Effect-Directed Synthesis of PhoP/PhoQ Inhibitors to Regulate Salmonella Virulence.

Salmonella spp. are among the leading bacterial causes of foodborne infections. The PhoP/PhoQ two-component regulatory system serves as a master virulence regulator in Salmonella. Although PhoP/PhoQ represents an ideal target for disarming Salmonella virulence, it has very few inhibitors reported so far. We describe a novel platform by which an inhibitor was selected out of around 185 compounds directly from reaction media containing thiosemicarbazones and mono-, di-, and trihydrazones. To achieve this, tandem library preparation, thin-layer chromatography (TLC) bioautography, and effect-directed deconvolution were applied. We illustrate the potential of this effect-directed synthesis for the identification of new useful bioactive compounds for the food field.

The Rcs signal transduction pathway is triggered by enterobacterial common antigen structure alterations in Serratia marcescens.

The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to the Serratia marcescens motile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis of wec cluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of the S. marcescens Rcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected in wec mutant strains in other pathogenic bacteria.

Histone-like nucleoid-structuring protein (H-NS) regulatory role in antibiotic resistance in Acinetobacter baumannii.

In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes involved in pathogenesis and stress response. In addition, H-NS inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with ß-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.

PhoQ is an unsaturated fatty acid receptor that fine-tunes Salmonella pathogenic traits.

The Salmonella enterica PhoP/PhoQ two-component signaling system coordinates the spatiotemporal expression of key virulence factors that confer pathogenic traits. Through biochemical and structural analyses, we found that the sensor histidine kinase PhoQ acted as a receptor for long-chain unsaturated fatty acids (LCUFAs), which induced a conformational change in the periplasmic domain of the PhoQ protein. This resulted in the repression of PhoQ autokinase activity, leading to inhibition of the expression of PhoP/PhoQ-dependent genes. Recognition of the LCUFA linoleic acid (LA) by PhoQ was not stereospecific because positional and geometrical isomers of LA equally inhibited PhoQ autophosphorylation, which was conserved in multiple S. enterica serovars. Because orally acquired Salmonella encounters conjugated LA (CLA), a product of the metabolic conversion of LA by microbiota, in the human intestine, we tested how short-term oral administration of CLA affected gut colonization and systemic dissemination in a mouse model of Salmonella-induced colitis. Compared to untreated mice, CLA-treated mice showed increased gut colonization by wild-type Salmonella, as well as increased dissemination to the spleen. In contrast, the inability of the phoP strain to disseminate systemically remained unchanged by CLA treatment. Together, our results reveal that, by inhibiting PhoQ, environmental LCUFAs fine-tune the fate of Salmonella during infection. These findings may aid in the design of new anti-Salmonella therapies.

mgtA Expression is induced by rob overexpression and mediates a Salmonella enterica resistance phenotype.

Rob is a member of the Sox/Mar subfamily of AraC/XylS-type transcriptional regulators implicated in bacterial multidrug, heavy metal, superoxide, and organic solvent resistance phenotypes. We demonstrate that, in Salmonella enterica, Rob overexpression upregulates the transcription of mgtA, which codes for the MgtA Mg2+ transporter. mgtA was previously characterized as a member of the Mg2+-modulated PhoPQ regulon. Here we demonstrate that Rob (but not its paralog protein SoxS or MarA) is able to induce mgtA transcription in a PhoP-independent fashion by binding to a conserved Mar/Sox/Rob motif localized downstream of the PhoP-box and overlapping the PhoP-dependent transcriptional start site. We found that Rob-induced mgtA expression confers low-level cyclohexane resistance on Salmonella. Because mgtA intactness is required for Rob-induced cyclohexane resistance, provided the AcrAB multidrug efflux pump can be expressed, we postulate that MgtA is involved in the AcrAB-mediated cyclohexane detoxification mechanism promoted by Rob in Salmonella.

Induction of RpoS degradation by the two-component system regulator RstA in Salmonella enterica.

Bacterial survival in diverse and changing environments relies on the accurate interplay between different regulatory pathways, which determine the design of an adequate adaptive response. The proper outcome depends on a precise gene expression profile generated from the finely tuned and concerted action of transcriptional factors of distinct regulatory hierarchies. Salmonella enterica serovar Typhimurium harbors multiple regulatory systems that are crucial for the bacterium to cope with harsh extra- and intracellular environments. In this work, we found that the expression of Salmonella RstA, a response regulator from the two-component system family, was able to downregulate the expression of three RpoS-controlled genes (narZ, spvA, and bapA). Furthermore, this downregulation was achieved by a reduction in RpoS cellular levels. The alternative sigma factor RpoS is critical for bacterial endurance under the most-stressful conditions, including stationary-phase entrance and host adaptation. Accordingly, RpoS cellular levels are tightly controlled by complex transcriptional, translational, and posttranslational mechanisms. The analysis of each regulatory step revealed that in Salmonella, RstA expression was able to promote RpoS degradation independently of the MviA-ClpXP proteolytic pathway. Additionally, we show that RstA is involved in modulating Salmonella biofilm formation. The fact that the RpoS-modulated genes affected by RstA expression have previously been demonstrated to contribute to Salmonella pathogenic traits, which include biofilm-forming capacity, suggests that under yet unknown conditions, RstA may function as a control point of RpoS-dependent pathways that govern Salmonella virulence.

The H box-harboring domain is key to the function of the Salmonella enterica PhoQ Mg2+-sensor in the recognition of its partner PhoP.

In two-component signaling systems, the transduction strategy relies on a conserved His-Asp phosphoryl exchange between the sensor histidine kinase and its cognate response-regulator, and structural and functional consensus motifs are found when comparing either the diverse histidine kinases or response regulators present in a single cell. Therefore, the mechanism that guarantees the specific recognition between partners of an individual pair is essential to unequivocally generate the appropriate adaptive response. Based on sequence alignments with other histidine kinases, we dissected the Salmonella enterica Mg2+-sensor PhoQ in different subdomains and examined by in vivo and in vitro assays its interaction with the associated response regulator PhoP. This signal transduction system allows Salmonella to withstand environmental Mg2+ limitation by triggering gene expression that is vital throughout the infective cycle in the host. Using resonant mirror biosensor technology, we calculated the kinetic and equilibrium binding constants and determined that the His-phosphotransfer domain is essential for the PhoQ specific recognition and interaction with PhoP. Additionally, we show the role of this domain in the bimolecular transphosphorylation and provide evidence that this region undergoes dimerization.