The LysR-type transcriptional regulator VirR is required for expression of the virulence gene vapA of Rhodococcus equi ATCC 33701

Abstract

The virulence of the intracellular pathogen Rhodococcus equi in foals is dependent on the presence of an 81-kb virulence plasmid encoding the virulence protein VapA. Expression of this protein is induced by exposure to oxidative stress, high temperatures, and low pHs, which reflect the conditions encountered by R. equi when it enters the host environment. The aim of this study was to determine whether the LysR-type transcriptional regulator VirR, which is encoded by the virulence plasmid, is required for the expression of vapA. It was shown that the virR gene is cotranscribed with four downstream genes, one of which encodes a two-component response regulator. The expression of VapA, as monitored by Western blotting, was completely dependent on the presence of virR. Maximal expression was observed when vapA was present together with the complete virR operon, suggesting that at least one of the virR operon genes, in addition to virR, is required for the expression of vapA to wild-type levels. The transcriptional start site of vapA was determined to be a cytidine located 226 bp upstream from the vapA initiation codon. His-tagged VirR protein was expressed in Escherichia coli and purified by nickel affinity chromatography. DNA binding studies showed that purified VirR binds to a DNA fragment containing the vapA promoter. We therefore conclude that VirR is required for the activation of vapA transcription.

FIG. 1.

Transcriptional organization of virR gene cluster. (A) Genetic organization of DNA region harboring ORF3 to ORF9. The arrow above the map denotes the direction of transcription of the polycistronic message. Arrows below the illustration indicate oligonucleotide primers used for RT-PCR. (B) Results of RT-PCR analyses. Each oligonucleotide pair was used in three amplification reactions, with 2 μl of the reverse transcriptase-containing reaction (cDNA), without reverse transcriptase (−RT), and with R. equi ATCC 33701 genomic DNA (DNA). The oligonucleotide pairs used were 003R and 004R (i), 004F and 005R (ii), 005F and 006R (iii), 006F and 007NR (iv), 007NF and 008R (v), and 008F and 009R (vi). The size of each band is indicated.

FIG. 2.

Determination of vapA transcriptional start site in R. equi ATCC 33701. Fluorescent primer extension was performed with the Cy5-labeled primer CY5VAPA200R and 5 μg of total cellular RNA extracted from R. equi grown under vapA-inducing conditions (37°C, pH 6.5). CY5VAPA200R is complementary to a sequence 131 bp downstream from the vapA initiation codon. (A) Cy5-labeled primer extension product combined with DNA size standards and analyzed with the CEQ 8000 fragment analysis system. (B) Nucleotide sequence obtained by using VAPA200R. A dideoxy sequencing reaction mix was spiked with the Cy5-labeled primer extension product. The arrow indicates the transcriptional start site where the Cy5-labeled cDNA and the sequencing product overlapped. (C) Sequence of vapA promoter region. The transcriptional start site (+1) and putative −10 and −35 regions are boxed, and putative LysR motifs (T-N₁₁-A) are indicated with brackets.

FIG. 3.

Effect of virR and the virR operon on expression of vapA. The expression of VapA was determined by Western blotting with VapA monoclonal antibodies. (A) Lanes 1 and 2, R. equi wild-type strain grown at 37°C and pH 6.5 (lane 1) and at 30°C and pH 8.0 (lane 2); lanes 3 and 4, R. equi (P⁻) harboring pRvip10 (virR vapA) grown at 37°C and pH 6.5 (lane 3) and at 30°C and pH 8.0 (lane 4); lane 5, R. equi (P⁻) grown at 37°C and pH 6.5. Each lane was loaded with 2 μg of protein. (B) Lanes 1 and 2, R. equi wild-type strain grown at 37°C and pH 6.5 (lane 1) and at 30°C and pH 8.0 (lane 2); lanes 4 and 5, R. equi (P⁻) harboring pForlan21 (virR operon and vapA) grown at 37°C and pH 6.5 (lane 4) and at 30°C and pH 8.0 (lane 5); lane 3, R. equi (P⁻) grown at 37°C and pH 6.5. Each lane was loaded with 2 μg of protein.

FIG. 4.

Coomassie brilliant blue-stained denaturing polyacrylamide gel showing cell extracts of E. coli BL21 harboring pET3b (lane 1) or pET3bvirRhis (lane 2). VirR-His was purified by Ni²⁺ affinity chromatography (lane 3). The sizes of the molecular mass standards are shown in kilodaltons (lane 4).

FIG. 5.

EMSA analysis of VirR binding to the vapA promoter region. Various concentrations of VirR were incubated with 2 ng of radiolabeled DNA (262 bp) containing the vapA promoter region. The amount of protein added to each lane was as follows: lane 1, radiolabeled DNA fragment only; lane 2, 50 ng; lane 3, 100 ng; lane 4, 200 ng; lane 5, 300 ng; and lane 6, 400 ng. Protein-DNA complexes are indicated with black arrows. Nonbound DNA is indicated with a gray arrow.