Audrey Caro

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.

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.

Characterization of the Population of the Sulfur-Oxidizing Symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by Single-Cell Analyses†

ABSTRACT We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 μm in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.

Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.

Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are human pathogens. Little is known about these Vibrio spp. in the coastal lagoons of France. The purpose of this study was to investigate their incidence in water, shellfish and sediment of three French Mediterranean coastal lagoons using the most probable number-polymerase chain reaction (MPN-PCR). In summer, the total number of V. parahaemolyticus in water, sediment, mussels and clams collected from the three lagoons varied from 1 to >1.1 × 10³ MPN/l, 0.09 to 1.1 × 10³ MPN/ml, 9 to 210 MPN/g and 1.5 to 2.1 MPN/g, respectively. In winter, all samples except mussels contained V. parahaemolyticus, but at very low concentrations. Pathogenic (tdh- or trh2-positive) V. parahaemolyticus were present in water, sediment and shellfish samples collected from these lagoons. The number of V. vulnificus in water, sediment and shellfish samples ranged from 1 to 1.1 × 10³ MPN/l, 0.07 to 110 MPN/ml and 0.04 to 15 MPN/g, respectively, during summer. V. vulnificus was not detected during winter. V. cholerae was rarely detected in water and sediment during summer. In summary, results of this study highlight the finding that the three human pathogenic Vibrio spp. are present in the lagoons and constitute a potential public health hazard.

Plasticity of symbiont acquisition throughout the life cycle of the shallow-water tropical lucinid Codakia orbiculata (Mollusca: Bivalvia)

In marine invertebrates that acquire their symbionts from the environment, these are generally only taken up during early developmental stages. In the symbiosis between lucinid clams and their intracellular sulfur-oxidizing bacteria, it has been shown that the juveniles acquire their symbionts from an environmental stock of free-living symbiont forms, but it is not known if adult clams are still competent to take up symbiotic bacteria from the environment. In this study, we investigated symbiont acquisition in adult specimens of the lucinid clam Codakia orbiculata, using transmission electron microscopy, fluorescence in situ hybridization, immunohistochemistry and PCR. We show here that adults that had no detectable symbionts after starvation in aquaria for 6 months, rapidly reacquired symbionts within days after being returned to their natural environments in the field. Control specimens that were starved and then exposed to seawater aquaria with sulfide did not reacquire symbionts. This indicates that the reacquisition of symbionts in the starved clams returned to the field was not caused by high division rates of a small pool of remaining symbionts that we were not able to detect with the methods used here. Immunohistochemistry with an antibody against actin, a protein involved in the phagocytosis of intracellular bacteria, showed that actin was expressed at the apical ends of the gill cells that took up symbionts, providing further evidence that the symbionts were acquired from the environment. Interestingly, actin expression was also observed in symbiont-containing cells of untreated lucinids freshly collected from the environment, indicating that symbiont acquisition from the environment occurs continuously in these clams throughout their lifetime.

Effects of Long-Term Starvation on a Host Bivalve (Codakia orbicularis, Lucinidae) and Its Symbiont Population

ABSTRACT The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.

Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia Orbicularis (Bivalvia: Lucinidae)

ABSTRACT The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.

Survie et maintien de la virulence de Salmonella Typhimurium VNC exposée simultanément à trois facteurs stressants expérimentaux

Abstract The release of enteric pathogenic bacteria in aquatic environments poses the problem of the fate of these bacteria under the effects of environmental factors (solar radiation, salt concentration, temperature, nutrient level, pH, competition). Frequently, these bacterial cells, potentially pathogenic, enter into a non-culturable state on routine bacteriological plating media. However, the use of direct detection methods DAPI stained cells allows the visualization of these Viable but Non Culturable cells VNC. But, beyond the characterization of the viability of the cells electron transport activity, metabolic activity, membrane integrity, structure and/or quantity of DNA, what happens with the virulence of these cells? This problem was experimentally investigated according to the bacterial model Salmonella Typhimurium. The virulence of this strain, which is the agent of the murine typhoid, was evaluated on a mouse model. Experimentally, the effects of some environmental factors on the survival and on the maintenance of virulence of Salmonella Typhimurium were measured in microcosms exposed to UV radiation (four germicidal lamps 8 mW s −1 cm −2 , wave length: 254 nm, salt concentration (Sea Salt Sigma, 37, nutrient starvation. The microcosms were simultaneously submitted to these three factors, with variable exposure times. For each of those times, the viability of the nonculturable cells which became nonculturable because of the exposure to the three factors was measured through different physiological states notable in the cells, after using different fluorescent dyes. The stained cells were observed by epifluorescence microscopy and analysed by image cytometry. So, the cellular populations are characterised by enumeration of respiring bacteria CTC, [39], metabolising bacteria YEK, [22] modified, bacteria owning an undamaged cytoplasmic membrane LD, Live/Dead BacLight Viability Kit. Molecular Probes Inc.; we also determined the quantity and/or structure of DNA of the cells fluorescence level of DAPI stained cells), After exposure to the three factors for one hour 13.56 J cm −2 , while the plate count cell density rapidly decreased from #10 7 cells mL −1 to0.1 cell mL −1 , physiological states of these viable but non-culturable cells are similar to those of non-exposed cells. On the other hand, after exposure for three hours, only 10 % of the cells deposit a CTC formazan-crystal and 20 % are substrate responsive cells enlarged cells in presence of Yeast Extract and Cephalexin: YEC. Half of the cellular population presents an undamaged cytoplasmic membrane and the level of fluorecence of DAPI stained cells is close to 85 %, which shows that the DNA of these cells is weakly damaged. After exposure to the three experimental factors for24 hours 315 J cm −2 , weak replies to the physiological tests used in this study to characterize the viability of the non-culturable cellular population are observed CTC: 4 %; YEC: 2 %; LD: 11.8 % while the fluorescence level of DAPI stained cells remains firm at 80 %. At the same time, the virulence expression of VNC cells of Salmonella Typhimurium, evaluated by intraperitoneal injection to the mouse route which excludes uncontrolled parameters, unlike the per os route does not seem to be correlated with the cellular viability such as it has been evaluated in this study. A 30 min exposure (6.73 J cm −2 ) to the three environmental factors, leading to the non-culturability of almost the entire exposed cell population 0.08 culturable cell mL −1 whereas the level of viability of those culturable cells is closed to the one of non-exposed cells. The injection of 1000 of those cells 0.001 culturable cells in 100 μL inoculated into the mouse a group of ten mice does not cause any mortality four weeks post-inoculation, whereas the injection of the same dose of non-exposed cells leads to the death of all mice in the group one week post-inoculation. According to our preliminary experiments on Salmonella Typhimurium, the loss of the state of culturability and the loss of virulence towards mice by intra-peritoneal route, because of the exposure to associated effects of UV irradiation 254 nm, salinity 37 and nutrient starvation, seem to be concomitant.

