Frédérique Le Roux

Ecological Populations of Bacteria Act as Socially Cohesive Units of Antibiotic Production and Resistance

Toxic Neighborhood Bacterial populations are often considered to be driven by gene-centric, selfish dynamics. Superficially, antibiotic production fits this picture as individuals can gain most benefit by inhibiting or killing close relatives with high niche overlap. Contrary to that notion, Cordero et al. (p. 1228; see the Perspective by Morlon) show that bacteria in the wild form social units in which antibiotic production and resistance leads to cooperation within, and antagonism between, populations. A combination of high-throughput interaction screening, molecular genetics, and genomics revealed that antibiotics are produced by only a few members of each population, while all other members are resistant. In the past, lack of knowledge of the ecological structure of microbial populations has led to interpretations of antibiotic production and resistance as being largely driven by short-lived, cyclic invasions of populations by antibiotic-producing resistant bacteria. This work shows that structured, socially cohesive bacterial populations exist in the wild and form organizational patterns similar to those of animal and plant populations. Natural antibiotics enforce competition between, rather than within, bacterial populations. In animals and plants, social structure can reduce conflict within populations and bias aggression toward competing populations; however, for bacteria in the wild it remains unknown whether such population-level organization exists. Here, we show that environmental bacteria are organized into socially cohesive units in which antagonism occurs between rather than within ecologically defined populations. By screening approximately 35,000 possible mutual interactions among Vibrionaceae isolates from the ocean, we show that genotypic clusters known to have cohesive habitat association also act as units in terms of antibiotic production and resistance. Genetic analyses show that within populations, broad-range antibiotics are produced by few genotypes, whereas all others are resistant, suggesting cooperation between conspecifics. Natural antibiotics may thus mediate competition between populations rather than solely increase the success of individuals.

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

ABSTRACT Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

Comparative analysis of Vibrio splendidus-related strains isolated during Crassostrea gigas mortality events.

French mollusc production is based mainly on the Pacific cupped oyster, Crassostrea gigas. Since 1991, annual mass mortality of juveniles has been reported during summer months. These recurring episodes concern professionals who fear that like Portugese oyster, C. angulata, C. gigas could in turn disappear following one of these epizooties. Previously, bacteriological analysis of moribund oyster juveniles yielded an isolate of a Vibrio splendidus biovar II strain, named TNEMF6. This isolate was demonstrated to be pathogenic to Crassostrea gigas spat by experimental challenge. To study the association between summer oyster mortality and presence of TNEMF6 cluster strains, Vibrionaceae fauna were isolated from infected spat along the French Atlantic coast between 1997-1998. Strains related to V. splendidus biovar II were selected. Comparison with TNEMF6 was performed by classical biochemical tests and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) of SSU rDNA, rpoD, and gyrB genes. Genomic similarities were confirmed by DNA/DNA hybridization. Only one strain out of 14, TNNIII7, was found to be closely related to the pathogenic bacteria. Neither the phenotypic nor the genotypic markers used in this study were able to distinguish pathogenic from non-pathogenic strains of the widespread V. splendidus. However, future genetic comparisons of TNEMF6 and TNNIII7 is likely to reveal genes involved in pathogenicity.

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus.

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg-EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD.

Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity.

Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrates mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.

Cg-TIMP, an inducible tissue inhibitor of metalloproteinase from the Pacific oyster Crassostrea gigas with a potential role in wound healing and defense mechanisms1

We have cloned and characterized a cDNA encoding Cg‐TIMP, the first tissue inhibitor of metalloproteinase identified in mollusks. The isolated cDNA encodes a protein of 221 residues that has a domain organization similar to that of vertebrate TIMPs including a signal sequence, and the 12 cysteines characteristic of the TIMP signature. Analysis of Cg‐TIMP expression in adult oyster tissues, by Northern blot and in situ hybridization, indicates that Cg‐TIMP was only expressed in hemocytes which are the key components of defense mechanisms in mollusks. We also observed that Cg‐TIMP mRNA accumulated during shell damage and bacterial challenge. This pattern of expression suggests that Cg‐TIMP may be an important factor in wound healing and defense mechanisms.

DNA Probes As Potential Tools for the Detection of Marteilia refringens

Abstract: Since its first description, the paramyxean parasite Marteilia refringens has been recognized as a significant pathogen of bivalve mollusks. The existence of a complex life cycle was postulated by many authors. Here we report the development of DNA-based detection assays as powerful tools to elucidate the Marteilia refringens life cycle. After alignment of the Marteilia refringens ribosomal DNA small subunit sequence with those of various eukaryotic organisms, polymerase chain reaction primers were designed. Specific primers were used to amplify DNA extracted from purified Marteilia refringens and infected hosts. The specificity of amplified fragments was confirmed by Southern blotting with an oligoprobe. For in situ hybridization, four probes were tested for specific detection of 18S rRNA isolated from Marteilia refringens and other eukaryotic cells by Northern blotting. The most specific probe, Smart 2, was successfully used to detect Marteilia refringens by in situ hybridization in infected oysters and mussels.

The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas

Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 microM Cg-Def defensin and Cg-BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg-Def and Cg-BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP-induced damages was independent of the ompU-mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. splendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus-induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan-Meier survival curves, P < 0.01). Moreover, in co-infection assays, the ompU mutant was out competed by the wild-type strain with competitive indexes in the range of 0.1-0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.

Molecular Evidence for the Existence of Two Species of Marteilia in Europe

Abstract Marteilia refringens is one of the most significant pathogens of bivalve molluscs. Previous sequencing of the small subunit ribosomal RNA gene of M. refringens isolates derived from the infected mussels (Mytilus edulis and Mytilus galloprovinciallis) and the oyster (Ostrea edulis) in Europe did not reveal genetic polymorphisms despite indications from epizootiological data that distinct types may exist. We investigated the existence of polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes. The sequences of this region proved to be clearly dimorphic among Marteilia from five sampling sites. The distribution of the two genetic types, named “O” and “M”, appeared to be linked to the host species, oysters and mussels, respectively. We therefore support the recognition of two species of Marteilia in Europe and propose that the “O” type corresponds to M. refringens and the “M” type to M. maurini.

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus

ABSTRACT Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Δvsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Δvsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Δvsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.