Activation of the CpxRA System by Deletion of cpxA Impairs the Ability of Haemophilus ducreyi To Infect Humans

Abstract

Haemophilus ducreyi must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the H. ducreyi genome and negatively regulates the lspB-lspA2 operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame H. ducreyi cpxA deletion mutant, 35000HP⌬cpxA. In human inoculation experiments, 35000HP⌬cpxA formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the lspB-lspA2 promoter was downregulated in 35000HP⌬cpxA, confirming that CpxR was activated. Deletion of cpxA downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in trans restored parental phenotypes. 35000HP⌬cpxA is the first H. ducreyi mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.

Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

“…3). In Haemophilus ducreyi, it regulates proteins involved in resistance to phagocytosis, affecting the ability of H. ducreyi to infect humans (4).…”

Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions

“…, where E is the amplification efficiency (equal to 10 Ϫ1/slope ) and ⌬C T is the change in cycle threshold (10). For induction of FLAG-tagged RpoE expression, 35000HP(pDG11) and 35000HP(pDG10) were grown to mid-log phase (OD 660 ϭ 0.2) and then treated with 200 ng/ml of ATc, harvested, and analyzed as described above.…”

“…The reporter constructs were then electroporated into 35000HP(pDG17). As a control, a reporter containing the putative lspB promoter region was also electroporated into 35000HP-(pDG17) (10). Whole-cell lysates were prepared from each transformant harvested at 0, 30, 60, and 120 min after RpoE induction and analyzed by Western blotting using monoclonal antibodies specific to GFP (Clontech) and the peptidoglycan-associated protein (PAL) (10).…”

“…As a control, a reporter containing the putative lspB promoter region was also electroporated into 35000HP-(pDG17) (10). Whole-cell lysates were prepared from each transformant harvested at 0, 30, 60, and 120 min after RpoE induction and analyzed by Western blotting using monoclonal antibodies specific to GFP (Clontech) and the peptidoglycan-associated protein (PAL) (10). For each strain, the level of expression of GFP protein normalized to PAL was determined by densitometry using ImageJ software (35).…”

“…The H. ducreyi genome encodes homologues of CpxRA, which is the only obvious intact TCS encoded in the genome. Activation of CpxR by deletion of cpxA downregulates the majority of its targets, including multiple virulence determinants, and attenuates the virulence of H. ducreyi in humans (10)(11)(12). However, deletion of cpxR does not affect the expression of virulence determinants or reduce the virulence of the organism in humans, suggesting that CpxRA is dispensable for H. ducreyi infection (13).…”