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

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 nutrients for growth. In manuscript II “Characterization of the growth and virulence of a Vibrio anguillarum citrate synthase mutant”, the role of glutamate auxotrophy during V. anguillarum M93Sm infection was further characterized. A citrate synthase (gltA) deletion mutant was created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout. The ΔgltA mutant exhibited a decreased final cell density when grown in LB20 that resulted from the exhaustion of glutamate from the media. There was no significant decrease in the expression of the three hemolysin genes by the ΔgltA mutant when detected by qRT-PCR or mortality during challenge experiments. A ΔgltA mutant capable of growing in minimal media was isolated and shown to have a spontaneous mutation in the transcriptional activator of 2-methylcitrate synthase (prpR). This mutation resulted in an increase in expression of 2-methylcitrate synthase (prpC). The ΔgltA prpR(R66L) mutant was characterized with regard to growth in complex media and exhibited a growth advantage compared to the ΔgltA mutant after 24 h in spleen extract medium. Further, after growing 120 h in spleen extract medium, colonies of ΔgltA mutants were shown to be capable of growing in minimal media. ΔprpC and a ΔgltA ΔprpC mutants were created and characterized with regard to growth in minimal and complex media and virulence in juvenile rainbow trout. The ΔgltA ΔprpC mutant had no growth advantage in spleen extract medium compared to the ΔgltA but was still as virulent as the wild type against rainbow trout. As expected, the ΔprpC mutant was similar to the wild type in regards to both growth in minimal and complex media and virulence against rainbow trout. The data strongly suggests that simple starvation for αketoglutarate derivatives (glutamate and glutamine) is not directly linked to attenuation of virulence as previously proposed. Additionally, spontaneous mutations can occur that compensate for the original gene deletion if the new mutation can replace or bypass the lost metabolic reaction and results in a growth advantage. In manuscript III “Characterization of Vibrio anguillarum NB10Sm TCA cycle mutants” the role of central metabolism in virulence was examine in the O1 serotype strain of V. anguillarum NB10Sm. A V. anguillarum NB10Sm icd mutant was created, characterized for growth in complex media and demonstrated to be as virulent as the wild type in juvenile rainbow trout. Several additional central metabolism single and double mutants were created in the following genes cra, gltA, Δicd gltA, sucA, sucC, sdhC, ΔfrdA, ΔfrdA sdhC, and fumA and characterized with regard to growth in complex media. Two mutants (ΔsucA and ΔfrdA ΔsdhC) that demonstrated a significantly reduced growth yield compared to the wild type were further characterized with regard to their growth in several forms of complex media, expression of virulence genes, and virulence in juvenile rainbow trout. The data strongly suggest that there is no correlation between a lower growth yield in vitro and a decrease in virulence in vivo. Even though M93Sm and NB10Sm are same species, mutations made in the same TCA cycle genes can cause drastically difference results in regards to growth and virulence.