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Applied and Environmental Microbiology, September 2008, p. 5750-5758, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01043-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

DjlA, a Membrane-Anchored DnaJ-Like Protein, Is Required for Cytotoxicity of Clam Pathogen Vibrio tapetis to Hemocytes{triangledown}

Fatma Lakhal,1 Stéphanie Bury-Moné,1,{dagger} Yanoura Nomane,1 Nelly Le Goïc,2 Christine Paillard,2 and Annick Jacq1*

Institut de Génétique et Microbiologie, UMR8621, CNRS, Université Paris-Sud XI, Bâtiment 400, Centre Scientifique d'Orsay, 91405 Orsay Cedex, France,1 LEMAR, UMR 6539, CNRS, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Technopole Brest-Iroise, Place Copernic, 29280 Plouzané, France2

Received 9 May 2008/ Accepted 11 July 2008


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ABSTRACT
 
DjlA is an inner membrane cochaperone belonging to the DnaJ family, which has been shown to be involved in Legionella sp. pathogenesis. In this study, we explored the role of this protein in the physiology and virulence of Vibrio tapetis, the etiological agent of brown ring disease (BRD) in Manila clam (Ruditapes philippinarum). Analysis of the djlA locus in V. tapetis revealed a putative organization in an operon with a downstream gene that we designated duf924Vt, which encodes a conserved protein with an unknown function and has homologues in bacteria and eukaryotes. djlA mutants displayed a reduced growth rate and showed an important loss of cytotoxic activity against R. philippinarum hemocytes in vitro, which could be restored by extrachromosomal expression of wild-type djlAVt but not duf924Vt. These results are in keeping with the potential importance of DjlA for bacterial pathogenicity and open new perspectives for understanding the mechanism of action of this protein in the novel V. tapetis-R. philippinarum interaction model.


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INTRODUCTION
 
Vibrio tapetis CECT4600 (formerly Vibrio Predominant 1 or VP1) was isolated in 1990 at Landeda (North Finistère, France) and was characterized as the etiological agent of brown ring disease (BRD) in the Japanese clam Ruditapes philippinarum (6, 37). BRD appeared in 1987 in Brittany (France), where this species of clam was introduced for aquaculture. Since then, the disease has been found in several marine bivalves from the United Kingdom, Ireland, Spain, Italy, and Korea (4, 17, 25, 41). In addition, more recently, V. tapetis was isolated from the corkwing wrasse (Symphodus melops) suffering from vibriosis (22). In clams, moribund animals exhibit a brown organic deposit consisting of conchiolin, after which the disease is named, between the edge of the shell and the pallial line, and the major site of infection is the extrapallial space and the periostracal lamina (38-40). Clam sensitivity to V. tapetis seems to vary according to the geographic origin of the clams. French Manila clam populations are more susceptible to BRD than populations in the United States (1), and recently, similar findings were reported for Galician-grown Manila clams compared to Irish-grown clams (16). In addition, intraspecific molecular typing showed that there are several genetic groups that strongly correlate with the host species, suggesting that genetic modifications are responsible for host specificity (43).

Like the defense strategies of many invertebrates, the clam defense strategy involves biochemical and hematological changes. Hemocytes present in the hemolymph constitute the main line of defense against infections. Infected R. philippinarum has a higher total hemocyte count and lysozyme activity, particularly in the extrapallial fluids, which are in contact with the site of infection. An increased number of granulocytes, a particular type of hemocytes with phagocytic activity, is important for the resistance of R. philippinarum to infection by V. tapetis, and severely diseased clams exhibit a decreased hemocyte count and decreased phagocytic activity (1, 2). Hemocytes from American-grown R. phillipinarum had higher phagocytic activity against V. tapetis than their counterparts from French-grown clams (1). More recently, Allam and Ford (2) showed that V. tapetis and its extracellular products were able to inhibit the phagocytic activity and to decrease the viability of clam hemocytes. In addition, hemocyte susceptibility to V. tapetis cytotoxic activity correlated with susceptibility to the disease (2). After incubation with V. tapetis, hemocytes become rounded and lose their filopods, as well as their adhesion properties, suggesting that there is cytoskeleton rearrangement upon contact with the bacterium (11). It is likely that the observed cytotoxicity of V. tapetis to clam hemocytes plays an important role in virulence. Hence, in addition to in vivo tests, an in vitro test relying on the capacity of V. tapetis to decrease hemocyte adhesion has been developed to evaluate V. tapetis pathogenicity for clams (3, 11).

