The Effects of TCA Cycle Mutations on the Virulence of Vibrio Aguillarum Strains M93SM and NB10SM

Abstract

Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans. Mortality rates range from 30% to 100% and systemic infection usually causes fish to die within 1-4 days of initial infection. The primary routes of infections are through the skin, gills and intestines. Chemotactic motility and the metalloprotease EmpA have been shown to be important virulence factors during the invasion stage while the siderophore anguibactin, flagellin subunits and lipopolysaccharides were shown to be important for persistence in the host during the post-invasion stage. Three secreted proteins that are cytotoxic against epithelial cells and erythrocytes have been characterized in V. anguillarum: the HlyA homolog Vah1, the phospholipase Plp, and the MARTX toxin RtxA. Previous research has demonstrated that mutations in vah1 and/or plp resulted in slight attenuation against juvenile Atlantic salmon (Salmo salar); however, rtxA mutants were avirulent. Expression of the cytotoxins are under control of the transcriptional activator HlyU and the repressor H-NS. Additionally, a V. anguillarum hns mutant showed attenuation in virulence when injected intraperitoneally, suggesting that proper coordination of gene expression is an important factor during the post-invasion stage. In manuscript I “Isocitrate dehydrogenase mutation in Vibrio anguillarum results in virulence attenuation and immunoprotection in rainbow trout (Oncorhynchus mykiss)”, seven central metabolism mutants were created in the M93Sm strain and characterized with regard to growth in minimal and complex media, expression of virulence genes and virulence in juvenile rainbow trout. Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum experimental challenge. The icd mutant did not demonstrate a significant decrease in the expression of the three hemolysin genes was detected by qRT-PCR. Only the icd mutant exhibited a significantly decreased growth yield in complex media that was directly related to the amount of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex and minimal media and pathogenicity in rainbow trout. The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate derivatives (glutamate and glutamine), caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease may be dependent upon the availability of host-supplied