD, then they were diluted to obtain an OD below 1.0. Activities in Miller units were determined with the following formula: 1000 X ((OD420nm – 1.75 OD550nm)/ (OD600nm X reaction time)).
Epithelial cell experiments. Caco-2 epithelial cells (ATCC#HTB-37TM) were purchased from ATCC. Cultured Caco-2 epithelial cells were maintained in
Eagle’s Minimal Essential Medium with 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 20% fetal bovine serum and antibiotics at 37°C with 5.0% CO2 as indicated by ATCC.
Cells were grown to confluency and passaged twice to make a branch for each cell type all at the same passage number. The same passage number was used for all experiments. To prepare cells for invasion, cultured cells were grown to
90-100% confluence then passaged at a high cell density 90%-100% confluence to reach 90-100% confluence overnight. Caco-2 cells were plated into 24-well plates in complete growth medium without antibiotics. Bacteria were grown overnight in LB medium with antibiotic supplementation at 37°C with shaking.
Bacteria were subcultured into LB medium without antibiotics and grown
70 overnight at 37°C without shaking. Bacteria were washed once with phosphate
buffered saline (PBS) and subcultured into complete cell culture growth medium
without fetal bovine serum and antibiotics. Medium was removed from the
cultured cells and the bacterial suspension was added at a MOI of 10:1. Plates
were centrifuged at 800 rpm for 10 min at room temperature and then incubated
at 37°C, 5.0% CO2 for desired time periods. At each time point, cultured cells
were washed three times with PBS and treated with gentamicin (10 mg/ml) for 2
hours to kill extracellular bacteria. After two and six hr, the cultured cells were
washed three times with PBS and lysed with 0.1% Triton X-100 detergent for 5
min. The medium was collected, diluted, plated onto LB plates and grown
overnight at 37°C for cfu determination after 18-24 hr incubation. The number of
bacteria obtained from within the cultured cells was divided by the total number
of bacteria added to the cultured cells to obtain the “percent gentamicin
protected”.
Immunohistochemistry for LAMP-1. Four well chamber slides (Nalge Nuno
International #154917) were treated with poly-L-lysine for 24 hours at 37°C at
5.0% CO2. Caco-2 epithelial cells were plated at 90-100% confluency into the
chamber slides and allowed to grow for 1-2 days for complete adherence. Caco-
2 cells were infected for 1 hour with wild type S. Typhimurium. After one hour
cells were washed 3 times with PBS and grown in the presence of gentamicin
(10 mg/ml) for 6 hours. Cells were washed three times for 5 min with PBS, fixed
with 2.5% paraformaldehyde for 10 minutes at 37°C. Then cells were washed three times for 5 min with PBS, permeabilized with 0.2% saponin (with 10%
71 secondary animal serum), and washed again three times for 5 min with PBS.
Then cells were blocked with PBS containing 10% secondary animal serum
(Jackson Immunoresearch #017000121), 1% BSA, and 0.1% saponin for 1 hour.
LAMP-1 antibody (Affinity Bioreagents #PA1-654A) was diluted (1:50, 1:100,
1:1000) into blocking solution and incubated on cells overnight at 4°C. Cells were washed three times for 5 min with PBS. Secondary antibody labeled with
Alexa Fluor 594 (Invitrogen # A21207) was diluted (1:500) into blocking solution and incubated for 1 hour at room temperature. Cells were washed three times for 5 min with PBS. Slides were mounted with Vectashield mounting medium containing DAPI (counterstain for nucleus) (Vector Labs#H-1200), dried for 30 minutes, and viewed under a microscope.
Macrophage experiments. J774A.1 macrophages (ATCC#TIB-67) were purchased from ATCC. J774A.1 macrophage-like cells were maintained in
Dulbecco’s Modified Eagle Medium with glucose (4.5g/L), 10% fetal bovine serum, sodium pyruvate (0.1g/L) and antibiotics. Cells were grown to confluency and passaged twice to make a branch for each cell type all at the same passage number. The same passage number was used for all experiments. To prepare cells for invasion, cultured cells were grown to 90-100% confluence, then passaged at a high cell density 500,000 cells per well. Macrophage-like cells were plated into 48-well plates in complete growth medium without antibiotics.
The more crowded the cells the better they survive. Bacteria were grown overnight in LB medium with antibiotic supplementation at 37°C with shaking.
Bacteria were subcultured into LB medium without antibiotics and grown
72 overnight at 37°C without shaking. Bacteria were washed once with PBS,
resuspended into mouse serum with complement (Innovative Research#IMS-
COMPL-25ml), incubated at 37°C for 20 min, and added to complete cell culture
growth medium with fetal bovine serum and without antibiotics. Medium was
removed from the cultured cells and the bacterial suspension was added at a
MOI of 10:1. Plates were centrifuged at 1000 rpm for 10 min at room
temperature and then incubated at 37°C, 5.0% CO2 for 15-30 min. To obtain the
number of bacteria that entered the macrophages, cultured cells were washed
three times with PBS and lysed with 1.0% Triton X-100 detergent. The medium
was collected, diluted, and plated. At each time point, cultured cells were
washed three times with PBS and treated with gentamicin (12 μg/ml) to kill extracellular bacteria. After two and ten hours, the cultured cells were washed three times with PBS, and lysed with 1.0% Triton X-100 detergent for 5 min. The medium was collected, diluted, and plated onto LB plates and grown overnight at
37°C for cfu determination after 18-24 hr incubation. The number of bacteria obtained from within the cultured cells was divided by the total number of bacteria that entered the cultured cells to obtain the “percent gentamicin protected”.
Bacterial strains and plasmids using in Chapter 5. Bacterial strains used in
chapter 5 are listed in Table 5.1, p.150. Unless otherwise indicated, all bacterial
strains are derived from Salmonella enterica serovar Typhimurium SL1344. To
create the corB, corC, and corD strains, each gene was deleted using the
technique of Datsenko and Wanner (38). Deletions were confirmed by PCR.
73 The high-frequency generalized transducing bacteriophage P22 HT 105/1, int-
201 was used to transduce the corB, corC, and corD deletions into MM2089 thus
creating strains MM3238, MM3239, and MM3240; phage-free, phage-sensitive
transductants were purified using successive rounds of purification on EBU agar
(126). The Escherichia coli corA and Bacillus subtilis mgtE genes were cloned from E. coli DH5α cells (MM3097) and Bacillus subtilis genomic DNA (ATCC#
23857D) respectively into a pBADMycHis(a) vector (Invitrogen) creating pBADECcorA and pBADBSmgtE. Restriction sites for insertion into this vector were XhoI and EcoRI. An additional stop codon was also added to prevent the
MycHis tag from being added. These plasmids were shuttled into the
restrictionless MM1242 strain and then to MM2242, creating strains MM3218 and
MM3217 respectively.
Atomic absorption. Bacteria were grown overnight in LB with antibiotics at
37°C with shaking. Cells were then washed in the cold three times with cold N- minimal medium and then subcultured into LB or N-minimal medium containing
0.4% glucose, 0.1% casamino acids all without antibiotics. N-minimal medium
samples had either 10 µM, 10.0 mM, or 100 mM MgSO4 added and were
incubated at 37°C with shaking. At desired time points, 1.0 ml of each culture
was collected, and pelleted through a 2:1 dibutyl/dioctyl phthalate solution to strip
extracellular water. The phthalate solution was removed, and 1.0 N nitric acid
was used to digest the pellet. Samples were sonicated for 30 seconds in a water
bath sonicator and read by atomic absorption spectrometry. A standard curve of
74 known concentrations of Mg2+ was used to obtain the mass content within the
cells. Values obtained were corrected for OD600nm.
Luciferase assays. Cells were grown in LB with antibiotic supplementation
overnight at 37°C with shaking, spun down in the cold (4°C) and washed 3 times
in cold N minimal medium. Cells were subcultured into either LB or into N-
minimal with 0.1% casamino acids, 0.4% glucose. Either 10 μM or 10 mM
MgSO4 was added to the N-minimal cultures and all cells were grown at 37°C
with shaking. Time points were taken at approximately 2, 4, 6, and 20 hr. Cells
were diluted 1:100 in N minimal medium and 50 μl of cell suspension was added
to 500 μl luciferase reaction buffer (0.01% dodecyl aldehyde in 50 mM sodium
phosphate, pH 7.5). Reactions were vortexed for 10 seconds and read on a
liquid scintillation counter for 30 sec. Relative light units (RLU) were obtained
from the scintillation counter and these values were corrected for OD600nm.
Western blots used in Chapter 5. Triplicate aliquots were grown as described
for luciferase assays to log or stationary phase in the various media. Cells were
pelleted and resuspended in 1.0 ml of N-minimal medium. Resuspended cells
were lysed by sonication for 30 sec, and protein concentrations were determined
using the Bradford protein assay (BioRad). Ten μg of total protein was loaded
onto 12% SDS polyacrylamide gels. Gels were electrophoresed and transferred
onto nitrocellulose (Schleicher & Schuell, Keene, N.H.). The CorA antibody (177) was used at a dilution of 1:10,000 and a DnaK antibody (Biorad) was used at a dilution of 1:5000. Secondary horseradish peroxidase linked anti-rabbit and anti- mouse antibodies (Amersham) were used at dilutions of 1:10,000. Proteins were
75 visualized using enhanced chemiluminescence as recommended by the
manufacturer (Amersham). Western blots were scanned and quantitated by
densitometry using DnaK as loading control.
Transport assays used in Chapter 5. Bacteria were grown as described above
for luciferase assays. For the MgtE experiment, cells were grown in LB overnight
with antibiotics at 37°C, subcultured, and grown overnight in LB with 1 mM
arabinose at 37°C. The uptake of 63Ni2+ (GE Healthcare, Piscataway, NJ) and
57Co2+ were assayed instead of Mg2+ uptake, as 28Mg2+ is extremely expensive
and difficult to obtain. Methods for transport have been described in detail above.
76 Chapter 3
Fe2+ toxicity and CorA studies
Introduction
Mg2+ homeostasis is poorly understood (47,56,108,118,130,143,151-153).
Almost nothing is known about eukaryotic Mg2+ transport proteins, and relatively
little is known, even with respect to bacteria, about regulation of influx and efflux
systems or intracellular buffering of Mg2+ (34,62,77,120,176,179). Three
structurally distinct systems for Mg2+ influx have been identified in bacteria:
MgtA/B, CorA, and MgtE. The MgtA/B class is the least abundant, present
largely in enterobacteria and some other gram-negative species. In contrast,
both the CorA and the MgtE Mg2+ influx systems are present across a wide
spectrum of bacteria. CorA is somewhat less abundant than MgtE in the archaea
and has only distant functional homologs in the eukarya (103,172,173,175).
Judging on the basis of available genomic sequence data, CorA is a primary constitutive Mg2+ uptake system in about half of all eubacteria and in a substantial
minority of archaea.
Salmonella enterica serovar Typhimurium carries members of two of the
three classes of Mg2+ uptake systems described above, namely, a single CorA transporter and two Mgt transporters (104,176). MgtA is the endogenous P-type
ATPase Mg2+ transporter of S. enterica serovar Typhimurium and other
enterobacteria (180,181,188). S. enterica serovar Typhimurium MgtB is encoded
on a pathogenicity island (17), is 50% identical to MgtA, and has close homologs
in only a small number of bacterial species. Both P-type ATPase systems are
77 repressed in cells grown in normal laboratory medium but are induced markedly
in cells exposed to low extracellular Mg2+ concentrations (138,180,187). This
induction is mediated by the PhoPQ two-component regulatory system
(62,182,202). The CorA Mg2+ transporter appears to be constitutively expressed
(28,180,187). CorA is a novel transport protein in that it lacks homology to any
2+ other known transporter. It mediates Mg influx with a K0.5 (extracellular ion
concentration for half-maximal influx) of 10 µM. CorA also mediates influx of Ni2+
2+ and Co with K0.5 values of 200 and 20 µM, respectively. Transport is not
significantly inhibited by Ca2+, Mn2+, or Zn2+ (78,172,179) but can be potently and
selectively inhibited by several cation hexaammines that mimic the fully hydrated
Mg2+ cation (109).
Previous studies by Hantke and by Chamnongpol and Groisman were
interpreted as implicating CorA in iron transport and thus in iron toxicity. Hantke
reported that addition of extracellular Fe2+ to S. enterica serovar Typhimurium
and E. coli cultures resulted in a rapid onset of toxicity and cell death (85).
Mutation of corA was reported to make the cells markedly resistant to this Fe2+- mediated toxicity. This phenotype was hypothesized to be from an ability of CorA
to mediate the influx of Fe2+. Hantke also reported that a corA strain accumulated
less 55Fe2+ than the wild type. Chamnongpol and Groisman (28) subsequently reported that a mutation in phoP rendered S. enterica serovar Typhimurium extremely sensitive to Fe2+-mediated toxicity. A corA phoP double mutant was
relatively resistant to Fe2+ toxicity. In contrast to Hantke's results, however,
Chamnongpol and Groisman reported little or no difference in sensitivity to Fe2+
78 between wild-type and corA S. enterica serovar Typhimurium strains. In accord
with Hantke's hypothesis, Chamnongpol and Groisman (28) also explained the
Fe2+-dependent phenotypes by suggesting that CorA mediates Fe2+ influx.
However, neither group demonstrated directly that Fe2+ could interact with the
CorA transport system. Therefore we tested a corA strain. Our results showed
that it does not exhibit alterations in Fe2+ availability or toxicity from CorA-
mediated Fe2+ uptake, since, according to a direct transport assay, CorA cannot
transport Fe2+.
79 Results
Effect of mutation of corA on survival after exposure to iron. During
encounter with host cells, S. enterica serovar Typhimurium encounters significant
stress due to exposure to increased reactive oxygen and nitrogen species
(50,98). In addition, stress due to iron limitation is well known to affect virulence.
A reported phenotype of a corA strain is a lack of sensitivity to Fe2+ toxicity (85).
Therefore, the survival rates of wild-type (strains SL1344, LT2, and 14028s) and
corA and phoP strains were determined by challenge with different
concentrations of Fe2+ in liquid medium and protocols adapted both from Hantke and from Chamnongpol and Groisman (Figure 3.1, p.91 and Table 3.1, p.89). A
marked increase in Fe2+ sensitivity in three different phoP strains, as had been
reported by Chamnongpol and Groisman (28), was observed; however, altered
sensitivity to iron as a function of corA allele status, as reported by Hantke, could
not be demonstrated (Figure 3.1, p.91 and Table 3.2, p.90). Only a slight effect
of a 20 to 30% difference in survival could be observed for wild-type compared to
corA cells. Hantke observed 1 to 2 log differences. A similar lack of effect of a
corA mutation on iron sensitivity was also noted by Chamnongpol and Groisman.
Iron toxicity was independent of S. enterica serovar Typhimurium strain
background, whether LT2, SL1344, or 14028s (Table 3.2, p.90). Similar results
were also obtained from plate diffusion assays used to detect iron toxicity (data
not shown).
Reported changes in Fe2+ sensitivity from both Hantke and Chamnongpol
and Groisman were attributed to an ability of the CorA transporter to mediate
80 influx of Fe2+. However, these interpretations were inferred from indirect assays rather than from direct demonstration of Fe2+ interaction with CorA. We therefore
examined the effect of expression of CorA on Fe2+ accumulation.
CorA and iron accumulation. The only characterized primary Fe2+ (ferrous)
uptake system in S. enterica serovar Typhimurium, E. coli, and Helicobacter
pylori is FeoB (86,101,201). Although other transport systems such as MntH and
SitABCD transport Fe2+ when it is present at very high concentrations, Fe2+ is not
their primary substrate cation. We therefore measured the ability of wild-type,
corA, feoB, and corA feoB strains to accumulate iron.
If CorA provides any significant amount of Fe2+ uptake, its mutation should
diminish total Fe2+ accumulation after growth on typical laboratory medium.
While iron uptake results differed between experiments, the average amount (n =
11) of 55Fe2+ accumulation did not differ significantly between a wild-type versus a
corA strain or between a wild-type and an feoB strain (Figure 3.2, p.92). While
Hantke observed no difference in Fe2+ uptake between wild-type and feoB
strains, he did report a difference between wild-type and corA strains. We
suggest that this difference in results can be attributed to the very high variability
in iron accumulation from experiment to experiment, as indicated by the error
bars in Figure 3.2, p.92. We examined several parameters to reduce variability in
an attempt to better detect a possible difference in iron accumulation, including
type of buffer, presence or absence of Mg2+, iron concentration, use of EDTA in
wash buffers, and time of incubation, but could never detect consistent
differences or markedly reduce variability, most likely because iron binds tightly to
81 many biological molecules and cannot be readily removed without more severe
(and likely deleterious) means. Similar issues arise in measurement of 45Ca2+
uptake, as this lab has previously noted (78). Regardless, under the growth and
assay conditions used, the presence or absence of CorA had no apparent effect
on Fe2+ accumulation, an indication that CorA cannot mediate the uptake of iron.
Interestingly, by an unknown mechanism, the corA feoB strain accumulated more
Fe2+ than the wild type or the single mutants (Figure 3.2, p.92).
Effect of iron on CorA-mediated transport. We next determined the ability of
Fe2+ and Fe3+ to interact with CorA. If CorA is capable of transporting iron, then
iron must inhibit the uptake of other CorA substrates. 63Ni2+ has been used as a
surrogate due to the high cost and poor availability of 28Mg2+ (78,95,179). The
2+ 2+ apparent K0.5 of Mg for CorA is 10 to 20 µM, while that of Ni is 200 µM. If the
63 2+ total [ Ni ] in the assay solution is equal to the K0.5, then the concentration of a
63 2+ cation required to inhibit 50% of control Ni uptake (IC50) is exactly double the
true Ki for that cation by simple mass action law consideration. The ability of
Mg2+ with and without 1 mM ascorbate, Fe2+ with 1 mM ascorbate, and Fe3+ to inhibit 63Ni2+ uptake was measured for MM2089, a wild-type SL1344 S. enterica
serovar Typhimurium strain. Although this strain carries functional mgtA and mgtCB alleles in addition to corA, corA is constitutively expressed, and cells for
assay were grown overnight in LB, which contains sufficient Mg2+ to completely repress transcription of mgtA and mgtCB (174,187).
Neither Fe2+ nor Fe3+ could inhibit 63Ni2+ uptake via CorA at pH 7.0. At concentrations up to 20 µM Fe3+ (data not shown) or 100 µM Fe2+ (Figure 3.3,
82 p.93), neither iron species had any effect on 63Ni2+ uptake. At the assay pH of
7.0, higher concentrations of Fe2+ and Fe3+ precipitated during filtering; they did
not precipitate in stock solutions (Figure 3.3, p.193). Thus, it could not be determined whether very high concentrations of iron could inhibit CorA at pH 7.0.
Nonetheless, biological systems are highly unlikely to encounter free iron concentrations of this magnitude; therefore, for practical purposes, iron is not an
effective inhibitor of the CorA Mg2+ transporter. We also performed the same experiment at pH 5.8 since S. enterica serovar Typhimurium can encounter acid
conditions in some growth situations. Acid pH increases the apparent Vmax of
2+ CorA about twofold (177,186) and slightly decreases the K0.5 for Ni to about 100
µM. Again, neither Fe2+ nor Fe3+ had any effect on uptake via CorA at concentrations of up to 300 µM Fe2+ (Figure 3.3, p.93) or 300 µM Fe3+ (data not
shown). Since we have previously shown that S. enterica serovar Typhimurium
LT2, 14028s, and SL1344 strains have identical CorA transport properties and
since coding and promoter sequences are identical for all three strains, these
results are strain independent.
Effect of Mg2+, Co (III) hexaammine, and CorA on 55Fe2+ uptake. Mg2+
55 2+ inhibited uptake of Fe (1 µM) in wild-type cells with a K0.5 of 50 to 100 µM, a
2+ 2+ result several fold higher than the K0.5 of Mg for CorA. Moreover, Mg inhibition of 55Fe2+ uptake was identical in a corA strain (Figure 3.4, p.194), an
indication that the ability of Mg2+ to inhibit 55Fe2+ uptake was not due to interaction
with the CorA transporter. The ability of Mg2+ to inhibit 55Fe2+ uptake presumably
reflects a low-affinity interaction of Mg2+ with one or more iron transporters.
83
Finally, Co (III) hexaammine is a selective inhibitor of CorA, with a Ki of 1
µM. However, its half-maximal inhibitory concentration for inhibition of 55Fe2+
uptake was around 1 mM, about 3 logs higher than its Ki for CorA (Figure 3.4,
p.194). These data clearly indicate that Co (III) hexaammine does not affect
55Fe2+ uptake except at concentrations far higher than its effect on CorA.
84 Discussion
Chemistry of Fe2+ and Mg2+. A cation that is a substrate for a transport system
must in turn inhibit the transport of any other substrate cation. However, from the
viewpoint of the respective chemical properties of Fe2+ and Mg2+, it is highly
unlikely that Fe2+ would be transported by a system capable of selectively
interacting with and transporting Mg2+. Mg2+ has a far larger hydration shell than
does Fe2+. Mg2+ is a very hard Lewis acid, whereas Fe2+ is an intermediate-to- soft Lewis acid. While Mg2+ readily interacts with carboxylate and phosphate
anions in solution or on proteins (16,124), its transport by CorA (177,186) or by
MgtB (D.G. Kehres and M. E. Maguire, unpublished observations) does not
require electrostatic interactions, in apparent contrast to transport of iron
(26,113,162,212). Mg2+ is invariably hexacoordinate, with resulting Mg2+-ligand
bond angles of 90 ± 5° in the octahedron (35). In contrast, Fe2+ can be tetra- or
hexacoordinate, with far more flexibility in bond angles (35). Iron prefers in many
cases to bind to sulfur-containing ligands, whereas Mg2+ binds almost exclusively
to oxygen-containing ligands (35,124). Finally, although Mg2+ does not possess d orbital electrons, the other two known substrates of CorA, Co2+ and Ni2+, are high-spin d7 and d8 coordinate ligands whereas Fe2+ is usually a low spin d6 ligand in biological complexes (35,43,124). Overall, this combination of properties makes Fe2+ a very unlikely substrate for CorA. This does not preclude
however simple inhibition of transport by a non-substrate cation, since an
inhibitory cation need only interact with a part of the transport protein.
85 Our results differ from those of Hantke (85), who reported that mutation of
corA made E. coli and S. enterica serovar Typhimurium relatively resistant to
Fe2+-mediated killing. Neither we nor Chamnongpol and Groisman (28) observed any difference in Fe2+ toxicity between a wild-type and a corA strain of S. enterica
serovar Typhimurium. We have no explanation for this discrepancy. We and
Chamnongpol and Groisman previously (28) also observed a difference in iron
toxicity between phoP and wild-type strains. (These authors also reported that
introduction of a corA mutation into a phoP strain restored wild-type Fe2+ resistance, a phenotype we did not investigate.) However, an interpretation from
such experiments that corA cells were not transporting iron because they were
resistant to iron toxicity is premature without direct transport assays.
Furthermore, our data do not show that a corA strain accumulates less iron than a
wild-type strain, again differing from Hantke's results. We attribute this latter
difference to the highly variable nature of iron transport (see error bars in Figure
3.2 p.192). During preliminary experiments, transport assay conditions were
adjusted considerably in an effort to minimize such variability with 55Fe2+, with
incomplete success. Subsequently, of 11 independent experiments in which we
evaluated 55Fe2+ uptake, a corA strain accumulated slightly more iron than wild-
type in 5 experiments, slightly less iron in 4 experiments, and an approximately
equivalent amount in 2 experiments. Thus, it would be possible to observe a
decrease (or an increase) in iron uptake in a corA strain if only a few experiments were performed. These results are in contrast to those comparing wild-type or corA strains with a corA feoB strain, for which 10 or 11 experiments showed a
86 marked increase in iron uptake in the corA feoB strain compared to the results
seen with the others.
2+ Regardless of these accumulation experiments, the K0.5 of Mg inhibition
of 55Fe2+ uptake is many fold greater than would be expected if Mg2+ were
interacting with CorA, the dose response curve for Mg2+ inhibition of 55Fe2+ uptake is independent of the presence of the CorA transporter, and the ability of the CorA-selective inhibitor Co (III) hexaammine to inhibit 55Fe2+ uptake does not
correlate with its Ki for inhibition of CorA (Figure 3.4, p,194). Thus, even with the
experimental limitations noted, mutation of corA has no significant effect on
55Fe2+ uptake. These conclusions are emphasized by the demonstration, by
direct transport assay, that Fe2+ does not affect CorA-mediated transport of a known substrate, 63Ni2+ (Figure 3.3, p.193). One substrate of a transporter must inhibit flux of another substrate. Fe2+ does not inhibit CorA-mediated transport of
63Ni2+ even at high non-physiological iron concentrations (Figure 3.3, p.193).
Thus, we conclude that CorA cannot transport Fe2+.
Despite the reasonable presumptions that CorA might be considered a
"housekeeping" system and, further, that its mutation leads to no significant Mg2+-
dependent growth phenotype, loss of corA gives a surprising array of
phenotypes. These include altered transcription of PhoPQ-regulated genes,
altered expression of genes encoded by Salmonella pathogenicity island I,
increased sensitivity to heat shock and peroxide, and diminished virulence (See
Chapters 4 and 5). Thus, while it is possible that loss of corA may be a cause of
87 one or more phenotypes related to iron (28,85), the association is indirect and does not arise from an ability of CorA to transport iron.
88 TABLE 3.1. Bacterial strains used in this chapter
Reference or Strain Relevant Genotype Source MM385 S. Typhimurium LT2 corA::Tn10 This lab
MM1267 S. Typhimurium 14028s phoP E.A. Groismana
MM1269 S. Typhimurium 14028s (wild type) E.A. Groisman
MM1272 S. Typhimurium 14028s phoP E.A. Groisman
MM2089 S. Typhimurium SL1344 (wild type) B.B. Finlayb
MM2242 corA52::Tn10U16U17 MM385 x MM2089
MM2291 S. Typhimurium LT2 This lab
MM2363 S. Typhimurium SL1344 phoP M. Mahanc
MM2985 S. Typhimurium SL1344 UfeoB This study MM2242 X MM2986 corA52::Tn10U16U17 UfeoB MM2985 a Department of Molecular Microbiology, Washington University of St. Louis, St.
Louis, MO b Biotechnology Laboratory, University of British Columbia, British Columbia,
Canada c Department of Molecular, Cellular and Developmental Biology, University of
California Santa Barbara, Santa Barbara, CA
89 Table 3.2. Effect of Fe2+ on viability
[Fe2+] MM2089 MM1269 MM2242 MM1267 MM1272 MM2363 μM (SL1344) (14028s) (SL1344 (14028s (14028s (SL1344 corA) phoP) phoP) phoP) 10 56 ± 2 62 ± 6 45 ± 13 7 ± 3 32 ± 2 17 ± 4 100 57 ± 1 42 ± 3 35 ± 2 0.3 ± 0.2 1.5 ± 1 0.15 ± 0.1 500 44 ± 4 37 ± 1 21 ± 3 0 0.4 ± 0.1 0.3 ± 0.2
Values represent the average ± range of duplicates. Cells were grown and exposed to the indicated concentration of Fe2+ as indicated in Materials and
Methods. The data was calculated as the percent viability without added iron.
This experiment is representative of 2 additional experiments and of further experiments with different times of exposure and iron concentrations.
90
150
125
100
75 Percent Survival Wild type SL1344 50 SL1344 corA Wild type LT2 25 0 10 20 30 40 50 60 70
Time (minutes)
Figure 3.1. Fe2+ toxicity in S. Typhimurium. Iron toxicity was measured in wild type SL1344, LT2, and SL1344 corA strains grown overnight in tryptone yeast extract medium resuspended in HEPES after 0, 5, 20, 40, and 60 minute incubations with 30 μM Fe2+ in 1 mM ascorbate. A 20-30% decrease in survival
occurred for both wild type strains whereas the corA strain showed no decreased
survival. Data are an average of two independent experiments.
91
150
100
50
Percent Wild type Uptake
0 e oB oB e typ corA f fe d A Wil cor
Figure 3.2. Fe2+ uptake in S. Typhimurium. Iron accumulation was measured
in wild type, corA, feoB, and corA feoB strains after 20 min incubation using 1 μM
55Fe2+ and no Mg2+ as described in Materials and Methods. Ascorbate was
present at 1 mM throughout. Preliminary experiments had shown that maximal intracellular 55Fe2+ was reached after 15-20 min incubation. The data are the
average of 11 experiments. There is no significant difference in uptake between
any of the strains (P>0.05) except for the corA feoB strain which exhibits about a
50% increase in total accumulation compared to the other three strains (paired t
test, p<0.01).
92
140
120
uptake
2+ 100
Ni 63 80
60 Mg2+ + Asc
Mg2+ 40 Fe2+ + Asc
Percent of total 20 Fe2+ (pH 5) 0 0 -8 -7 -6 -5 -4 -3 -2 [Cation] (M)
Figure 3.3. Effect of iron on CorA-mediated transport. Using a final Ni2+ concentration of 200 μM, 63Ni2+ uptake was measured in wild type cells
(MM2089) as previously described (78,179). The effect of Fe2+ with 1 mM
ascorbate at pH 7 (■) and Fe2+ at pH 5.8 (□) is shown. Inhibition by Mg2+ with (●)
and without (○) 1 mM ascorbate is shown for comparison. Iron solutions were made fresh immediately before each experiment. The experiment shown is representative of three additional experiments. For each of the iron dose response curves, iron precipitated at concentrations higher than the last point shown. Experiments using Fe3+ also showed no inhibition (data not shown).
93
100
80
60 (Percent of Control)
40
Uptake Wild type + Mg2+ 2+ 20 Wild type + CoHex Fe
55 corA + Mg2+ 0 0 -6 -5 -4 -3 -2 -1
Log [Inhibitor] (M)
Figure 3.4. Effect of Mg2+ on 55Fe2+ uptake. The ability of Mg2+ (●) and Co
(III)hexaammine (■) in a wild type strain (MM2089) and of Mg2+ (○) in a corA
strain (MM2242) to inhibit 55Fe2+ uptake was measured in the same manner as
for 63Ni2+ uptake (Chapter 2). The final 55Fe2+ concentration was 1 μM.
Ascorbate was present at 1 mM. The Mg2+ data shown are the averages of at
least 4 independent experiments. The average initial uptake with no Mg2+
55 2+ present was approximately 1.03 nmol of Fe /OD600nm. Error bars are not
shown for clarity but were generally slightly larger than the size of the symbols.
For Co (III)hexaammine, the data is from a single experiment representative of
one additional experiment.
94 Chapter 4
Studies elucidating the role of CorA in Salmonella pathogenesis
Introduction
Salmonella enterica serovar Typhimurium causes infections that usually appear
as a gastrointestinal ailment in over 1.2 million Americans per year. Infection
begins with oral consumption of live bacteria in contaminated food. In the small
intestine, the bacteria are actively taken up by M cells of the Peyer’s patches
and/or mononuclear phagocytes. A T3SS carried on SPI1 delivers effector
proteins to the M cell to aid in invasion (58,76). Survival within the macrophage
phagosome requires another T3SS encoded on SPI2 (93,139,166). Thus, S.
Typhimurium’s environment fluctuates markedly during its pathogenic lifecycle
and requires the coordination of expression of a multitude of virulence effectors
and housekeeping genes. Furthermore, these gene products form dynamic
signaling networks that interact to control the infection process.
Mg2+ signaling has been shown to play an important role in virulence of
many bacteria (62). PhoP/PhoQ is a two component system that senses certain
stimuli including low Mg2+, antimicrobial peptides, and pH (4,131,183).
PhoP/PhoQ is essential to the virulence process and is involved in the regulation
of Salmonella survival in macrophages (52,58,75,131). PhoPQ activation
increases transcription of a number of genes including mgtA and mgtB which
encode two Mg2+ transport systems of S. Typhimurium expressed only in
response to low Mg2+ (4,55,62,122,132,174,188). This ability to sense Mg2+ is
95 required for Salmonella to survive within the host; thus, extracellular Mg2+ is associated with virulence.
Since Mg2+ transport is induced by PhoPQ, it is logical to ask if
intracellular Mg2+ of the bacterium is involved in virulence. To obtain Mg2+, S.
Typhimurium has three Mg2+ transport systems: CorA, the primary Mg2+ channel
is constitutively expressed while the MgtA and MgtB transporters are only expressed in response to PhoPQ activation
(4,55,62,92,95,122,132,174,180,187). CorA lacks homology to any other known protein and is widespread throughout the bacteria and archaea (96,118,122,172).
MgtA and MgtB are similar to eukaryotic P-type ATPases and are less widely distributed (121,122,180,188). Induction of MgtA and MgtB during infection implicates these Mg2+ transporters in virulence (64,174), we show here that mutation of corA results in a decrease in virulence after either oral or intraperitoneal infection.
96 Results
The corA strain is attenuated in the mouse. We wished to determine the virulence of a corA mutant as compared to wild type. Wild type killed all mice after either oral or intraperitoneal administration. When a corA mutant was administered to mice by the oral route, 50% of the mice survived and by the intraperitoneal route, 30% survived (Figure 4.1, p.120, and 4.2, p. 121). Also, the onset of illness and death was delayed as compared to wild type. A plasmid carrying the corA allele was able to completely rescue the virulence defect.
Thus, corA is required for full virulence in the mouse.
The transcriptome of a corA strain reveals repression of virulence effectors. To obtain a more global understanding of possible alterations in a corA strain, a microarray comparing corA and wild type strains was conducted using cells grown to log phase (Table 4.2, p.107-111) and stationary phase
(Table 4.3, p.112-119) in LB. Table 4.2 and Table 4.3 list the genes that were upregulated or repressed in the corA strain sorted by function or putative function. The array results for log phase cells were confirmed for selected genes by reverse transcription of isolated RNA followed by qPCR (Figure 4.3, p.122) and are discussed next. The array results for stationary phase cells were not replicated by reverse transcription of isolated RNA followed by qPCR and are discussed later.
