Correspondence: Makoto Ito, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: +81 92 642 2898; fax: +81 92 642 2907; e-mail: email@example.com
Vibrios, distributed in marine and brackish environments, can cause vibriosis in fish and shellfish under appropriate conditions. Previously, we clarified by thin-layer chromatography (TLC) overlay assay that 35S-labeled Vibrio trachuri adhered to GM4 isolated from red sea bream intestine. However, whether GM4 actually functions on epithelial cells as an attachment site for vibrios still remains to be uncovered. We found that six isolates, classified as V. harveyi, V. campbellii, and V. splendidus, from intestinal microflora of red sea bream adhered to GM4 but not galactosylceramide (GalCer) by TLC-overlay assay. Tissue-overlay assays revealed that V. harveyi labeled with green fluorescent protein (GFP) adhered to epithelial cells of red sea bream intestine where GM4 and GalCer were found to be distributed on the top layer of actin filaments by immunohistochemical analysis using corresponding antibodies. The number of adhering vibrios was diminished by pretreatment with anti-GM4 antibody, but not anti-GalCer antibody. These results clearly indicate that vibrios adhere to epithelial cells of red sea bream intestine utilizing GM4 as an attachment site.
Vibriosis, one of the prevalent diseases in fish and shellfish in aquaculture, is caused by vibrios such as Vibrio harveyi (trachuri) (Iwamoto et al., 1995; Austin & Zhang, 2006), V. campbellii (Haldar et al., 2011), V. splendidus (Sugumar et al., 1998; Gatesoupe et al., 1999), and V. anguillarum (Toranzo & Barja, 1990). The disease occurs in aquaculture in salt and brackish water in European countries, North America, Australia, and Japan (Simidu & Tsukamoto, 1985; Muroga, 2001; Rigos & Katharios, 2010). Vibrios, distributed in environmental waters as well as healthy fish intestine, can cause the disease in immunocompromised hosts (Conte, 2004).
The first step in the bacterial infection is the attachment of bacteria to the surface of epithelial cells of host animals, which facilitates colonization on or penetration of the cells (Rottner et al., 2005). The apical surface of epithelial cells has animal- and/or tissue-specific glycoconjugates, such as glycosphingolipids (GSLs) and glycoproteins; the GSLs are known to be significant receptors for bacteria invading host cells and tissues (Karlsson et al., 1992; Varki, 1993; Dwek, 1996). The affinity of bacteria for host cells/tissues mainly depends on the structure of GSL-sugar chains (Fantini et al., 2000).
Although accumulating evidence indicates the integral role for GSLs in mammals as a receptor for bacteria (Karlsson, 1986; Brennan et al., 1991; Backhed et al., 2002), there are few reports focusing on the interaction between marine bacteria and GSLs in host fish (Irie et al., 2004; Chisada et al., 2005; Matsunaga et al., 2011). We revealed by thin-layer chromatography (TLC) overlay assay that 35S-labeled V. trachuri adhere to GM4 (NeuAcα2-3Galβ1-1′Cer) from red sea bream intestine (Chisada et al., 2005). Vibrio trachuri, a junior subjective synonym for V. harveyi (Thompson et al., 2002), is pathogenic under considerable conditions (Iwamoto et al., 1995). Furthermore, several type-cultures of vibrio causing vibriosis adhered to GM4 and GM3 (NeuAcα2-3Galβ1-4Glcβ1-1′Cer) extracted from the intestinal tracts of seven typical species of fish (P. major, Seriola quinqueradiata, Seriola lalandi, Seriola dumerili, Trachurus japonicus, Scomber japonicas, and Paralichtys olivaceus) used in aquaculture in Japan; however, vibrios hardly adhered to GalCer (Galβ1-1′Cer) and GlcCer (Glcβ1-1′Cer), a core structure of GM4 and GM3, respectively (Matsunaga et al., 2011). This result indicated that the minimum structure of GSL-sugars required for adherence of vibrios was NeuAcα2-3Galβ1- at the nonreducing end of the GSLs. However, whether these GSLs actually function as an attachment site for vibrios on the epithelial cells of fish intestines still remains to be uncovered.
