Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence

Abstract

Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40 degrees C compared to cells grown at 30 degrees C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter P(hupA), and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection.

FIG. 1.

Dependency of the V. vulnificus hupA expression on temperatures and iron levels. (A and B) Wild-type V. vulnificus was grown in iron-replete medium (LBS) at different temperatures as indicated, and then samples removed at an optical density at 600 nm of 0.6 were analyzed for protein profiles (A) and the hupA transcript (B). (A) A protein spot whose abundance increased in the V. vulnificus cells grown at 40°C is indicated with arrows and was subjected to matrix-assisted laser desorption ionization-time of flight MS analysis. (B) Total RNAs isolated as described in Materials and Methods were separated (bottom) and then hybridized to HUPAP, a ³²P DNA probe (top). The relative levels of the hupA transcripts are presented relative to the level of the hupA in cells grown at 40°C. The two bands represent rRNAs (bottom), and the molecular size markers (Invitrogen) and the hupA transcripts are shown in kilobases. (C) Total RNAs were isolated from V. vulnificus grown either in iron-replete medium (+) or in medium low in iron (−) transferred to nylon membrane (Roche, IN). The relative levels of the hupA transcripts are presented relative to the level of hupA in cells grown with medium low in iron at 40°C. The hupA transcripts in the RNAs were determined by Northern slot blot analyses using the HUPAP as a ³²P DNA probe.

FIG. 2.

Effect of CRP on the cellular level of the hupA transcript and P_hupA activity. Cultures of the wild type and crp mutant KC74 were grown at 40°C with either iron-replete medium or medium low in iron, and total RNAs were isolated as described in Materials and Methods. (A) The hupA transcripts in the RNAs were determined by Northern slot blot analyses using the HUPAP as a ³²P DNA probe. +, iron-replete medium; −, medium low in iron. The relative levels of the hupA transcripts are presented relative to the level of the hupA in the wild type grown with medium low in iron. (B) P_hupA activities were determined by primer extension of the RNA derived from each strain grown with medium low in iron. Lanes G, A, T, and C represent the nucleotide sequencing ladders of pSM0610. The relative levels of the P_hupA activity are presented relative to the level of the P_hupA activity in the wild type. The asterisk indicates the transcription start site. WT, wild type; crp, crp mutant.

FIG. 3.

Gel mobility shift assay results for binding of CRP to the hupA regulatory region. A 350-bp DNA fragment of the upstream region of P_hupA was radioactively labeled and then used as a probe DNA. (A) The radiolabeled fragments (7 nM) were mixed with increasing amounts of CRP as indicated: 0, 10, 20, 40, and 80 nM of CRP in lanes 1 to 5, respectively. (B) For competition analysis, the same, but unlabeled, DNA fragment was used as a competitor. Various amounts of the competitor DNA were added to a reaction mixture containing 7 nM labeled DNA prior to the addition of CRP. Lanes 1 to 5, probe DNA incubated with 80 nM of CRP and 0, 7, 14, 28, and 35 nM of the competitor DNA, respectively, as indicated. B, bound DNA; F, free DNA.

FIG. 4.

DNase I protection analysis for identification of CRP binding site and sequence analysis of the hupA upstream region. (A) The ³²P-labeled 350-bp hupA regulatory region was incubated with increasing amounts of CRP and then digested with DNase I. Lane 1, no CRP added; lanes 2 to 6, CRP at 50, 100, 150, 200, and 250 nM, respectively. Lanes G, A, T, and C represent the nucleotide sequencing ladders of pOH0801. The hypersensitivity and protection in the presence of CRP are indicated by thick lines and open boxes, respectively. (B) The transcription start site is indicated by a bent arrow (P_hupA). The sequences proposed for the binding sites of CRP are shown in a shaded box. The conserved nucleotide sequences for the binding of CRP and Fur are indicated above the V. vulnificus DNA sequence in uppercase letters. The positions of the putative −10 and −35 regions are underlined with dotted lines for the promoter P_hupA. The ATG translation initiation codon and putative ribosome-binding site (SD) are indicated in bold. ORF, open reading frame.

FIG. 5.

Cultures of the wild-type (WT), hupA mutant SM02, and complemented strains were grown on iron-depleted medium with 10 μM hemin at 40°C. OD₆₀₀, optical density at 600 nm.

FIG. 6.

Effect of hupA mutation on virulence of V. vulnificus toward INT-407 cells. INT-407 cells were infected with the wild-type (WT), hupA mutant SM02, or complemented strains at various MOIs for 1.5 h (A) or at an MOI of 30 for various incubation times (B). The cell cytotoxicity was determined by an LDH release assay. Data are means ± SEM from three independent experiments. *, P < 0.01; **, P < 0.1, relative to groups infected with the wild-type V. vulnificus at each MOI or each incubation time.

FIG. 7.

Expression of rtxA and growth rates of the wild type (WT) and hupA mutant SM02 during infection. (A) The relative levels of rtxA expression in the wild type and SM02 were determined by quantitative real-time PCR analysis. Details for preparation of total cellular RNA and real-time PCR are given in Materials and Methods, and the expression levels of rtxA were normalized to the 16S rRNA expression level. (B) Growth of the strains during the infection of INT-407 cells was monitored. The strains were used to infect the INT-407 cells at an MOI of 30, and then bacterial cells in the supernatant were determined by counting CFU on LBS agar plates at time intervals as indicated. Data are means ± SEM from three independent experiments.