The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus

Abstract

Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.

FIG. 1.

The VV20481, VV20480, and rtxA1 genes are transcribed as an operon. (A) Small arrows represent the location of primers. Primer P1 was for the RT reaction using RNA extracted from V. vulnificus CMCP6 cells. The generated cDNA was used as a template in PCR using primer P2 or P3 combined with primer P1. (B) RT-PCR results using different primer combinations. The primers used were the following: lanes 1 and 2, P1 and P2; lanes 3 and 4, P1 and P3. The RT enzyme was omitted in the reactions of lanes 2 and 4. MW, DNA molecular weight marker.

FIG. 2.

Cytotoxicity of mutants in hlyU and other genes regulated by HlyU. Bacteria were incubated with HeLa cells at a multiplicity of infection of 10 for 90 min at 37°C with 5% CO₂; then the released lactate dehydrogenase in the supernatant was measured by a CytoTox96 Non-Radioactive Cytotoxicity Assay kit. Error bars represent the means ± standard deviations of percent cytotoxicity from triplicate experiments. WT, V. vulnificus CMCP6; MQ1/pMMB-HLYU, ΔhlyU mutant with complementing plasmid pMMB-HLYU; MQ1/pMMB208, ΔhlyU mutant with empty vector pMMB208; MQ2-1, rtxA1 mutant with insertion of the suicide plasmid pDM4 in the rtxA1 gene; MQ2-2, wild-type strain regenerated by eliminating pDM4 from the rtxA1 gene.

FIG. 3.

Gel mobility shift assay for the binding of the HlyU protein to the VV20481-VV20480-rtxA1 promoter region. (A) Location of the DNA probes (a, b, c, and d). (B) The labeled DNA probe d (4 nM) was mixed with increasing amounts of the HlyU protein. Lanes 1 to 6 contain, respectively, 0, 10, 50, 90, 150, 300 nM HlyU. (C) For competition analysis, the unlabeled specific DNA was used as a competitor. Before the addition of 150 nM HlyU, increasing amounts of unlabeled competitor DNAs were added to the reaction containing a 4 nM concentration of the labeled DNA probe. Lanes 1 to 4 contain, respectively, 1, 10, 50, and 300 nM unlabeled competitor DNAs.