SummaryThe PhoPQ two-component system of the intracellular pathogen Salmonella enterica senses and controls resistance to a-helical antimicrobial peptides (AMPs) by regulating covalent modifications of lipid A. A homologue of the phoPQ operon was found in the genome of the murine enteric extracellular pathogen, Citrobacter rodentium. Here we report that C. rodentium PhoPQ was apparently unable to mediate activation of target genes in the presence of a-helical AMPs. However, these AMPs activated C. rodentium PhoPQ expressed in a S. enterica DphoPQ mutant. Analysis of the outer membrane (OM) fractions of the C. rodentium wild-type and DphoPQ strains led to the identification of an omptin family protease (CroP) that was absent in DphoPQ. Deletion of croP in C. rodentium resulted in higher susceptibility to a-helical AMPs, indicating a direct role of CroP in AMP resistance. CroP greatly contributed to the protection of the OM from AMP damage by actively degrading a-helical AMPs before they reach the periplasmic space. Accordingly, transcriptional activation of PhoP-regulated genes by a-helical AMPs was restored in the DcroP mutant. This study shows that resistance to a-helical AMPs by the extracellular pathogen C. rodentium relies primarily on the CroP OM protease.

The PmrAB two-component system of enterobacteria regulates a number of genes whose protein products modify lipopolysaccharide (LPS). The LPS is modified during transport to the bacterial outer membrane (OM). A subset of PmrAB-mediated LPS modifications consists of the addition of phosphoethanolamine (pEtN) to lipid A by PmrC and to the core by CptA. In Salmonella enterica, pEtN modifications have been associated with resistance to polymyxin B and to excess iron. To investigate putative functions of pEtN modifications in Citrobacter rodentium, ⌬pmrAB, ⌬pmrC, ⌬cptA, and ⌬pmrC ⌬cptA deletion mutants were constructed. Compared to the wild type, most mutant strains were found to be more susceptible to antibiotics that must diffuse across the LPS layer of the OM. All mutant strains also showed increased influx rates of ethidium dye across their OM, suggesting that PmrAB-regulated pEtN modifications affect OM permeability. This was confirmed by increased partitioning of the fluorescent dye 1-N-phenylnaphthylamine (NPN) into the OM phospholipid layer of the mutant strains. In addition, substantial release of periplasmic ␤-lactamase was observed for the ⌬pmrAB and ⌬pmrC ⌬cptA strains, indicating a loss of OM integrity. This study attributes a new role for PmrAB-mediated pEtN LPS modifications in the maintenance of C. rodentium OM integrity.

No abstract

The prpZ gene cluster consists of three ORFs coding for proteins with homology to eukaryotic-type Ser/Thr protein phosphatases 2C (prpZ) and Ser/Thr protein kinases (prkY and prkX). This cluster is present in the sequenced genomes of Salmonella enterica serovar Typhi (S. Typhi) strains Ty2 and CT18. This study investigated the genetic organization of this gene cluster, its regulation and its putative involvement in virulence. The three genes are transcribed as a polycistronic mRNA as demonstrated by reverse transcriptase (RT)-PCR. Analysis of a prpZ::lacZ transcriptional fusion showed that the prpZ locus is expressed throughout the growth phase. LacZ activity and real-time RT-PCR showed that transcription of the mRNA is negatively regulated upon exposure of cells to HOCl and, to a lesser extent, hydrogen peroxide. A deletion mutant of the prpZ gene cluster showed a significantly lower level of survival than the parental strain Ty2 in human macrophages at 48 h postinfection. Together these data suggest that prpZ, prkY and prkX are virulence genes that may be part of a signaling pathway controlling long-term survival of S. Typhi in host cells.

Outer 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 cptA. IMPORTANCE 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.