Whereas some vibrios, such as Vibrio cholerae and Vibrio vulnificus, are human pathogens, the vast majority of vibrios are commensals or pathogens of fish and shellfish, although human pathogenicity can also be associated with the ingestion of contaminated seafood. The diversity of pathogenicity mechanisms and the basis for host specificity in vibrios have yet to be explored. It was recently shown that in a Vibrio splendidus strain pathogenic for the oyster Crassostrea gigas, the secreted metalloprotease Vsm is important for extracellular product toxicity but not for virulence of the bacterium in an oyster infection model (26). For V. tapetis, potential virulence factors, such as pili and secreted activities (for instance hemolysin), have been described (7, 40). However, the roles of these factors in virulence have not been demonstrated genetically. Given the importance of BRD as an emerging disease in shellfish, it is important to perform genetic studies to investigate at the molecular level the basis for V. tapetis virulence. We initiated such a study and validated our approach by exploring the potential role of DjlA in pathogenicity.

DjlA (DnaJ-like protein A), a member of the DnaJ/Hsp40 family and the third DnaK/Hsp70 partner in Escherichia coli, is an inner membrane-anchored bacterial cochaperone (13, 19). Only a few phenotypes have been associated with djlA inactivation in E. coli, but this has been done only in the presence of another mutation either affecting dnaJ (18, 20) or generating envelope stress (8, 45). The absence of any strong defect in a djlA single mutant has precluded identification of a specific function for the membrane cochaperone DjlA relative to the other cytoplasmic DnaK partners. However, our group and others have demonstrated that moderate overproduction of DjlA induces the Rcs pathway, which comprises a complex phosphorelay system (10, 12). Previous studies have reported the involvement of DjlA in the virulence of Legionella sp. (33, 50). In this paper, we describe an analysis of the role of DjlA in the physiology and hemocyte cytotoxicity of V. tapetis.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions.
The main characteristics of the bacterial strains used in this study are shown in Table 1. V. tapetis strain CECT4600 and derivatives of it were grown at 18°C with agitation in saline Luria-Bertani broth (SLB) (10 g Bacto peptone per liter, 5 g yeast extract per liter, 20 g NaCl per liter). For growth on solid medium, 15 g of agar per liter was added to SLB broth. For cytotoxicity and pathogenicity tests, V. tapetis strains were grown at 18°C with agitation in Zobell medium (4 g peptone per liter, 1 g yeast extract per liter, 0.1 g ferric phosphate per liter, 30 g sea salt per liter). Alternatively, in some experiments (see Fig. 4), strains were grown on Zobell plates for 72 h, scraped cells were resuspended in filtered sterile seawater, and the optical densities at 600 nm of the suspensions were determined.


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  		TABLE 1. Strains used in this study


Figure 4
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  		FIG. 4. Effect of wild-type djlA and duf924 on the cytotoxic activity of VT16 (djlA). The plus and minus signs indicate that the cells were grown in the presence and in the absence of 0.1% arabinose, respectively, prior to incubation with the hemocytes. Homogeneous groups that were significantly different (P < 0.05, multiple-range test) are indicated by different letters. The bars indicate the means of three independent experiments. The error bars indicate standard deviations. WT, wild type.

E. coli strains were grown at 37°C in LB broth or on LB agar (29). For selection of V. tapetis transconjugants, antibiotics were used if they were needed at the following concentrations: 4 µg/ml of chloramphenicol (higher concentrations that were used initially were found to significantly inhibit V. tapetis growth even when the organism carried the cat gene), 50 µg/ml of streptomycin, and 250 µg/ml of kanamycin. For selective growth of E. coli strains, antibiotics were used when they were required at the following concentrations: 25 µg/ml of chloramphenicol, 25 µg/ml of kanamycin, and 100 µg/ml of ampicillin. When necessary, thymidine and diaminopimelate (DAP) were added to growth media at a final concentration of 0.3 mM. The PBAD promoter was induced by addition of 1% L-arabinose to the media.

Molecular techniques, PCR, and sequencing.
Standard procedures were used for small-scale plasmid preparation, endonuclease digestion, ligation, agarose gel electrophoresis, elution of DNA fragments from agarose gels, and E. coli transformation (44). Midi Qiagen columns were used for large-scale plasmid preparation. A genomic DNA extraction kit (Sigma) was used for rapid preparation of chromosomal DNA from V. tapetis strains. PCR were carried out according to the manufacturer's recommendations using either Taq DNA polymerase (Fermentas) or Dynazyme (Finnzyme), when a high-fidelity enzyme was required (i.e., when PCR products were to be sequenced or cloned). PCR products and plasmid inserts were sequenced with an ABI 310 automated DNA sequencer (Applied Biosystems) or by Genome express (France). Primers used in this study are shown in Table 2 and were obtained from Sigma Genosys, Operon, or Eurogentec. As a rule, all cloned inserts were sequenced in both directions to ensure accuracy.