In this study, we isolated, from intestinal microflora of red sea bream without symptoms of vibriosis, six strains of bacteria, which preferentially adhered to GM4 not GalCer, both major GSLs of red sea bream intestines. These six strains were classified to be either V. harveyi, V. campbellii, or V. splendidus. Tissue-overlay assays using GFP-labeled V. harveyi and frozen sections of red sea bream intestine revealed that V. harveyi adhered to epithelial cells of red sea bream intestine utilizing GM4 as an attachment site. This study helps to understand the mechanism by which vibrios invade the intestinal tracts of their host fish and should facilitate the development of probiotics and drugs which prevent the adhesion of vibrios to the intestinal tract, leading to the prevention of vibriosis.
Materials and methods
Red sea bream (Pagrus major) were purchased from a fish market in Fukuoka, Japan. GalCer and GM4 were isolated from the intestinal tract of red sea bream (Chisada et al., 2005). [35S] methionine was obtained from MP Biochemicals, Inc. (Irvine, CA). Silica gel 60 TLC plates were purchased from Merck KGaA (Darmstadt, Germany). Plastic TLC plates (POLYGRAM SIL G) for TLC-overlay assays were obtained from MACHEREY-NAGEL Co. (Duren, Germany). Sep-Pak Plus Silica Cartridge and Sep-Pak Plus Waters Accell QMA Cartridge were purchased from Waters Co. (Milford, MA). Anti-GM4 polyclonal antibody was obtained from Matreya LLC (Pleasant Gap, PA). Anti-GalCer monoclonal antibody (AMR-20) was donated from Dr. T. Tai (Tokyo Metropolitan Institute of Medical Science, Japan). Alexa fluor 488 goat antirabbit IgG (H+L), Alexa fluor 488 goat antimouse IgM antibody, and Alexa fluor 568-conjugated phalloidin were purchased from Invitrogen Co. (Carlsbad, CA). To increase the intensity of signals of antibody staining, Can Get Signal® immunostain immunoreaction enhancer solution A or B (Toyobo Co., Ltd., Japan) was used. Gfp-donor Escherichia coli was donated from Dr. T. Sawabe (Hokkaido University) and Dr. E. V. Stabb (Georgia University).
The type culture strain of Vibrio harveyi (NBRC 15632) was obtained from the Department of Biotechnology, National Institute of Technology and Evaluation, Japan. Bacteria isolated from the intestinal lumen of red sea bream were stored in 15% glycerol at −80 °C in a deep freezer.
Culture and metabolic labeling of bacteria
Bacteria were cultured in PY-seawater medium composed of 1% polypeptone, 0.2% yeast extract, and 0.05% MgSO4.7H2O in 75% seawater (filtrated by 0.45 μm membrane filter), and 25% distilled water, pH 7.2. Radioisotope labeling of vibrios was conducted by culturing the bacteria in PY-seawater medium containing [35S] methionine (0.25 μCi mL−1) at 25 °C for 18 h.
Thin-layer chromatography (TLC)
GSLs were applied onto a glass or a plastic TLC plate, which was developed with chloroform/methanol/0.02% CaCl2 (5/4/1, v/v/v) (solvent I). All GSLs were visualized by spraying the plate with orcinol-H2SO4 reagent.
The adherence of bacteria to GSLs was examined by TLC-overlay assay as described in (Karlsson, 1986; Backhed et al., 2002) with minor modifications. Briefly, 20 nmol of each GSL was applied to a plastic TLC plate, which was developed with solvent I. After drying, the plate was dipped in diethylether/n-hexane (1/5, v/v) containing 0.5% (w/v) polyisobutylmethacrylate for 1 min. After drying, the plate was soaked in PBS containing 2% casein and 0.1% cold methionine for 2 h at room temperature (RT), and then incubated with 3 mL of [35S]-labeled bacterial suspension (1.5 × 109 cells mL−1 of PBS, 2700 dpm mL−1). Following incubation at 25 °C for 20 h, the plastic plate was gently washed with phosphate buffer containing 3% NaCl and finally exposed to an imaging plate. The radioactivity of GSLs due to adherence of [35S]-labeled bacteria was quantified with an imaging analyzer, FLA-5000 (Fuji Film Co., Kanagawa, Japan).
