Identification and regulation of hemolysin genes of Vibrio anguillarum

Author

Ling Li, University of Rhode Island

Date of Award

2009

Degree Type

Dissertation

First Advisor

David R. Nelson

Abstract

Vibrio anguillarum is a causative agent of vibriosis. One hemolysin gene, vah1, does not account for all hemolytic activity. Further studies identified a putative rtx operon with six genes (rtxACHBDE). Mutation in both vah1 and any of rtx genes resulted in a hemolysin negative mutant. Vah1 and RtxA each have cytotoxie activity against fish cells. Finally, virulence examination in fish suggested that RtxA is a major virulence factor for V. anguillarum. Additionally, RtxA is a MARTX toxin, and causes fish cell rounding and death. A conserved domain in the RtxA was identified to be a cysteine protease (CPD). CPD was activated by cell lysate, but not by GTP. The cleavage site for CPD was determined and suggested that CPD processed the full RtxA into the 120 kDa carboxyl portion of RtxA Va, which released into bacterial culture supernatant after cleavage. Further study revealed that a cell rounding domain (CRD) between the CPD and the C-repeat regions resulted in the loss of both hemolytic activity and ASK cell rounding demonstrated that CRD is required for the functions of the RtxA Va toxin. The plp gene is one component in the vah1 cluster, and considered as a repressor of Vah1. Plp had a distinct effect to the different erythrocytes. The recombinant Plp protein (rP1p) was purified and re-folded. The active rPlp showed a relative heat stable phospholipase A2 activity. Fatherly, we demonstrated that Plp is a secretory protein, and specifically lyse the fish erythrocytes due to its specific activity on phosphotidylcholine, but no activity on other phospholipids. Here, we also identify a positive hemolysin regulator gene, the hlyU gene, from V. anguillarum. Mutation in hlyU resulted in a decrease of hemolytic activity, but no effect on cytotoxicity. RT-PCR data revealed that the expression of rtx genes was deceased in the hlyU mutant. Interestingly, the expression of vah1 and plp was not effect in the hlyU mutant, but significantly increased in a hlyU complement strain. Finally, we also mapped the transcriptional start sites in the intergenic regions of both vah1/plp and rtxH/rtxB genes, demonstrating that all hemolysin promoters are σ 70 type.

Recommended Citation

Li, Ling, "Identification and regulation of hemolysin genes of Vibrio anguillarum" (2009). Open Access Dissertations. Paper 2339.
https://digitalcommons.uri.edu/oa_diss/2339