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  		TABLE 2. Oligonucleotides used in this study

DNA sequence amplification and analysis of the djlA locus.
Primers used for PCR amplification of V. tapetis djlA and its flanking genes were designed using the corresponding region of the closely related organism V. splendidus strain LGP32, which was kindly made available by Didier Mazel and Frédérique Le Roux (Institut Pasteur, France). The V. tapetis djlA locus was analyzed using DNAStrider (CEA, France). Protein similarity searches were performed using BLASTP at NCBI.

Plasmid construction.
Table 3 and Fig. 1 show the plasmids used in this study, and Table 2 shows the primers. To generate pGEB14, a 454-bp fragment of djlAVt ({Delta}djlA [Fig. 1]) was amplified by PCR using V. tapetis CECT4600 genomic DNA as the template and primers {Delta}djlAvt-1 and {Delta}djlAvt-2 (Table 2), digested with the SmaI and SacI enzymes, and ligated into pSW23T (15) digested by the same enzymes. To generate pGEB33, a 1,470-bp fragment encompassing oriVR6K{gamma}, oriTRP4, the multiple-cloning site, and {Delta}djlAVt was amplified by PCR using pGEB14 as the template and primers SW1-BamHI and SW2-NotI, digested with BamHI and NotI, and ligated to an 820-bp fragment corresponding to the aph(3')II gene (Kmr cassette) that was amplified by PCR using pZE21 (27) as the template and primers Kana-BamHI and Kana-NotI and digested with the BamHI and NotI enzymes. To generate pGEB29, a 205-bp fragment containing the RP4 origin of transfer (oriTRP4) was amplified by PCR using pSW23T as the template and primers oriT-HindIII-1 and oriT-HindIII-2, digested by HindIII, and religated with HindIII-digested pBAD33. The orientation of oriT was determined by sequencing the insert and is indicated in Fig. 1. To generate pGEB21, a 1,087-bp fragment containing the bla gene (Ampr) obtained by digestion of pBAD18 (21) by ClaI was introduced into the ClaI site of pGEB29. The djlAVt, duf924Vt, and djlAVt-duf924Vt genes were PCR amplified using V. tapetis CECT4600 genomic DNA as the template and primers VtdjlAD-SacI and VtdjlAR-SalI, primers Vtduf924D-SacI and Vtduf924R-SalI, and primers VtdjlAD-SacI and Vtduf924R-SalI, respectively. The PCR products were digested by SacI and SalI and religated into pGEB21 that was digested by the same enzymes, generating pGEB22, pGEB23, and pGEB24, respectively.


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  		TABLE 3. Plasmids used in this study


Figure 1
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  		FIG. 1. Schematic diagrams of the plasmids used in this study. The designations of genes of interest and relevant loci are indicated. {Delta}djlA, 454-bp internal djlAVt fragment (see Materials and Methods).

β-Galactosidase assays.
Derivatives of GEB495 (Table 1), which contains a cpsB10::lacZ fusion, carrying plasmids pGEB21 to pGEB24 were used to assay the effect of DjlA (E. coli and V. tapetis) on activation of the RcsC/D/B pathway. The β-galactosidase activities of 24-h plate cultures grown at 30°C in the absence or presence of L-arabinose (0.1%) were assayed as described by Clarke et al. (14). The results were expressed in Miller units (29).

Conjugation.
Since repeated attempts to transform V. tapetis by electroporation or by chemical methods have been unsuccessful (data not shown), recombinant DNA was introduced into V. tapetis by conjugation. In most experiments, β2163 (Table 1) was used as the donor strain. β2163 requires DAP for growth and can be counterselected in SLB not supplemented with DAP. In addition, β2163 carries the pir gene and can be used to propagate plasmids that rely on the R6K{gamma} origin for replication. However, because this strain is resistant to kanamycin, we used E. coli S17-1 {lambda}pir (46) as the donor strain for pGEB33. Although carrying a streptomycin resistance gene, at 18°C and in the presence of 50 µg/ml of streptomycin, a concentration to which V. tapetis is naturally resistant, this strain produces only background growth, on which large transconjugant colonies can be easily identified. Both S17-1 {lambda}pir and β2163 carry on their chromosomes the RP4 conjugation function and mobilize plasmids carrying oriTRP4.

Initially, conjugation was carried out as described below at two temperatures (18 and 25°C). The efficiency of conjugation was found to be approximately 10–2 at both temperatures, and subsequent conjugation was done at 18°C. Transconjugants were selected in the presence of 4 µg/ml of chloramphenicol, since a higher concentration was found to inhibit V. tapetis growth, even in the presence of the plasmid.

Donor (E. coli with a mobilizable plasmid) and recipient (V. tapetis and derivatives) strains were grown to stationary phase in the presence of the appropriate antibiotic at 37 and 18°C, respectively. In the first conjugation protocol, overnight cultures of donor and recipient strains were diluted 100-fold in broth and grown to optical densities at 600 nm of 0.3 and 0.6, respectively. Conjugation experiments were performed by using the filter mating procedure described by Biskri et al. (5) with a donor/recipient ratio of at least 1/8. Conjugation was performed overnight on filters incubated at 18°C on SLB plates supplemented with DAP if the donor strain was a dap mutant. The protocol was later modified so that 10 µl of the donor strain and 10 µl of the recipient strains were mixed and directly spotted on plates. Cells were then resuspended in 700 µl of SLB, and a 100-µl aliquot of undiluted cells or of a 1/10 dilution was spread on SLB plates in the presence of the appropriate antibiotic(s). Transconjugants were purified twice on selective medium at 18°C. Identification of these transconjugants as V. tapetis was confirmed by an absence of growth at 37°C and by performing 16S rRNA gene PCR using primers VtR and VtF (Table 2). The presence in the transconjugants of the transferred plasmid was verified by plasmid DNA extraction and restriction mapping. V. tapetis djlA mutants were verified by PCR using primers Vt-djlA-U-SacI and Vt-duf924-R-SalI flanking the genomic djlA gene and by Western blotting with antibodies directed against E. coli DjlA as described by Clarke et al. (13).

In vitro cytotoxicity assays and in vivo virulence assays.
Hemocyte cytotoxicity assays were performed as described by Choquet et al. (11). Briefly, the hemolymph of R. philippinarum individuals was collected through the hinge ligament from the posterior adductor muscle (34). Samples from five clams were pooled, and the hemocytes were then incubated for 3 h at 18°C with (at a ratio of 25 bacteria per hemocyte) or without bacteria in the well of a microtiter plate. For each strain, the experiment was done in triplicate. The amount of nonadherent hemocytes in the supernatant (stained with the fluorescent dye SYBR green 1[Molecular Probes, Eugene, OR]) was calculated using the count time and flow rate of the cytometer, as estimated by the method of Marie et al. (28). The results were expressed as the ratio of the number of nonadherent cells in the presence of bacteria to the number of nonadherent cells after incubation with filtered sterile seawater. A strain was considered noncytotoxic when this ratio was ≤1. In one experiment (see Fig. 4), the statistical significance of the data was analyzed using analysis of variance and a multirange analysis (least significant difference). Differences were considered statistically significant if the P value was <0.05.

For in vivo experiments, two modes of injection were used: (i) injection of V. tapetis strains into the adductor muscle, which induced clam mortality in a few days (3) and (ii) inoculation of a fixed number of bacteria (5 x 107 bacteria) into the pallial cavity, which resulted in BRD symptoms after a few weeks (35). Injected clams (two sets of 50 individuals) were examined for BRD symptoms 1 month after inoculation.

Nucleotide sequence accession number.
The nucleotide sequence of the djlAVt-duf924Vt region has been deposited in the GenBank database under accession number EU303302.


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RESULTS
 
Identification and sequence analysis of V. tapetis djlA locus.
In order to sequence the V. tapetis djlA gene, primers were designed using the most conserved regions (coding sequences) of djlA and its flanking genes in the corresponding region of V. splendidus strain LGP32 (Frédérique Le Roux and Didier Mazel, personal communication). A 1,952-bp amplified product was obtained, and its sequence was determined (GenBank accession number EU303302). Analysis of the sequence revealed the presence of two open reading frames (ORFs). The first ORF encodes a 282-amino-acid protein with 76 and 52% identity and 85 and 68% similarity to the DjlA proteins of V. splendidus 12B01 (accession number ZP_00992441) and E. coli K-12 (accession number NP_414597), respectively. This result, as well as results presented below, confirmed that this protein is DjlA (designated DjlAVt here). The second ORF encodes a 178-amino-acid protein belonging to the DUF924 family (InterPro: IPR010323), which comprises a number of conserved proteins with unknown functions, and we designated this protein Duf924Vt. A 123-bp intergenic region separates the two genes, and no Rho-independent terminator could be detected in this region, suggesting that the djlA and duf924 genes form an operon. However, this structure is not evolutionarily conserved, even among vibrios, since this genetic organization is present only in V. splendidus and V. tapetis. In the other vibrios sequenced so far, duf924 appears to be located on chromosome II, whereas djlA is on chromosome I.

Phylogenetic trees for DjlAVt and Duf924Vt were in good agreement with each other, as well as with that for the vibrio 16S rRNA genes (data not shown).

DjlAVt can activate the Rcs pathway in E. coli.
We and other groups have reported that moderate (5- to 10-fold) overproduction of E. coli djlA (djlAEc) was able to activate the Rcs system, a phosphorelay that controls the expression of the cps operon, which is responsible for the production of the capsular exopolysaccharide colanic acid (8, 12). Zuber and coworkers also demonstrated that Coxiella burnetti DjlA was able to induce the Rcs system in E. coli (51). To examine whether DjlAVt, alone or in conjunction with DUF924Vt, was able to activate the Rcs pathway in E. coli, we constructed pGEB22, pGEB23, and pGEB24 (Table 3 and Fig. 1), in which djlAVt, duf924Vt, or djlAVt-duf924Vt was placed under control of the PBAD promoter in pGEB21, which is a derivative of pBAD33 (Table 3 and Fig. 1) (21). These plasmids were introduced into GEB495, an E. coli strain that carries a cps::lacZ fusion, whose expression is a reporter for Rcs activation (12). Cells were grown in the absence or in the presence of arabinose, and β-galactosidase activity was assayed (see Materials and Methods). The effect of djlAVt and duf924Vt induction was compared with the effect of djlAEc induction (pPSG961-31 [Table 3]). As shown in Fig. 2, the results showed that overexpression of djlAVt significantly increased (more than 10-fold) E. coli cpsB gene expression, albeit at a level one-fourth to one-fifth the level obtained after induction of djlAEc and in a way independent of duf924.


Figure 2
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  		FIG. 2. DjlAVt induces cps-lacZ fusion expression in E. coli. β-Galactosidase activity was assayed as described in Materials and Methods after 24 h of growth at 30°C in the absence (– ARA) or in the presence (+ ARA) of arabinose of GEB495 carrying pGEB21 (empty vector) or plasmids in which either djlAEc, djlAVt, duf924Vt, or both djlAVt and duf924Vt were placed under control of the PBAD promoter. The bars indicate the means of at least three independent experiments. The error bars indicate standard deviations.

Construction and phenotypic analysis of V. tapetis djlA mutants.
To analyze the effect of inactivation of djlA on V. tapetis phenotypes, we constructed two djlA mutant strains by insertional mutagenesis, using a replicative R6K plasmid, pSW23T. This suicide vector carries an R6K{gamma} origin of replication and an RP4 oriT transfer origin (15). Replication of R6K plasmids is dependent on the pir-encoded {Pi} protein (23). Hence, after transfer by conjugation into V. tapetis, which does not have the pir gene, this plasmid behaves as a suicide vector. The presence in the selective medium of antibiotics for which a resistance gene is carried by the plasmid selects integration events through homologous recombination, provided the plasmid carries a region of homology with the targeted gene. A 454-bp fragment corresponding to the central part of the djlAVt gene was introduced into pSW23T as described in Materials and Methods, generating pGEB14, which carries a Cmr gene. pGEB33 is similar to pGEB14, but the Cmr cassette is replaced by a Kmr cassette (Fig. 1). V. tapetis djlA mutants were obtained with both plasmids, and correct insertion of the plasmid at the djlA locus was verified by PCR (see Materials and Methods). We designated these mutants VT4 (Cmr) and VT16 (Kmr), respectively. The absence of the DjlA protein was confirmed in the VT4 mutant by Western blotting with antibodies raised against DjlAEc, which can react with DjlAVt (Fig. 3a).


