Unit: Biology of Gram-positive Pathogens
Director: Patrick TRIEU-CUOT
The main goals of our research activity aim at elucidating new pathways/mechanisms involved in the pathogenesis of low GC% Gram positive pathogens. We believe that the in-depth understanding of infectious processes will contribute to the development of new therapeutics or innovative tools for the treatment, prevention and control of infections due to Gram positive bacteria. We have chosen Staphylocccus aureus and Streptococcus agalactiae, as models of extracellular human pathogens and Listeria monocytogenes as a model intracellular pathogen.
Our main research topics are: 1) bacterial surface components (lipoteichoic acids and surface proteins) involved in the interactions with the host, 2) gene regulation and expression of virulence genes in relation with stress response and environmental adaptation (two-component regulatory systems).
Bacterial surface components involved in virulence
During or following translocation, the signal peptides of lipoproteins are modified by the diacylglyceryl transferase Lgt prior cleavage by the signal peptidase Lsp. The genes encoding Lgt and Lsp were inactivated in order to determine the contribution of the lipoproteins to the virulence of S. agalactiae and a double mutant Δlgt/lsp was also constructed. Western blot analysis of two prototype lipoproteins acting as adhesins (Lmb and ScaA) revealed that these proteins were only detected in the membrane fractions of WT and Lsp- strains. Interestingly, they were massively released in the culture supernatant of the Δlgt and Δlgt/lsp mutants and were absent in the membrane, cell-wall, and cytoplasmic fractions of the corresponding bacterial lysates. All mutants displayed altered adhesion to fibronectin and fibrinogen. However, only the Δlsp and Δlgt/lsp mutants were severely impaired in their ability to bind human epithelial cell lines and were strongly reduced in virulence in a neonate rat infection model. These results suggest that Lsp is required for biosynthesis of the active form of (an) as yet unknown lipoprotein(s) essential for GBS virulence.
Incorporation of D-Ala residues into lipoteichoic acids (LTA) requires the activity of four proteins encoded by the dlt operon: DltA is a cytoplasmic D-ala-D-alanyl carrier protein ligase which catalyzes D-alanylation of the D-alanyl carrier protein DltC; DltB is a transmembrane protein thought to be involved in efflux of D-Ala-DltC to the acylation site; and DltD, a protein whose function and cellular localization are unknown. Formation of D-Ala esters of LTAs is essential for virulence of extra- (S. agalactiae and S. aureus) and intracellular (L. monocytogenes) Gram-positive pathogens and DltA-D therefore constitute potential targets for new antimicrobial agents. By using complementary approaches, we showed that: 1) DltD is anchored in the cytoplasmic leaflet of the membrane via its NH2 extremity, 2) the acidic carrier DltC (pI = 3.9) is efficiently exported to the site of esterification by DltB and might be retained in the cell-wall through interactions with positively charged components. Thus, DltC appears to be the only extracellular potential target for inhibitors. It remains to be determined if the transfer of D-Ala to the LTAs is catalyzed by an enzyme.
Gene regulation and stress response
The degU gene of L. monocytogenes appears to encode an orphan response regulator since the degS histidine kinase gene is not present in the genome, and there are no orphan histidine kinase genes. A significant decrease (11-fold) in the LD50 of the Δ degU mutant strain, compared to that of the otherwise isogenic parental EGDe, was observed in a murine model of infection. We carried out a transcriptome analysis with the ΔdegU mutant and approximately 81 genes were identified as positively controlled by DegU, while 6 were repressed (Photo 1). Most of these genes are of unknown function and their role in the pathogenesis of L. monocytogenes is currently being investigated. The L. monocytogenes DegU protein was overproduced and purified. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified DegU to the promoter region of its own gene, where it acts as a repressor of its own synthesis.
The two-component regulatory system CovS/CovR is a pleiotropic regulator of the virulence factors of S. pyogenes,. The orthologous system was inactivated in S. agalactiae and the resulting mutant displayed hyper-hemolytic activity and was hyper-adherent to all epithelial cell lines tested (A549, Hela, Caco-2 and L2) (Photo 2). However, its virulence was dramatically reduced in an intraperitoneal-injection model in neonatal rats and we showed that it was unable to grow in human serum. A comparative transcriptome analysis of S. agalactiae WT and ΔcovRS mutant strains revealed that 76 genes were repressed whereas 63 were activated when this regulatory system was active. Purified CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses.
We have investigated the role and regulation of the S. aureus clp genes encoding subunits of the Clp ATP-dependent protease. By primer extension analysis and DNaseI footprinting studies, we showed that CtsR of S. aureus controls the stress-induced expression of clpP, clpB and clpC by direct and specific binding to their promoter regions. ClpP, ClpC, ClpB and ClpX, and to a lesser extent, ClpL, were required for intracellular multiplication of S. aureus within bovine mammary MAC-T epithelial cells. In addition to virulence factor production, the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data indicate that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.
Photo 1: Transcriptome analysis in Listeria monocytogenes. The entire 3000 gene complement of Listeria monocytogenes is present on a high density membrane DNA array as double-stranded DNA fragments. Red spots indicate genes whose expression is repressed, green spots those whose expression is activated, yellow spots thos whose expression does not vary under the two conditions tested, and black spots represent genes that are not expressed under the experimental conditions used.
Photo 2: Phenotype of the global two-component ΔcovSR mutant of S. agalactiae: hyper-hemolytic on blood agar plates and hyper-adherence on epithelial cells
Keywords: Gram positive bacteria, gene expression, virulence factors, stress response, signal transduction