SPI1 encodes a T3SS required for entry into epithelial M cells (58,76).
Components of SPI1 (e.g., invA, invB, invH) are repressed in a corA strain (Table
4.2, p.107-111). Western blots against InvH and SipC, SPI1 proteins, confirm
97 that protein expression is also decreased in a corA strain (Figure 4.4, p.123).
Antibodies were obtained from Simon Daefler and his protocols indicate that to
obtain the most robust expression of SPI1 proteins, cells should be grown to 0.5
OD600nm units in LB pH 6.0 and shifted to LB pH 8.0 and grown for an additional
hour. The pH shift experiments indicated that expression of SPI1 was altered in
a corA strain (Figure 4.5, p.124). However at some point, despite being able to
initially replicate these experiments, these experiments could no longer be
replicated to obtain a difference between a corA strain and wild type. Thus, the
method used to grown cells for the microarray (in 25 ml LB in 125 ml flasks to log
phase) was used to grow cells for this experiment. Using this growth method, the
defects of a corA strain in altered expression of SPI1 were replicated.
Additional changes in mRNA expression are apparent in a corA strain that
also have relevance to virulence. Several SPI2 genes (e.g., sseE, sseC, sscB)
required for survival within the macrophages (93,139,166) are also repressed in
a corA strain (Table 4.2, p.107-111). Some flagellar genes are required for S.
Typhimurium virulence (23). Flagellar genes (e.g., fljB, fliN, fliM) are also
repressed in a corA strain (Table 4.2, p.107-111). Consistent with this decreased
expression, a swimming assay using cells grown on LB plates with a low
percentage agar (Figure 4.6, p.125) show that a corA strain cannot swim as well
as wild type. Altogether, these results suggest that CorA is important for optimal
expression of multiple virulence effectors.
Of the 87 genes upregulated in a corA strain, about 20% are membrane or
putative membrane proteins. Two classes of membrane proteins that stand out
98 are sets of genes required for enterochelin-dependent iron uptake (entE, entC, fepA, etc) and curli production. Both iron transport and curli expression have been closely associated with virulence, although it is unclear how their upregulation in a corA strain is related to the virulence defect (57,192,194). We confirmed upregulation of siderophores by a liquid chrome azurol S (CAS) assay
(3). The CAS assay is a colormetric assay. If iron is bound by CAS, the solution remains blue. However, if siderophores are present and bind the iron, the solution turns orange. The upregulation of curli was confirmed by a Congo red binding assay (74) (Figures 4.7, p.126 and 4.8, p.127). The altered transcription of multiple inner and outer membrane proteins suggests that significant modification of both the inner and outer membrane results from mutation of corA.
Despite all these changes in membrane proteins, Caco-2 epithelial cells produce similar cytokines (TGFβ1, IL1β, and TNFα) in response to corA and wild type cells (Figures 4.9, p.128, 4.10, p.129, and 4.11, p.130). Thus, corA cells do not appear to be recognized differently from wild type by at least epithelial cells. The two other Mg2+ transporters, MgtA and MgtA, of Salmonella were also found to be upregulated in a corA mutant. The microarray only picked up mgtA and mgtC, but since mgtB and mgtC are co-transcribed, mgtB is most likely up as well.
Moreover, transcription of mgtA, mgtB, and mgtC was measured through chromosomal β-galactosidase reporters within the genes (Figure 4.12, p.131).
All three were upregulated in a corA mutant compared to wild type.
Microarray results from wild type and corA cells grown to stationary phase in LB are presented in Table 4.3, p.112-119. There is little overlap between log
99 and stationary phase results. The stationary phase data indicate that ribosomal proteins (e.g. rpsM, rpsF, rplT) are downregulated in a corA mutant. No other functional group could be separately from the set of genes identified. Several
DNA binding proteins (e.g. ttrR, phoB, and spvR), some of which are transcriptional activators were increased in a corA strain. Also, some ABC transport proteins (e.g. sfbC, mglB, and hisP) were increased in the corA strain.
The validity of these arrays is questionable. The raw signal from the slides was weak, thus many spots were not assigned intensity values. Moreover, the microarray results were not replicated by RTqPCR; opposite results were obtained for 4 of the 8 genes tested when compared to the array. Thus follow-up phenotype assays, as completed for log phase, were not completed for stationary phase cells. Overall, due to the inconsistent data no hypotheses or connections to the virulence phenotype can be discerned.
CorA is required for invasion and replication within epithelial cells. The microarray and Western blot results indicated that SPI1 encoded effectors were downregulated. These results suggest that epithelial cell invasion might be compromised in a corA strain. Therefore, a corA strain was tested for its ability to interact with cultured Caco-2 intestinal epithelial cells. corA cells have a significantly decreased ability to invade Caco-2 epithelial cells (Figure 4.13, p.132). In addition, by 4 to 6 hr post infection, a corA strain is 50% defective for replication inside Caco-2 epithelial cells compared to replication of wild type cells
(Figure 4.14, p.133). Thus, CorA is required for optimal invasion and replication within Caco-2 epithelial cells. The positive control for defective invasion, an invA
100 strain almost completely fails to invade and replicate within Caco-2 epithelial cells
(Figure 4.14, p.133). The corA defect can be rescued by complementation with a
functional S. Typhimurium corA allele (Figure 4.14, p.133).
Formation of Sifs (Salmonella induced filaments) is required for replication
within epithelial cells, thus Sif formation was visualized in wild type and a corA
strain. Immunohistochemistry using antibodies against Lamp-1 (a lysosomal
marker present on Sifs) was conducted (Figure 4.15, p.134). However, no conclusive data could be obtained.
CorA is weakly required for survival within macrophage-like cells. Since the microarray data indicated a corA mutant has SPI2 defects as well, we tested a corA strain’s capacity to enter and survive within cultured J774A.1 macrophage- like cells. Macrophage-like cells phagocytose corA cells with a similar efficiency as wild type cells (Figure 4.16, p.135). By 8-10 hr post infection, a corA strain is weakly decreased for survival inside J774A.1 macrophage-like cells compared to wild type cells (Figure 4.16, p.135). A phoP strain, a positive control, survives two-fold less in J774A.1 macrophage-like cells compared to wild type (Figure
4.16, p.135). Thus down-regulated expression of SPI2 in a corA strain minimally affects interactions with macrophages.
101 Discussion
We have found that a corA S. Typhimurium strain is attenuated for virulence in the mouse following both oral and intraperitoneal inoculation routes.
We conducted microarray experiments comparing wild type and corA strains to further examine this phenotype. The results suggest that CorA is required at multiple points during the infection process.
Genes downregulated in a corA strain.
Transcription of several genes encoding components of two pathogenicity islands and the flagellar regulon are repressed in a corA mutant during log phase growth in LB. Specifically, multiple genes encoding the T3SS resident in SPI1 show decreased transcription. Western blots of selected proteins of this T3SS confirmed the prediction of the microarray experiments that protein expression had been altered. The invasion and replication defects of the corA strain in
Caco-2 epithelial cells are consistent with the decreased expression of the T3SS encoded on SPI1 since these proteins are required for efficient invasion of epithelial cells (58,76).
In addition to decreased expression of SPI1, components of SPI2 are also transcriptionally repressed in a corA mutant. SPI2 is thought to be of primary importance for survival within macrophages (93,139,166). The decreased SPI2 expression is consistent with the attenuation of a corA strain when mice are infected via the intraperitoneal route, bypassing the trans-epithelial route.
However, there is only a weak effect on survival within the J774A.1 macrophage- like cell line. SPI2 has been shown to be expressed inside epithelial cells
102 alongside SPI1 (127). Moreover, SPI2 has been shown to be important for the development of enterocolitis, not just systemic disease (32). Thus, downregulation of SPI2 in a corA mutant may contribute to the epithelial cell phenotype as well as the weak macrophage phenotype. Alternatively, corA may also be needed at some later stage of infection.
Flagellar genes are repressed in a corA mutant. Consistent with the decreased expression of flagellar genes, a swimming assay comparing wild type and corA strains indicates that a corA strain does not swim as well as wild type
(Figure 4.6, p.125). Flagella may have some virulence properties that could aid in adherence, in bacterial movement to evade immune cells, or with movement
(chemotaxis) towards beneficial surroundings. However, only a handful of flagellar genes have been shown to be essential for Salmonella virulence; thus it is not clear if the swimming defect impacts virulence.
Microarrays were also conducted for wild type and corA cells grown to stationary phase in LB (Table 4.3, p.112-119). There was no overlap between genes downregulated in log and stationary phase. Most of the genes identified do not fit into a specific category. One group stood out, proteins which make up the 30S and 50S ribosomal subunits. It is unclear why these proteins would be decreased in a corA strain.
Genes upregulated in a corA strain.
A number of genes are also upregulated in a corA strain grown to log phase in LB (Table 4.2, p.107-111), including those encoding the enterochelin iron uptake system, curli, and many inner and outer membrane proteins. The
103 altered transcription of multiple inner and outer membrane proteins suggests that
significant modification of both the inner and outer membrane results from
mutation of corA. Despite these substantial changes in Salmonella membrane
proteins, Caco-2 epithelial cells produce a similar amount of TNFα, IGFβ and IL-
1 cytokines in response to corA and wild type strains (Figures 4.9, p.128, 4.10,
p.129, and 4.11, p.130) suggesting that host cell recognition is not altered.
Although iron is essential for virulence (57,192), it is unclear why a single
iron uptake system should be upregulated and whether its upregulation is
relevant to attenuation of a corA strain. A significant remodeling of both
membranes would seem to result from the loss of corA. However, without more
information on the function of these membrane proteins, no conclusions can be
made as to their role in the corA virulence phenotype. In stationary phase cells
grown in LB, several transcriptional activators and ABC transport proteins are
upregulated in a corA strain. The role they play in corA virulence phenotype is
also unclear.
Extracellular Mg2+ is important in terms of directly regulating mgtA and mgtCB expression. A mgtA mgtB double mutant is attenuated in the mouse after oral gavage despite the continued presence of a functional corA allele (D.G.
Kehres and M.E. Maguire, unpublished observations). Extracellular Mg2+ also
interacts with the PhoPQ two component system, which is essential for virulence
(59,75,131). Thus, both extracellular Mg2+ and all three Mg2+ transporters of S.
Typhimurium are important for virulence, adding additional complexity to the
network(s) responsible for Mg2+ homeostasis.
104 Our results indicate that CorA is required for virulence in some manner
presumably to modulate transport and cellular Mg2+ content. Given the variety of
pathways affected by loss of CorA, we hypothesize that CorA may be part of a
broader signaling network within the cell. Chapter 5 focuses on understanding
the possible mechanism(s) of CorA’s involvement in virulence and the data
suggest that regulation of CorA is important for S. Typhimurium virulence.
Current experiments are focused on understanding this apparent regulation of
CorA expression and function and on delineation its connection to S.
Typhimurium virulence.
105 Table 4.1. Bacterial strains and plasmids used in this study.
Strain Genotype Source
MM1364 SL1344 invA::Tn10::phoA R. Maurera Salmonella enterica Serovar Typhimurium B.B. MM2089 SL1344 Finlayb MM2242 SL1344 corA52::Tn10 16 17 J. Lina
MM2320 SL1344 corA52::Tn10 16 17 pJL10 J. Lina
MM2249 SL1344 mgtC::lacZ J. Lina
MM2250 SL1344 mgtA::lacZ J. Lina
MM2251 SL1344 mgtB::lacZ J. Lina
MM2257 SL1344 mgtC::lacZ corA52::Tn10 16 17 J. Lina
MM2258 SL1344 mgtA::lacZ corA52::Tn10 16 17 J. Lina
MM2259 SL1344 mgtB::lacZ corA52::Tn10 16 17 J. Lina J. S. MM3220 14028s phoP::Tn10cam Gunnc WN152 14028s phoP ::Tn10dCm 131 aCase Western Reserve University, Cleveland, OH 44106, USA. bMichael Smith Laboratories, University of British Columbia, Vancouver, BC V6T
1Z4, Canada. cCenter for Microbial Interface Biology, The Ohio State University, Columbus, OH
43210, USA.
106 Table 4.2. Differences in gene expression between wild type and corA strains.
A cDNA microarray was used to determine genes differentially repressed or upregulated in a corA strain compared to a wild type strain. RNA was isolated from cells grown to log phase in LB. Genes with altered expression and the fold change are indicated and are placed into categories based on function or putative function. Only expression changes with p values less then 0.05 are listed.