Figure 3
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  		FIG. 3. Effect of inactivation of djlA on the cytotoxicity of V. tapetis for R. philippinarum hemocytes. (a) Western blot analysis of DjlA expression in V. tapetis parental and djlA VT4 mutant strains. Wild-type strain CECT4600 (VT1) and a mutant djlA strain (VT4) were analyzed by Western blotting using antibodies raised against the E. coli DjlA protein. The band corresponding to DjlA is indicated by an arrow. The upper band corresponds to a putative sodium dodecyl sulfate-resistant dimer of the DjlA protein (49). The sizes of the molecular mass markers are indicated on the right. (b) Microscopic observation after 3 h of incubation of hemocytes with filtered sterile seawater (FSSW), V. tapetis CECT 4600 (WT), or the djlA mutant (djlA–). (c) Cytotoxicity to hemocytes of V. tapetis wild-type (WT) and djlA mutant strains. The results are expressed as the ratio of the number of nonadherent cells after incubation with the bacteria to the number of nonadherent cells after incubation with filtered sterile seawater and are the means of three different experiments.

Both djlA mutant strains exhibited significantly reduced growth rates; the generation time for a mutant at 18°C was 4 h, compared to 2 h for the wild-type strain. In addition, we found that VT4 was not able to kill R. phillipinarum when it was injected into the adductor muscle and was not able to induce BRD when it was injected into the extrapallial space, as described in Materials and Methods (data not shown).

V. tapetis djlA mutant has reduced cytotoxic activity against R. philippinarum hemocytes in vitro.
Since the in vivo phenotypes of the djlA mutant could be due to the decreased growth rate rather than to a direct effect of the absence of DjlA, we examined the effect of the mutation on the cytotoxic activity of V. tapetis against clam hemocytes. Choquet et al. (11) demonstrated that V. tapetis induces morphological alterations, such as cell rounding and swelling, which can be observed by microscopy, as well as a loss of adhesion properties. Counting of nonadherent cells can be performed by flow cytometry, as described in Materials and Methods (11, 24). Microscopic observations showed that hemocyte incubation with wild-type V. tapetis resulted in cell rounding and loss of cytoplasmic extensions (Fig. 3b). As a consequence, the cells lost the ability to adhere to the plastic of the microplate (Fig. 3c). Hemocytes incubated with the djlA mutant were much less affected. They had a less swollen phenotype with filopods that were still clearly visible, as observed in the control sample incubated with filtered sterile seawater (Fig. 3b). Similar results were obtained with the Kmr djlA mutant (data not shown).

Consistent with microscopic observations, nonadherent cell ratios (i.e, the ratios of nonadherent cells in the presence of the mutant to nonadherent cells in the presence of filtered sterile water) showed that djlA inactivation led to an important decrease in the cytotoxic effects of the bacterium on R. philippinarum hemocytes. Indeed, the mutant strain, with a nonadherent cell ratio of 1, compared with a ratio of 2.5 for the wild-type strain, had no cytotoxic activity (Fig. 3c).

DjlA, but not Duf924, is required for cytotoxicity.
Together, the results described above provided a strong indication that DjlA is involved in V. tapetis cytotoxicity. However, since djlA might be in an operon with duf924, at least some of the observed phenotypes could be due to a polar effect on duf924 of the insertion in the djlA gene. To determine the roles of both genes, we introduced by conjugation pGEB21 (empty plasmid), pGEB22 (djlA), pGEB23 (duf924), and pGEB24 (djlA-duf924), all of which carried a Cmr gene, into VT16 (djlA Kmr). As a control, the same plasmids were introduced into the wild-type strain. The initial experiments, performed using the gene encoding the green fluorescent protein under control of the PBAD promoter, verified that this promoter was functional and fully inducible by arabinose in V. tapetis. In addition, no expression of green fluorescent protein was obtained in the absence of arabinose, indicating that this promoter is tightly regulated in this organism (data no shown).

We first examined if expression of djlA or duf924 or both could restore the normal growth rate in the mutant. Surprisingly, addition of arabinose to the medium did not restore the growth rate in any case, although in the case of pGEB22 and pGEB24, expression of DjlA could be detected by Western blotting (data not shown). We also did not observe any toxicity or slow growth of the wild-type strain in the presence of arabinose with any of the plasmids.

