A Haemophilus ducreyi CpxR deletion mutant is virulent in human volunteers

Abstract

Haemophilus ducreyi 35000HP contains a homolog of the CpxRA 2-component signal transduction system, which controls the cell envelope stress response system in other gram-negative bacteria and regulates some important H. ducreyi virulence factors. A H. ducreyi cpxR mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule formation rate in 5 volunteers was 33% (95% confidence interval [CI], 1.3%-65.3%) at 15 parent sites and 40% (95% CI, 18.1%-61.9%) at 15 mutant sites (P = .35). Thus, the cpxR mutant was not attenuated for virulence. Inactivation of the H. ducreyi cpxR gene did not reduce the ability of this mutant to express certain proven virulence factors, including the DsrA serum resistance protein and the LspA2 protein, which inhibits phagocytosis. These results expand our understanding of the involvement of the CpxRA system in regulating virulence expression in H. ducreyi.

Figure 1.

Protein expression and inhibition of phagocytosis by wild-type and mutant strains of Haemophilus ducreyi. A, Western blot analysis of protein expression by wild-type 35000HP (lane 1), the 35000HPΩ12 mutant (lane 2), the 35000HPΔcpxR mutant (lane 3), and the 35000HP cpxA::cm mutant (lane 4). The specific antigen reactive with each primary antibody is listed to the right of each panel. B, Ability of these 4 H. ducreyi strains to inhibit the phagocytic activity of murine J774A.1 macrophages, as measured by the uptake of immunoglobulin G–opsonized latex beads. A representative experiment is shown. The multiplicity of infection (MOI) used for each strain is listed at the top of each column.

Figure 2.

Expression of DsrA and serum resistance by wild-type and mutant strains of Haemophilus ducreyi. A, Representative Western blot analysis of whole cell lysates of 35000HP (lane 1), 35000HPΔcpxA (lane 2), 35000HPΔcpxR (lane 3), and the dsrA mutant FX517 (lane 4) probed with both polyclonal DsrA antiserum and the PAL-specific monoclonal antibody 3B9. The positions of the DsrA and PAL proteins are indicated. B, Serum bactericidal activity assays. The percentage survival of 35000HP, 35000HPΔcpxA, 35000HPΔcpxR, and FX517 in 50% normal human serum (NHS), calculated as (geometric mean colony-forming units [CFUs] in NHS/geometric mean CFUs in heat-inactivated NHS) × 100. Values represent means ± standard deviations of 5 independent experiments. After adjustment for multiple comparisons, differences in these assays were considered significant at P ≤ .012.