Recognition of Antimicrobial Peptides by a Bacterial Sensor Kinase

SummaryPhoQ is a membrane bound sensor kinase important for the pathogenesis of a number of Gram-negative bacterial species. PhoQ and its cognate response regulator PhoP constitute a signal-transduction cascade that controls inducible resistance to host antimicrobial peptides. We show that enzymatic activity of Salmonella typhimurium PhoQ is directly activated by antimicrobial peptides. A highly acidic surface of the PhoQ sensor domain participates in both divalent-cation and antimicrobial-peptide binding as a first step in signal transduction across the bacterial membrane. Identification of PhoQ signaling mutants, binding studies with the PhoQ sensor domain, and structural analysis of this domain can be incorporated into a model in which antimicrobial peptides displace divalent cations from PhoQ metal binding sites to initiate signal transduction. Our findings reveal a molecular mechanism by which bacteria sense small innate immune molecules to initiate a transcriptional program that promotes bacterial virulence

Mutational Analysis of the Residue at Position 48 in the Salmonella enterica Serovar Typhimurium PhoQ Sensor Kinase

The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg(2+) depletion. The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor. This mutation has been shown to increase net phosphorylation of the PhoP response regulator. We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]). Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro. Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype. In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg(2+), in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype. By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity. In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity. Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states

PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production in Citrobacter rodentium

International audienceOuter membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium ΔpmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA) and cptA OMV production in strains lacking either pmrC (eptA) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC (eptA) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that β-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC (eptA) and cptAIMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria

The Virulence Effect of CpxRA in Citrobacter rodentium Is Independent of the Auxiliary Proteins NlpE and CpxP

Citrobacter rodentium is a murine pathogen used to model the intestinal infection caused by Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC), two diarrheal pathogens responsible for morbidity and mortality in developing and developed countries, respectively. During infection, these bacteria must sense and adapt to the gut environment of the host. In order to adapt to changing environmental cues and modulate expression of specific genes, bacteria can use two-component signal transduction systems (TCS). We have shown that the deletion of the Cpx TCS in C. rodentium leads to a marked attenuation in virulence in C3H/HeJ mice. In E. coli, the Cpx TCS is reportedly activated in response to signals from the outer-membrane lipoprotein NlpE. We therefore investigated the role of NlpE in C. rodentium virulence. We also assessed the role of the reported negative regulator of CpxRA, CpxP. We found that as opposed to the ΔcpxRA strain, neither the ΔnlpE, ΔcpxP nor the ΔnlpEΔcpxP strains were significantly attenuated, and had similar in vivo localization to wild-type C. rodentium. The in vitro adherence of the Cpx auxiliary protein mutants, ΔnlpE, ΔcpxP, ΔnlpEΔcpxP, was comparable to wild-type C. rodentium, whereas the ΔcpxRA strain showed significantly decreased adherence. To further elucidate the mechanisms behind the contrasting virulence phenotypes, we performed microarrays in order to define the regulon of the Cpx TCS. We detected 393 genes differentially regulated in the ΔcpxRA strain. The gene expression profile of the ΔnlpE strain is strikingly different than the profile of ΔcpxRA with regards to the genes activated by CpxRA. Further, there is no clear inverse correlation in the expression pattern of the ΔcpxP strain in comparison to ΔcpxRA. Taken together, these data suggest that in these conditions, CpxRA activates gene expression in a largely NlpE- and CpxP-independent manner. Compared to wildtype, 161 genes were downregulated in the ΔcpxRA strain, while being upregulated or unchanged in the Cpx auxiliary protein deletion strains. This group of genes, which we hypothesize may contribute to the loss of virulence of ΔcpxRA, includes T6SS components, ompF, the regulator for colanic acid synthesis, and several genes involved in maltose metabolism

Both group 4 capsule and lipopolysaccharide O-antigen contribute to enteropathogenic Escherichia coli resistance to human α-defensin 5.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5

Capsule production and HD-5 resistance in EPEC and EHEC.

<p>(A) Transcription of <i>gfcA</i> in the EPEC and EHEC wild-type strains was quantified by qPCR. Data shown (2^-ΔCT x 10³) are representative of <i>gfcA</i> gene expression normalized against 16S RNA gene expression. Results are expressed as means ± SEs of triplicate samples. Asterisks indicate statistical significance; **, <i>P</i> <0.01 by paired <i>t</i> test. (B) Capsule staining of EPEC and EHEC wild-type strains, capsules are visualized by negative staining at a magnification of 100 X. Images shown are representative of at least ten fields of view from three independent experiments. (C) Survival of EPEC and EHEC wild-type cells in the presence of 5 µM HD-5. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance; **, <i>P</i> <0.01 by two-way ANOVA and Bonferroni's multiple comparison <i>post </i><i>hoc</i> test.</p

HD-5 and HD-6 do not synergize.

<p>Survival of EPEC wild-type, ∆<i>gfcA</i>, ∆<i>gfcA</i>(p<i>gfcA</i>) strains (A) and EPEC wild-type, ∆<i>waaL</i>, ∆<i>waaL</i>(p<i>waaL</i>), ∆<i>gfcA</i>∆<i>waaL</i> and ∆<i>gfcA</i>∆<i>waaL</i>(p<i>waaL</i>) strains (B) in the presence of 4 µM HD-6. (C) Survival of EPEC wild-type, ∆<i>gfcA</i>, ∆<i>gfcA</i>(p<i>gfcA</i>), ∆<i>waaL</i> and ∆<i>waaL</i>(p<i>waaL</i>) in the presence of 5 µM HD-5 and 4 µM HD-6. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance; **, <i>P</i><0.01; ****, <i>P</i> <0.0001 by one-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p

The O-antigen promotes resistance to HD-5.

<p>Survival of the indicated EPEC strains in the presence of LL-37 (A) and HD-5 (B and C) at the indicated concentrations. Results are expressed as means ± SEs of triplicate samples. Data shown are representative of at least three independent experiments. Asterisks indicate statistical significance; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; ****, <i>P</i> <0.0001 by two-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p

Expression of surface structures by EPEC.

<p>Transcription of the indicated genes was quantified by qPCR. Data shown (2^-ΔCT x 10³) are normalized against transcription of the 16S RNA gene. Results are expressed as means ± SEs of triplicate samples. Asterisks indicate statistical significance; ***, <i>P</i> <0.001 by one-way ANOVA and Bonferroni’s multiple comparison <i>post </i><i>hoc</i> test.</p