Identification of cptA, a PmrA-regulated locus required for phosphoethanolamine modification of the Salmonella enterica serovar typhimurium lipopolysaccharide core

In response to the in vivo environment, the Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) is modified. These modifications are controlled in part by the two-component regulatory system PmrA-PmrB, with the addition of 4-aminoarabinose (Ara4N) to the lipid A and phosphoethanolamine (pEtN) to the lipid A and core. Here we demonstrate that the PmrA-regulated STM4118 (cptA) gene is necessary for the addition of pEtN to the LPS core. pmrC, a PmrA-regulated gene necessary for the addition of pEtN to lipid A, did not affect core pEtN addition. Although imparting a similar surface charge modification as Ara4N, which greatly affects polymyxin B resistance and murine virulence, neither pmrC nor cptA plays a dramatic role in antimicrobial peptide resistance in vitro or virulence in the mouse model. Therefore, factors other than surface charge/electrostatic interaction contribute to resistance to antimicrobial peptides such as polymyxin B.     

Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening

Salmonella enterica polymyxin B (PM) resistance is modulated mainly by substitutions of the acyl chains and the phosphate groups on the lipid A moiety of lipopolysaccharide. These modifications are mediated by genes under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems. In this study, a deletion in the gene encoding the alternative σ⁵⁴ factor, rpoN , was shown to increase PM resistance without affecting protamine sensitivity. The results presented here showed that the increased polymyxin resistance observed in the Δ rpoN mutant occurs through a PmrA/PhoP-independent pathway. Downregulation of one or more genes belonging to the RpoN regulon may provide an additional mechanism of defence against membrane-permeabilizing antimicrobial peptides that helps the pathogen to survive in different environments.     

Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection

Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid-like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig-14 and virK genes of Salmonella enterica are important in bacterial resistance to anti-microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon-gamma (IFN-gamma) is crucial to controlling replication of mig-14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig-14 contributes to long-term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig-14 and virK contribute to the survival of Salmonella in macrophages treated with IFN-gamma and are necessary for resistance to cathelin-related anti-microbial peptide (CRAMP), an anti-microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig-14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig-14 is an inner membrane-associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig-14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig-14 in anti-microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.     

PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A

Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated. The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium. Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance. Thus, P. aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions.     

Resistance to antimicrobial peptides contributes to persistence of Salmonella typhimurium in the C. elegans intestine

The human pathogen Salmonella typhimurium can colonize, proliferate and persist in the intestine causing enteritis in mammals and mortality in the nematode Caenorhabditis elegans. Using C. elegans as a model, we determined that the Salmonella pathogenicity islands-1 and -2 (SPI-1 and SPI-2), PhoP and the virulence plasmid are required for the establishment of a persistent infection. We observed that the PhoP regulon, SPI-1, SPI-2 and spvR are induced in C. elegans and isogenic strains lacking these virulence factors exhibited significant defects in the ability to persist in the worm intestine. Salmonella infection also leads to induction of two C. elegans antimicrobial genes, abf-2 and spp-1, which act to limit bacterial proliferation. The SPI-2, phoP and Delta pSLT mutants are more sensitive to the cationic peptide polymyxin B, suggesting that resistance to worm's antimicrobial peptides might be necessary for Salmonella to persist in the C. elegans intestine. Importantly, we showed that the persistence defects of the SPI-2, phoP and Delta pSLT mutants could be rescued in vivo when expression of C. elegans spp-1 was reduced by RNAi. Together, our data suggest that resistance to host antimicrobials in the intestinal lumen is a key mechanism for Salmonella persistence.     

Structural characterization of lipo-oligosaccharide (LOS) from Yersinia pestis: regulation of LOS structure by the PhoPQ system

The two-component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhimurium. In Yersinia pestis, which expresses lipo-oligosaccharide (LOS), a PhoPQ regulatory system has been identified and an isogenic mutant constructed. To investigate potential modifications to LOS from Y. pestis, which to date has not been fully characterized, purified LOS from wild-type plague and the phoP defective mutant were analysed by mass spectrometry. Here we report the structural characterization of LOS from Y. pestis and the direct comparison of LOS from a phoP mutant. Structural modifications to lipid A, the host signalling portion of LOS, were not detected but analysis of the core revealed the expression of two distinct molecular species in wild-type LOS, differing in terminal galactose or heptose. The phoP mutant was restricted to the expression of a single molecular species, containing terminal heptose. The minimum inhibitory concentration of cationic antimicrobial peptides for the two strains was determined and compared with the wild-type: the phoP mutant was highly sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ regulatory system and the ability to alter LOS structure may be required for survival of Y. pestis within the mammalian and/or flea host.     

A Lactobacillus acidophilus strain of human gastrointestinal microbiota origin elicits killing of enterovirulent Salmonella enterica Serovar Typhimurium by triggering lethal bacterial membrane damage

The human gastrointestinal microbiota produces antagonistic activities against gastrointestinal bacterial pathogens. We undertook a study to investigate the mechanism(s) by which a Lactobacillus acidophilus strain of human microbiota origin antagonizes the gram-negative enteroinvasive pathogen Salmonella enterica serovar Typhimurium. We showed that the cell-free culture supernatant of L. acidophilus strain LB (LB-CFCS) induced the following effects in S. enterica SL1344: (i) a decrease in intracellular ATP that paralleled bacterial death, (ii) the release of lipopolysaccharide, (iii) permeabilization of the bacterial membrane, and (iv) an increase in the sensitivity of Salmonella to the lytic action of sodium dodecyl sulfate. Finally, we showed using two mutant strains of Salmonella, PhoP MS7953s and PmrA JKS1170, that the two-component regulatory systems PhoP-PhoQ and PmrA-PmrB that regulate the mechanisms of resistance to antibacterial agents in Salmonella did not influence the anti-Salmonella effect of LB-CFCS.     

Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification

Salmonella enterica serovar Typhimurium remodels the lipid A component of lipopolysaccharide, a major component of the outer membrane, to survive within animals. The activation of the sensor kinase PhoQ in host environments increases the synthesis of enzymes that deacylate, palmitoylate, hydroxylate, and attach aminoarabinose to lipid A, also known as endotoxin. These modifications promote bacterial resistance to antimicrobial peptides and reduce the host recognition of lipid A by Toll-like receptor 4. The Salmonella lipid A 3-O-deacylase, PagL, is an outer membrane protein whose expression is regulated by PhoQ. In S. enterica serovar Typhimurium strains that had the ability to add aminoarabinose to lipid A, 3-O-deacylated lipid A species were not detected, despite the PhoQ induction of PagL protein expression. In contrast, strains defective for the aminoarabinose modification of lipid A demonstrated in vivo PagL activity, indicating that this membrane modification inhibited PagL's enzymatic activity. Since not all lipid A molecules are modified with aminoarabinose upon PhoQ activation, these results cannot be ascribed to the substrate specificity of PagL. PagL-dependent deacylation was detected in sonically disrupted membranes and membranes treated with the nonionic detergent n-octyl-beta-d-glucopyranoside, suggesting that perturbation of the intact outer membrane releases PagL from posttranslational inhibition by aminoarabinose-containing membranes. Taken together, these results suggest that PagL enzymatic deacylation is posttranslationally inhibited by membrane environments, which either sequester PagL from its substrate or alter its conformation.     

Quantitative proteomic analysis of Salmonella enterica serovar Typhimurium under PhoP/PhoQ activation conditions

The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg(2+) concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg(2+) concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg(2+) concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg(2+) concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg(2+)-concentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella's adaptation to the host environment.