We then asked if cytotoxicity to hemocytes could be restored. Indeed, since gene expression in the plasmids used depended on induction by arabinose, only in vitro tests could be carried out. Mutant cells with the various plasmids were grown in the presence or in the absence of arabinose, and a fixed amount of cells was incubated with R. philippinarum hemocytes in microtiter plates as described above (see Materials and Methods). As shown in Fig. 4, the presence of arabinose in the medium could, in a statistically significant way (P < 0.05, as determined by a multiple-range test), restore the wild-type level of cytotoxic activity to the plasmid-containing mutant only when wild-type djlA was present (pGEB22 and pGEB24); a slightly higher increase was observed in the case of pGEB24, the djlAVt-duf924Vt-containing plasmid, suggesting that there is possible synergy between the two genes. However, most of the virulence attenuation observed in the mutant appeared to be due to the lack of DjlA, since duf924 expression alone did not result in recovery of cytotoxic activity. Thus, these results confirmed the key role that DjlA seems to play in V. tapetis cytotoxicity.


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DISCUSSION
 
We report here successful use of tools enabling genetic manipulation of V. tapetis, such as transformation through conjugation, inducible gene expression, and mutagenesis. Initial attempts to introduce DNA by electroporation and by chemical methods were unsuccessful. There could be a number of reasons for this. One possibility is that the replicons that we used were not functional in V. tapetis. However, this is not the case for p15A replicons, since they could be successfully introduced through conjugation. Other replicons based on the ColE1 origin of replication have been successfully used in other vibrios, and they are likely to be able to replicate in V. tapetis. A more likely explanation involves the type of resistance cassette used. For instance, in the case of tetracycline, the presence of the resistance cassette failed to provide resistance to this antibiotic in V. tapetis (data not shown). So far, in terms of antibiotic cassettes suitable for genetic manipulation of V. tapetis, cat (Cmr) and aph(3')II (Kmr) can be used. Although the presence of an ampicillin resistance cassette, in pGEB21 for instance, could confer an appropriate level of resistance once the cassette was introduced into the strain (resistance to up to 100 µg/ml, whereas the wild-type strain is inhibited by 2.5 µg/ml of ampicillin), surprisingly, we failed to select transconjugants using this cassette.

V. tapetis presents several other particularities. If the level of resistance conferred by the cat gene is low, with concentrations greater than 6 µg/ml inhibiting growth, this organism has a high natural level of kanamycin resistance, making it necessary to use a kanamycin concentration higher than 200 µg/ml. V. tapetis is also inherently resistant to 50 µg/ml of streptomycin. Several members of the multidrug and toxic compound extrusion family have been described in vibrios: Vibrio parahaemolyticus possesses at least two Na+/drug antiporter multidrug efflux proteins, VmrA (9) and NorM (31), and non-O1 V. cholerae contains eight members of this transporter family (47). In particular, NorM of V. parahaemolyticus and V. cholerae can confer elevated resistance to kanamycin and streptomycin (32, 47). Therefore, multidrug and toxic compound extrusion pumps might be responsible for the natural kanamycin and streptomycin resistance of V. tapetis.

These genetic tools were used to explore the role of DjlA in the physiology and virulence of V. tapetis. Amplification and sequencing of the djlA region with primers chosen from conserved regions of V. splendidus LGP32 (since the genome sequence of V. tapetis is not available yet) revealed the presence of an ORF that we designated duf924 situated downstream and in the same orientation as djlAVt. This ORF encodes a bacterial DUF924 domain protein with an unknown function. The gene that is further downstream of duf924 is transcribed in the opposite direction from the genome and cannot be part of the putative operon. The putative organization in the operon containing djlAVt and duf924Vt could suggest a common function. However, this structure is not evolutionarily conserved; among sequenced Vibrio species, only V. splendidus has the same genetic organization, suggesting that the operon is a recently formed operon that could contain functionally unrelated genes (42).

DjlAVt was found to be functional in E. coli, as indicated by its ability to induce expression of the cps operon as a reporter of the activation of the Rcs pathway, although it does so less efficiently than E. coli DjlA itself (Fig. 2). This is not surprising since the domains which are known to be important for activation of the Rcs system, such as the transmembrane domain or the J-domain (12, 49), are quite conserved (data not shown).