Identification of bacteria
Bacteria were identified based on biochemical and physiological properties and a 16S rRNA gene analysis. The former was basically performed according to Bergy's Manual of Determinative Bacteriology. The 16S rRNA gene analysis was conducted as described in (Lane et al., 1985). An approximately 1500-bp fragment of the 16S rRNA gene was amplified using universal primers p27f (5′-AGAGTTTGATCMTGGCTCAG-3′; position 8-27; E. coli numbering) and p1492r (5′-GGCTACCTTGTTACGACTT-3′; position 1492-1474). PCR products were excised from the 1.0% agarose gel and sequenced directly. The following sequencing primers were used:
r1L (5′-GTATTACCGCGGCTGCTGG-3′; position 536-518), r2L1 (5′-CATCGTTTACGGCGTGGAC-3′; position 821-803), r2L2 (5′-GACTACCAGGGTATCTAA-3′; position 805-786), r3L (5′-TTGCGCTCGTTGCGGGACT-3′; position 1111-1093), r4L (5′-ACGGGCGGTGTGTACAAG-3′; position 1406-1389), rE1L (5′-GTAGGAGTCTGGACCGTGT-3′; position 345-327), f1L (5′-GAGTTTGATCCTGGCTCAG-3′; position 9-27), f2L (5′-CCAGCAGCGGCGGTAATAG-3′; position 518-536), f3L (5′-GTCCCGCAACGAGCGCAAC-3′; position 1094-1112). 16S rDNA sequences were analyzed by BLASTn search through the National Center for Biotechnology Information GenBank database.
Samples were fixed with 4% paraformaldehyde in PBS at 4 °C overnight, washed with PBS, and then immersed in 15% sucrose in PBS containing 0.02% NaN3 at 4 °C for 1 h. Sample was then transferred into 30% sucrose in the same solution and incubated at 4 °C overnight. Samples were embedded in OCT compound (Sakura Finetechnical, Japan), frozen at −30 °C for 1 h, and then kept at −80 °C until use. The frozen materials were serially sectioned 10-μm-thick using a Cryostat (Leica CM1900, Leica Microsystems, Nussloch, Germany). After drying, sections were blocked with 2% BSA in PBS at RT for 2 h. Subsequently, the sections were incubated with anti-GM4 rabbit polyclonal antibody (1:100 dilution) or anti-GalCer mouse monoclonal antibody (1 : 200 dilution) at 4 °C overnight, followed by Alexa fluor 488-conjugated goat antirabbit IgG antibody (1 : 3000) or Alexa fluor 488-conjugated goat antimouse IgM antibody at RT for 2 h. These antibodies were diluted at the ratio indicated in parentheses with Can Get Signal immunostain immunoreactions enhancer solution A (for anti-GalCer antibody) or B (for anti-GM4 antibody), which contains Alexa fluor 568-conjugated phalloidin (200 mU mL−1) for counterstaining β-actin filaments of intestinal villi. Each section was washed five times with PBS containing 0.25% skim milk. Immunostained sections were mounted with fluoromount and observed under a confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).
Generation of GFP-labeled V. harveyi
Vibrio harveyi was labeled with green fluorescent protein (GFP) by the conjugative transfer of a plasmid containing the gfp gene from E. coli CC118λpir strain carrying pEVS104 (tra trb Knr) and pKV111 (gfp Cmr) (Sawabe et al., 2006). The gfp-donor E. coli strain was cultured in LB broth containing 20 μg mL−1 chloramphenicol and 40 μg mL−1 kanamycin at 37 °C overnight, while the V. harveyi recipient strain was cultured in PY-seawater medium at 25 °C overnight. Each of the cultures (100 μL) was mixed in a 1.5-mL tube and centrifuged (at 15 000 g at 25 °C for 3 min) to pellet these cells. The pellet was washed twice with PY-seawater medium to remove antibiotics and resuspended in 10 μL of the medium. The suspension was spotted onto a PY-seawater agar plate containing 0.5% sodium alginate. After incubation at 20 °C for 1–2 days, the bacteria were collected into a 1.5-mL tube and suspended in 700 μL of PY-seawater medium. One hundred microliters of the suspension was plated onto a PY-seawater agar plate containing 0.5% sodium alginate and 20 μg mL−1 chloramphenicol or a TCBS agar plate (Nissui Seiyaku, Tokyo, Japan) containing 20 μg mL−1 chloramphenicol at 20 °C. The green fluorescence of each colony was examined under a MZFLIII fluorescence microscope (Leica, Germany) with the Cool Pix 990 (Nikon, Japan) camera system or a confocal laser-scanning microscope (Digital Eclipse C1). GFP-expressing V. harveyi was purified by a streaked plate method using a TCBS agar plate at 20 °C.
