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Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

ABSTRACT: Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ?cslA and ?cslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (?cslA ?cslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ?cslA ?cslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes.

SUBMITTER: Li N

PROVIDER: S-EPMC4592881 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

Li Nan N Qin Ting T Zhang Xiao Lin XL Huang Bei B Liu Zhi Xin ZX Xie Hai Xia HX Zhang Jin J McBride Mark J MJ Nie Pin P

Applied and environmental microbiology 20150807 21

Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to s ...[more]

PMID: 26253667

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Project description:BackgroundFlavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067 in rainbow trout (Onconrhynchus mykiss) and used high-resolution scanning electron microscopy to identify the fine structures of the cells grown in liquid and on agar. We also analysed the proteins secreted extracellularly and in membrane vesicles to identify possible virulence factors.ResultsOnly the Rhizoid morphotype was virulent in rainbow trout. Under electron microscopy, the cells of Rhizoid and Soft morphotypes were observed to display an organised structure within the colony, whereas in the Rough type this internal organisation was absent. Planktonic cells of the Rhizoid and Rough morphotypes produced large membrane vesicles that were not seen on the cells of the Soft morphotype. The vesicles were purified and analysed. Two proteins with predicted functions were identified, OmpA and SprF. Furthermore, the Rhizoid morphotype secreted a notable amount of a small, unidentified 13 kDa protein absent in the Rough and Soft morphotypes, indicating an association with bacterial virulence.ConclusionsOur results suggest three factors that are associated with the virulence of F. columnare: the coordinated organisation of cells, a secreted protein and outer membrane vesicles. The internal organisation of the cells within a colony may be associated with bacterial gliding motility, which has been suggested to be connected with virulence in F. columnare. The function of the secreted 13 kDa protein by the cells of the virulent morphotype cells remains unknown. The membrane vesicles might be connected with the adhesion of cells to the surfaces and could also carry potential virulence factors. Indeed, OmpA is a virulence factor in several bacterial pathogens, often linked with adhesion and invasion, and SprF is a protein connected with gliding motility and the protein secretion of flavobacteria.

Project description:Flavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare.As a first step toward developing a robust set of genetic tools for F. columnare, a protocol was developed to introduce the E. coli - Flavobacterium shuttle vector pCP29 into F. columnare strain C#2 by conjugal mating at an efficiency of 1.5 x 10(-3) antibiotic-resistant transconjugants per recipient cell. Eight of eleven F. columnare strains tested were able to receive pCP29 using the protocol. pCP29 contains the cfxA and ermF genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin. This is in contrast to other Flavobacterium species where ermF-based erythromycin resistance is strong. The green fluorescent protein gene (gfp) was introduced into F. columnare strains under the control of two different native Flavobacterium promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn4351 was successfully introduced into F. columnare, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 microg ml(-1)) were used, and high levels of background growth occurred. These results demonstrate that Tn4351 functions in F. columnare but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in F. columnare.The conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of F. columnare. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen.

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Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

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Gene deletion strategy to examine the involvement of the two chondroitin lyases in Flavobacterium columnare virulence.

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