Gwendoline Kint

The proteome of Salmonella Typhimurium grown under in vivo-mimicking conditions.

To successfully infect a host, it is a prerequisite for enteric pathogens such as Salmonella enterica serovar Typhimurium to adapt to their environment, in casu the gastrointestinal tract. The adoption of an appropriate lifestyle is triggered by environmental signals such as the low oxygen availability and high osmolarity prevalent in the gut. In order to gain more insight in the changes that are induced when S. Typhimurium is adapting to these particular conditions, we used 2‐D DIGE technology to investigate the combined effect of low oxygen tension and high osmolarity on the proteome of S. Typhimurium SL1344 compared to standard laboratory conditions. As a validation of the 2‐D DIGE technique, preferential protein labeling by the Cy‐dyes was assessed and proved to be negligible. The differentially expressed proteins identified reflect very well the applied culture conditions. Furthermore, reported transcriptional changes and observed changes at the translational level show overlap. Among the metabolic processes that are upregulated under in vivo‐mimicking conditions are anaerobic fumarate respiration and the utilization of 1,2‐propanediol. We also provide evidence that S. Typhimurium expresses an arginine deiminase pathway for the catabolism of L‐arginine. The increased activity of this pathway was biochemically validated. Finally, also proteins involved in quorum sensing and virulence are differentially expressed under in vivo‐mimicking conditions. These conditions offer possibilities as a simplified model system for the host environment given the high overlap of identifications in our study and reported genuine in vivo studies, respectively.

The small regulatory RNA molecule MicA is involved in Salmonella enterica serovar Typhimurium biofilm formation.

BackgroundLuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.ResultsAnalysis of additional S. Typhimurium luxS mutants indicated that the LuxS enzyme itself is not a prerequisite for a wild type mature biofilm. However, in close proximity of the luxS coding sequence, a small RNA molecule, MicA, is encoded on the opposite DNA strand. Interference with the MicA expression level showed that a balanced MicA level is essential for mature Salmonella biofilm formation. Several MicA targets known to date have previously been reported to be implicated in biofilm formation in Salmonella or in other bacterial species. Additionally, we showed by RT-qPCR analysis that MicA levels are indeed altered in some luxS mutants, corresponding to their biofilm formation phenotype.ConclusionsWe show that the S. Typhimurium biofilm formation phenotype of a luxS mutant in which the complete coding region is deleted, is dependent on the sRNA molecule MicA, encoded in the luxS adjacent genomic region, rather than on LuxS itself. Future studies are required to fully elucidate the role of MicA in Salmonella biofilm formation.

2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

BackgroundQuorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.ResultsDifferential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-βla and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of β-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis.Conclusion2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.

FabR regulates Salmonella biofilm formation via its direct target FabB

BackgroundBiofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics.ResultsAll the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation.ConclusionOverall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies.