H-NS Is a Negative Regulator of the Two Hemolysin/Cytotoxin Gene Clusters in Vibrio anguillarum

Abstract

Hemolysins produced by Vibrio anguillarum have been implicated in the development of hemorrhagic septicemia during vibriosis, a fatal fish disease. Previously, two hemolysin gene clusters responsible for the hemolysis and cytotoxicity of V. anguillarum were identified: the vah1-plp gene cluster and the rtxACHBDE gene cluster. In this study, 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 nearly wildtype levels. Furthermore, expression of rtxA, rtxH, rtxB, vah1, and plp increased in the hns mutant and decreased in the hnscomplemented mutant strain compared to expression in the wild-type strain. Additionally, experiments using DNase I showed that purified recombinant H-NS protected multiple sites in the promoter regions 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 upregulation 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. Vibrio anguillarum is the causative agent of vibriosis, a fatal hemorrhagic septicemic disease. V. anguillarum infects more than 50 fresh-and saltwater fish species, including various species of economic importance to the larviculture and aquaculture industries, such as salmon, rainbow trout, turbot, sea bass, sea bream, cod, eel, and ayu (1). Infections by this bacterium have mortality rates of 30% to 100%, resulting in severe economic losses to aquaculture worldwide (2).The ability of V. anguillarum to infect and cause disease in fish is dependent upon several virulence factors and their proper regulation (3). One of these virulence factors is hemolytic activity. In V. anguillarum M93Sm, there are two known gene clusters that encode at least three hemolysins (4, 5). Rock and Nelson (4) reported that the vah1-plp hemolysin gene cluster (Fig. 1A) contains at least two genes, vah1 and plp, that affect hemolytic activity. Vah1 (encoded by vah1) is a putative pore-forming hemolysin causing vacuolization of target cells that has strong amino acid sequence identity to Vibrio cholerae El Tor hemolysin (hlyA) and V. fluvialis hemolysin (5). Mutations in the divergently transcribed plp gene result in both increased expression of vah1 and increased hemolysis of sheep's blood, suggesting that Plp (encoded by plp) is a putative repressor of vah1 transcription (4). Restoration of plp by complementation restores the wild-...

The Transcriptional Regulator HlyU Positively Regulates Expression of exsA , Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus

“…3) and therefore strongly implied a role in exsA regulation. In silico analysis of Vp0529 using NCBI BLAST indicated sequence similarity to HlyU, a DNA binding protein of many Vibrio species that acts as a positive regulator of virulence genes (27)(28)(29)(30)(31). Vp1133 encodes a histone-like nucleoid-structuring protein (H-NS) and has previously been implicated in exsA repression (32).…”

A comprehensive review of Vibrio (Listonella) anguillarum: ecology, pathology and prevention

“…The gene plp negatively regulates haemolytic expression of llp A and vah 1 (Rock & Nelson ) while the gene hly U – which is dependent upon its own positive regulation by the gene hns (Mou et al . ) – is responsible for the positive regulation of both vah 1 and rtx (Li et al . ); genetic regulation of the vah 2‐5 genes are unclear at the present time.…”

“…Vibrio anguillarum contains several virulence gene clusters that encode for haemolysin secretion including llpA, vah1 (Hirono et al 1996), vah2-5 and rtx (Rodkhum et al 2006;Li et al 2008). The gene plp negatively regulates haemolytic expression of llpA and vah1 (Rock & Nelson 2006) while the gene hlyUwhich is dependent upon its own positive regulation by the gene hns (Mou et al 2013)is responsible for the positive regulation of both vah1 and rtx ; genetic regulation of the vah2-5 genes are unclear at the present time. Negative regulation by plp is only partial to its role in V. anguillarum haemolysis as the gene additionally encodes for a specific phospholipase secretion which degrades phosphatidylcholine, a class of phospholipids that comprise approximately 58% of fish erythrocytes (Lee et al 1989;Li et al 2013a,b).…”

H-NS: an overarching regulator of the Vibrio cholerae life cycle

“…Transcriptional silencing of hlyA by H-NS was also reported in V. vulnificus [47] and V. anguillarum [48]. Expression of hlyA in V. cholerae is regulated by iron [49] and HlyU, a member of the SmtB/ArsR family of metalloregulators [50].…”