|Director : TRIEU-CUOT Patrick (email@example.com)|
Our research is directed mainly towards characterizing the virulence factors of Streptococcus agalactiae, a major cause of invasive infections in the neonate. To this end, genetic and cellular technics are combined with the use of a murine model to characterize the genes involved in the virulence of this bacterium. Beside this research, we also studied the dissemination of antibiotic resistance in the enterococci, streptococci, and related bacteria. Lastly, we have developed a novel genotypic method for accurate identification of Gram-positive bacteria (enterococci, streptococci, staphylococci,...) at the species level based on the rapid sequencing of the monocopy target gene sodA.
Virulence factors of Streptococcus agalactiae
Lancefield's group B Streptococcus (GBS), also referred to as S. agalactiae, is part of the normal flora colonizing the respiratory, gastrointestinal, and urogenital tracts of humans. It is the leading cause of invasive infections (septicemia, meningitis, and pneumonia) in neonates and is also responsible for invasive infections in adults, in particular in immuno-compromised hosts. GBS are subclassified into serotypes according to the immunologic reactivity of the polysaccharide capsule. Of the nine serotypes described so far, the types Ia, Ib, II, III, and V are responsible for the majority of neonatal human GBS disease. Serotype III GBS is particularly important because it causes a significant percentage of early-onset disease (infection occurring within the first week of life) and the majority of late-onset disease (infection occurring after the first week of life) in human neonates. It is also responsible for the majority (80%) of neonatal GBS meningitis cases.
The physiopathology of GBS infections implies that this bacteria i) can evade the host defense, ii) can adhere to and invade various types of epithelial cells, including those constituting the brain blood barrier, and iii) can rapidly adapt to various growth conditions (pH and temperature variations, nutritional starvation, etc...).
1. Contribution of the Mn-Cofactored Superoxide Dismutase (SodA) to the Virulence of S. agalactiae.
Superoxide dismutases convert the superoxide anions to molecular oxygen and hydrogen peroxide which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and, hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that GBS possesses a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis-complemented by integration into the chromosome of pAT113/Sp harboring the wild sodA gene. The SOD specific activity detected by gel analysis in cell-free extracts confirmed that active SODs were present in the parental and the complemented strains, but absent in the sodA mutant. The growth rate of the mutant in aerated medium was lower than that of the wild and complemented strains, whereas the growth rates of these strains were comparable in standing cultures. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after i.v. injection, the survival of the sodA mutant in the blood and the brain was markedly reduced in comparison to those of the parental and complemented strains, whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.
2. Biosynthesis of D-alanyl-lipoteichoic acid in S. agalactiae is regulated by a novel two-component regulatory system and contributes to virulence.
The dlt operon of gram-positive bacteria comprises four genes (dltA, dltB, dltC, and dltD) that catalysed the incorporation of D-alanine residues into the lipoteichoic acids (LTAs). We characterised the dlt operon of S. agalactiae which, in addition to the dltA-dltD genes, included two regulatory genes, designated dltR and dltS, located upstream of dltA. The gene dltR encodes a 224 amino acid putative response regulator belonging to the OmpR family of regulatory proteins. The gene dltS codes for a 395 amino acid putative histidine kinase thought to be involved in the sensing of environmental signals. The dlt operon of S. agalactiae is mainly transcribed from the PdltR promoter which directs synthesis of a 6.5-kb transcript encompassing dltR, dltS, dltA, dltB, dltC, and dltD. We demonstrated that PdltR is activated by DltR in D-Ala deficient LTA mutants resulting from insertional-inactivation of the dltA gene which encodes the cytoplasmic D-alanine-D-alanyl carrier ligase DltA. The DltA- mutant displayed the ability to form clumps in standing culture and an increased susceptibility to the cationic antimicrobial polypeptide colistin. We also reported that incorporation of D-Ala residues in the LTAs is important for the virulence of S. agalactiae since, in a mouse infection model, the DltA- mutant was eliminated more rapidly from the blood, brain, liver, and spleen than the wild type strain.
The dramatic clearance of the DltA- mutant in the blood was interpreted as resulting from an increased susceptibility to phagocytosis by polymorphonuclear cells and/or macrophages since the wild type strain and D-Ala deficient LTA mutant are similarly resistant to direct killing by mice or human serum in the absence of specific antibodies. Consistently, preliminary results have shown that this mutant is more susceptible to bacterial killing by murine macrophages than the wild type strain. Since the main phenotypic alterations caused by D-Ala ester deprivation in LTAs of S. agalactiae were an increased susceptibility to the cationic antimicrobial polypeptide colistin, a likely explanation for this observation is that the DltA- mutant is more susceptible to the murine defensins. An alternative possibility is that the mutant is more susceptible to the growth conditions (pH and oxidative stress) encountered in the phagolyzosome.
3. Structural analysis and functional analysis of the genome of S. agalactiae (In collaboration with Dr F. KUNST and Dr P. GLASER, Laboratoire de Séquençage des Micro-organismes Pathogènes, Pasteur Institute and with Dr T. MSADEK, Unité de Biochimie Microbienne, Pasteur Institute).
This research program was conceived to valorise at the best the availability of the genome of S. agalactiae that was sequenced by Dr F. KUNST and Dr P. GLASER (Laboratoire de Séquençage des Micro-organismes Pathogènes, Pasteur Institute). The three main objectives followed (resistance to oxidative stress, surface proteins, environmental adaptation and expression of virulence genes) were chosen because they constitute key elements in the comprehension of the physiopathology of GBS infections and for the characterization of the gene(s) involved in the crossing of the brain blood barrier. Furthermore, their elucidation may provide new targets to develop therapeutic treatments, in particular for the development of a vaccine.
A novel genotypic method for accurate identification of gram-positive cocci at the species level.
Several genotypic methods based on the analysis of PCR products derived from selected target DNA have been developed for bacterial species identification including the determination of the 16S rDNA sequence. In the latter case, however, the interpretation of these data may be complicated by the fact that closely related species may have identical 16S rDNA sequences or, alternatively, that divergent 16S rDNA sequences may exist within a single organism. To solve this problem, it is possible to use alternative monocopy target sequences which exhibit a higher divergence than that of the 16S rDNA. The sodA gene of the gram-positive cocci which encodes the manganese-dependent superoxide dismutase fulfills these criteria and we recently reported that sequencing of the sodA PCR product with the use of a single pair of degenerate primers constitutes a valuable approach to the genotypic identification of streptococcal and enterococcal species. We used the same universal primers to construct a sodA database of all coagulase-negative staphylococcal (CNS) type species described so far and we demonstrated the usefulness of this library for a rapid sequence-based identification method of CNS isolates. More recently, this approach enabled us to characterize 2 novel streptococcal species, Streptococcus lutetiensis et Streptococcus pasteurianae, which are phylogenetically related to Streptococcus bovis. These sodA databases might be used to develop a rapid microbial identification system based on the DNA chips technology.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
TRIEU-CUOT, Patrick (IP)
POYART, Claire (Necker)