Tissue-overlay assay using GFP-labeled V. harveyi
The adherence of V. harveyi to epithelial cells of the intestinal tract of red sea bream was examined using frozen sections of the intestinal tract and the GFP-labeled V. harveyi. The frozen sections (10 μm-thick) were blocked with 2.5% skim milk in PBS for 2 h at RT and then washed 5 times with 0.25% skim milk in PBS. The sections were stained with Alexa fluor 568-conjugated phalloidin (200 mU mL−1) at RT for 30 min. After three washes with the same solution, the sections were incubated with 100 μL of GFP-labeled V. harveyi (4.0 × 108 cells mL−1 of 0.25% skim milk in PBS) at RT for 3 h. For the control, sections were treated with anti-GM4 polyclonal antibody (×500 dilution) or anti-GalCer monoclonal antibody (×500) before incubation with the GFP-labeled bacteria. Then, the sections were washed 10 times by dipping them into PBS containing 0.25% skim milk. All sections were mounted with fluoromount and observed under a confocal laser-scanning microscope.
Isolation of bacteria adhering to GM4 from the red sea bream intestine
Previously, we reported that V. harveyi isolated from the intestine of red sea bream adhered to GM4 but not monohexosylceramide, although both are major GSLs in the red sea bream intestine (Chisada et al., 2005). In this study, to reveal the species of bacteria adhering to GM4 in intestinal lumens of the red sea bream, 100 strains of bacteria were isolated from five healthy red sea breams (12, 11, 30, 28, 19 strains were isolated from each trial) and then subjected to TLC-overlay assay. As shown in Table 1, six strains (tentatively named IB-3, 5, 6, 8, 9, and 13) were found to adhere to GM4, but these strains did not adhere to GalCer under the conditions used (Fig. 1).
Table 1. Identification of intestinal bacteria isolated from Pagrus major
Identification of 6 strains of bacteria adhering to GM4
Table 1 shows the biochemical and physiological properties of six strains of intestinal bacteria adhering to GM4. Based on these general characteristics, the strains were assigned to the genus Vibrio according to Bargey's Manual of Determinative of Bacteria. Furthermore, the sequences of 16S rRNA gene were the closest matches (>99%) with those of V. harveyi (four strains), V. campbellii (1 strain), and V. splendidus (one strain) in GenBank by analysis using BLASTn (Altschul et al., 1990). Phylogenetic tree of bacteria belonging to genus Vibrio with six isolates is shown in Supporting Information, Fig. S1.
Distribution of GM4 and GalCer on the luminal surface of the red sea bream intestine
The major GSLs of the intestinal tract of red sea bream were monohexosylceramide and GM4 (Chisada et al., 2005). The latter is the sialylated GalCer, in which an N-acetyl neuraminic acid binds to the C3 position of Gal via an alpha glycosidic linkage. The cellular localization of GM4 and GalCer, however, remains to be clarified. In this study, the localization of GM4 and GalCer in frozen sections of the intestinal tract was examined by an immunohistochemical approach using anti-GM4 and anti-GalCer antibodies. Strong signals for GM4 (green in Fig. 2b) and GalCer (green in Fig. 2j) were detected at the edge of the intestinal villus, as determined by the counterstaining of actin filaments with phalloidin (red in Fig. 2c and k). Merged images of GSLs and actin filaments confirmed that GM4 and GalCer were located on epithelial cells of the intestinal villus in the intestinal tract of red sea bream (Fig. 2d and l).
