Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors
Etienne Robino
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
The authors contributed equally to this workSearch for more papers by this authorAurore C. Poirier
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Present addresses: Department of School of Veterinary Medicine, University of Surrey, Guildford, UKThe authors contributed equally to this workSearch for more papers by this authorHajar Amraoui
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorSandra Le Bissonnais
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorAngélique Perret
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorCarmen Lopez-Joven
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorJean-Christophe Auguet
MARBEC, University of Montpellier, CNRS, Ifremer, IRD, Montpellier, France
Search for more papers by this authorTristan P. Rubio
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
MMSB UMR 5086, University of Lyon, Lyon, France.Search for more papers by this authorChantal Cazevieille
INM, University of Montpellier, Montpellier, France
Search for more papers by this authorJean-Luc Rolland
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorYann Héchard
EBI UMR CNRS 7267, University of Poitiers, Poitiers, France
Search for more papers by this authorDelphine Destoumieux-Garzón
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorCorresponding Author
Guillaume M. Charrière
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
For correspondence. E-mail [email protected]; Tel. +33(0)4-67-14-46-25.Search for more papers by this authorEtienne Robino
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
The authors contributed equally to this workSearch for more papers by this authorAurore C. Poirier
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Present addresses: Department of School of Veterinary Medicine, University of Surrey, Guildford, UKThe authors contributed equally to this workSearch for more papers by this authorHajar Amraoui
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorSandra Le Bissonnais
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorAngélique Perret
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorCarmen Lopez-Joven
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Universidad Austral de Chile, Valdivia, ChileSearch for more papers by this authorJean-Christophe Auguet
MARBEC, University of Montpellier, CNRS, Ifremer, IRD, Montpellier, France
Search for more papers by this authorTristan P. Rubio
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
MMSB UMR 5086, University of Lyon, Lyon, France.Search for more papers by this authorChantal Cazevieille
INM, University of Montpellier, Montpellier, France
Search for more papers by this authorJean-Luc Rolland
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorYann Héchard
EBI UMR CNRS 7267, University of Poitiers, Poitiers, France
Search for more papers by this authorDelphine Destoumieux-Garzón
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
Search for more papers by this authorCorresponding Author
Guillaume M. Charrière
IHPE UMR 5244, CNRS, Ifremer, University of Montpellier, University of Perpignan Via Domitia, Montpellier, France
For correspondence. E-mail [email protected]; Tel. +33(0)4-67-14-46-25.Search for more papers by this authorSummary
Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events and behave as facultative intracellular pathogen of oyster haemocytes. Herein, we wondered whether V. tasmaniensis LGP32 resistance to phagocytosis is specific to oyster immune cells or contributes to resistance to other phagocytes, like marine amoebae. To address this question, we developed an integrative study, from the first description of amoeba diversity in oyster farms to the characterization of LGP32 interactions with amoebae. An isolate of the Vannella genus, Vannella sp. AP1411, which was collected from oyster farms, is ubiquitous, and belongs to one clade of Vannella that could be found associated with Vibrionaceae. LGP32 was shown to be resistant to grazing by Vannella sp. AP1411 and this phenotype depends on some previously identified virulence factors: secreted metalloprotease Vsm and copper efflux p-ATPase CopA, which act at different steps during amoeba–vibrio interactions, whereas some other virulence factors were not involved. Altogether, our work indicates that some virulence factors can be involved in multi-host interactions of V. tasmaniensis ranging from protozoans to metazoans, potentially favouring their opportunistic behaviour.
Conflict of Interest
The authors declare that there are no conflict of interests related to this work.
Supporting Information
| Filename | Description |
|---|---|
| emi14770-sup-0001-FigureS1.pdfPDF document, 35.7 KB | Fig. S1. Phylogeny of the diversity of amoebae OTUs found by barcoding of the v7 region of the 18 s rDNA gene. Phylogenetic 18S rRNA-based trees of the Amoebozoa constructed with ARB software (http://www.arb-home.de (Ludwig et al., 2004)) loaded with the silva database (http://www.arb-silva.de). A base frequency filter was applied using the parsimony quick add marked tool implemented in ARB. All 18S rDNA gene sequences assigned to amoeba belonged to the Vannellidae family (written in red). |
| emi14770-sup-0002-FigureS2.pdfPDF document, 12 MB |
Fig. S2. Vannella sp. AP1411 isolated from Thau lagoon, France. (A) Different forms of Vannella sp. AP1411 under phase light microscopy. Trophozoite (left), pelagic (centre) and cystic form (right). Scale bar: 10 μm. (B) Transmission electron microscopy of trophozoite shape of the amoeba Vannella sp. AP1411. Whole cell overview showing nuclei (N) and several digestive vacuoles (DV) with or without E. coli SBS363 bacteria inside (B). Scale bar: 2 μm. |
| emi14770-sup-0003-FigureS3.pdfPDF document, 36.5 KB | Fig. S3. Quantification of Vannella sp. AP1411 phagocytosis by cytometry. (A) FSC/SSC Gate corresponding to amoebae (B) Determination of fluorescence background on amoebae alone. (C) Gate determination for GFP+ vibrios (Bacteria). (D) Measurement of the percentage of amoebae carrying GFP fluorescence after 3 days of co-culture with GFP-expressing vibrios. |
| emi14770-sup-0004-FigureS4.pdfPDF document, 41.8 KB |
Fig. S4 CusAB, SodA, Vsp, InhA, VipA1 and VipA2 are not involved in LGP32 resistance to grazing by Vannella sp. 1411. Bacterial resistance to grazing by Vannella sp. AP1411 of ΔcusAB, ΔsodA, Δvsp, ΔinhA, ΔvipA1 and ΔvipA2 mutant strains of LGP32 was assessed by measuring the fluorescence of the GFP-expressing bacteria, after contact with amoebae. Each condition was performed in triplicate and the results shown are the average of three independent experiments. Values are presented ± SEM. |
| emi14770-sup-0005-FigureS5.pdfPDF document, 42.8 KB | Fig. S5. Vsm or CopA do not affect the phagocytosis rate of LGP32 by the amoeba Vannella sp. AP1411. Grazing amoebae on GFP-vibrio LGP32wt, LGP32 ΔcopA or of LGP32 Δvsm were monitored by flow cytometry to estimate phagocytosis index per amoeba, which correspond to the average number of phagocyted bacteria per cell. The results shown are representative of two independent experiments. Data were analysed by one-way ANOVA with Friedman test. |
| emi14770-sup-0006-TableS1.docxWord 2007 document , 25.3 KB | Table S1. Sanger sequences of sampled marine amoebae |
| emi14770-sup-0007-Movie1.avivideo/avi, 535.7 KB | Supplementary Movie 1. Time-lapse microscopy movie of Vannella sp. AP1411 and LGP32wt lawn in co-culture. |
| emi14770-sup-0008-Movie2.avivideo/avi, 460.1 KB | Supplementary Movie 2. Time-lapse microscopy of Vannella sp. AP1411 and LGP32 ΔcopA lawn in co-culture. |
| emi14770-sup-0009-Movie3.avivideo/avi, 462.5 KB | Supplementary Movie 3. Time-lapse microscopy of Vannella sp. AP1411 and LGP32 Δvsm lawn in co-culture. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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