Regulation of expression of the Haemophilus ducreyi LspB and LspA2 proteins by CpxR

Abstract

The LspA1, LspA2, and LspB proteins of Haemophilus ducreyi comprise a two-partner secretion system that has been shown to be necessary for H. ducreyi to inhibit phagocytosis by immune cells in vitro. Inactivation of lspA1 resulted in increased levels of LspA2, suggesting that these two proteins are differentially controlled (C. J. Ward et al., Infect. Immun. 71:2478-2486, 2003). Expression of LspA2 but not LspA1 was shown to be both growth phase dependent and affected by the presence of fetal calf serum (FCS) in the growth medium. In addition, neither LspA1 nor LspA2 could be detected in culture supernatant fluid in the absence of FCS. DNA microarray analysis revealed that 324 H. ducreyi genes were differentially regulated after growth in the presence of FCS. Among these, the CpxRA two-component sensory transduction system was downregulated by the presence of FCS. Inactivation of cpxR resulted in increased expression of both LspB and LspA2. Electrophoretic mobility shift assays showed that a recombinant H. ducreyi CpxR protein bound the promoter region of the lspB-lspA2 operon. The cpxR and cpxA genes were shown to be part of an operon containing two additional genes in H. ducreyi 35000HP. This is the first description of a two-component sensory transduction system regulating a proven virulence factor of H. ducreyi.

FIG. 1.

Expression of LspA1 and LspA2 during growth of H. ducreyi in broth. (A) Growth of wild-type H. ducreyi 35000HP in CB. (B) Western blot-based detection of LspA1 and LspA2 proteins in whole-cell lysates using the LspA1-specific MAb 40A4 (top panel), the LspA2-specific MAb 1H9 (middle panel), and the PAL-specific MAb 3B9 (bottom panel). This latter antigen was used as a loading control. Cells were sampled every 2 h, beginning with the 2-h time point. Molecular mass position markers (in kilodaltons) are present on the left sides of these three panels.

FIG. 2.

Effect of FCS on growth and LspA protein expression. (A) Wild-type H. ducreyi 35000HP cells were grown in CB with FCS (•) and without FCS (○). (B) Western blot-based detection of LspA1 and LspA2 proteins in whole-cell lysates (WCL) and CCS from H. ducreyi 35000HP cells grown in CB with FCS (panels 1, 3, 5, and 7) or without FCS (panels 2, 4, 6, and 8) using the LspA1-specific MAb 40A4 (panels 1 to 4) and the LspA2-specific MAb 1H9 (panels 5 to 8). Molecular mass position markers (in kilodaltons) are present on the left sides of these eight panels. It should be noted that both LspA1 and LspA2 frequently appeared smeared or as multiple bands in Western blot analysis (63). (C and D) Real-time RT-PCR measurement of relative expression levels of lspA1 (▪) and lspA2 (░⃞) in wild-type H. ducreyi 35000HP cells grown in CB−FCS compared to CB+FCS in two separate experiments. Expression of gyrB was used to normalize the amount of cDNA per sample. These two experiments were performed independent of those used to measure protein expression. The bracket bars denote the highest and lowest values.

FIG. 3.

Real-time RT-PCR analysis of the relative levels of expression of selected H. ducreyi genes in the presence of FCS. The data indicate relative expression levels in H. ducreyi 35000HP cells grown in the presence of FCS compared to cells grown in the absence of FCS.

FIG. 4.

Characterization of the H. ducreyi 35000HP cpxR deletion mutant. (A) Southern blot analysis of EcoRV-digested chromosomal DNA from 35000HP (lanes 1 and 3) and the 35000HPΔcpxR mutant (lanes 2 and 4) probed with a cat gene fragment (lanes 1 and 2) and with a cpxR gene fragment (lanes 3 and 4). Size markers (in kb) are present on the left side of this panel. (B) Western blot analysis of whole-cell lysates from 35000HP (lane 1) and the 35000HPΔcpxR mutant (lane 2) probed with polyclonal antibody to the H. ducreyi CpxR protein. The arrow indicates the position of the CpxR protein. Molecular mass position markers (in kilodaltons) are present on the left side of this panel.

FIG. 5.

Protein expression by wild-type, mutant, and complemented mutant strains of H. ducreyi. Whole-cell lysates of 35000HP (lane 1), 35000HPΔcpxR (lane 2), 35000HPΔcpxR(pML125) (lane 3), and 35000HPΔcpxR(pLS88) (lane 4) were probed in Western blot analysis with the LspA1-specific MAb 40A4 (A), the LspA2-specific MAb 1H9 (B), polyclonal antiserum against LspB (C), polyclonal antiserum against CpxR (D), and the PAL-specific MAb 3B9 (E). The arrows indicate the relevant antigen in each panel. Molecular mass position markers (in kilodaltons) are present on the left sides of these five panels.

FIG. 6.

Interaction of CpxR with the promoter region of lspB. (A) Sequence logo for the CpxR recognition site based on 24 published E. coli CpxR binding sequences (17, 67). (B) Putative CpxR-binding consensus sequence upstream from the lspB ORF. (C) EMSA using 50 pmol purified His-tagged CpxR, together with the DIG-labeled lspB promoter region DNA (nt −157 to +43; panel 1), the DIG-labeled lspA1 promoter region DNA (nt −179 to +21; panel 2), and the DIG-labeled gyrB promoter region DNA (nt −154 to +47; panel 3).