Tissue-overlay assay using GFP-labeled V. harveyi
Adherence to the luminal surface was examined using GFP-labeled V. harveyi and frozen sections of the intestinal tract (Figs 3 and 4). It was observed that many GFP-labeled vibrios adhered to the edge of the intestinal villus (green in Fig. 3b), on counterstaining of actin filaments (red in Fig. 3c). Merged images showed many GFP-labeled bacteria attached to the intestinal villus (Fig. 3d and a magnified view in Fig. 3m). Pretreatment of the sections with anti-GM4 antibody decreased the number of vibrios on the intestinal villus (Fig. 3e–h and n). In contrast, anti-GalCer antibody did not decrease the number of vibrios adhered to the surface (Fig. 3i–l). Fig. 4 showed that the number of V. harveyi adhered to the intestinal villus was significantly decreased by treatment with anti-GM4, but not anti-GalCer, antibodies. Collectively, these results indicated that V. harveyi adhered to epithelial cells of the intestinal tract in red sea bream utilizing GM4 as an attachment site.
In this study, we isolated six strains of bacteria adhering to GM4 from the intestine of healthy red sea bream, which showed no symptoms of vibriosis (Fig. 1). These strains were found to be the closest matches to either V. campbellii, V. harveyi, or V. splendidus by 16S rRNA gene analysis (Table 1 and Fig. S1). These vibrio strains are known to be opportunistic bacteria causing vibriosis under stressful conditions such as overcrowding, overfeeding, water pollution, and extremely high/low temperature, which may trigger dysfunction of the immune system in host fish (Austin & Zhang, 2006; Magnadottir, 2006).
We showed for the first time that GM4 and GalCer were distributed at the edge of the intestinal villus (Fig. 2). Importantly, pretreatment with anti-GM4 but not anti-GalCer antibodies diminished the number of vibrios adhering to the intestinal villus (Fig. 3 and Fig. 4), indicating that vibrios utilize GM4 but not GalCer as an attachment site on the intestinal epithelial cells of fish. The present study suggests that these opportunistic vibrios, usually retained in the intestinal mucus layer of healthy fish, can pass through the mucosa under considerable conditions and invade the intestinal epithelial cells utilizing GM4 as an attachment site. The report that vibrios capable of adhering to epithelial cells could become a causative agent for vibriosis (Wang et al., 1998; Wang & Leung, 2000) would support this scenario.
The present study, however, does not reveal how opportunistic vibrios are retained in the intestinal tract. It is plausible that opportunistic vibrios remain in the mucus layers on the intestinal tract by adhering to mucus glycoproteins which share the same glycan structure found in the nonreducing end of GM3 and GM4. Actually, the NeuAcα2-3Gal- structure was found in not only mammal (Sata et al., 1989) but also fish mucus glycoproteins (Shephard, 1994; Neuhaus et al., 2007). Mucus layer, a reservoir of opportunistic vibrios, could stimulate the growth of some vibrios (Garcia et al., 1997).
In contrast to opportunistic vibrios, some strains of V. anguillarum are known to be acute-lethal pathogenic agents, which can cause vibriosis under experimentally normal conditions (Frans et al., 2011). These pathogenic vibrio strains possess specific proteases (mucinases) capable of degrading mucosal layers (Farrell & Crosa, 1991) and thus could reach the epithelial cells (O'Toole et al., 1999). However, opportunistic vibrios could not pass through the mucus layer. Because stress was found to cause a considerable reduction in the intestinal mucosal layer of farmed fish (Olsen et al., 2005; Magnadottir, 2006), opportunistic vibrios could reach the intestinal epithelial cells under such conditions.
In conclusion, the specific sugar sequence, NeuAcα2-3Galβ-, at the nonreducing end of GSLs functions as an attachment site for vibrios on the epithelial cells of fish intestinal tracts.
We thank Dr. T. Tai for donating anti-GalCer antibody. We also thank Dr. T. Sawabe and Dr. E. V. Stabb for providing gfp-donor E. coli.