Gene Gene Fold p value Description number symbol Change Genes repressed in ΔcorA SPI-1 STM2900 invH -3.2 0.03 Invasion protein STM2897 invE -2.9 0.01 Invasion protein Surface presentation of antigens; STM2895 invB -4.4 0.04 secretory proteins STM2896 invA -3.5 0.01 Invasion protein SPI-2 STM1402 sseE -3.6 0.01 Secretion system effector STM1400 sseC -2.6 0.05 Secretion system effector STM1397 sseA -4.5 0.01 Secretion system effector STM1403 sscB -2.1 0.05 Secretion system chaperone Secretion system apparatus: STM1414 ssaV -2.4 0.05 homology with the LcrD family of proteins STM1416 ssaO -2.3 0.03 Secretion system apparatus STM1411 ssaK -2.8 0.02 Secretion system apparatus STM1408 ssaI -3.1 0.02 Secretion system apparatus FLAGELLA and CHEMOTAXIS Flagellar synthesis: phase 2 STM2771 fljB -2.6 0.01 flagellin (filament structural protein) Flagellar biosynthesis, STM1977 fliN -2.1 0.05 component of motor switch and energizing Flagellar biosynthesis, STM1976 fliM -2.3 0.05 component of motor switch and energizing STM1975 fliL -2.3 0.03 Flagellar biosynthesis Methyl-accepting chemotaxis STM 1626 trg -2.8 0.04 protein III, ribose and galactose sensor receptor METABOLISM
107 Putative fructose-1,6- STM4078 yneB -2.3 0. 01 bisphosphate aldolase STM3202 ygiF -2.2 0.01 Putative cytoplasmic protein Putative ABC superfamily STM4076 ydeZ -2.1 0.01 (membrane), sugar transport protein Paralogous putative STM0962 ycaJ -2.2 0.03 polynucleotide enzyme Tetrathionate reductase STM1386 ttrS -2.6 0.02 complex: sensory transduction histidine kinase Putative 3-hexulose-6-phosphate STM3676 sgbU -2.3 0.02 isomerase Aspartokinase II in bifunctional STM4101 metL -2.5 0.03 enzyme: aspartokinase II; homoserine dehydrogenase II Dihydroxynaphtoic acid STM2307 menB -2.1 0.02 synthetase Octaprenyl diphosphate STM3305 ispB -2.3 0.03 synthase Fumarate reductase, anaerobic, STM4342 frdB -2.2 0.02 Fe-S protein subunit Putative carboxysome structural STM2456 eutL -2.5 0.04 protein, ethanolamine utilization Putative carboxysome structural STM2455 eutK -2.5 0.02 protein, ethanolamine utilization Sensory histidine kinase in two- component regulatory system STM4304 dcuS -2.1 0.01 with DcuR, senses fumarate/C4- dicarboxylate Outer membrane N-acetyl STM0570 apeE -2.2 0.03 phenylalanine beta-naphthyl ester-cleaving esterase OTHER STM1235 ymfB -2.0 0.03 Putative MutT-like protein STM3613 yhjJ -2.0 0.01 Putative Zn-dependent peptidase STM3187 ygiB -2.6 0.01 Putative inner membrane protein STM1804 ycgB -2.4 0.01 Putative cytoplasmic protein STM0308 yafV -2.3 0.01 Putative amidohydrolases Modulator of enterobacterial STM3919 wzzE -2.4 0.05 common antigen (ECA) polysaccharide chain length STM1298 topB -2.5 0.01 DNA topoisomerase III Sigma N (sigma 54) factor of RNA polymerase, pleiotropic STM3320 rpoN -2.4 0.05 functions (nitrogen metabolism, formate degradation, phage shock response) STM4461 pyrL -2.4 0.05 PyrBI operon leader peptide STM4580 nadR -2.0 0.01 NadAB transcriptional regulator P-type ATPase, high-affinity STM0705 kdpB -2.9 0.01 potassium transport system, B chain
108 Transcriptional repressor STM4094 cytR -3.6 0.01 (GalR/LacI family) Genes activated in ΔcorA ENTEROCHELIN Putative hydrolase of the STM2776 iroE 3.9 0.03 alpha/beta superfamily STM0586 fes 2.4 0.03 Enterochelin esterase Outer membrane porin, receptor for ferric enterobactin STM0585 fepA 3.2 0.01 (enterochelin) and colicins B and D 2,3-dihydroxybenzoate-AMP STM0596 entE 3.5 0.01 ligase Isochorismate synthetase, STM0595 entC 2.6 0.03 enterochelin biosynthesis 2,3-dihydro-2,3- STM0597 entB 2.2 0.01 dihydroxybenzoate synthetase, isochorismatase CURLI Putative transcriptional regulator STM1139 csgG 2.8 0.04 in curly assembly/transport, 2nd curli operon Curli production STM1140 csgF 2.3 0.04 assembly/transport component, 2nd curli operon Putative transcriptional regulator STM1142 csgD 2.1 0.03 (LuxR/UhpA family) Curlin major subunit, coiled STM1144 csgA 2.2 0.02 surface structures; cryptic OTHER MEMBRANE OR MEMBRANE ASSOCIATED STM2407 ypeC 2.3 0.03 Putative periplasmic protein Putative ABC-type STM2263 yojI 2.3 0.01 multidrug/protein/lipid transport system, ATPase component Putative MFS family transport STM4517 yjjO 2.2 0.01 protein STM4514 yjjH 2.0 0.01 Putative inner membrane protein Putative APC family, amino acid STM4345 yjeM 2.0 0.03 transport protein STM4332 yjeJ 2.3 0.01 Putative inner membrane protein STM3953 yigF 3.1 0.04 Putative inner membrane protein STM2983 ygdI 2.1 0.03 Putative lipoprotein STM2671 yfjR 3.2 0.01 Putative periplasmic protein Putative serine/threonine protein STM2520 yfgL 2.2 0.01 kinase Putative membrane STM2439 yfeL 2.5 0.02 carboxypeptidase (penicillin- binding protein) STM1214 ycfR 5.6 0.01 Putative outer membrane protein Putative outer membrane STM1212 ycfJ 2.4 0.04 lipoprotein STM0816 ybhS 3.6 0.04 Putative ABC superfamily
109 (membrane) transport protein Putative ABC-type multidrug STM0172 yadG 2.5 0.05 transport system, ATPase component Putative DMT superfamily STM4338 sugE 2.1 0.01 transport protein STM1881 yebF 2.9 0.03 Putative periplasmic protein ABC superfamily (atp_bind), STM1226 potA 2.2 0.04 spermidine/putrescine transporter ABC superfamily (membrane), STM1744 oppC 2.6 0.04 oligopeptide transport protein STM3764 mgtC 5.0 0.03 Mg2+ regulated protein STM4456 mgtA 2.1 0.01 P-type ATPase, Mg2+ transporter STM1240 envF 2.5 0.01 Putative envelope lipoprotein STM3224 ygjT 2.3 0.03 Putative resistance protein Putative alternative beta subunit STM1458 ydgM 2.1 0.03 of Na+-transporting NADH: ubiquinone oxidoreductase Suppressor of lon; inhibitor of cell division and FtsZ ring formation STM1071 sulA 3.1 0.01 upon DNA damage/inhibition, HslVU and Lon involved in its turnover LexA regulated gene, putative STM1321 ydjM 2.2 0.02 SOS response Putative fimbrial chaperone STM4573 stjC 2.5 0.02 protein STM3372 mreD 3.1 0.03 Rod shape-determining protein Cold shock-induced palmitoleoyl STM2401 ddg 2.3 0.05 transferase Suppressor of ompF assembly STM2120 asmA 2.3 0.01 strains STM3442 hopD 4.5 0.02 Leader peptidase HopD TRANSCRIPTION REGULATION Putative transcriptional repressor STM4127 yijC 2.4 0.01 (TetR/AcrR family) Putative transcriptional regulator, STM0256 yafC 2.3 0.01 LysR family Error-prone repair: SOS- response transcriptional STM1998 umuD 2.5 0.01 repressors (LexA homologs, RecA-mediated autopeptidases) Positive transcriptional regulator STM1982 rcsA 2.1 0.01 of capsular/exo- polysaccharide synthesis (LuxR/UhpA family) STM4402 ytfH 2.3 0.02 Putative transcriptional regulator STM3759 mart 2.0 0.04 Putative transcriptional regulator STM1924 flhC 2.3 0.04 Flagellar transcriptional activator OTHER Putative hydrolase of the HAD STM3950 yigB 2.6 0.02 superfamily
110 STM2801 ygaC 2.5 0.01 Putative cytoplasmic protein STM1059 ycbW 2.1 0.03 Putative cytoplasmic protein Putative hydrolase of the HAD STM0840 ybiV 2.8 0.04 superfamily Putative acyltransferase in STM2110 wcaF 3.3 0.01 colanic acid biosynthesis Hydoxyethylthiazole kinase (THZ STM2147 thiM 2.5 0.01 kinase) STM2387 sixA 2.1 0.01 Phosphohistidine phosphatase Putative membrane protein STM2215 Rtn 2.5 0.01 involved in resistance to lambda and N4 phages 50S ribosomal subunit protein STM3439 rplD 2.5 0.03 L4, regulates expression of S10 operon STM3922 rffG 2.3 0.01 dTDP-glucose 4,6-dehydratase STM2097 rfbB 2.1 0.05 dTDP-glucose 4,6-dehydratase STM2058 pduX 2.2 0.01 Propanediol utilization ribonucleoside-diphosphate STM2808 nrdF 2.3 0.04 reductase 2, beta subunit Putative cytoplasmic protein STM3325 yrbL 2.3 0.01
STM3023 yohL 2.2 0.01 Putative cytoplasmic protein NifU homologs involved in Fe-S STM2542 nifU 2.5 0.01 cluster formation Macrophage survival gene; STM1241 msgA 2.0 0.04 reduced mouse virulence Acyl-CoA synthetase (long-chain- STM1818 fadD 2.2 0.01 fatty-acid—CoA ligase) STM0635 lipB 2.2 0.03 Putative ligase STM0491 gsk 2.3 0.03 Inosine-guanosine kinase Bifunctional 5,10-methylene- tetrahydrofolate dehydrogenase STM0542 fold 2.0 0.01 and 5,10-methylene- tetrahydrofolate cyclohydrolase
111 Table 4.3. Differences in gene expression between wild type and corA strains.
A cDNA microarray was used to determine genes differentially repressed or upregulated in a corA strain compared to a wild type strain. RNA was isolated from cells grown to stationary phase in LB. Genes with altered expression and the fold change are indicated and are placed into categories based on function or putative function. Only expression changes with p values less then 0.05 are listed.
Gene number Gene Fold p value Description symbol Change Genes repressed in ΔcorA RIBOSOME STM3418 rpsM 2.4 0.01 30S ribosomal subunit protein S13 STM4391 rpsF 1.8 0.01 30S ribosomal subunit protein S6 STM1336 rplT 1.9 0.04 50S ribosomal subunit protein L20 STM1336 rplT 3.2 0.01 50S ribosomal subunit protein L20 STM3433 rplP 2.6 0.01 50S ribosomal subunit protein L16 STM3437 rplB 1.8 0.03 50S ribosomal subunit protein L2 OTHERS STM4531 yjiX 2.7 0.01 Putative cytoplasmic protein STM3646 yiaE 3.0 0.01 2-keto-D-gluconate reductase STM2957 ygcA 2.2 0.02 Putative RNA methyltransferase STM2438 yfeK 1.8 0.01 Putative periplasmic protein STM0940 ybjX 1.9 0.01 Homologue of virK STM0876 ybjN 2.9 0.05 Putative cytoplasmic protein STM0785 ybhE 2.0 0.03 Putative 3-carboxymuconate cyclase STM0667 ybeX 1.9 0.04 Putative CBS domain- containing protein STM0636 ybeD 1.6 0.05 Putative cytoplasmic protein STM0466 ybaZ 1.9 0.04 Putative methyltransferase STM0308 yafV 3.6 0.03 Putative amidohydrolases STM2103 wcaJ 1.8 0.04 Putative UDP-glucose lipid carrier transferase/glucose-1- phosphate transferase in colanic acid gene cluster
112 STM2781 virK 4.4 0.01 Virulence gene; homologous sequence to virK in Shigella STM3951 uvrD 1.4 0.01 DNA-dependent ATPase I and helicase II STM1683 tyrR 2.8 0.03 Transcriptional regulator of aromatic amino acid biosynthesis genes (aroF, aroG, tyrA) and aromatic amino acid transport, has intrinsic ATPase and phosphatase activity (EBP family) STM_PSLT100 traH 1.4 0.03 Conjugative transfer: assembly STM0447 Tig 2.7 0.04 Peptidyl-prolyl cis/trans isomerase, trigger factor; a molecular chaperone involved in cell division STM1401 sseD 1.9 0.02 Secretion system effector STM1295 sppA 1.4 0.02 Protease IV, a signal peptide peptidase STM3455 slyD 2.4 0.02 FKBP-type peptidyl prolyl cis- trans isomerase (rotamase) STM3741 rpoZ 1.8 0.02 RNA polymerase, omega subunit STM1349 Pps 1.5 0.05 Phosphoenolpyruvate synthase STM2501 Ppk 2.4 0.02 Polyphosphate kinase, component of RNA degradosome STM1326 pfkB 2.0 0.05 6-phosphofructokinase II STM0749 Pal 4.3 0.04 Tol protein required for outer membrane integrity, uptake of group A colicins, and translocation of phage DNA to cytoplasm STM1572 nmpC 1.4 0.03 New outer membrane protein; predicted bacterial porin STM2925 nlpD 2.2 0.01 Lipoprotein STM2543 nifS 3.7 0.02 Putative aminotransferase class-V STM0145 nadC 3.1 0.01 Quinolinate phosphoribosyltransferase STM0357 Mod 1.5 0.03 DNA methylase; restriction system STM2330 lrhA 1.9 0.04 NADH dehydrogenase transcriptional repressor (LysR family) STM1334.c infC 2.8 0.01 Protein chain initiation factor IF- 3 STM0953 infA 2.6 0.04 Protein chain initiation factor IF- 1 STM2283 glpT 2.5 0.04 MFS family, sn-glycerol-3-
113 phosphate transport protein STM3011 galR 1.8 0.01 Transcriptional repressor of galETK operon (GalR/LacI family) STM0219 Frr 1.7 0.04 Ribosome releasing factor STM0013 dnaJ 1.7 0.02 Heat shock protein, DnaJ and GrpE stimulates ATPase activity of DnaK STM3967 dlhH 1.8 0.04 Putative dienelactone hydrolase family STM2946 cysH 1.7 0.01 3'-phosphoadenosine 5'- phosphosulfate (PAPS) reductase STM3699 cysE 1.8 0.05 Serine acetyltransferase STM2021 cboQ 2.2 0.04 Synthesis of vitamin B12 adenosyl cobalamide precursor STM4122 argB 3.3 0.04 Acetylglutamate kinase STM4598 arcA 1.9 0.03 Response regulator (OmpR family) in two-component regulatory system with ArcB (or CpxA), regulates genes in aerobic pathways STM1567 adhP 1.4 0.04 Alcohol dehydrogenase, propanol preferring Genes activated in ΔcorA DNA-BINDING STM2145 yegW 2.0 0.01 Putative regulatory protein, gntR family STM1487 ynfL 1.5 0.02 Putative transcriptional regulator, LysR family STM1588 yncC 1.4 0.03 Putative regulatory protein, gntR family STM3790 uhpA 2.5 0.02 Response regulator (repressor) in two-component regulatory system wtih UhpB, regulates uhpT operon (LuxR/UhpA family) STM1387 ttrR 2.3 0.01 Tetrathionate reductase complex: response regulator STM2866 sprB 1.7 0.03 Transcriptional regulator STM_PSLT041 spvR 1.8 0.02 Salmonella plasmid virulence: regulation of spv operon, lysR family STM0397 phoB 2.6 0.02 Response regulator in two- component regulatory system with PhoR (or CreC), regulates pho regulon (OmpR family) STM0450 Lon 2.2 0.02 DNA-binding, ATP-dependent protease la; cleaves RcsA and SulA, heat shock k-protein
114 (DNA binding activity) STM0702 kdpE 2.9 0.03 Response regulator in two- component regulatory system with KdpD, regulates kdp operon encoding a high-affinity K translocating ATPase (OmpR family) STM3908 ilvY 1.5 0.03 Positive regulator for ilvC (LysR family) STM2867 hilC 1.5 0.03 Bacterial regulatory helix-turn- helix proteins, araC family STM2813 emrR 2.8 0.01 Transcriptional repressor of emrAB operon (MarR family) STM0313 dinP 2.0 0.01 DNA polymerase IV, devoid of proofreading, damage-inducible protein P ABC TRANSPORT STM0512 sfbC 1.7 0.02 Putative binding-protein- dependent transport systems inner membrane component STM4063 Sbp 1.4 0.05 ABC superfamily (bind_prot), sulfate transport protein STM2190 mglB 1.6 0.02 ABC superfamily (peri_perm), galactose transport protein STM2351 hisP 1.9 0.02 ABC superfamily (atp_bind), histidine and lysine/arginine/ornithine transport protein STM1954 fliY 2.7 0.01 Putative periplasmic binding transport protein
OTHERS STM4172 zraP 1.8 0.04 Zinc-resistance associated protein STM4016 yshA 2.7 0.05 Putative outer membrane protein STM3325 yrbL 2.1 0.05 Putative cytoplasmic protein STM3317 yrbK 1.7 0.04 Putative inner membrane protein STM3185 yqiE 1.7 0.03 Putative resistance protein STM2964 yqcB 1.9 0.01 Putative synthase STM1984 yodD 2.5 0.02 Putative cytoplasmic protein STM1823 yoaH 2.7 0.05 Putative cytoplasmic protein STM1237 ymfC 1.6 0.03 Putative ribosomal large subunit pseudouridine synthase STM4437 yjgA 2.4 0.01 Putative cytoplasmic protein STM4018 yihP 1.5 0.02 Putative GPH family transport protein STM3971 yigP 1.4 0.01 Putative inner membrane protein
115 STM3969 yigN 1.5 0.01 Putative inner membrane protein STM3582 yhhT 1.7 0.01 Putative PerM family permease STM3579 yhhQ 2.1 0.05 Putative integral membrane protein STM3363 yhcO 1.8 0.04 Putative cytoplasmic protein STM3361 yhcN 2.6 0.02 Putative outer membrane protein STM3107 yggN 1.8 0.02 Putative periplasmic protein STM2388 yfcX 1.8 0.02 Paral putative dehydrogenase STM2123 yegE 1.8 0.05 Putative PAS/PAC domain; Diguanylate cyclase/phosphodiesterase domain 1, Diguanylate cyclase/phosphodiesterase domain 2, STM1903 yecE 1.4 0.04 Putative cytoplasmic protein STM1902 yecD 2.1 0.03 Putative isochorismatase STM1848 yebS 2.2 0.01 Putative inner membrane protein STM1880 yebE 1.8 0.05 Putative inner membrane protein STM1466 ydgA 1.9 0.05 Putative periplasmic protein STM1684 ycjF 1.8 0.02 Putative inner membrane protein STM1811 ycgN 1.5 0.01 Putative cytoplasmic protein STM1164 yceB 1.7 0.01 Putative outer membrane lipoprotein STM1164 yceB 1.6 0.01 Putative outer membrane lipoprotein STM1164 yceB 1.6 0.01 Putative outer membrane lipoprotein STM0986 ycaQ 1.8 0.02 Putative cytoplasmic protein STM0661 ybeK 1.6 0.03 Putative purine nucleoside hydrolase STM0500 ybbJ 1.5 0.02 Putative Membrane protein implicated in regulation of membrane protease activity STM0498 ybaR 2.4 0.02 Putative copper-transporting ATPase STM0262 yafS 2.7 0.02 Putative SAM-dependent methyltransferase STM0120 yabC 1.7 0.03 Putative S-adenosyl methionine adenyltransferase STM0006 yaaJ 1.5 0.03 Putative AGCS family, alanine/glycine transport protein STM2114 wcaB 2.3 0.01 Putative acyl transferase in colanic acid biosynthesis STM3590 uspB 2.4 0.01 Universal stress protein B, involved in stationary-phase
116 resistance to ethanol STM2647 Ung 1.4 0.01 Uracil-DNA-glycosylase STM2669 tyrA 1.4 0.02 Bifunctional: chorismate mutase T; prephenate dehydrogenase STM_PSLT102 traS 1.8 0.02 Conjugative transfer: surface exclusion STM3825 tort 1.5 0.01 Periplasmic sensor in multi- component regulatory system with TorS (sensory kinase) and TorR (regulator), regulates tor operon STM2784 tctE 1.6 0.04 Tricarboxylic transport: regulatory protein STM2967 Syd 3.8 0.04 Interacts with secY STM0177 stiA 1.7 0.03 Putative fimbrial subunit STM_PSLT038 spvC 1.5 0.01 Salmonella plasmid virulence: hydrophilic protein STM0800 slrP 1.4 0.03 Leucine-rich repeat protein STM3675 sgbH 1.7 0.01 Putative 3-hexulose-6- phosphate isomerase STM0408 secF 1.7 0.02 Preprotein translocase, IISP family, membrane subunit STM2222 rsuA 1.4 0.05 16S rRNA pseudouridylate 516 synthase STM3483 Rpe 1.5 0.01 D-ribulose-5-phosphate 3- epimerase STM3710 rfaD 3.3 0.04 ADP-L-glycero-D- mannoheptose-6-epimerase STM3594 prlC 1.7 0.03 Oligopeptidase A STM1064 pqiB 1.5 0.02 Paraquat-inducible protein B STM4148 nusG 1.9 0.04 Component in transcription antitermination STM2808 nrdF 1.6 0.03 Ribonucleoside-diphosphate reductase 2, beta subunit STM2255 napC 1.5 0.01 Periplasmic nitrate reductase, cytochrome c-type protein STM0928 nanH 1.8 0.03 Sialidase (neuraminidase) STM0124 murF 1.4 0.05 D-alanine:D-alanine-adding enzyme STM1153 msyB 4.0 0.01 Acidic protein suppresses mutants lacking function of protein export STM2310 menF 1.8 0.01 Isochorismate synthase (isochorismate hydroxymutase 2), menaquinone biosynthesis STM0215 Map 1.5 0.01 Methionine aminopeptidase STM2472 maeB 2.1 0.03 Paral putative transferase STM2855 hypB 1.6 0.03 Hydrogenase-3 accessory protein, assembly of metallocenter
117 STM0788 hutG 2.2 0.04 Formimionoglutamate hydrolase STM1107 hpaX 1.8 0.04 4-hydroxyphenylacetate catabolism STM4363 hflK 1.4 0.03 With HflC, part of modulator for protease specific for FtsH phage lambda cII repressor STM0936 Hcr 1.6 0.04 NADH oxidoreductase for hcp gene product STM1165 grxB 2.0 0.04 Glutaredoxin 2 STM3655 glyS 1.4 0.05 Glycine tRNA synthetase, beta subunit STM0525 glxK 1.8 0.01 Glycerate kinase II STM2282 glpQ 1.7 0.04 Glycerophosphodiester phosphodiesterase, periplasmic STM3538 glgB 2.0 0.02 1,4-alpha-glucan branching enzyme STM3247 garK 1.6 0.01 Glycerate kinase STM2206 fruF 1.6 0.05 Phosphoenolpyruvate- dependent sugar phosphotransferase system, EIIA 2 STM1959 fliC 2.9 0.01 Flagellar biosynthesis; flagellin, filament structural protein STM1174 flgB 1.7 0.04 Flagellar biosynthesis, cell- proximal portion of basal-body rod STM3470 Fic 1.6 0.02 Putative cell filamentation protein, stationary phase induced gene, affects cell division STM4034 fdhE 1.6 0.03 Putative formate dehydrogenase formation protein STM2468 eutQ 1.5 0.01 Putative ethanolamine utilization protein STM3070 Epd 1.6 0.02 D-erythrose 4-phosphate dehydrogenase STM2519 engA 2.0 0.05 Putative GTP-binding protein STM2814 emrA 2.2 0.01 Multidrug resistance secretion protein STM4334 Efp 1.5 0.03 Elongation factor P (EF-P) CHIP7_STM1985 dsrA 2.0 0.05 A small RNA antisilencer of the H-HS-silenced rdsA gene in E. coli STM0966 dmsC 1.6 0.01 Anaerobic dimethyl sulfoxide reductase, subunit C STM2984 csdA 1.4 0.01 Putative selenocysteine lyase STM0630 crcB 1.7 0.03 High-copy crc-csp restores normal chromosome
118 condensation in presence of camphor or mukB mutations STM1315 celD 1.7 0.02 Transcriptional repressor of cel operon (AraC/XylS family) STM1314 celC 1.9 0.03 PTS family, sugar specific enzyme III for cellobiose, arbutin, and salicin STM4130 btuB 1.4 0.04 Outer membrane receptor for transport of vitamin B12, E colicins, and bacteriophage BF23 STM4339 Blc 1.6 0.03 Outer membrane lipoprotein (lipocalin) STM4120 argE 1.5 0.04 Acetylornithine deacetylase STM2264 alkB 2.0 0.03 DNA repair system specific for alkylated DNA STM2124 alkA 2.0 0.03 3-methyl-adenine DNA glycosylase II, inducible STM3390 acre 1.7 0.01 Transmembrane protein affecting septum formation and cell membrane permeability STM2337 ackA 1.6 0.01 Acetate kinase A (propionate kinase 2) STM0955 Aat 1.4 0.01 Leucyl, phenylalanyl-tRNA- protein transferase
119
100
80
60
40
survival Percent MM2089 (7E9) MM2089 (7E8) 20 MM2242 (5E9) MM2242 (5E8) MM2320 (7E8) 0 0 10 20 Days
Figure 4.1. Attenuation of a corA strain after oral administration. Mice were infected by oral gavage (with either wild type (MM2089), a corA strain (MM2242), or a corA strain with a plasmid carrying a functional corA allele (MM2320) as described in Chapter 2.
