Unit: Necker-Institut Pasteur joint Streptococcus and Enterococcus Laboratory

Director: Patrick TRIEU-CUOT

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. The Necker-Institut Pasteur joint Streptococcus and Enterococcus Laboratory was closed on December 31, 2003.

Virulence factors of Streptococcus agalactiae

Lancefield's group B Streptococcus (GBS), also referred to as S. agalactiae, is a normal constituent of the human digestive flora that might colonize the urogenital tract of woman and the respiratory tract of newborns. 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. Genome sequence of S. agalactiae NEM316 (In collaboration with Dr F. KUNST and Dr P. GLASER, Laboratoire de Séquençage des Micro-organismes Pathogènes, Institut Pasteur and with Dr T. MSADEK, Unité de Biochimie Microbienne, Institut Pasteur).

The GBS strain sequenced was NEM316 (CIP 82.45, ATCC 12403). This strain, responsible for a fatal septicemia, belongs to the capsular serotype III, is susceptible to antibiotics, can be modified genetically (electroporation, conjugation), and is virulent in a murine infection model. The genome of this strain is 2,211,485 base pairs long and contains 2118 protein coding genes. Fifty five percent of the predicted genes have an ortholog in the Streptococcus pyogenes genome, representing a conserved backbone between these two streptococci. Among the genes in S. agalactiae that lack an ortholog in S. pyogenes, 50% are clustered within fourteen islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared to other pathogenic streptococci (S. mutans, S. pyogenes and S. pneumoniae), S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.

2. Esterification of lipoteichoic acids and virulence of S. agalactiae

D-alanylation of lipoteichoic acids (LTAs) allows Gram-positive bacteria to modulate their surface charge, regulate ligand binding, and control the electromechanical properties of the cell wall. We thus investigated the role of D-alanyl LTAs in the virulence of S. agalactiae. We demonstrated that a DltA- isogenic mutant which is unable to synthesize D-alanyl LTAs displayed an increased susceptibility to host defense peptides such as human defensins and animal-derived cationic peptides. Consistently, the mutant strain was more susceptible to killing by mice bone marrow-derived macrophages and human neutrophils than the wild-type strain. In addition, the virulence of the DltA- mutant was severely impaired in mouse and neonatal rat models. We observed that this mutant was eliminated more rapidly than the wild-type strain from the lung of three-week old mice inoculated intranasally and, consequently, was unable to induce a pneumonia. Finally, after intravenous injection of three-week old mice, the survival of the DltA- mutant was markedly reduced in the blood and the brain in comparison to that of the wild-type strain. We hypothesize that the decreased virulence of the DltA- mutant was a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D-alanylation of the LTAs contributes to the virulence of S. agalactiae

3. Surface proteins and virulence of S. agalactiae

Surface proteins of pathogenic bacteria play an important role during the infectious process by mediating interactions between the pathogen and the host cells and/or evasion from the host defense. We studied the proteins which are covalently linked to the bacterial cell wall via a carboxylic LPXTG motif. The protease-transpeptidase reaction required for linkage is catalyzed by a membrane-associated protein named sortase (SrtA). The role of the cell wall anchored surface proteins in the virulence of S. agalactiae was thus envisaged by two complementary approaches (construction of a sortase-deficient mutant, inactivation of genes encoding LPXTG proteins). Analysis of the genome of NEM36 enabled the characterization of a gene encoding a protein displaying 55% and 30% of identity with SrtA of Streptococcus gordonii and of Staphylococcus aureus, respectively. An SrtA- mutant of S. agalactiae NEM316 was unable to anchor properly the LPXTG proteins Alp2 and ScpB to the cell surface, was impaired for binding to fibronectin and displayed a significant reduction in adherence to human (A549, HeLa, Caco-2) or murine (L2) epithelial cell lines compared to the isogenic wild-type strain. The virulence of the SrtA- mutant was not significantly different from that of the wild-type strain in neonatal rat models (LD50 and lung colonization) but this strain displayed a 4-log reduction in its capacity to colonize the intestine of gnotobiotic mice in a competition assay. It is therefore tempting to speculate that SrtA is required for rectal and vaginal GBS carriage in human.

The screening in silico of NEM316 genome revealed the presence of 30 genes encoding putative surface proteins bearing the carboxylic LPXTG motif. The distribution of 21 of these genes was studied by PCR in a panel of 99 unrelated GBS strains isolated from non-invasive (70 strains from vaginal carriage or urinary tract infection) or invasive (29 strains from blood culture or meningitidis) infections. Among these genes, 6 were present in all strains tested. We are currently investigating the function of the corresponding proteins and the possibility to use them as vaccine targets.

Keywords: Streptococcus agalactiae, genomics, gene expression, virulence, molecular taxonomy

Activity Reports 2003 - Institut Pasteur

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