Créé(e) 07/05/2015 Mis à jour 07/07/2017

Emergence of invertebrate pathogens in natural Vibrio populations: ecology, evolution and pathogenesis

Our team integrated the Research Unit - “Integrative Biology of Marine Models Laboratory” in January 2014 and now includes two researchers in molecular microbiology (1DR and 1CR Ifremer), a technician (AI CNRS ), a post doc in bioinformatic and two PhD students . Our research concerns the evolution of vibrio virulence into the wild and has been supported by 3 ANRs that we coordinate. As our team is proficient with the genetic of environmental vibrios, we are also involved in a strong collaborative network to validate hypothesis in ecology and evolution . In addition, we initiated and coordinate a “European network on vibrio research”

Climate change has caused a worldwide increase in reports of vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals (1). In addition, the rapid growth of aquaculture has been the source of anthropogenic changes on a massive scale. Animals have been displaced from their natural environments, farmed at high densities and exposed to environmental stresses, including antibiotic treatment. Unfortunately but not surprisingly, marine farming areas constitute ideal locations for the study of the emergence of pathogens in real time. While the studies of animal pathogens have benefited from the era of genomics, the search for pathogenesis determinants is often biased by what is known from human pathogens (e.g. V. cholerae), precluding the discovery of new mechanisms specific to marine animal species. Overall our results show that vibrios from the wild (as opposed to laboratory model strains) are pertinent to address basic questions such as evolutionary and ecological dynamics of pathogens, as well as how they are a source of original molecular mechanisms for virulence, cell to cell interaction and genetic regulation.

Vibrio is one of the best-described marine bacterial groups in evolutionary ecology. They can be easily isolated and cultured, allowing multilocus or whole genome sequencing to obtain fine-scale genetic resolution among individual isolates. A series of studies (2) has shown that despite a high genetic diversity, these bacteria are divided into phylogenetic groups sharing a lifestyle (planktonic or associated) and preferences for habitat (organic particles, phyto or zooplankton) (3). Gene transfer appears more frequent within these groups than between groups (4) and the production of "public goods" govern social cohesion between strains (5,6). Thus, these phylogenetic groups satisfy the concept of species and provide a framework to investigate the functional unit of pathogenesis, i.e. a clone that emerges after a recent acquisition of virulence genes (7), a species with virulence encoded by the core genome (8) or a consortium (9).

Investigating “vibrio virulence into the wild” requires an animal model of infection. Of the recent work aiming to improve the in vivo model, standardization of animal hatching seems to have promising perspectives. In particular, we currently use specific pathogen-free (SPF) juvenile oysters for experimental ecology and infection (10). Indeed, these animals can be deployed in the environment to compare Vibrio populations in seawater and oysters before and during disease events and isolate colonizers. Then, thousands of these standardized animals can be used in the laboratory to assess the virulence of hundreds isolates (8,9,11).

An important axis of our work has been to develop genomic approaches (populational, comparative and functional) for environmental vibrios. More precisely, F. Le Roux coordinates the VibrioScope project integrated in the Genoscope-MicroScope platform (12) easing annotation and comparative genomics of vibrio species. We developed several genetic strategies to delete genes, genomic regions or cure plasmids in numerous strains. This includes suicide vectors carrying new markers for counter selection of allelic exchange in Vibrio for which the classical sacB toxin does not work (13), mariner transposon derivatives (TnSeq) to screen a collection of mutants in an environment of interest (14) and derivatives of plasmids originally isolated from Vibrio to express gene in trans (11,15). Finally we are currently working on methods to investigate vibrio population dynamic considering that 16S rRNA sequences collapse the majority of population into only 2-3 taxa. This includes illumina barcoding using vibrio core genes, qPCR and FISH.

