| Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis, which is a major problem for the aquaculture industry world wide. Previously, a 255 bp gene fragment of V. anguillarum was obtained from a virulence related suppression subtractive hybridization (SSH) library. In this study, the complete gene sequence was obtained by long and accurate PCR (LAPCR). After sequence analysis and homologous comparison, this gene was revealed to encode a membrane-bound lytic murein transglycosylase D (MltD). Bioinformatic analysis showed that the MltD consist of 523 amino acids, comprises a signal peptide, a transglycosylase domain and three LysM domains, and this structure is very similar with that in Escherichia coli, except that the latter consist of 452 amino acids and only has two LysM repeats at C-terminal. The MltD protein in V. anguillarum is supposed to be stable in physical properties, and hydrophilic; a lipoprotein signal peptide is at the N-terminal; except the signal peptide, the rest are non-cytoplasmic; the whole molecule shows solid antigenicity; secondary structure is predominated by a helix and random coil; the protein sequence is highly consistent with the putative MltD proteins in other Vibrio sp.MltD gene was subcloned into an expression plasmid pET-32a (+), and overexpressed in E. coli strain BL21 (DE3). The expressed fused MltD protein was purified by Ni 2+-affinity chromatography, and the molecular weight of recombinant protein is assessed to be about 70.2 kDa after SDS-PAGE analysis, which is consistent with the therotical molecular weight. The results of enzymic activity shows that both of the crude enzyme extract from E. coli BL21(DE3)/pET-32a(+)/mltD after induction by IPTG and purified MltD protein had hemolytic, phospholipase, gelatinase and diastase activities, whereas the enzymic activity of the crude enzyme extracted from BL21(DE3)/pET-32a(+) under the same conditions and the dialysate were negative results.A mltD mutant of pathogenic V. anguillarum CW-1 was constructed by homologous recombination, and the production of extracellular gelatinase and protease decreased markedly compared with those of the wild type strain; and the haemolytic activity was totally lost. Hyperosmotic challenge and antibiotic sensitivity assay showed that the resistance of mltD mutant against high salts concentration; and rocephin, fortun, cefobid, gentamycin, kanamycin and carbenicillin resistance were enhanced. But in the assay of acid-base change, the mutant showed a declining adaptability. The API 20E test showed that the mutant lost the ability to use glucose. Most importantly, virulence of the mltD mutant was enhanced compared with that of the wild-type, when it was inoculated intraperitoneally into zebra fish; the LD50 of the wild-type and the mutant were 3.92×103 CFU and 1.01×102 CFU per fish, respectively. During the infections to the gill cells of flounder, the cells which were infected by the mutant had a higher mortality than that were infected by the wild type cells of V. anguillarum, and this result was consistent with the result of infection to zebra fish.The function of the mltD gene only has been reported in E. coli and Helicobacter pylori. In Vibrio sp., only homologous sequences are found. This is the first report of mltD gene in V. anguillarum, and is the first discovery that MltD protein is involved in bacterial pathogenicity. Some suggestions regarding the function of MltD in the bacterial pathogenicity are also given.
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