Virulence factors of Lactococcus garvieae isolated from South African rainbow trout
Meyburgh, Cornelia Magdalena
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Bacterial infections cause severe economic loss in the rapidly growing aquaculture industry. Lactococcus garvieae, a Gram-positive bacterium, causes lethal, hyperacute septicaemia in numerous fish species of importance to aquaculture worldwide. The virulence factors of this pathogen is not well understood, although the exopolysaccharide (EPS) capsule is considered the most important virulence determinant. A conserved metabolic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), may function as a virulence factor of L. garvieae via the phenomenon of “moonlighting”, a term that refers to the ability of a protein to perform more than one function. Previous investigations have also identified L. garvieae GAPDH as an antigen, suggesting that GAPDH is localised to the extracellular environment. The general aim of this study was to improve the current understanding of factors contributing to L. garvieae virulence by means of the following objectives: Identify putative virulence factor genes of South African L. garvieae isolates by PCR and sequencing. Identify extracellular proteins from South African L. garvieae isolates. Clone, express and purify L. garvieae GAPDH. Identify putative ligands to recombinant GAPDH using a commercially available random peptide phage display library. Isolates obtained from diseased rainbow trout (Oncorynchus mykiss) from locations in present day Gauteng and Mpumalanga, South Africa, were screened for the presence of a set of putative virulence factor genes by PCR. The presence of EPS capsules were tested by negative staining and amplification of EPS biosynthesis genes by PCR. All virulence factor genes were found in the isolates tested, including an avirulent strain NCFB 657. However, negative staining indicated the absence of EPS capsules in the virulent isolates and NCFB 657 and EPS biosynthesis genes could not be amplified. Extracellular proteins detected and identified by LC/MS-MS included L-lactate dehydrogenase, enolase, 60 kDa chaperonin and phosphoenolpyruvate-protein phosphotransferase. The metabolic enzyme enolase is known to moonlight as a virulence factor in several streptococcal pathogens, similar to GAPDH.