WP4-T3 Characterization of pathogen virulence factors

Participants :

  • 1 - IFREMER
  • 2 - CSIC
  • 7 - UNIGE
  • 10 - CNRS

The virulence of selected pathogens will be studied in order to better understand pathogenesis and host/pathogen interactions. Studies will focus on some key virulent factors such as the adhesive properties (identification of surface ligands using adhesin-defective mutants, pili, capsule...), the secretion systems which play an essential role in toxicity (type III and IV) and also the siderophore biosynthesis and receptor systems which are essential for the multiplication of the bacteria into tissues.

Complete genome of some pathogenic Vibrio strain of bivalves (V. splendidus LGP32, V. aesturianus, V. harveyi, V. tapetis) have been sequenced and allow the identification of candidate virulence factors which might be important for pathogeny and/or host specificity, such as secretion system, RTX toxins, type IV pili, adhesins and iron acquisition systems. Complete genome sequence of new isolates of Vibrio which induce high mortalities
in oysters will be also performed by pyrosequencing in Genoscope (Evry, France). This will allow us providing a genomic comparative analysis at the species level in order to identify genes potentially involved in the strain pathogenicity. Defective mutants will be constructed and test to valid their role in pathogenicity. The complete sequence of strains will also facilitate the development of diagnostic tools to differentiate strains rapidly, such as
genotyping (WP2 - T3).

A secretomic differential approach, will aim at identifying ExtraCellularProducts (ECPs are produced and excreted by bacteria) from both pathogenic strains, as compared to avirulent strains. These experiments will consider different extracellular sub-fractions (experimental procedures will derive from those described in Benachour et al., 2009; Barbey et al., 2009). ECPs will be identified by mass spectrometry (LC ESI MS2 for whole sub-fractions, and MALDI-TOF/TOF after electrophoretic and/or chromatographic separation). These results will be compared to the theoretical (in silico) secretome derived from genome analysis. The actual role of ECPs in the virulence of the different vibrio strains will be evaluated by

  • (i) inactivating the corresponding genes, and
  • (ii) testing the effect of purified ECPs on different aspects of the host immune.

Adhesion to bivalve tissues of selected isolates of V. splendidus and V. aesturianus will be assessed by the use of either radio-labelled or GFP labelled bacteria. Interactions with hemocytes in the presence and in the absence of hemolymph components will be also studied. The role played by specific ligands in Vibrio interactions with different bivalve substrates will be evaluated by an approach similar to that used for chitin, testing adhesion efficiency of selected adhesive strains in the presence and in the absence of substrate analogues (e.g., carbohydrates). The presence of genes encoding for known ligands will be also evaluated. Special attention will be given to the study of the role of adhesion in Vibrio pathogenicity for bivalves, focusing on N-acetyl glucosamine binding ligands, which play an important role in V. cholerae adherence to different substrates, in the environment and animal host, acting as a virulence trait. When applicable, the role in adhesion of the main identified ligand(s) will be confirmed by construction of mutant(s) and comparison of their adherence efficiency with that of the parent, both in experimental infection and in vitro experiments. Adhesive properties of reference Vibrio strains and selected isolates from WP3 will be tested.

Another aspect will be the identification and characterization of specific virulence factors expressed in the first steps of infection and also the siderophore biosynthesis and receptor systems which are essential for the multiplication of the bacteria into tissues.

Microarrays for Vibrio will be constructed to determine the effect of the host immune effectors on the bacterial transcriptome. Alternativly, according to the evolution of sequencing techniques, next generation sequencing will be used for comparative transcriptomics. The role of identified virulence factors will be also studied by constructing deletion mutants and analysing their properties in vivo and in vitro.

The identification of bacterial factors that enable oyster pathogenic Vibrio to escape from the oyster immune system will be studied. Moreover, their role in the virulence will be determined using experimental infections.

Concerning viral infections, we will explore how OsHV-1 is able to infect oysters through experimental trials (WP4 - T1). Kinetics of virus genes will be analysed in order to define the viral and host proteins involve in virus recognition, virus entry and virus replication. In particular, the expression of virus transmembrane proteins and virus inbibitor apoptosis proteins (IAP) will be studied at different tilmes in OsHV-1-challenged oysters and mussels.