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 of intracellular pathogen.
Our main research topics are: 1) bacterial surface components (lipoteichoic acids and surface proteins) involved in the interactions with the host, 2) metabolic adaptation and virulence, and 3) 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
Genome datamining for genes encoding sortases in Gram-positive bacteria led us to propose the existence of four classes of sortases designated A, B, C and D (Dramsi et al., 2005). An in silico genome analysis indicated that S. agalactiae (Group B streptococci, GBS) strain NEM316 encodes five putative sortases including the major SrtA enzyme and four class C sortases. We previously characterized the role of SrtA which likely anchors most if not all 35 LPXTG-containing proteins encoded by NEM316 (Lalioui et al., 2005). The genes encoding GBS class C sortases are tandemly arranged in two different loci, srtC1-C2 and srtC3-C4, possessing similar genetic organization and thought to be involved in pilus biosynthesis. Each pair of sortase genes is flanked by LPXTG-protein encoding genes, two upstream and one downstream, and a divergently transcribed regulator located at its 5' extremity of this putative operon. We demonstrated that NEM316 expresses only the srtC3-C4 locus which encodes three surface proteins (Gbs1474, Gbs1477, and Gbs1478) that polymerize to form appendages ressembling pili. Structural and functional analysis of this locus revealed that: 1) the transcriptional regulator RogB is required for expression of the srtC3-C4 operon; 2) Gbs1477 and either SrtC3 or SrtC4 are required for pilus biogenesis; and 3) GBS NEM316 pili are composed of at least three LPXTG-proteins, Gbs1477, Gbs1474, and Gbs1478. Gbs1477 constitutes the major component of the pilus (Fig. 1) whereas Gbs1474, a minor associated component, likely anchors the pilus to the cell wall, and Gbs1478 is thought to be the adhesin. We also demonstrate a certain flexibility in pilus biosynthesis as pili-like structures are formed in the absence of either the basal subunit (Gbs1474) or the adhesin (Gbs1478). This study opens new perspectives in bacteria-host interactions in pathogenic streptococci.
Metabolic adaptation and virulence
To date, three strategies used by GBS to deal with environmental oxygen and ROS have been genetically and biochemically characterized: 1) ROS detoxification rely on the manganese-dependent superoxide dismutase (Mn-SodA) and we previously demonstrated that a sodA-disrupted mutant was highly susceptible to macrophage killing and survived poorly in the blood of mice infected intravenously; 2) ROS protection involves the characteristic carotenoid pigment, which constitutes a physical barrier at the surface of GBS and contributes to survival against ROS-generating agents such as those produced by macrophage (Liu et al., Proc. Natl. Acad. Sci. USA, 2004, 101-14491); 3) like all streptococcal pathogens, GBS was considered as an aerotolerant but nonrespiring, fermenting and acid-producing bacterium. However, we demonstrated that GBS was able to undergo a respiration metabolism if supplied with two essential respiratory components found in the host, menaquinone (vitamin K) and heme. These components activate an electron transport chain, which uses cytochrome bd quinol oxidase as terminal oxidoreductase. Respiration metabolism conferred a markedly improved capacity of GBS to persist during prolonged growth, and to eliminate O2 from its environment. Respiration-defective strains showed reduced growth in human blood and were attenuated for virulence in a neonatal rat sepsis model. Based on these findings, we suggested that respiration metabolism facilitates GBS dissemination and virulence by promoting its survival in blood.
Gene regulation and stress response
The degU gene of L. monocytogenes appears to encode an orphan response regulator since the cognate 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. DegU negatively regulates its own synthesis in L. monocytogenes, in contrast to the situation in B. subtilis and is essential for bacterial motility at 25°C. Indeed, DegU is required for expression of several motility and chemotaxis genes, including the flaA and motAB genes which are expressed at 25°C but not at 37°C. Furthermore, expression of DegU is required for the formation of efficient biofilms by Listeria monocytogenes (adherence to plastic surfaces). We have inactivated the phosphorylation site of DegU in vivo and shown that the protein retains much of its activity, indicating that the unphosphorylated form is active.
The YycG/YycF two-component system is very highly conserved and appears to be specific to low G+C Gram-positive bacteria, including several pathogens (Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes). This system is required for cell viability, although the basis for this and the nature of the YycF regulon remained elusive. Definition of a potential YycF recognition sequence allowed us to identify likely members of the YycF regulon in S. aureus. In order to test our predictions, we placed the yycF operon of S. aureus under the control of an inducible promoter, and confirmed that it is essential for cell viability. The YycG and YycF proteins of S. aureus were overproduced in E. coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNaseI footprinting assays were used to show direct binding in vitro of purified YycF to the promoter regions of the ssaA and isaA genes, encoding potential surface antigens, as well as that of the lytM gene encoding a peptidoglycan hydrolase.
More recently, we have used fluorescence microscopy to show loss of cell viability in the absence of YycG/YycF, and shown using Real-Time PCR that the system positively activates expression of the ssaA, isaA and lytM genes, as well as that of several potential virulence genes whose promoter sequence is preceded by a potential YycF binding site. Finally, we have shown that there is a direct relationship between the amount of YycF in the cell and adherence of the bacteria to inert polystyrene surfaces, i.e. the ability to form biofilms.
The B. subtilis Clp ATP-dependent protease plays a central role in the signal transduction network controlling stationary phase responses such as competence, degradative enzyme synthesis, stress response and sporulation. We previously reported that the B. subtilis clpP mutant is non-motile as judged by swarm plate assays and displays a highly filamentous morphology, growing as long chains of elongated cells during the exponential growth phase. This suggested that expression of chemotaxis, motility and/or autolysin genes, most of which require the sigma D alternative sigma factor for their expression, might be affected in the clpP mutant. Fluorescence microscopy revealed very similar phenotypes for the clpP and sigD mutants of B. subtilis: chromosome segregation and septation occur normally, but the cells fail to separate, suggesting that expression of a specific sigma D-dependent autolysin may be affected in the clpP mutant (Figure 2). Among these, the major autolysin LytF plays an important role in cell separation. We showed that lytF is not expressed in the clpP mutant. By placing the lytF gene under the control of a sigma D-independent inducible promoter, we were able to show that increasing concentrations of LytF within the cell progressively reversed the filamentous phenotype of the clpP mutant. Sigma D activity, and thus lytF expression, is known to be controlled by the FlgM anti-sigma factor in B. subtilis. Accordingly, a flgM mutation completely reverses the clpP filamentous phenotype, suggesting that the FlgM anti-sigma factor may be specifically degraded by the Clp ATP-dependent protease, and that its accumulation in the clpP mutant leads to inhibition of sigma D activity, and thus a loss of motility, chemotaxis and cell separation.
Figure 1: Immunolocalization of the pilin Gbs1477 on S. agalactiae strain NEM316 cell surface by electron microscopy. Bacteria were stained with rabbit antiserum against Gbs1477 and 10-nm colloidal gold-conjugated mouse anti-rabbit IgG antibody. Samples were viewed by scanning electron microscopy (Electron microscopy platform, IP).
Figure 2: Phenotype of the B. subtilis ÆclpP under fluorescence microscopy showing the cell separation defect : membranes are stained in pink and nucleic acids in blue.
Keywords: Gram positive bacteria, gene expression, virulence factors, stress response, signal transduction