120
100 MM2089 (700) MM2242 (900) 80 MM2320 (850)
60
40
survival Percent
20
0 0 10 20 Days
Figure 4.2. Attenuation of a corA strain after intraperitoneal administration.
Mice were infected by intraperitoneal injection with either wild type (MM2089), a
corA strain (MM2242), or a corA strain with a plasmid carrying a functional corA
allele (MM2320) as described in Chapter 2.
121
4
2
0
-2
-4
Fold change -6
-8
-10
iroE entE invA invB invH fepA entC sseE ssaK csgA
Figure 4.3. RTqPCR of selected genes. Total RNA was isolated from wild type
(MM2089) and corA strains (MM2242) grown in LB to log phase. RNA was reverse transcribed, and qPCR using SYBR green was conducted on the resulting cDNA. Data were analyzed and fold changes as compared to a dnaK
internal control are plotted. The qPCR is the average of two independent
experiments completed in triplicate. p values indicate t tests comparing wild type
(MM2089) and corA strains (MM2242) normalized to a dnaK control.
122
WT ΔcorA WT ΔcorA
DnaK
CorA
SipC
InvH
Figure 4.4. SPI1 Western blot. Western blots of SPI1 proteins were performed using total protein from wild type (MM2089) and a corA strain (MM2242) after growth in LB medium to log phase. The Western blots are representative of four independent experiments. Two independent experiments are represented in the figure.
123
Figure 4.5. SPI1 pH shift Western blot. Western blots of SPI-1 proteins were performed using total protein from wild type (MM2089) and a corA strain
(MM2242) after pH shift.
124
Figure 4.6. Motility assay. Wild type (MM2089) (left) and corA (MM2242)
(right) strains were plated at the center of swim agar plates and allowed to grow at room temperature for 20 hr. The data are representative of three independent experiments.
125
0.3 WT ΔcorA
0.2 640nm OD
0.1
0.0 2 hours 3 hours 4 hours
Figure 4.7. CAS assay. Wild type and the corA mutant were grown in LB to log
phase. Cells were spun down and supernatants were filtered with a 0.45 um
filter. Then chrome-S-azurol solution was added at a 1:1 dilution to culture
supernatants and incubated at room temperature for 2, 3, and 4 hours. At the
time points indicated, solutions were read at OD640 nm.
126 100
80
60 relative tocontrol 40 500nm OD
20
0 Control Wild type ΔcorA
Figure 4.8. Congo red binding assay. Wild type and the corA mutant were grown to log phase in LB. Cultures were centrifuged and pellets were resuspended in 0.002% Congo red in PBS and incubated at 37°C for 1-2 hours.
Congo red solution alone was also incubated as a control. All samples were read at OD500nm on a spectrophotometer with PBS used to blank.
127 ΔinvA WT ΔcorA
1 ΔcorA pCorA
β 150
100 pg per ml of TGF of ml pg per
50 1 2 3 4 5 6 Hours
Figure 4.9. ELISA for TGFβ1. Caco-2 epithelial cells were infected for 1 hour by an invA mutant, wild type, a corA mutant, and a corA mutant pCorA. Media
was removed 1, 2, 4, and 6 hours after infection, filtered, and levels of TGFβ1
was measured by ELISA.
128 150 ΔinvA WT ΔcorA ΔcorA pCorA β 100
pg per ml per pg IL1 50
0 1 2 3 4 5 6 Hours
Figure 4.10. ELISA for IL1β. Caco-2 epithelial cells were infected for 1 hour by an invA mutant, wild type, a corA mutant, and a corA mutant pCorA. Media was removed 1, 2, 4, and 6 hours after infection, filtered, and levels of IL1β was measured by ELISA.
129 25 α
15
5 ΔinvA pg per ml of TNF WT ΔcorA ΔcorA pCorA
-5 1 2 3 4 5 6 Hours
Figure 4.11. ELISA for TNFα. Caco-2 epithelial cells were infected for 1 hour by an invA mutant, wild type, a corA mutant, and a corA mutant pCorA. Media was removed 1, 2, 4, and 6 hours after infection, filtered, and levels of TNFα was measured by ELISA.
130 5 20 ΔmgtC::lacZ ΔmgtC::lacZ ΔmgtA::lacZ ΔmgtA::lacZ 4 ΔmgtB::lacZ ΔmgtB::lacZ
3 -galactosidase β -galactosidase β 10 2
1 activitycompared to wild type fold change in change fold activity compared to wild type wild to compared activity fold change in change fold
0 0 2422 2422 Time (Hours) grown in LB Time (Hours) grown in low Mg
Figure 4.12. LacZ reporter assay. Wild type and corA strains grown in LB
(repressing conditions) or low Mg (activating conditions) carrying β-galactosidase reporters of mgtC, mgtB, and mgtB. Activities for β-galactosidase reporters in the corA strain were divided by activities for β-galactosidase reporters in the wild type strain as indicated by fold change on the y-axis.
131
2.0
1.5
p < 0.03
1.0
0.5
Percent gentamicin protected bacteria 0.0 Wild type corA
Figure 4.13. Epithelial cell invasion. Epithelial cell invasion by wild type
(MM2089) and a corA strain (MM2242). S. Typhimurium strains were allowed to invade Caco-2 epithelial cells for 1 hr before being washed. A corA strain with a plasmid carrying a functional corA allele (MM2320) and a invA strain (MM1364) were used as controls. Invasion assays are an average of five independent experiments.
132
20
15
10
5
Percent gentamicin protected bacteria 0 Wild type invA corA corA/pCorA
Figure 4.14. Replication within epithelial cells. Epithelial cell invasion by wild type (MM2089) and a corA strain (MM2242). S. Typhimurium strains were allowed to invade Caco-2 epithelial cells for 1 hr before being washed and allowed to replicate for 6 hr in the presence of gentamicin. A corA strain with a plasmid carrying a functional corA allele (MM2320) and a invA strain (MM1364) were used as controls. Replication assays are an average three independent experiments.
133
Figure 4.15. Immunohistochemistry for LAMP1. Immunohistochemistry of
Caco-2 epithelial cells invaded by wild type bacteria. Caco-2 cells are stained with Lamp-1 antibody to indicate lysosomal membranes (red) and DAPI (4',6- diamidino-2-phenylindole) to indicate the DNA in the nucleus (blue).
134
100
80
Wild type ΔcorA Survival Percent 60 ΔcorA pCorA ΔphoP
40 0 2 4 6 8
Time (hours)
Figure 4.16. Macrophage survival. Macrophage entry and survival by wild type (MM2089) and a corA strain (MM2242). S. Typhimurium strains were allowed to invade J774A.1 macrophage-like cells for 15-30 min before being washed and allowed to replicate for 8-10 hr in the presence of gentamicin. A corA strain with a plasmid carrying a functional corA allele (MM2320) and a phoP strain (MM3220) were used as controls. Entry and survival assays are an average of three independent experiments.
135 Chapter 5
Studies elucidating the regulation of CorA in Salmonella
Introduction
Mg2+ is implicated in several stages of S. Typhimurium infection. For example,
exposure to low extracellular Mg2+ results in activation of the PhoP/Q two
component system and consequently markedly alters expression of many genes
necessary for virulence including but not limited to SPI1, SPI2, host antimicrobial
peptide resistance, bile resistance, and biofilm formation (4,62,131). mgtA and
mgtB which encode two inducible Mg2+ transport systems are also PhoP/Q regulated (62,180,187). However, the primary source of intracellular Mg2+ in S.
Typhimurium and many other bacteria and archaea is the CorA Mg2+ channel
(96,103,118,122,172). Chapter 4 outlined studies examining the effect of
mutation of corA on virulence and gene expression. A strain lacking corA was
attenuated in mice after both oral and intraperitoneal infection routes and was
defective for invasion and replication within Caco-2 epithelial cells. Microarray
data indicated that multiple metabolic pathways are affected by corA mutation.
The simplest explanation for these defects would be that in the absence of CorA,
the organism cannot obtain sufficient intracellular Mg2+ for optimal virulence
despite having two other Mg2+ transporters. However, the results outlined below
show that intracellular Mg2+ is relatively unaffected by loss of corA. The virulence
phenotype and changes in gene expression are related to the presence of a
functional CorA protein and most likely regulation of CorA rather than the actual transport process.
136 Results
A functional and evolutionarily related CorA is required for replication of S.
Typhimurium within Caco-2 epithelial cells.
Since CorA is the primary Mg2+ channel in Salmonella, one reasonable
hypothesis for a virulence defect is that a corA strain has insufficient intracellular
levels of Mg2+. To address this question we measured total intracellular Mg2+ content by atomic absorption. Mg2+ content was measured in N-minimal medium
with 10 μM or 100 mM Mg2+ and in LB. Total intracellular Mg2+ was similar in N- minimal medium with 10 μM Mg2+ and LB media but was about 2 fold increased
in cells grown in N-minimal medium with 100 mM Mg2. Regardless of the
absolute level of Mg2+ however, total intracellular Mg2+ did not differ between wild
type and corA strains under any growth condition (Figure 5.1, p.151). Thus, a
corA strain does not appear to lack Mg2+. One caveat to this experiment is that
only “total” intracellular Mg2+ is being measured by atomic absorption. It is
possible that the level of “free” Mg2+ is altered in a corA strain, but there is
currently no known method to accurately measure intracellular “free” Mg2+ in
actively growing cells.
We next determined whether increased Mg2+ transport could rescue the corA phenotype. CorA from Escherichia coli rescues the defect as well as S.
Typhimurium CorA (Figure 5.2, p.152). However, E. coli CorA and S.
Typhimurium CorA differ by only eight amino acids, all conservative substitutions.
We then determined whether a more phylogenetically distant CorA could rescue the corA phenotype. CorA from Methanococcus jannaschii cannot rescue the
137 replication defect (Figure 5.3, p.153) even though it supports substantial Mg2+ uptake when expressed in S. Typhimurium (173). MgtE is another class of Mg2+ transporter in prokaryotes, structurally and mechanistically unrelated to CorA
(88,178,191). MgtE from Bacillus subtilis is functional in S. Typhimurium (Figure
5.4, p.154); however, the B. subtilis MgtE also cannot rescue the corA defect
(Figure 5.2, p.152). Thus, only an evolutionarily similar CorA can rescue the invasion/replication defect; moreover, simply supplying Mg2+ via another
transporter also cannot rescue the invasion phenotype. These results imply that
the CorA protein itself is what is important for virulence.
We next tested whether the S. Typhimurium CorA needed to be functional
to rescue the invasion/replication defect. We compared our collection of S.
Typhimurium CorA mutants (177,186) to the recent crystal structure of
Thermatoga maritima CorA (47,47,118,118,143,143) and chose two S.
Typhimurium CorA mutants with alanine substitutions at positions F266 and
P269 (186). Residues F266 and P269 are approximately one helical turn apart
on transmembrane segment one which forms the pore of the channel. Previous
transport experiments with these mutants indicated that CorA is expressed at
approximately wild type levels and that each mutant exhibits 40-50% wild type
level of transport. However, when expressed in a strain (MM281) lacking all three Mg2+ transporters, the strain requires 0.25 mM Mg2+ for growth in N-
minimal medium whereas a strain expressing wild type CorA requires only 0.01
mM Mg2+. This phenotype implies that the F266A CorA and P269A CorA
138 channels cannot close properly and are partially open or “leaky”, thus making it more difficult for the strain to acquire and retain sufficient Mg2+.
We expressed these two mutant corA alleles in the corA strain (which still retains functional alleles of mgtA and mgtB). Neither of these mutant CorA proteins could rescue the invasion/replication defects in Caco-2 cells (Figure 5.5, p.155). Thus, CorA must be functional for S. Typhimurium to be fully virulent, but in addition the channel apparently must be able to close normally.
Next we took advantage of cobalt (III) hexaammine, a selective competitive inhibitor of CorA influx (109). In a strain lacking mgtA and mgtB, chronic inhibition with cobalt (III) hexaammine is bacteriostatic. We reasoned that giving wild type cells a maximum inhibitory dose of cobalt (III) hexaammine would mimic a closed channel but one which has the potential to function normally. Chronic (overnight) or acute (immediately before infection) exposure of wild type S. Typhimurium to cobalt (III) hexaammine to inhibit Mg2+ uptake had no effect on invasion of and/or replication within Caco-2 epithelial cells (Figure
5.6, p.156). The results suggest that significant flux of Mg2+ through CorA is not essential for the invasion/replication defect.
