Patrice Got

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.

Phytoplankton distribution and productivity in a highly turbid, tropical coastal system (Bach Dang Estuary, Vietnam)

Phytoplankton diversity, primary and bacterial production, nutrients and metallic contaminants were measured during the wet season (July) and dry season (March) in the Bach Dang Estuary, a sub-estuary of the Red River system, Northern Vietnam. Using canonical correspondence analysis we show that phytoplankton community structure is potentially influenced by both organometallic species (Hg and Sn) and inorganic metal (Hg) concentrations. During March, dissolved methylmercury and inorganic mercury were important factors for determining phytoplankton community composition at most of the stations. In contrast, during July, low salinity phytoplankton community composition was associated with particulate methylmercury concentrations, whereas phytoplankton community composition in the higher salinity stations was more related to dissolved inorganic mercury and dissolved mono and tributyltin concentrations. These results highlight the importance of taking into account factors other than light and nutrients, such as eco-toxic heavy metals, in understanding phytoplankton diversity and activity in estuarine ecosystems.

Collapse of a Planktothrix agardhii perennial bloom and microcystin dynamics in response to reduced phosphate concentrations in a temperate lake

Planktothrix agardhii dynamics, microcystin concentration and limnological variables were monitored every 2 weeks for 2 years (2004-2006) in a shallow hypereutrophic artificial lake (BNV, Viry-Châtillon, France). Time-series analysis identified two components in the P. agardhii biomass dynamics: (1) a significant decreasing trend in P. agardhii biomass (65% of the overall variance) and (2) a residual component without significant seasonal periodicity. A path-analysis model was built to determine the main factors controlling the P. agardhii dynamics over the period studied. The model explained 66% of P. agardhii biomass changes. The decreasing trend in P. agardhii biomass was significantly related to a decrease in the PO4(3-) concentration resulting from an improved treatment of the incoming watershed surface water. The residual component was related to zooplankton dynamics (cyclopoid abundances), supporting the hypothesis of a top-down control of P. agardhii, but only when the biomass was low. Forty-nine percent of the variability in the microcystin (MC) concentration (min:<0.1 microg equivalent MC-LR L(-1); max: 7.4 microg equivalent MC-LR L(-1)) could be explained by changes in the P. agardhii biomass. The highest toxin content was observed when P. agardhii biomass was the lowest, which suggests changes in the proportion of microcystin-producing and -nonproducing subpopulations and/or the physiological status of cells.

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.

Bacterial carbon dependence on freshly produced phytoplankton exudates under different nutrient availability and grazing pressure conditions in coastal marine waters.

The effects of grazing pressure and inorganic nutrient availability on the direct carbon transfer from freshly produced phytoplankton exudates to heterotrophic bacteria biomass production were studied in Mediterranean coastal waters. The short-term incorporation of ¹³C (H¹³CO₃) in phytoplankton and bacterial lipid biomarkers was measured as well as the total bacterial carbon production (BP), viral lysis and the microbial community structure under three experimental conditions: (1) High inorganic Nutrient and High Grazing (HN + HG), (2) High inorganic Nutrient and Low Grazing (HN + LG) and (3) under natural in situ conditions with Low inorganic Nutrient and High Grazing (LN + HG) during spring. Under phytoplankton bloom conditions (HN + LG), the bacterial use of freshly produced phytoplankton exudates as a source of carbon, estimated from ¹³C enrichment of bacterial lipids, contributed more than half of the total bacterial production. However, under conditions of high grazing pressure on phytoplankton with or without the addition of inorganic nutrients (HN + HG and LN + HG), the ¹³C enrichment of bacterial lipids was low compared with the high total bacterial production. BP therefore seems to depend mainly on freshly produced phytoplankton exudates during the early phase of phytoplankton bloom period. However, BP seems mainly relying on recycled carbon from viral lysis and predators under high grazing pressure.

Accucary of epifluorescence microscopy counts for direct estimates of bacterial numbers

Abstract The aim of this study was to determine the extent and source of inaccuracies for total counts of microorganisms by filtration followed by epifluorescent microscopy. The extent of these inaccuracies were determined by comparing the microscopic method with viable plate counts while their possible sources were investigated using flow cytometry and fluorescent microbeads. One factor responsible for these accuracies was found to be the microscope conversion factor which did not take into account the total filter surface area on which the bacteria were impacted. Approximately a two-fold underestimation was observed when the filtration area rather than the effective area over which the cells are disturbed were compared. Thus it is recommended that the latter be accurately measured when calculating the conversion factor in order to reduce the inaccuracy of direct microscopic counts.

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

ABSTRACT The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.

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.

Effects of sewage discharges on microbial components in tropical coastal waters (Senegal, West Africa)

Coastal ecosystems are frequently submitted to anthropogenic pressure but little is known about how the dynamics of aquatic communities can be altered. The impact of urban and industrial discharges on microbial plankton was studied in Hann Bay near Dakar (Senegal) on the Atlantic Ocean. Spatial patterns were studied using three transects, with a total of 20 stations, during two periods in May and November (before and after the seasonal upwelling), revealing a clear contrast between near shore stations and the intermediate and seaward stations. In November, phytoplankton were associated with dissolved nutrient availability (e.g. with nitrate, r = 0.76) whereas in May, phytoplankton were more correlated with microbial variables (e.g. with heterotrophic nanoflagellates, r = 0.63). Most samples (40) failed to meet the quality levels for the faecal indicator bacteria (FIB) defined by the European Union bathing water quality directive. The topography plays a major role in water circulation explaining the presence of FIB at the seaward stations. The high prevalence of FIB during the two periods suggests chronic pollution and a potential risk to recreational swimmers and fish consumers in Hann Bay. Thus, as demonstrated in various temperate systems, the decline of water quality constitutes a serious problem in many West African countries.