Characterization of the CpxRA regulon in Haemophilus ducreyi

Abstract

The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

FIG. 1.

Characterization of the H. ducreyi cpxA deletion mutant. (A) Schematic of the cpxRA locus in the wild-type strain 35000HP, the 35000HPΔcpxR deletion mutant (35), and the 35000HPΔcpxA deletion mutant. (B) Growth of the wild-type parent strain 35000HP (closed circles) and the cpxA deletion mutant (open circles) in broth. Results from a representative experiment are shown. (C) Autoagglutination rates for the wild-type parent strain 35000HP (closed circles) and the cpxA deletion mutant (open circles). (D) Photograph of suspensions of the wild-type parent strain 35000HP (1) and the cpxA deletion mutant (2) after 3 h (at the end of the autoagglutination assay for which results are presented in panel C). (E) Total cell protein profiles for the wild-type parent strain 35000HP (lane 1) and the cpxA deletion mutant (lane 2) as determined by resolving proteins by SDS-PAGE and staining with Coomassie blue. The white arrow indicates the OmpP2B protein, which is overexpressed in the mutant, and the black arrow indicates the DsrA protein, which is present in the wild-type strain and missing in the mutant.

FIG. 2.

Deletion of cpxA or cpxR results in dysregulation of expression of several H. ducreyi gene products involved in virulence. H. ducreyi strains grown for 8 h in CB were used for preparation of whole-cell lysates or for RNA extraction. (A) Western blot analysis of whole-cell lysates of 35000HP (lane 1), 35000HPΔcpxR (lane 2), 35000HPΔcpxR(pML125) (lane 3), 35000HPΔcpxR(pLS88) (lane 4), and 35000HPΔcpxR(pML154) (lane 5); primary antibodies are indicated to the right of each panel. The PAL monoclonal antibody 3B9 (56) was used as a loading control for both panels A and C. It should be noted here that both LspA1 and LspA2 typically appear diffuse or exhibit a multiple banding pattern in Western blot analysis (35, 61). Black arrows on the left in panels A and C indicate the position of the relevant antigen. (B) Real-time RT-PCR showing transcript levels for dsrA, flp1, lspA1, lspA2, and lspB in 35000HPΔcpxR relative to 35000HP. (C) Western blot analysis of 35000HP (lane 1), 35000HPΔcpxA (lane 2), 35000HPΔcpxA(pML141) (lane 3), 35000HPΔcpxA(pLS88) (lane 4), and 35000HPΔcpxA(pML153) (lane 5); primary antibodies are indicated to the right of each panel. (D) Real-time RT-PCR showing transcript levels for dsrA, flp1, lspA1, lspA2, and lspB in 35000HPΔcpxA relative to 35000HP.

FIG. 3.

Real-time RT-PCR analysis of selected gene expression in mutant and recombinant strains of H. ducreyi. (A) Genes in 35000HPΔcpxA exhibiting upregulation, downregulation, or no change relative to those in 35000HP in a DNA microarray analysis were analyzed by real-time RT-PCR. (B) Genes in 35000HPΔcpxR(pML125) exhibiting upregulation, downregulation, or no change relative to those in 35000HPΔcpxR(pLS88) in a DNA microarray analysis were analyzed by real-time RT-PCR. These data are the means of results from three independent experiments.

FIG. 4.

CpxR interacts with the promoter regions of genes encoding known and putative H. ducreyi virulence factors but not with the promoter regions of homologs of E. coli cell envelope stress response genes. (A) Nucleotide sequences of the promoter regions for the H. ducreyi lspB, ompA2, ompP2A, flp1, dsrA, and fimA genes. Putative consensus CpxR binding sequences are shown in bold and underlined. Numbers in parentheses indicate the position of the first base of the first repeat (left) and the last base of the last repeat (right) relative to the predicted translation initiation codon. (B and D) Results from EMSAs using 50 pmol of purified rCpxR-His together with the DIG-labeled promoter region DNA. Nucleotide sequences of the primers used to PCR amplify the promoter regions are listed in Table 2. (B) Results from EMSAs using the promoter regions of gyrB, lspA1, lspB, ompA2, ompP2A, flp1, dsrA, and fimA. The lspB, lspA1, and gyrB promoter regions were used as positive (i.e., lspB) or negative (i.e., lspA1 and gyrB) controls in every assay. The minus sign indicates that no CpxR protein was present, whereas the plus sign indicates the presence of CpxR protein. (C) Real-time RT-PCR analysis of gene expression from lspA1, lspB, dsrA, flp1, degP, dsbA, ppiD, psd, and secA in H. ducreyi ΔcpxA relative to wild-type H. ducreyi. (D) Results from EMSAs using the promoter regions of the following H. ducreyi genes: degP, dsbA, ppiD, psd, and secA.