Comparative modifications in bacterial gill-endosymbiotic populations of the two bivalves Codakia orbiculata and Lucina pensylvanica during bacterial loss and reacquisition

Until now, the culture of sulphur-oxidizing bacterial symbionts associated with marine invertebrates remains impossible. Therefore, few studies focused on symbionts physiology under stress conditions. In this study, we carried out a comparative experiment based on two different species of lucinid bivalves (Codakia orbiculata and Lucina pensylvanica) under comparable stress factors. The bivalves were starved for 6 months in sulphide-free filtered seawater. For C. orbiculata only, starved individuals were then put back to the field, in natural sediment. We used in situ hybridization, flow cytometry and X-ray fluorescence to characterize the symbiont population hosted in the gills of both species. In L. pensylvanica, no decrease in symbiont abundance was observed throughout the starvation experiment, whereas elemental sulphur slowly decreased to zero after 3 months of starvation. Conversely, in C. orbiculata, symbiont abundance within bacteriocytes decreased rapidly and sulphur from symbionts disappeared during the first weeks of the experiment. The modifications of the cellular characteristics (SSC--relative cell size and FL1--genomic content) of the symbiotic populations along starvation were not comparable between species. Return to the sediment of starved C. orbiculata individuals led to a rapid (2-4 weeks) recovery of symbiotic cellular characteristics, comparable with unstressed symbionts. These results suggest that endosymbiotic population regulation is host-species-dependent in lucinids.

Oyster Farming, Temperature, and Plankton Influence the Dynamics of Pathogenic Vibrios in the Thau Lagoon

Vibrio species have been associated with recurrent mass mortalities of juvenile oysters Crassostrea gigas threatening oyster farming worldwide. However, knowledge of the ecology of pathogens in affected oyster farming areas remains scarce. Specifically, there are no data regarding (i) the environmental reservoirs of Vibrio populations pathogenic to oysters, (ii) the environmental factors favoring their transmission, and (iii) the influence of oyster farming on the persistence of those pathogens. This knowledge gap limits our capacity to predict and mitigate disease occurrence. To address these issues, we monitored Vibrio species potentially pathogenic to C. gigas in 2013 and 2014 in the Thau Lagoon, a major oyster farming region in the coastal French Mediterranean. Sampling stations were chosen inside and outside oyster farms. Abundance and composition of phyto-, microzoo-, and mesozooplankton communities were measured monthly. The spatial and temporal dynamics of plankton and Vibrio species were compared, and positive correlations between plankton species and vibrios were verified by qPCR on isolated specimens of plankton. Vibrio crassostreae was present in the water column over both years, whereas Vibrio tasmaniensis was mostly found in 2013 and Vibrio aestuarianus was never detected. Moreover, V. tasmaniensis and V. crassostreae were found both as free-living or plankton-attached vibrios 1 month after spring mortalities of the oyster juveniles. Overall, V. crassostreae was associated with temperature and plankton composition, whereas V. tasmaniensis correlated with plankton composition only. The abundance of Vibrio species in the water column was similar inside and outside oyster farms, suggesting important spatial dispersion of pathogens in surrounding areas. Remarkably, a major increase in V. tasmaniensis and V. crassostreae was measured in the sediment of oyster farms during cold months. Thus, a winter reservoir of pathogenic vibrios could contribute to their ecology in this Mediterranean shellfish farming ecosystem.