Patrick Trieu-Cuot

Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease

Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty‐five per cent 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 14 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 with other pathogenic streptococci, S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.

Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the sodA Gene as a Target

ABSTRACT Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodAint) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60gene sequences. Sequence analysis revealed that the staphylococcalsodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that thesodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.

Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens

Antimicrobial drugs targeting the reportedly essential type II fatty acid synthesis (FASII) pathway have been recently acclaimed for their efficacy against infections caused by multiresistant Gram-positive bacteria. Our findings show that the strategy for antibiotic development based on FASII pathway targets is fundamentally flawed by the fact that exogenous fatty acids fully bypass inhibition of this pathway in both in vitro and in vivo conditions. We demonstrate that major Gram-positive pathogens—such as streptococci, pneumococci, enterococci and staphylococci—overcome drug-induced FASII pathway inhibition when supplied with exogenous fatty acids, and human serum proves to be a highly effective source of fatty acids. For opportunist pathogen Streptococcus agalactiae, growth in serum leads to an overall decrease of FASII gene expression. No antibiotic inhibitor could have a stronger effect than the inactivation of the target gene, so we challenged the role of FASII using deletion mutants. Our results unequivocally show that the FASII target enzymes are dispensable in vivo during S. agalactiae infection. The results of this study largely compromise the use of FASII-based antimicrobials for treating sepsis caused by Gram-positive pathogens.

Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes

A strain of Listeria monocytogenes, isolated from a patient with meningoencephalitis, was resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. The genes conferring resistance to these antibiotics were carried by a 37-kb plasmid, pIP811, that was self-transferable to other L monocytogenes cells, to enterococci-streptococci, and to Staphylococcus aureus. The efficacy of transfer and the stability of pIP811 were higher in enterococci-streptococci than in the other gram-positive bacteria. As indicated by nucleic acid hybridisation, the genes in pIP811 conferring resistance to chloramphenicol, erythromycin, and streptomycin were closely related to plasmid-borne determinants that are common in enterococci-streptococci. Plasmid pIP811 shared extensive sequence homology with pAM beta 1, the prototype broad host range resistance plasmid in these two groups of gram-positive cocci. These results suggest that emergence of multiple antibiotic resistance in Listeria spp is due to acquisition of a replicon originating in enterococci-streptococci. The dissemination of resistance to other strains of L monocytogenes is likely.

Assembly and role of pili in group B streptococci

Streptococcus agalactiae[group B streptococcus (GBS)] is the leading cause of neonatal pneumonia, sepsis and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes five putative sortases, including the major class A sortase enzyme and four class C sortases. The genes encoding the class C sortases are tandemly arranged in two different loci, srtC1‐C2 and srtC3‐C4, with a similar genetic organization and are 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 regulatory gene located upstream from this locus. We demonstrated that strain NEM316 expresses only the srtC3‐C4 locus, which encodes three surface proteins (Gbs1474, Gbs1477 and Gbs1478) that polymerize to form appendages resembling pili. Structural and functional analysis of this locus revealed that: (i) the transcriptional activator RogB is required for expression of the srtC3‐C4 operon; (ii) Gbs1477, and either SrtC3 or SrtC4 are absolutely required for pilus biogenesis; and (iii) GBS NEM316 pili are composed of three surface proteins, Gbs1477, the bona fide pilin which is the major component, Gbs1474, a minor associated component, and Gbs1478, a pilus‐associated adhesin. Surprisingly, pilus‐like structures can be formed in the absence of the two minor components, i.e. the putative anchor Gbs1474 or the adhesin Gbs1478. Adherence assays showed that Gbs1478 confers adhesive capacity to the pilus. This study provides the first evidence that adhesive pili are also present in Gram‐positive pathogens.

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram‐positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D‐alanine residues into the cell wall‐associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D‐Ala‐deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA− mutant did not show any morphological alterations and its growth rate was similar to that of the wild‐type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA− mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow‐derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA− mutant to non‐phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D‐alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.

Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and Description of Streptococcus pseudopneumoniae sp. nov.

ABSTRACT We have identified an unusual group of viridans group streptococci that resemble Streptococcus pneumoniae. DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the S. oralis-S. mitis group of viridans group streptococci. We suggest that this novel species be termed Streptococcus pseudopneumoniae. A combination of phenotypic and genetic reactions allows its identification. S. pseudopneumoniae strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO2 but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of S. pneumoniae. Genetic tests for pneumolysin (ply) and manganese-dependent superoxide dismutase (sodA) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and S. pneumoniae.

Shuttle vectors containing a multiple cloning site and a lacZα gene for conjugal transfer of DNA from Escherichia coli to Gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.

The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates

Lethal meningitis triggered by the hypervirulent group B streptococcus clone ST-17 is mediated by a novel surface protein called HvgA.

Dual Role for Pilus in Adherence to Epithelial Cells and Biofilm Formation in Streptococcus agalactiae

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation.