Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology, Molecular Biology and Biochemistry

First Advisor

David R. Nelson

Abstract

Vibrio anguillarum is the causative agent of vibriosis, a fatal hemorrhagic septicemic disease. V. anguillarum infects more than 50 fresh and salt-water fish species including various species of economic importance to the larviculture and aquaculture industry. In vibriosis, V. anguillarum invades its host fish through the intestine and skin. Infected fish usually die with systemic infection of V. anguillarum. Bacterial hemolysins are exotoxins that cause lysis of erythrocytes in the host and thus the release of the intracellular heme, and are therefore identified as important virulence factors. Moreover, hemolysins are known to also cause lysis in other cell types, including mast cells, neutrophils and polymorphonuclear cells. Two hemolysin gene clusters, vah1-plp and rtxACHBDE, have been previously identified and described. The activities of the protein encoded by the plp gene were not known.

In the first manuscript, we describe the biochemical activities of the plp-encoded protein and its role in pathogenesis. The plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), which has homology to lipolytic enzymes containing the catalytic site amino acid signature SGNH. Hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that Plp has different effects against erythrocytes from different species. His6-tagged recombinant Plp protein (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5–9 and at temperatures of 30-64°C. Divalent cations and metal chelators did not affect activity of rPlp. We also demonstrated that Plp was secreted using thin layer chromatography and immunoblot analysis. Additionally, rPlp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rPlp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. Further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxA and/or vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.

In the next manuscript, we used degenerate PCR to identify a positive hemolysin regulatory gene, hlyU, from the unsequenced V. anguillarum genome. The hlyU gene of V. anguillarum encodes a 92-amino acid protein and is highly homologous to other bacterial HlyU proteins. An hlyU mutant was constructed, which exhibited ~5-fold decrease in hemolytic activity on sheep blood agar with no statistically significant decrease in cytotoxicity of the wild type strain. Complementation of the hlyU mutation restored both hemolytic and cytotoxic activity. Both semi-quantitative RT-PCR and real time RT-qPCR were used to examine expression of the hemolysin genes under exponential and stationary phase conditions in wild type, hlyU mutant, and hlyU complemented strains. Compared to the wild type strain, expression of rtx genes deceased in the hlyU mutant while expression of vah1 and plp was not affected in the hlyU mutant. Complementation of the hlyU mutation restored expression of the rtx genes and increased vah1 and plp expression to levels higher than in the wild type. The transcriptional start sites in the intergenic regions of both vah1/plp and rtxH/rtxB genes were determined using 5’-RACE and the binding sites for purified HlyU was discovered using DNA gel mobility shift experiments and DNase protection assays.

In the third manuscript, we identified the hns gene, which encodes the H-NS protein, and acts as a negative regulator of both gene clusters. The V. anguillarum H-NS protein shares strong homology with other bacterial H-NS proteins. An hns mutant exhibited increased hemolytic activity and cytotoxicity compared to the wild type strain. Complementation of the hns mutation restored hemolytic activity and cytotoxicity levels to near wild type levels. Further, expression of rtxA, rtxH, rtxB, vah1 and plp increased in the hns mutant, and decreased in the complemented hns mutant strain when compared to the wild type strain. Additionally, experiments using DNase I, showed that purified recombinant H-NS protected multiple sites in the promoter region of both gene clusters. The hns mutant also exhibited significantly attenuated virulence against rainbow trout. Complementation of the hns mutation restored virulence to wild type levels, suggesting that H-NS regulates many genes that affect fitness and virulence. Previously, we showed that HlyU is a positive regulator of expression for both gene clusters. In this study, we demonstrate that up-regulation by hlyU is hns-dependent, suggesting that H-NS acts to repress or silence both gene clusters, and HlyU acts to relieve that repression or silencing.

In the last manuscript, we aimed to create avirulent and immunogenic V. anguillarum strains that can be used as a live vaccine for fish, without knocking out any of the hemolysin genes. For this purpose, six genes (mdh, icd, sucA, sucC, sdhC, and fumA) encoding enzymes in tricarboxylic acid (TCA) cycle and one gene (cra) encoding a fructose metabolism repressor were identified and mutated in wild type V. anguillarum M93Sm (serotype O2a). Among all mutants, icd mutant showed high attenuation of virulence and lowest cell density limit in two forms of rich media. All mutants exhibited the same or higher levels of hemolysin gene expression compared to wild type during log phase. Further, fish that were pre-treated by immersion with icd mutant protected rainbow trout from the subsequent challenge of V. anguillarum M93Sm; and fish that were pre-treated with injection of the icd mutant elicited cross-serotype immunity against the subsequent challenge of V. anguillarum NB10 (serotype O1). The results suggest the TCA cycle mutation approach is likely to be an easy method to construct modified live vaccine for a wide variety of pathogenic bacteria.

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