Cloning And Characterization Of Possible Virulence Factors Of Flavobacterium Columnare

Columnaris is a world-wide bacterial fish disease, causing heavy economical losses in aquaculture industry. The bacterial pathogen, Flavobacterium columnare belongs to the family Flavobacteriaceae. The infection of the bacterium may cause severe gill-rotting and sometimes skin ulceration. Bacterial gill-rot disease is epidemic in China, and a major disease in several major cultured fish species in China, including the grass carp Ctenopharyngodoe idellus and the mandarin fish Siniperca chuatsi. The present study, by using bacteria isolated from outbreaks of columnaris disease in China as the pathogen, aimed to identify genes of possible virulence factors of the bacteria, and functions of some of these identified genes were also examined. The bacterium causing columnaris disease was identified first as Myxococcus piscicola Lu, Nie & Ko, 1975 from diseased grass carp in China. In order to clarify the identity of this bacterium, 16S rDNA and 16S-23S rDNA intergenic spacer region sequences were cloned from the so-called M. piscicola. As revealed by the high percentage of boostrap values with other species in the genus Flavobacterium and other strains of F. columnare isolated in other countries, the bacterium, M. piscicola causing columnaris disease isolated from grass carp should be a synonym of Flavobacterium columnare, and the most virulent strain isolated from grass carp, i.e. the G₄ strain was employed in the present study. In order to identify genes encoding the outer membrane proteins (OMPs) of F. columnare G₄, the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. A total of 36 genes were obtained, and 11 of which are possible virulence factors which can be divided into four categories: 1) gliding related genes, 2) genes related to eliminating the killing ability of the host, 3) membrane-associated proteases, and 4) extracellular protease and collagenase. The gliding related gene gldH and its role in gliding motility, chitin utilization and adherence was examined in F. columnare G4. Western blotting analysis using rabbit antisera against recombinant GldH showed that the protein is localized in the outer membrane fraction of the bacteria. The defined deletion of the chromosomal copy of gldH in wild-type F. columnare G4 has led to the establishment of a gldH deficient mutant. Functional experiments have shown that the gldH mutant loses its abilities in cell gliding, colony spreading, chitin digestion, and adherence to gill tissues. The mutant obtained from defined gene deletion in the present study represents the first description of an adhesion-defective mutant for F. columnare, and the techniques involved in the present study may provide basis for further study on other genes of F. columnare and also other Gram-negative bacterial pathogens. Gene and functional analysis were also conducted for the glycosaminoglycan-degrading enzyme chondroitin AC lyase from F. columnare G4. The chondroitin AC lyase gene was obtained by degenerated primers and then genomic walking methods. The gene, cslA was then successfully expressed in E. coli and recombinant chondroitin AC lyase, rChonAC was purified, with the lytic activity of rChonAC analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G4. The low level of lytic activity as observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare. In addition, histological and cytological studies on the developing thymus and pronephros of the mandarin fish Siniperca chuatsi (Perciformes: Teleostei) were also conducted in the present study. The thymus of the mandarin fish was examined by light and transmission electron microscopy to understand its formation and cellular composition. Obvious thymus was found packed with lymphocytes on 7 dph. The thymus of S. chuatsi was connected with the extension of the third, fourth and fifth branchial pouches throughout the early development, and remained its superficial position in the adults, with perforations observed on its surface. In thymus of the adultfish, thymic epithelial cells (TECs) characteric of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular, and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and incomplete blood-thymus barrier found in the inner zone. Taken together, thymus development in mandarin fish agrees, to some extent, with ontogenetic patterns observed in other fish species. The renal tubules and lymphoblast-like cells could be observed one day after hatching. And the developing process could be subjectively divided into three stages. At the first stage, only renal tubules could be observed, and the pronephros functions as urinary organ; with pronephros development, more lymphoid cell appears, and the pronephros seems bifunctional, i.e. it functions as an urinary organ and a lymphoid organ as well; then with it comes to 35 dph, the number of the renal tubule decreased rapidly, and the pronephros is occupied completely by lymphoid cells. In summary, possible virulence genes of F. columnare G4 have been screened successfully by using antisera developed against the OMPs of the bacterium, and a total of 15 such genes have been sequenced. Techniques for defined gene knock-out have been established for understanding the function of the possible genes. The present study thus provide a foundation for further investigation on the virulence factors of F. columnare G4 and their interactions with fish hosts.