Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium

BACKGROUND: The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions. RESULTS: We performed a systematic analysis of the regulation of type III effectors during growth in vitro. We have tested the ability of seven regulatory genes to regulate ten effector genes. Each regulator was expressed in the absence of the other six to avoid cascade effects. Our results confirm and extend the previously reported regulation of TTSS1 and TTSS2 effectors by InvF-SicA and SsrB respectively. CONCLUSION: The set of strains constructed for this study can be used to quickly and systematically study the regulation of newly identified effector genes of Salmonella enterica. The approach we have used can also be applied to study complex regulatory cascades in other bacterial species

E. coli K-12 and EHEC Genes Regulated by SdiA

Background: Escherichia and Salmonella encode SdiA, a transcription factor of the LuxR family that regulates genes in response to N-acyl homoserine lactones (AHLs) produced by other species of bacteria. E. coli genes that change expression in the presence of plasmid-encoded sdiA have been identified by several labs. However, many of these genes were identified by overexpressing sdiA on a plasmid and have not been tested for a response to sdiA produced from its natural position in the chromosome or for a response to AHL. Methodology/Principal Findings: We determined that two important loci reported to respond to plasmid-based sdiA, ftsQAZ and acrAB, do not respond to sdiA expressed from its natural position in the chromosome or to AHLs. To identify genes that are regulated by chromosomal sdiA and/or AHLs, we screened 10,000 random transposon-based luciferase fusions in E. coli K-12 and a further 10,000 in E. coli O157:H7 for a response to AHL and then tested these genes for sdiAdependence. We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA. Gene regulation by sdiA of E. coli is only partially dependent upon AHL. Conclusions/Significance: The genes of E. coli that respond to plasmid-based expression of sdiA are largely different than those that respond to chromosomal sdiA and/or AHL. This has significant implications for determining the true function o

SdiA, an N-Acylhomoserine Lactone Receptor, Becomes Active during the Transit of Salmonella enterica through the Gastrointestinal Tract of Turtles

encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species..We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals

Salmonella enterica Serovar Typhimurium Can Detect Acyl Homoserine Lactone Production by Yersinia enterocolitica in Mice▿

LuxR-type transcription factors detect acyl homoserine lactones (AHLs) and are typically used by bacteria to determine the population density of their own species. Escherichia coli and Salmonella enterica serovar Typhimurium cannot synthesize AHLs but can detect the AHLs produced by other bacterial species using the LuxR homolog, SdiA. Previously we determined that S. Typhimurium did not detect AHLs during transit through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during transit through turtles colonized by Aeromonas hydrophila, leading to the hypothesis that SdiA is used for detecting the AHL production of other pathogens. In this report, we determined that SdiA is activated during the transit of S. Typhimurium through mice infected with the AHL-producing pathogen Yersinia enterocolitica. SdiA is not activated during transit through mice infected with a yenI mutant of Y. enterocolitica that cannot synthesize AHLs. However, activation of SdiA did not confer a fitness advantage in Yersinia-infected mice. We hypothesized that this is due to infrequent or short interactions between S. Typhimurium and Y. enterocolitica or that the SdiA regulon members do not function in mice. To test these hypotheses, we constructed an S. Typhimurium strain that synthesizes AHLs to mimic a constant interaction with Y. enterocolitica. In this background, sdiA+ S. Typhimurium rapidly outcompetes the sdiA mutant in mice. All known members of the sdiA regulon are required for this phenotype. Thus, all members of the sdiA regulon are functional in mice

Regulation of <i>acrA</i> by <i>sdiA</i>.

<p>A chromosomal merodiploid <i>acrA</i>⁺/<i>acrA</i>-<i>lacZY</i> fusion was constructed in a Δ<i>lac</i> mutant <i>E. coli</i> strain (JLD370), and in an isogenic <i>sdiA</i> mutant (JLD373). Additionally, derivatives of the <i>sdiA</i> mutant were constructed that contained either a low copy number vector expressing <i>sdiA</i> (pCX16) or the vector control (pGB2). The strains were subcultured 1:100 into LB broth containing either 1 µM oxoC6 or EA. The cultures were incubated with shaking at 30°C (A) and 37°C (B). Samples were removed from the cultures at time points for β-galactosidase assays. Each strain was assayed in triplicate and error bars represent standard deviation. * denotes p<0.05 compared to the adjacent solvent control.</p

Acid resistance of <i>E. coli</i> K-12 and EHEC.

<p>Cells were grown in LB glucose with 1 µM oxo-C6 or 0.1% EA at either 37°C or 30°C and then subcultured into pre-warmed MEM with glucose and glutamate at pH 2.0 with continued incubation at the same temperature. Resistance to the acid challenge was determined by plating for cfu/ml every hour for two hours. <i>E. coli</i> K-12 wild-type MG1655 and <i>sdiA</i> mutant JNS21 at 37°C (A) and 30°C (B). EHEC wild-type 700927 and <i>sdiA</i> mutant DL1 at 37°C (C) and 30°C (D). Each strain was assayed in triplicate and error bars represent standard deviation.</p

Regulation of <i>ftsQAZ</i> by <i>sdiA</i>.

<p>A chromosomal <i>ftsQAZ</i>-<i>lacZ</i> fusion was constructed in a Δ<i>lac</i> mutant <i>E. coli</i> strain (JLD3011), and an isogenic <i>sdiA</i> mutant (JLD3013). Additionally, derivatives of the <i>sdiA</i> mutant were constructed that contained either a low copy number vector expressing <i>sdiA</i> (pCX16) or the vector control (pGB2). The strains were subcultured 1∶100 into LB broth containing either 1 µM oxoC6 or EA. The cultures were incubated with shaking at 30°C (A) and at 37°C (B). Samples were removed from the cultures at time points for β-galactosidase assays. Each strain was assayed in triplicate and error bars represent standard deviation. * denotes p<0.05 compared to the adjacent solvent control.</p

Antibiotic resistance of <i>E. coli</i> K-12, EHEC and <i>S.</i> Typhimurium grown in motility agar at 37°C.

<p>Strains were grown in LB 0.3% motility agar with either 1 µM oxoC6 or 0.1% EA and a dilution series of each antibiotic tested. The minimum inhibitory concentration was read from the well in which no visible growth was seen at the inoculation point. In panel A, <i>S.</i> Typhimurium was not tested because the <i>sdiA</i> plasmid carries a gene for chloramphenicol resistance. Each strain was assayed in triplicate and error bars represent standard deviation.</p

Figure 7

<p>A) Acid fitness island of <i>E. coli</i>. The transposon insertion in <i>E. coli</i> K-12, AL4001, is within <i>gadW</i> at nucleotide 3662317 of Genbank accession number U00096. The transposon insertions in the EHEC strains are shown on the same map but the nucleotide positions are from Genbank accession number BA000007. JLD605 is within <i>gadE</i> at nucleotide 4401036; JLD607 is within <i>yhiD</i> at nucleotide 4397949; JLD610 is within <i>hdeA</i> at nucleotide 4398821. B) JLD604 is just upstream of ECs2675 in the anti-sense orientation at nucleotide 3662317.</p