To mutagenize djlAVt, we used a strategy which was initially described by Miller and Mekalanos (30) and relies on the use of a suicide vector carrying a truncated version of the targeted gene, which can then be disrupted by insertion of the plasmid through homologous recombination. This technique has been used with considerable success, but it has several potential drawbacks. (i) Reversion can occur due to a single recombination event, making the presence of a selective antibiotic in the culture necessary. This could minimize the phenotype during in vivo experiments. However, this does not seem to have been a problem in our experiments. (ii) The insertion can have a polar effect on a downstream gene. (iii) The insertion can generate a partial duplication of the targeted gene. In our case, this duplicated truncated gene would have encoded two-thirds of the DjlA protein (i.e., a truncated version deleted of the J-domain). Toxic expression of such a truncated version of DjlA, which is potentially able to interact with substrates via its transmembrane domain (49) but is not functional as a cochaperone, could explain the slow-growth phenotype of the mutant. It should be noted that complete djlA null mutations did not affect growth either in E. coli or in Legionella sp. Indeed, such a toxic effect of a truncated version of DjlA might be dominant negative and could account for the absence of complementation by the wild-type djlA gene. An alternative explanation is that the growth defect is due to a polar effect of the mutation on the expression of duf924. However, in such a case, complementation of the growth defect by Duf924 would be expected.

Despite these drawbacks, the phenotypic analysis of the mutant clearly points to a potentially important role for djlA in pathogenicity. In vivo experiments (inoculation into the extrapallial cavity or injection into the adductor muscle) showed that V. tapetis djlA mutants were not able to induce BRD and to kill R. philippinarum. However, the observed attenuation in virulence may be a consequence of the growth defect rather than a consequence of a specific effect of the absence of DjlA. Hence, in vitro experiments were carried out, using a set amount of bacteria (see Materials and Methods). The djlA mutant cannot induce rounding of hemocytes (Fig. 3b) or inhibit hemocyte adhesion properties, an ability which specifically reflects V. tapetis cytotoxic activity and which other vibrios, such as V. splendidus, which is pathogenic to oysters, do not display (11). The loss of cytotoxicity to clam hemocytes in the mutant could by itself account for the absence of virulence in vivo, given the importance of hemocytes in clam defenses. Only the expression of DjlA from pGEB22 or pGEB24 could rescue the phenotype by restoring cytotoxicity to the wild-type level (Fig. 4). In addition, the fact that in vitro toxicity can be restored independent of a wild-type growth rate indicates that this phenotype is not due to the growth defect of the mutant.

A role of DjlA in bacterial pathogenicity has been reported previously: it is essential for intracellular growth of Legionella dumoffii (33) and of Legionella pneumophila, where it is proposed to play a role in assembly of the dot/icm type IV secretion system, which is responsible for secretion of effectors necessary for intracellular survival and multiplication in macrophages (33, 50). In the case of V. tapetis, it was recently shown that incubation of this bacterium with R. philippinarum hemocytes could specifically inhibit their phagocytic ability (2). Rounding of the hemocytes and loss of filopods upon interaction with the pathogen suggest that there is actin rearrangement. It is tempting to speculate that these effects are mediated by secreted effectors. DjlAVt is an excellent candidate to promote, through its chaperone activity, assembly of the system(s) responsible for secreting such cytotoxic factors.

There is currently a lot of interest in the genomes of vibrios, and a number of genomic sequences have been determined. In parallel, to fully explore the biodiversity of this ecologically important genus (48), many species of which are responsible for diseases both in humans and in animals, such as corals, mollusks, and fish, and to understand the full repertoire of host-pathogen interactions, it is important to develop genetic analysis of nonmodel bacteria to make them amenable to functional analysis. The work presented here is a first step in this direction in the case of V. tapetis.


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ACKNOWLEDGMENTS
 
We thank Frédérique Le Roux and Didier Mazel for kindly providing plasmids, strains, and the unpublished sequence of the djlA region of V. splendidus LGP32 and for many helpful discussions. We thank Nolwenn Trinkler and Marie-Agnes Travers for help with in vivo experiments and flow cytometry. We thank Candice Rigoulay and Philippe Bouloc for stimulating discussions and critical reading of the manuscript.

This study was carried out with financial support from the CNRS, Université Paris-Sud (UMR8621), and the Groupement d'Intérêt Scientifique "Institut de Génomique Marine." Fatma Lakhal was a recipient of a fellowship from the Agence Universitaire de la Francophonie.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut de Génétique et Microbiologie, Bâtiment 400, Université Paris-Sud, Orsay 91405 Cedex, France. Phone: 33-1 69 15 57 17. Fax: 33-1 69 15 66 78. E-mail: annick.jacq{at}igmors.u-psud.fr Back

{triangledown} Published ahead of print on 18 July 2008. Back

{dagger} Present address: LBPA, CNRS-UMR 8113, E.N.S. Cachan, 61 avenue du Président Wilson, 94235 Cachan, France. Back


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Applied and Environmental Microbiology, September 2008, p. 5750-5758, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01043-08
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