Analysis of natural infection dynamics, population genomics and molecular genetics has already provided important insights on oyster disease. First, virulence can coincide with species delineation, suggesting that specific species are reservoirs of pathogens (11,15). Future work will explore the dynamics of these species in oyster environment. We are particularly interesting by the role of phages in controlling pathogenic populations. Second, population diversity may increase the severity of pathogenesis. For example, V. tasmaniensis and V. crassostreae have been associated to diseased oysters and found to co-occur at the individual level (11). Infection with V. tasmaniensis involves an intracellular phase in hemocytes and resistance to antimicrobial peptides, reactive oxygen species and copper (16,17). Infection with V. crassostreae relies at least partially on distinct genes encoding for unknown functions. Hence oysters can be infected by species with different and potentially additive virulence mechanisms. Consistent with the hypothesis of a "shared weapons" experimental infections have demonstrated that some strains are moderately virulent when injected into animals individually, and display heightened virulence in mixed experimental infections (8,18). A promising extension of this work will be to investigate cooperation within and between vibrio species in the oyster disease.

1 Le Roux, F. et al. The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis (Paris, 11-12th March 2015). Front Microbiol 6, 830, doi:10.3389/fmicb.2015.00830 (2015).

2 Shapiro, B. J. & Polz, M. F. Microbial Speciation. Cold Spring Harb Perspect Biol 7, a018143, doi:10.1101/cshperspect.a018143 (2015).

3 Hunt, D. E. et al. Resource partitioning and sympatric differentiation among closely related bacterioplankton. Science 320, 1081-1085, doi:10.1126/science.1157890 (2008).

4 Shapiro, B. J. et al. Population genomics of early events in the ecological differentiation of bacteria. Science 336, 48-51, doi:10.1126/science.1218198 (2012).

5 Cordero, O. X., Ventouras, L. A., Delong, E. F. & Polz, M. F. Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations. Proceedings of the National Academy of Sciences of the United States of America 109, 20059-20064, doi:10.1073/pnas.1213344109 (2012).

6 Cordero, O. X. et al. Ecological populations of bacteria act as socially cohesive units of antibiotic production and resistance. Science 337, 1228-1231, doi:10.1126/science.1219385 (2012).

7 Goudenege, D. et al. Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits. Isme J 7, 1985-1996, doi:10.1038/ismej.2013.90 (2013).

8 Lemire, A. et al. Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters. Isme J 9, 1523-1531, doi:10.1038/ismej.2014.233 (2014).

9 Le Roux, F., Wegner, K. M. & Polz, M. F. Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals. Trends Microbiol, doi:10.1016/j.tim.2016.03.006 (2016).

10 Petton, B. et al. Crassostrea gigas mortality in France: the usual suspect, a herpes virus, may not be the killer in this polymicrobial opportunistic disease. Front Microbiol 6, 686, doi:10.3389/fmicb.2015.00686 (2015).

11 Bruto, M. et al. V. crassostreae, an oyster benign colonizer that turned into a pathogen after being invaded by a plasmid. Isme J in press.

12 Vallenet, D. et al. MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. Nucleic Acids Res 41, D636-647, doi:10.1093/nar/gks1194 (2013).

13 Le Roux, F., Binesse, J., Saulnier, D. & Mazel, D. Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector. Appl Environ Microbiol 73, 777-784 (2007).

14 Takemura, A. Environmental microbiology (in review).

15 Le Roux, F., Davis, B. M. & Waldor, M. K. Conserved small RNAs govern replication and incompatibility of a diverse new plasmid family from marine bacteria. Nucleic Acids Res 39, 1004-1013, doi:10.1093/nar/gkq852 (2011).

16 Duperthuy, M. et al. Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus. Proceedings of the National Academy of Sciences of the United States of America 108, 2993-2998, doi:10.1073/pnas.1015326108 (2011).

17 Vanhove, A. S. et al. Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics. Environ Microbiol, doi:10.1111/1462-2920.13083 (2015).

18 Gay, M., Renault, T., Pons, A. M. & Le Roux, F. Two vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations. Diseases of aquatic organisms 62, 65-74, doi:10.3354/dao062065 (2004).