We next examined whether Mg2+ efflux via CorA was relevant. When extracellular [Mg2+] is below 1 mM, no Mg2+ efflux via CorA or any other system can be detected using 28Mg2+ as tracer (72). However, when cells are exposed to high (>5 mM) extracellular Mg2+, CorA mediates 28Mg2+ efflux. We have previously described three additional loci, corB, corC, and corD, that markedly increase the extracellular Mg2+ concentration required to elicit CorA mediated
139 efflux (72). S. Typhimurium invasion of and replication within Caco-2 epithelial
cells was unaffected by mutation of any of these loci (Figure 5.7, p.157). Thus,
neither the corB, corC, or corD loci nor CorA-mediated efflux appear to be
important for the invasion/replication phenotype.
CorA-mediated influx is regulated.
A previous study by Chamnongpol and Groisman indicated that CorA-
mediated influx was increased in a phoP strain compared to wild type cells but
that CorA protein level was not altered (28). This result implies that Mg2+ influx
via CorA is being regulated in some manner in a phoP strain. However, since
PhoP is crucial for multiple signaling cascades in Salmonella, a phoP strain is not
the ideal background in which to assess CorA regulation. Therefore, we
investigated the possible regulation in wild type and corA bacteria. We also
utilized a phoP and phoQ strains, since results from these strains should
replicate published data.
Wild type, corA, phoP, and phoQ strains were grown in different media
through log phase and into stationary phase. Experiments were conducted in
SL1344 and 14028s strain backgrounds and similar results were obtained for
both. Cells were grown in either N-minimal medium with 10 μM MgSO4 (low Mg) or 10 mM MgSO4 (high Mg), or in LB. corA transcription was measured using a
low copy plasmid-borne luciferase reporter driven by the endogenous corA
promoter. Total CorA protein was determined with anti-CorA Western blots.
Mg2+ influx was quantified by determining the initial rate of uptake of 63Ni2+ via
140 CorA. Finally, total intracellular Mg2+ content was measured by atomic
absorption.
For wild type and the corA strain, the general pattern of corA transcription
versus time was essentially identical in all three growth media (Figure 5.8,
p.158); transcription was highest during early log phase and decreased markedly
by stationary phase. Moreover, neither the pattern nor the relative amount of
transcription from the corA promoter was altered in the absence of a functional
corA allele. These results suggest that transcription of corA is sensitive to growth
rate, and they confirm previous data that corA transcription is independent of
extracellular Mg2+ concentration (174,187). phoP and phoQ are required to achieve wild type levels of corA transcription when grown in low Mg (Figure 5.9,
p.159). Without them, transcription of corA is delayed. Only in a phoP mutant
does it match wild type levels and only in stationary phase. Transcription of corA
is increased in the phoQ mutant when grown in LB or high Mg. Thus, phoP and
phoQ are most likely regulating corA transcription in low Mg; this regulation is
most likely indirect, as corA has never been picked up previously by other
screens for PhoPQ regulated genes.
In low Mg, wild type stationary phase cells contain more CorA protein than
log phase cells (Figure 5.10, p.160). However, transport of 63Ni2+ is decreased in stationary phase (Figure 5.12, p.162). Thus protein content does not correlate with transport. Furthermore, total intracellular Mg2+ content is the same between wild type log and stationary phase cells grown in low Mg (Figure 5.16, p.166).
Levels of CorA protein measured in stationary phase in phoP and phoQ mutants
141 are lower in low Mg2+ then wild type (Figure 5.11, p.161), this correlates with the
decreases seen in transcription of corA. Transport was increased 3 to 11 fold in
phoP and phoQ strains when grown in low Mg in both growth phases, confirming
Chamnongpol and Groisman’s data (Figure 5.13, p.163). Mg2+ content was
increased in the phoP and phoQ strains when grown in low Mg which
corresponds with the increase in transport (Figure 5.17, p.167).
After growth in LB, CorA protein content (Figure 5.10, p.160) is increased
in stationary phase as is transport of 63Ni2+ in wild type cells (Figure 5.12, p.162).
However, Mg2+ content is significantly lower in stationary phase despite
increased levels of CorA protein and transport (Figure 5.16, p.166). No differences in the amount of CorA protein, CorA transport, or Mg2+ content exist between wild type, phoP, and phoQ strains when grown in LB (Figures 5.11, p.161, 5.13, p.163, and 5.17, p.167).
Finally, in high Mg similar amounts of CorA protein are found in stationary phase versus log phase wild type cells (Figure 5.10, p.160). Yet stationary phase cells do not transport any 63Ni2+ (Figure 5.12, p.162). The lack of uptake
via CorA in the high Mg stationary phase cells is not due to a general lack of
energy or transport since these cells exhibit a substantial rate of manganese (as
54Mn2+) uptake via the MntH and SitABCD Mn2+ transporters (Figure 5.14, p.164).
Moreover, despite the lack of transport in the stationary phase cells, they contain
much more intracellular Mg2+ than cells grown to stationary phase in other media
(Figure 5.16 p.166).
142 Next, the lack of 63Ni2+ transport by wild type cells in stationary phase
when grown in high Mg was addressed. One general difference between log and
stationary phase cultures is the pH of the growth medium changes. N-minimal
medium is typically buffered with 20 mM Tris to a pH of 7.0, and by overnight the
pH of this medium drops to 4.0. Thus, we assessed whether the pH of the
medium was affecting transport in stationary phase. Several growth media based on N-minimal medium were made with varying buffering capacities. The most buffered cultures were able to maintain an overnight pH of around 6.0
(Figure 5.15, p.165). Moreover, the most buffered cultures transported more
63Ni2+ (Figure 5.15, p.165). Thus by inhibiting the 3 unit pH drop of the growth
medium, the cells were still able to transport 63Ni2+. Thus, the lack of transport in
regular stationary phase cultures (Figure 5.15, p.165) could be a result of low pH
in minimal medium. Supporting these results are the observation that pH of LB remains at 7.0 in stationary phase cultures; these cells have robust transport
(Barrow et al JBac 1996). Overall, these results suggest that the CorA protein present in stationary phase cultures is functional but does not actively transport
63Ni2+ because CorA transport is affected by pH.
Overall, these data show that changes in the levels of CorA protein in S.
Typhimurium do not always result in a corresponding change in either the initial
rate of cation influx via CorA or intracellular Mg2+ content. For example, even
though CorA transport is completely undetectable in stationary phase cells grown in high Mg, CorA protein is present in an amount similar to the same cells during
log phase. Further the lack of transport in the stationary phase cells does not
143 alter total intracellular Mg2+ compared to log phase cells. In addition, PhoP/Q is
somehow regulating corA transcription and function in low Mg. Taken together these results strongly suggest that CorA function is regulated. Whether this regulation involves a posttranslational modification such as phosphorylation, interaction with another protein, allosteric modulation, or a combination will be the subject of future experiments.
144 Discussion
The role of Mg2+ versus CorA.
A corA strain is attenuated for virulence in the mouse and defective for
invasion and replication within Caco-2 epithelial cells. We therefore conducted
experiments with the Caco-2 cells to examine potential reasons why loss of corA
results in these phenotypes. The overall results indicate that the presence of an
evolutionarily related and functional CorA protein itself is essential for virulence.
Surprisingly, intracellular Mg2+ content does not seem to be related to the
invasion and replication deficit since total intracellular Mg2+ content was not
significantly different between wild type and a corA mutant. MgtE, a Mg2+ transporter from B. subtilis fails to complement the corA virulence phenotype despite its robust transport in S. Typhimurium. Chronic or acute inhibition of
CorA by high concentrations of the selective inhibitor cobalt (III) hexaammine did not elicit the invasion/replication phenotype suggesting that a significant amount of Mg2+ flux through CorA is not essential. Thus, these results suggest that the
CorA protein but not intracellular Mg2+ or Mg2+ influx via CorA is important for
Salmonella interaction with epithelial cells.
While the overall funnel-shaped structure of T. maritima CorA is presumably conserved between E. coli, M. jannaschii, S. Typhimurium and presumably the rest of the CorA family (118,123), only E. coli CorA complements the invasion/replication phenotype. S. Typhimurium and E. coli CorA differ by only eight residues, whereas the membrane domain of M. jannaschii is only 22% homologous to S. Typhimurium and its soluble domain is merely 12%
145 homologous. Therefore, the protein has to look very much like S. Typhimurium
CorA in surface detail and spatial placement of individual residues to complement. Thus, we conclude that the CorA protein must be phylogenetically related to the S. Typhimurium CorA.
We further addressed CorA function by examining efflux. CorA-mediated efflux that is elicited at high extracellular Mg2+ concentrations appears
unnecessary for invasion and replication within Caco-2 epithelial cells since a strain carrying a mutation in the corB, corC, or corD genes had no effect compared to wild type (Figure 5.7, p.157).
The F266A and P269A mutants of CorA have 40-50% wild type level of transport but appear to leak Mg2+ presumably because they cannot close
properly. These mutants do not rescue the invasion/replication defect (Figure
5.5, p.155) implying that CorA must not only be functional but also be able to
close normally. Supporting this hypothesis is chronic or acute inhibition of CorA by cobalt (III) hexaammine, which leaves CorA functional, but mimics a closed conformation. The inhibitor treated wild type cells invade and replicate comparably to untreated wild type cells in Caco-2 epithelial cells (Figure 5.6, p.156). Therefore, our overall conclusions are that a CorA protein must be evolutionarily related, functional, and be able to achieve a closed state to rescue the invasion/replication defect. These results further imply the functional regulation of CorA was important for Salmonella virulence within epithelial cells.
Regulation of CorA.
146 Based on the above conclusions, we investigated the regulation of CorA
transcription and translation. While the levels of corA mRNA did not vary greatly
among wild type and corA cells grown in any test medium (Figure 5.8, p.158),
CorA protein levels varied markedly (Figure 5.10, p.160). This was true whether
measured in stationary phase or in log phase, although mRNA levels were consistently markedly lower in stationary phase than in log phase. For wild type
cells in contrast to mRNA content, the amount of CorA protein did not correlate
either with the rate of CorA mediated transport or with Mg2+ content (Figure 5.10,
p.160, 5.12, p.162, and 5.16, p.166).
In phoP and phoQ strains, corA mRNA was decreased when grown in low
Mg and only reached wild type levels upon entry into stationary phase. Thus,
phoP and phoQ are required for expression of corA when grown in low Mg
(Figure 5.9, p.159). In LB and high Mg, corA mRNA was decreased at the 2 hr
time point in the phoP and phoQ strains. However corA mRNA did increase over
time in both strains. Moreover in the phoQ strain, corA mRNA was several fold
increased over wild type by stationary phase. CorA protein was decreased in the phoP and phoQ strains when grown in low Mg, which is consistent with the decreased transcription of corA under these same conditions (Figure 5.11, p.161). Interestingly, in the phoP and phoQ strains, the rate of CorA mediated transport and Mg2+ content appear to correlate, however these measurements
did not correlate with CorA protein (Figures 5.13, p.163 and 5.17, p.167). In
phoP and phoQ strains, transcription and protein are decreased, but uptake is
increased as well as Mg2+ content. This is observed during growth in low Mg.
147 Thus phoP and phoQ are somehow involved in regulating corA transcription and
CorA protein in low Mg.
One would expect that an increased level of CorA protein would be
accompanied by a similar increase in the initial rate of Mg2+ influx, but the data
are counter to this expectation. For example, in stationary phase cells grown in
high Mg, CorA is present, but no Mg2+ influx can be detected (Figure 5.12,
p.162). In sharp contrast, Mg2+ influx is very high during stationary phase in cells
grown in LB, even though protein levels and Mg2+ content are relatively low
compared to the other conditions tested. Interpretation of these results is not
confounded by the presence of wild type alleles of the mgtA and mgtB Mg2+ transporters since in both high Mg and in LB, both genes are virtually completely repressed (180,187,188). Even in low Mg, where mgtA and mgtB are expressed at low levels, the rate of CorA-mediated transport is comparable and CorA protein levels are far higher than in cells grown in other media.
Further, Mg2+ content does not correlate with the expression of CorA.
Although content is highest in cells grown in high Mg in both log and stationary
phase, CorA protein levels are relatively low compared to other growth
conditions. The increased intracellular Mg2+ content in high Mg must be the
result of CorA-mediated influx since expression of MgtA and MgtB is completely
repressed under this growth condition. Interestingly, these data also argue that
the ability of the CorA Mg2+ channel to mediate Mg2+ efflux at high extracellular
Mg2+ concentrations is not physiologically relevant. Since exposure to 10 mM
extracellular Mg2+ should elicit significant efflux via CorA, cells grown in high
148 Mg2+ concentrations might be expected to have decreased rather than markedly increased cellular Mg2+ content.
Finally, these results indicate that either translation of corA mRNA or CorA
protein stability as well as CorA function are regulated in some manner. The regulation of expression must be at the translational or posttranslational level
since, despite markedly different CorA protein content in cells grown in various
medium, both the pattern of mRNA expression and the relative amounts of
mRNA are virtually identical under all conditions tested. However, current data
do not speak to the mechanism by which translation and protein stability are
regulated.
Functional regulation of CorA is inferred from the mismatches in CorA
protein content, CorA transport, and total intracellular Mg2+ content. In turn, this
implies that CorA is subject to posttranslational modification, allosteric
modification, interacts with another protein, or a combination of these regulatory
mechanisms. Mg2+ itself can likely be eliminated as a potential allosteric modifier
since there is no obvious correlation of function with intracellular or extracellular
Mg2+ concentration. This interpretation of the data further implies that CorA is a
part of a broader signaling network within the cell. Data in this chapter (also see
Chapter 4) are all consistent with a role for CorA and Mg2+ homeostasis in
several major pathways in S. Typhimurium including some which impact
virulence. Current experiments are focused on understanding this apparent
regulation of CorA expression and function and to delineate its connection to S.
Typhimurium virulence.
149
Table 5.1: Bacterial strains used in Chapter 5.
Strain Genotype Source MM1242 JR501 MM1270 14028s phoQ::kan E.A. Groismana MM1364 SL1344 invA::Tn10::phoA R. Maurerb MM2089 Salmonella enterica Serovar Typhimurium B.B. Finlayc SL1344 MM2242 SL1344 corA52::Tn10 16 17 J. Linb MM2320 SL1344 corA52::Tn10 16 17 pJL10 J. Linb MM3097 Escherichia coli DH5α MM3203 SL1344 corA52::Tn10 16 17 pMetCorA This study MM3217 SL1344 corA52::Tn10 16 17 pBADBSmgtE This study MM3218 SL1344 corA52::Tn10 16 17 pBADECcorA This study MM3220 14028s phoP::cam J.S. Gunnc MM3227 SL1344 corA52::Tn10 16 17 pCorAF266A This study MM3228 SL1344 corA52::Tn10 16 17 pCorAP269A This study MM3238 SL1344 corB::kan This study MM3239 SL1344 corC::kan This study MM3240 SL1344 corD::kan This study MM3252 SL1344 pTTCAlux 187 MM3253 SL1344 corA52::Tn10 16 17 pTTCAlux This study MM3254 14028s phoQ::cam pTTCAlux This study MM3256 14028s phoP::cam pTTCAlux This study aDepartment of Molecular Microbiology, Howard Hughes Medical Institute,
Washington University School of Medicine, Campus Box 8230, 660 South Euclid
Avenue, St. Louis, MO 63110, USA.
bCase Western Reserve University, Cleveland, OH 44106, USA.
cBiotechnology Laboratory, University of British Columbia, British Columbia,
Canada
cThe Center for Microbial Interface Biology; Department of Molecular Virology,
Immunology and Medical Genetics; and Department of Internal Medicine,
Division of Infectious Diseases, The Ohio State University, Columbus, OH 43210,
USA.
150
Figure 5.1. Total intracellular Mg2+ content. Total intracellular Mg2+ content was measured by atomic absorption after growing cells in either LB or N-minimal medium with 10 μM or 100 mM MgSO4. Data represent the average of three
samples per strain.
151
p>0.75
20
15 F p<0.04
10 p<0.007
5
Percent gentamicin protected bacteria 0 WT ΔcorA ΔcorA + ΔcorA + pBSMgtE pECCorA
igure 5.2. Replication within epithelial cells. S. Typhimurium were allowed to invade Caco-2 epithelial cells for 1 hr, and intracellular bacteria were left to replicate for 6 hr in the presence of gentamicin. p values indicate t tests comparing wild type (MM2089) to the other strains. Data represent the average of three independent experiments.
152 WT ΔcorA pMetCorA 40 ΔinvA
20 Percent gentamicin protected bacteria protected gentamicin Percent 0 1 2 3 4 Hours
Figure 5.3. Epithelial cell invasion time course. S. Typhimurium were allowed to invade Caco-2 epithelial cells for 1 hr, and intracellular bacteria were left to replicate for 2 and 4 hrs in the presence of gentamicin. Data are representative of one of two independent experiments.
