PmrAB-regulated LPS phosphoethanolamine modifications have multiple roles in «Citrobacter rodentium»

Gram-negative bacteria have the ability to modify their lipopolysaccharide (LPS) in response to changing environmental conditions.The principal regulator of LPS modifications in the Enterobacteriaceae is the PmrA-PmrB (PmrAB) two-component system, which is activated by micromolar concentrations of Fe 3+ and Al 3+ and mildly acidic pH.PmrAB mediates the transcription of pmrC and cptA, encoding phosphoethanolamine (pEtN) transferases that catalyze the addition of pEtN to the 1-and 4'-phosphoryl groups of the lipid A moiety and to the phosphoryl group of heptose-I of the core moiety of LPS, respectively.The first objective was to determine if PmrC-and CptA-mediated pEtN modifications play a role in maintaining outer membrane integrity in C. rodentium under PmrAB-inducing conditions.To assess the role of pEtN modifications in outer membrane integrity, C. rodentium ∆pmrAB, ∆pmrC, ∆cptA and ∆pmrC∆cptA strains were generated.The ∆pmrC, ∆pmrC∆cptA and ∆pmrAB demonstrated increased susceptibility to hydrophobic antibiotics, whereas only the ∆pmrC∆cptA and ∆pmrAB strains displayed a loss of OM integrity under PmrAB-inducing conditions.The second objective was to determine the mechanism by which PmrC-and CptA-mediated pEtN modifications maintain outer membrane integrity under PmrAB-inducing conditions.Whole-cell lysate LPS extractions performed on ∆waaL strains grown in the presence of micromolar concentrations of iron demonstrated that the ∆waaL∆pmrC∆cptA strain accumulates free lipid A, which is a measure of a defect in LPS turnover.Using sucrose density gradient centrifugation, it was shown that the ∆waaL∆pmrC∆cptA strain accumulated LPS throughout its cell envelope in the presence of micromolar iron, whereas the ∆waaL strain had most of its LPS in the fractions corresponding to the outer membrane, suggesting that pEtN modifications are involved in the transfer of LPS from the IM to the OM.The third objective was to determine whether the response to iron results in the formation of OM vesicles (OMVs).It was determined that OM vesiculation does occur in the ix CONTRIBUTIONS TO ORIGINAL SCIENTIFIC KNOWLEDGE 1.We show that PmrC and CptA maintain OM integrity in C. rodentium.2. We demonstrate that PmrC and CptA confer resistance to hydrophobic antibiotics.3. We show that PmrC and CptA promote lipid A turnover.4. We demonstrate that lack of PmrC and CptA results in free lipid A accumulation in the presence of iron.5. We demonstrate that PmrC and CptA promote transfer of LPS to the OM. 6.We show that iron increases Gram-negative bacterial resistance to hydrophobic antibiotics in a concentration-dependent manner.7. We demonstrate that iron promotes OM vesiculation.8.We show that PmrC and CptA confer protection against OMVs.

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