153
200 ΔcorA pBSMgtE WT ΔcorA 150
100
Uptake Normalized to WT 2+
Co 50 57
Percent 0 0 10 -5 10 -4 10 -3 10 -2 10 -1 [Co2+] (M) Figure 5.4. 57Co2+ uptake. Inhibition of 57Co2+ uptake by various concentrations of CoCl2 was measured in wild type (MM2089), a corA strain (MM2242), and a corA strain with a plasmid carrying a functional B. subtilis mgtE gene (MM3228).
The data are normalized to the amount of 57Co2+ uptake in wild type cells in the absence of additional CoCl2. The data shown are a single experiment representative of two independent experiments.
154
Wild type corA corA pF266AcorA pP269A
40
20 p<0.025 p<0.023
p<0.029
Percent gentamicin protected bacteria 0 WT ΔcorA ΔcorA + ΔcorA + pCorAF266A pCorAP269A
Figure 5.5. Replication within epithelial cells. S. Typhimurium were allowed to invade Caco-2 epithelial cells for 1 hr, and intracellular bacteria were left to replicate for 6 hr in the presence of gentamicin. p values indicate t tests comparing wild type (MM2089) samples to each other and corA (MM2242) samples to each other. A single experiments is shown representative of 2 additional experiments.
155 Wild type Wild type ChronicWild type Acute corA corA chronic corA acute
p>0.15 No treatment
20 Chronic Co(III) Hexaammine Acute Co(III) Hexaammine
15 p>0.78
10
p>0.42 p>0.31
5
bacteria protected Percent gentamicin 0 Wild type ΔcorA
Figure 5.6. Replication within epithelial cells. S. Typhimurium were allowed to invade Caco-2 epithelial cells for 1 hr, and intracellular bacteria were left to replicate for 6 hr in the presence of gentamicin. Bacterial cells were treated either chronically (overnight during growth) or acutely (immediately prior to invasion) with cobalt (III) hexaammine. In both cases, cobalt (III) hexaammine was maintained in the media during invasion. p values indicate t tests comparing wild type (MM2089) to the other strains. Data represent the average of two independent experiments.
156
Wild type corA corB corC corD
p>0.70
40 p>0.25
p>0.55
30
20 P<0.03
10
bacteria protected gentamicin Percent 0 WT ΔcorA ΔcorB ΔcorC ΔcorD
Figure 5.7. Replication within epithelial cells. S. Typhimurium were allowed to invade Caco-2 epithelial cells for 1 hr, and intracellular bacteria were left to replicate for 6 hr in the presence of gentamicin. p values indicate t tests comparing wild type (MM2089) to the other strains. A single experiments is shown representative of 3 additional experiments.
Wild type ΔcorA
157 1010 10uM 10uM LBuM LBuMLBLB1010 10mM 10mM mM mM1010 10uM 10uM LBuM LBuMLBLB1010 10mM 10mM mM mM low Mg 120000 LB high Mg
600nm 80000
RLU per OD 40000
0 Wild type ΔcorA Figure 5.8. corA transcription. CorA expression during growth for cells grown in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10 mM Mg2+ (high
Mg). corA transcription was determined using a luciferase reporter at 2, 4, 6, and
20 hr. Data represent an average of four independent experiments.
158 17.5 low Mg LB 7.5 high Mg 4
3 normalized WT to
600nm 2
1 RLU perRLU OD
0 WT ΔcorA ΔphoQ ΔphoP
Figure 5.9. corA transcription. CorA expression during growth for cells grown in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10 mM Mg2+ (high
Mg). corA transcription was determined using a luciferase reporter at 2, 4, 6, and
20 hr. Data represent an average of four independent experiments. Data were all normalized to wild type.
159 low Mg log low Mg stat LB log LB stat high Mg log high Mg stat
p<0.01 Log 2.0 Stationary
1.5
1.0 p<0.02
0.5 p>0.78
CorA protein normalized DnaKto protein 0.0 low Mg2+ LB high Mg2+
Figure 5.10. CorA protein content. CorA expression during growth for cells grown in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10 mM
Mg2+ (high Mg). CorA protein levels were measured by anti-CorA Western blots.
CorA protein levels were normalized to DnaK protein, a loading control.
Measurements were made on cells sampled at mid-log phase and after entry into stationary phase, p values indicate t tests comparing log to stationary phase within the same medium, and the data represent an average of three independent experiments.
160 WT ΔcorA ΔphoQ ΔphoP 1.0
0.5 CorA protein normalized to DnaK and wild type wild and DnaK to normalized protein CorA 0.0 low Mg LB high Mg
Figure 5.11. CorA protein content. CorA expression during growth for cells grown in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10 mM
Mg2+ (high Mg). CorA protein levels were measured by anti-CorA Western blots.
CorA protein levels were normalized to DnaK protein, a loading control.
Measurements were made on cells sampled entry into stationary phase, the data represent an average of three independent experiments, and the data were all normalized to wild type.
161 low Mg log low Mg stat LB log LB stat high Mg log high Mg stat 15000
Log
600nm Stationary p<0.002
10000 per OD 2+
Ni 63
5000
p<0.04
Total cpm uptake p<0.04 0 low Mg2+ LB high Mg2+
Figure 5.12. Total uptake of 63Ni2+. CorA function during growth for cells grown
in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10 mM Mg2+ (high
Mg). The initial rate of CorA-mediated influx was determined using 63Ni2+.
Measurements were made on cells sampled at mid-log phase and after entry into
stationary phase, p values indicate t tests comparing log to stationary phase
within the same medium, and the data represent an average of three
independent experiments.
162 12.5 WT
per ΔcorA
2+ ΔphoQ ΔphoP Ni
63 7.5 normalized to WT 2.5 600nm OD Total cpm uptake cpm Total
-2.5 low Mg log low Mg stat LB log LB stat high Mg log high Mg stat
Figure 5.13. Total uptake of 63Ni2+. CorA function during growth for cells grown
in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10 mM Mg2+ (high
Mg). The initial rate of CorA-mediated influx was determined using 63Ni2+.
Measurements were made on cells sampled at mid-log phase and after entry into
stationary phase, the data represent an average of three independent
experiments, and the data were all normalized to wild type.
163 120 WT ΔcorA 100
80 uptake 2+
Mn 60 54
40 Percent 20
0 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 2+ [Cd ] M
Figure 5.14. 54Mn2+ uptake. Inhibition of 54Mn2+ uptake by various concentrations of CdCl2 was measured in wild type (MM2089), and a corA strain
(MM2242) grown in high Mg. The data shown are a single experiment
representative of two independent experiments.
164
9 Transport pH 600nm 6000 8
perOD 7 2+ pH Ni 4000 63 6
5 2000
4 Total cpm uptake
0 3 50 mM Tris 40 mM Tris 20 mM Tris 50 mM Mes 40 mM Mes 20 mM Mes 100 mM Tris 100 mM Mes 50/50mM both 20/20mM both 10/10mM both Growth medium
Figure 5.15. pH dependence on 63Ni2+ uptake. The initial rate of CorA- mediated influx was determined using 63Ni2+ and pH was measured prior to transport. Wild type cells were grown to stationary phase in high Mg in various buffered media.
165
Figure 5.16. Total intracellular Mg2+ content. CorA function during growth for
cells grown in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10
mM Mg2+ (high Mg). Mg2+ content was determined by atomic absorption.
Measurements were made on cells sampled at mid-log phase and after entry into
stationary phase, p values indicate t tests comparing log to stationary phase
within the same medium, and the data represent an average of three
independent experiments.
166
Figure 5.17. Total intracellular Mg2+ content. CorA function during growth for
cells grown in either LB or N-minimal medium with 10 μM Mg2+ (low Mg) or 10
mM Mg2+ (high Mg). Mg2+ content was determined by atomic absorption.
Measurements were made on cells sampled at mid-log phase and after entry into
stationary phase, the data represent an average of three independent
experiments, and the data were normalized to wild type.
167 Chapter Six
Conclusions and Future Directions
Summary
Mg2+ homeostasis is poorly understood in any cell type, bacterial, archaeal or eukaryotic. In bacteria, Mg2+ has been linked to virulence. However, what
role Mg2+ plays in virulence is still unknown. Extracellular Mg2+ is a cue for the
two component system PhoPQ. PhoPQ regulates many signaling pathways
which are involved in virulence, as well as Mg2+ transport. Salmonella has three
Mg2+ transport systems: CorA, MgtA, and MgtB. mgtA and mgtB are directly
regulated by PhoPQ and only expressed in response to low Mg2+. mgtA is further regulated at the transcriptional level by a riboswitch for intracellular Mg2+.
This work shows that corA expression is also regulated, because in phoP and
phoQ mutants transcription of corA is altered (Chapter 5).
Prior to this work, CorA was thought to function simply as a housekeeping gene. However this work has shown that CorA is required for the full virulence of
S. Typhimurium (Chapter 4). Previous data from other groups hypothesized that
CorA may be functionally regulated by, and transport, Fe2+ (28,85). This work
also shows that CorA does not transport Fe2+ (Chapter 3) and that transport by
CorA is regulated (Chapter 5), though not by Fe2+. The regulation Mg2+ transport
through multiple mechanisms implies that maintaining Mg2+ homeostasis is
critical for proper cell function. Moreover, this work shows that Mg2+ homeostasis
is a complex, regulated process. Overall, studies on Mg2+ signaling and/or
168 homeostasis in the bacterium could eventually result in establishing a new
therapeutic target against bacterial pathogenesis.
Regulation of corA transcription
As demonstrated in Chapter 5, CorA expression and function appear to be
regulated. Levels of corA transcription are similar for wild type and corA cells grown in low Mg, LB, and high Mg and thus does not vary with different Mg2+ concentrations in the growth medium. Transcription is highest at the beginning of log phase and steadily declines by stationary phase. However transcription of corA in low Mg appears dependent on the presence of phoP or phoQ. Moreover, transcription of corA is dysregulated in the phoQ strain in LB and high Mg; it is increased seven-fold compared to wild type. This pattern of dysregulated transcription is what would be expected for a PhoPQ regulated gene in a phoP or phoQ mutant.
The initial question is whether the phoPQ effect is direct or indirect. The corA promoter does not contain a PhoP box, but not all PhoPQ regulated genes have PhoP boxes (see Table 1.2, p.45-46) so this is not definitive evidence.
Thus, the first step would be to conduct either a ChIP or DNase footprinting assay to determine if PhoP binds the corA promoter. However, this seems unlikely since the effect is more pronounced in the phoQ strain. It is possible the
PhoQ could signal to another transcriptional activator besides PhoP to regulate corA transcription; however, there is no evidence in the literature that PhoQ by itself can signal through another protein. There could be an interesting dynamic of multiple transcriptional regulators binding to or interacting to regulate the corA
169 promoter. The possibility of multiple interacting transcriptional factors affecting corA transcription will have to be considered when interpreting the data obtained from future experiments.
Together PhoP and PhoQ are known to crosstalk with other transcriptional activators/regulators, such as HilA, PmrA, PmrD, SlyA and SsrB. Thus, the next logical step would be to determine whether mutants of these other activators affect transcription of corA. Knockout mutants for hilA, pmrA, pmrD, slyA, and ssrB would be generated (38), and the corA luciferase reporter plasmid transformed into these strains for measurement of luciferase activity under appropriate growth conditions. Any mutants with altered transcription compared to wild type would be considered a potential regulator of corA transcription.
Positive results would be followed up by a ChIP or DNase footprinting assay to determine if the regulator binds the corA promoter.
If no hits are obtained from the known regulators, then a transposon mutagenesis approach would be used to screen for other potential regulators of corA transcription. Transposons are used to randomly mutagenize a strain of bacteria. This mutagenesis creates a pool of mutated strains, each of which has a single random transposon insertion somewhere in the Salmonella chromosome. Then, an assay is designed to screen these different mutants for a phenotype of interest, in this case altered transcription of corA. For such an assay, two parameters need to be chosen: the background strain used for mutagenesis and the type of the screen used to identify specific mutants.
170 The wild type strain or the phoQ mutant would be chosen for random mutagenesis with transposons, because mutants with increased or decreased transcription of corA compared to wild type or the phoQ mutant are of interest.
The corA promoter driving a reporter on a plasmid would be introduced into the pool of transposon mutants. Because a plate based assay would be the easiest way to screen for mutants, β-galactosidase would be chosen as the reporter.
Colonies in which corA transcription is occurring may express β-galactosidase, and when grown on plates containing the β-galactosidase’s substrate, 5-bromo-
4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) should turn blue. Those not transcribing corA and thus not expressing β-galactosidase should remain white.
Thus, colonies with increased expression of corA should appear darker blue and decreased expression of corA should appear lighter blue or white. In liquid cultures using wild type cells, transcription of corA is highest in early log phase and steadily decreases by stationary phase. However, a colony on a plate is not really in either log phase or stationary phase. Thus, it is unknown what phenotype wild type cells will have on a plate. A control assay comparing wild type cells carry the β-galactosidase reporter plated on low Mg, LB, and high Mg plates will be conducted. If wild type cells turn blue on one of the tested plate media, then β-galactosidase will be used as the reporter.
The pool of mutants carrying the β-galactosidase reporter plasmid would then be screened on X-gal plates to identify mutants with altered (increased or decreased compared to wild type control) transcription of corA. Those mutants with altered transcription would be grown in liquid low Mg, LB, and high Mg
171 media. Samples would be taken at 2, 4, 6, and 20 hr. These samples would
screened by a β-galactosidase liquid assay using another β-galactosidase
substrate, ortho-Nitrophenyl-β-galactoside (ONPG). If transcription were still
altered compared to wild type, then selected mutants would be sequenced to
identify the transposon’s point of insertion. The points of insertion could be
problematic as transposon could insert into an operon disrupting multiple genes
or put genes out of frame. Thus controls to ensure that the insertion is responsible for the altered transcription need to be conducted. One control is to transduce the insertion would into a wild type strain using the P22 bacteriophage.
Once the transduction is verified by PCR, the β-galactosidase reporter plasmid would be reintroduced and another β-galactosidase assay would be conducted to verify that transcription is truly altered. Also, a plasmid to complement the transposon mutation would be introduced to further verify the mutation is responsible for the phenotype. phoP and phoQ should be identified during this screen and would serve as positive controls that the mutagenesis and screening assay is working appropriately. This type of screen may be demanding, but is commonly used in microbiology.
Further steps would involve determining how this mutation effects corA
transcription. The gene product could affect transcription by directly binding the
corA promoter or it could be involved in a more complex signaling network that
results in transcription of corA. If a protein directly interacts with the promoter
then homology searches could reveal that it is similar to other transcriptional
regulators. Thus, to assess whether the protein could directly regulate corA
172 transcription by binding to the corA promoter, either a ChIP or DNase footprinting assay would be conducted. Since phoP and phoQ have been shown to be involved in corA transcription, the regulatory pathway would have to be further examined (see below).
A second possibility is that the insertion disrupts transcription of a protein that itself signals or is part of a cascade of proteins that signal the transcriptional activator of corA. If this is the case then the signaling cascade would need to be further dissected. PhoQ has been shown to regulate corA transcription in all media tested (Chapter 5). However, PhoQ is a membrane protein and most likely does not bind DNA directly. Thus, PhoQ has be to signaling to another protein which is then responsible for either directly or further signaling another protein(s) to directly activate or inhibit corA transcription (see Figure 6.1, p.188).
By generating different sets of double and triple mutants and growing them in low
Mg, LB, and high Mg, a signaling pathway involved in regulating corA transcription would be explored.
A third possibility is that the insertion changes β-galactosidase mRNA or protein stability in the cell and mRNA for β-galactosidase or the protein itself is degraded. The insertion site may give clues as to whether this is occurring. For example, if a mRNA stability protein is disrupted, then mRNA stability would be altered. mRNA stability is discussed below. However, results indicating that the degradation of β-galactosidase is being affected would not be relevant.
If no plate phenotype is obtained for wild type cells, then β-galactosidase plate assays would not work. Thus, an alternative screen would be set-up.
173 Liquid screening of hundreds of transposon mutants using β-galactosidase is
tedious and slow. Thus, a luciferase reporter would be used, as an alternative
for the plate assays. Again, the wild type strain will be mutagenesized with
transposons, but this time a corA promoter driven luciferase gene on a plasmid
will be introduced. Individual colonies will then be subcultured in low Mg, LB, and
high Mg in 96 well plates. A control experiment would be conducted to ensure
that wild type behaves the same in 96 well plate format as it does when grown in
a 10 ml culture tube. At 2, 4, 6, and 20 hr, a luciferase assay will be conducted
and the plates will be read on a luminometer. Similar steps as those described
above will be completed for mutants identified through this screen.
Another method to study the regulation of corA transcription would be to
dissect the corA promoter by generating a series of promoter truncations and/or
mutants within the corA luciferase reporter plasmid. These plasmids would be
transformed into both wild type and the phoQ mutant. Then, specific sections of promoter necessary for transcription and/or for the phoQ effect would be identified by using either the β-galactosidase or luciferase assay as described above. Since this assay does not require screening hundreds of mutants, the reporter used is not as important. One caveat to this method is that only a region of the promoter necessary for corA transcription would be identified and not the regulator involved. Ideally but not likely to work, a PubMed search of the literature using the crucial sequence could lead to identification of transcriptional motif and regulator. Alternatively, the sequence could be used for a nucleotide blast search of the Salmonella genome to identify other genes with this
174 sequence. This search could lead to a discovery of a gene whose regulation is
known and thus could lead to identification of a regulator. For confirmation,
potential regulators would be knocked-out and these knockouts would be
screened for luciferase or β-galactosidase activity. Finally, similar assays as
described above would be completed to assess relevance.
Regulation of corA mRNA and/or CorA protein stability
CorA protein levels do not correspond with the changes in transcription
and are somewhat dependent on extracellular Mg2+ levels. For example, wild type cells have similar levels of transcription/translation in the different media tested yet the amount of protein varies. Specifically, CorA protein levels are highest in low Mg2+ and lowest in high Mg2+. This result led to the conclusion that
either mRNA translation/stability and/or protein stability are regulated. The most
likely explanation for the differences in corA transcription and CorA protein
content is that corA mRNA stability is regulated based on the following rationale.
Transcription and translation of the luciferase reporter are at its highest after 2 hr
of growth whereupon they steadily decline into stationary phase. However CorA
protein content increases from log to stationary phase in cells grown in low Mg
and LB. It seems logical that the stability of the mRNA is responsible for the
increase in corA protein, because transcription and translation may slow down in
stationary phase, but that does not mean the corA mRNA is no longer in the cell.
Since protein increases from log to stationary phase, CorA protein most likely is
not being degraded during that transition. However, the level of degradation of
CorA protein could have decreased. Overall, the discrepancies between corA
175 transcription and CorA protein content could be readily explained by corA mRNA
stability without invoking any other regulatory factors. The set of experiments,
described below are designed to examine the above conclusion.
The best approach would be to isolate total RNA from wild type cells
grown in low Mg, LB, and high Mg at many time points, such as 0 min,15 min, 30
min, 1 hr, 3 hr, 5 hr, 7 hr, 10 hr, 12 hr, 15 hr, 18 hr, and 20 hr after growth. This
mRNA would be used for RTqPCR of the corA gene; alternatively Northern blots
could be completed. This will provide an accurate measure of the amount of
corA mRNA in the different media and establish a rate of transcription. Data
obtained from this experiment will be compared to CorA protein content (Figure
5.10, p.160).
Regulation of CorA function
Data presented in Chapter 5 reveal that CorA is functionally regulated. In
low Mg, CorA protein content increases in wild type cells grown from log to
stationary phase; however, transport decreases and Mg2+ content remains the same (Table 6.1, p.187). Similar mismatches occur for wild type cells grown in
LB and high Mg, as well as in phoP and phoQ mutants when grown in low Mg
(Chapter 5). CorA protein content does not correlate with CorA transport or with
Mg2+ content. These observations suggest that CorA function is being regulated
at some level. Possible regulatory mechanisms include allosteric modification of
CorA, a post-translational modification, or interaction with another protein(s).
More than one mechanism might be operative. Presumably one of these
regulatory mechanisms alters transport by CorA to maintain Mg2+ homeostasis
176 when cells are exposed to these different extracellular environments; this
regulation may be important for Salmonella virulence.
Allosteric regulation occurs when a ligand binds to a protein and alters its
conformation and/or function. Such modulation could cause CorA to either close
or open. Mg2+ would be the most logical candidate for such allosteric
modification of the CorA Mg2+ channel. However, from all of the data obtained
(Chapter 5) there is no indication the Mg2+ plays such a role. Neither
extracellular nor intracellular Mg2+ concentration appears to alter CorA function
from our data. Overall, Mg2+ serving as an allosteric modulator of CorA is too
simple of a model to explain the regulation of CorA function. There could be
another molecule in the cell that allosterically alters CorA function, but a potential
effector molecule is unclear at this point. Identifying a ligand that allosterically
regulates CorA would be a difficult process. For example, libraries of compounds
could be tested for their impact on CorA transport in a cell-free liposome system,
but without more information this type of assay is far too time-consuming. Other
methods for identifying an allosteric regulator that can be applied to other forms
of regulation are discussed later.
Post-translation modifications are chemical modifications of proteins, such
as phosphorylation. Typically an enzyme interacts with the target protein and chemically modifies the target. Another enzyme in the cell is typically able to remove that modification. The presence or absence of these modifiers results in conformational and functional changes to the protein. For CorA, this type of
177 modification could alter the probability that the channel would either close or
open.
To identify possible chemical modification of CorA, CorA protein would be
purified. During growth in high Mg in stationary phase, CorA protein is present,
but does not transport. Thus this growth condition would be ideal to identify a potential chemical modification to CorA, because the channel is present but not
functional. For comparison, CorA would also need to be purified from cells in
which CorA is functional, such as cells grown in LB. Mass spectrometry would be conducted to compare these two samples of purified CorA. However there are caveats to this assay. Using mass spectrometry to compare chemical modifications of the same protein is a difficult process. The percent of the protein that is covered during mass spectrometry can be low and modifications can be missed. This becomes increasingly difficult when comparing two proteins,
because the same residues need to be identified in both proteins. Moreover,
modifications can be potentially lost during purification process. A large amount
of very pure sample is required for this type of assay. However if a residue of
interest is obtained, the next series of experiments to verify the importance of this
residue for CorA function are easier. The residue would be mutated and
transport properties would be determined when cells were grown under the same
growth conditions.
An alternative could be to run protein gels of samples collected from wild
type or the phoQ mutant in the low Mg, LB, and high Mg which were grown to log
and stationary phase. Using anti-CorA Western blots, a shift in CorA protein may
178 be identified. A shift could be indicative of a modification to the CorA protein.
The percentage of acrylamide in the gels could be adjusted to determine an ideal
set-up to see the shift. Also, 2-D gels could be used to identify a CorA protein
shift.
One other potential regulatory mechanism is that CorA could interact with
another protein. That is, another protein binds or docks to CorA and regulates it
opening and/or closing. This protein might or might not also chemically modify
CorA during this process (see above). Either co-immunoprecipitation using a
anti-CorA antibody or a yeast two hybrid assay for membrane proteins can be
used to identify a protein which interacts with CorA.
For the co-immunoprecipitation assay, CorA would be co-
immunoprecipitated from cells grown to stationary phase in high Mg, because
under these conditions CorA is present, but does not transport. Alternatively, co-
immunoprecipitations could be conducted in a phoP or phoQ mutant, as these cells have increased transport when grown in low Mg, but decreased CorA
protein compared to wild type. The proteins obtained from the co-
immunoprecipitation would be electrophoresed on a protein gel and stained with
Coomassie blue. A Western blot would be conducted as a control for the
presence of CorA. If other proteins are obtained besides CorA, then either the
mixture of the proteins or individual proteins eluted from the gel would be
examined by mass spectrometry. Unlike identifying a chemical modification on a
protein, attempting to simply identify a protein using mass spectrometry is less
challenging. Less coverage is needed to begin to identify the interacting protein
179 as sequences obtained would only exist in some proteins. Also, more rounds of
mass spectrometry can aid in narrowing down the candidate pool. Then,
identified proteins would be directly tested for interactions with CorA. For
example, if a knockout of an interacting protein is generated, and this protein regulates CorA function, then transport in the knockout should be altered compared to wild type grown under the same conditions. Unfortunately, there is a major drawback to the co-immunoprecipitation assay. Kinetically, this type of assay should not work. Proteins that interact would have to have a very low affinity for them to interact long enough to conduct the purification. Moreover, not only does the kinetics of the protein protein interaction need to be considered, but also the kinetics of the CorA and the anti-CorA antibody interaction.
A yeast two hybrid assay to detect a protein that interacts with CorA is more complex since CorA is a membrane protein. A traditional two hybrid assay could be attempted using just the CorA soluble domain. However since soluble
CorA will not be in the membrane and part of the protein is missing, there is a
potential to miss the regulator. A modified yeast two hybrid for membrane
proteins might be the better alternative. This assay is based on a split ubiquitin
reporter protein, which will reassemble if the split ubiquitin is fused to two
proteins that interact. The reassembled ubiquitin is recognized by ubiquitin-
specific proteases. These proteases will cleave the C-terminally attached
reporter providing an immediate readout. The growth conditions under which to
measure this interaction include when cells are grown in high Mg to stationary
phase or grown in a phoP or phoQ mutant background, as regulation of CorA is
180 found under these conditions. If an interacting protein is identified, initial
experiments would verify that it regulates CorA function. A knockout of the
interacting protein would be made and this mutant strain would be tested for
altered transport of CorA.
Both the co-immunoprecipitation and the yeast two hybrid assay are rather
difficult and have low probabilities of obtaining desired results. An alternative
would be to browse the current microarray data obtained for wild type and corA
mutant. An inner membrane protein identified to be upregulated in the corA
mutant could be a potential binding partner for CorA. The genes for these
proteins could be knocked-out and their transport phenotypes could be assessed
after growth in low Mg, LB, or high Mg. Those with altered transport could serve
as a potential CorA interacting protein. Yeast two hybrid screens could then be
conducted with CorA and the protein identified to determine if they do interact.
If a protein that interacts with CorA and causes altered transport is identified, then an interesting experiment would be to co-crystallize CorA and this interacting protein. Co-crystallization of such a protein complex could further the
understanding of CorA function, since insight as to the mechanism by which
CorA opens and closes might be gleaned from such an experiment.
Listed below are alternatives methods to the approaches listed above to
identify a post-translation modification and protein-protein interaction, as well as
an approach to determine an allosteric ligand of CorA. One method would be to
set-up a screen using wild type bacteria and/or the phoP and phoQ mutants
could be mutagenized with a transposon. These mutants could then be
181 screened for altered transport. Mutants with altered transport may be involved in regulating CorA by any of the mechanisms described above. For example, if a metabolic enzyme is discovered in a screen, then the metabolite that enzyme produces could be an allosteric ligand for CorA. Thus, when the enzyme is absent the metabolite is not produced and CorA transport is altered. The problem with such an assay is throughput as it would require transport assay be performed on a multitude of mutants.
A different approach would be to conduct a microarray comparing wild type cells grown in LB and high Mg to stationary phase. Wild type cells grown in
LB to stationary phase have robust transport, while those grown in high Mg do not transport. Thus, a microarray comparing mRNA from these cells could reveal important changes in physiology that alter transport by CorA. A specific target is less likely to be discovered by this approach, but a general set of genes involved could be identified. Considerable changes are expected with a microarray as proteins from cells grown under these conditions are markedly different, as indicated by a protein gel stained with Coomassie blue (KM Papp-
Wallace, B Gorzelle, and ME Maguire, unpublished observations).
If the virulence phenotypes of the corA mutant are due to a lack of regulation of CorA protein, then another alternative assay could be conducted.
M. jannaschii CorA does not rescue invasion while S. Typhimurium CorA does.
Since S. Typhimurium CorA rescues, but M. jannaschii CorA does not then it could be assumed that M. jannaschii CorA is lacking important residues that mediate the rescue phenotype. For example, either a residue that is chemically
182 modified is not present in M. jannaschii or a stretch of protein is different that normally would interact with another protein. The sequences of these two proteins could be compared and those different between S. Typhimurium and M. jannaschii could be further examined. Chimeras made up of the S. Typhimurium and M. jannaschii CorA could be constructed to narrow down the region of importance. Specific residues that potentially are post-translationally modified could be mutated and transport ability assessed to identify a residue of importance. A peptide accompassing a region of importance could be used to purify an interacting protein. The peptide could be attached to a bead on a column and cell lysate from S. Typhimurium could be run through that column.
The interacting protein could then be purified. The one drawback to these assays are that the virulence must to linked to CorA regulation for these alternatives to work.
Regulation of Mg2+ and virulence
CorA function is necessary for invasion of and replication within Caco-2 epithelial cells (Chapter 5). In addition, our data indicate that these phenotypes require a CorA protein that is able to close. Moreover, the spatial arrangement of surface residues on the CorA protein is also important. These observations directly tie to the observations that CorA function appears to be regulated. Thus, it appears that regulation of CorA function is important for Salmonella interaction with epithelial cells. For example, when wild type cells are grown under conditions in which CorA transport regulation occurs, in high Mg to stationary
183 phase, they can no longer invade or replicate within Caco-2 epithelial cells
(Figure 6.2, p. 189).
Extracellular Mg2+ has been shown to be critical for regulating signaling
networks in the pathogen through the two component system PhoPQ (Chapter
1). We anticipate that CorA is part of a broader signaling network in the cell that
is closely linked to intracellular Mg2+ homeostasis. It is known that intracellular
Mg2+ directly regulates the transcription of at least one gene, mgtA, a Mg2+ transporter. Another hypothesis is that regulating Mg2+ homeostasis in the cell is
crucial to maintain appropriate signaling within the bacterium. As a result,
mutating or knocking out CorA, the primary Mg2+ channel alters signaling in the
cell, which is phenotypically displayed as a decrease in virulence. Thus,
regulation of CorA function is necessary for virulence. The most likely
mechanism is that CorA interacts with another protein and/or is chemical
modified. Identifying the mechanism of CorA regulation is essential to begin to interpret the role that Mg2+ plays in virulence.
Once a mechanism of regulation is determined, further experiments can
assess whether this regulation is tied to virulence. If a site of post-translation
modification is identified, then the site can be mutated on CorA. This CorA
mutant would then be tested for invasion and replication within Caco-2 epithelial
cells. If this mutant has a defect for interaction with epithelial cells, then the
regulatory mechanism of CorA is important. Moreover, such a mutant can be
compared to wild type in terms of survival after or intraperitoneal administration
184 to mice. Similar approaches can be used to assess a role in virulence if another
form of regulation is identified.
Conclusions and unanswered questions for the field
Many mysteries remain with PhoPQ. There appear to be several different
mechanisms by which PhoP interacts with the promoters of genes it regulates.
Conflict is present in the literature about which promoters PhoP can bind to.
Moreover, is phosphorylation of PhoP necessary for binding to its promoters?
While PhoPQ generally positively regulates SPI2 and negatively regulates SPI1,
there is some overlap, and the role of this differential regulation is unknown.
Moreover there is evidence that PhoPQ and SPI2 may act independently of one
another. Thus many questions remain unanswered about PhoPQ and virulence
as well.
The crystal structure of T. maritima CorA has been solved, thus opening
the door for further structure function studies. Many questions remain to be
answered about the mechanism of transport of CorA, but given the recent
structural information many will most likely be answered. Are N314, M302,
L294, and M291 functioning as gates to block the passage of Mg2+? Are there interactions between the ring of positive charge at the cytosolic membrane interface and the ring of negative charge created by the ends of the willow helices which influence the mechanism of transport? Are the five Mg2+s bound between D89 and D253 of adjacent monomers involved in the mechanism as well? How does efflux occur, and what role does it play physiologically? These
185 and several more questions need to be answered in order to determine how
cations get moved via CorA.
Not only are there structure function questions that remained to be
answered, the full role of CorA in general microbial physiology remains to be
determined. Mutation of corA and inhibition of CorA via selective antagonists can
rescue the Fe2+ sensitivity of the phoP and mgtA mgtCB mutants even though
2+ CorA does not transport Fe . Interestingly, the Vmax of CorA is dramatically
increased in these mutant backgrounds. Why? Moreover, exposure to
lactoperoxidase induces expression of corA, and corA mutants are hypersensitive to killing by lactoperoxidase. CorA is required for Salmonella virulence and is functionally regulated. The mechanism and physiological relevance of these observations are yet to be determined.
186 Table 6.1. Summary from data in Chapter five: The effect of different media on
CorA protein content, transport by CorA, and Mg2+ content during transition from log to stationary phase
Low Mg LB High Mg
Protein ↑ ↑ =
Transport ↓ ↑ ↓
Mg2+ Content = ↓ ↑
187
Figure 6.1. Regulation of corA transcription. In low Mg, phoQ is required for transcription of corA. In high Mg, loss of phoQ results in increased transcription of corA. The model is an schematic representation of how corA transcription may be being regulated in the different media. In low Mg, PhoP could phosphorylate Protein X triggering the release of Protein Z (A). Protein Z could promote the transcription of corA. In the absence of phoP, corA is not transcribed in low Mg (B). In high Mg, PhoP phosphorylates Protein X preventing the release of Protein Y, thus corA transcription is unaffected (C). If phoP is absent, transcription of corA is increased (D).
188 Wild type low Mg Wild type LB 3 Wild type high Mg ΔcorA low Mg ΔcorA LB ΔcorA high Mg
2
1 Percent gentamicin protected bacteria
0 1 hour 6 hours Time (hours)
Figure 6.2. Wild type and the corA mutant were grown in low Mg, LB, and high
Mg to stationary phase. These cells were used to invade Caco-2 epithelial cells.
Caco-2 epithelial cells were invaded for 1 hr and treated with gentamicin. 1 hr and 6 hr after gentamicin treatment Caco-2 epithelial cells were lysed and intracellular bacteria were plated. Colonies that formed were counted.
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