Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms

Abstract

The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

DksA and (p)ppGpp Have Unique and Overlapping Contributions to Haemophilus ducreyi Pathogenesis in Humans

“…Since both the dksA and (p)ppGpp°mutants were partially attenuated in humans, we determined the effects of their deficiencies on the expression of genes required for human infection. (p)ppGpp deficiency in strain 35000HP resulted in decreased expression of genes in the flp-tad operon, lspB-lspA2 operon, and the lspA1, hgbA, and csrA genes, which are all required for pustule formation in humans (Table 3) (26,40,45). Loss of (p)ppGpp increased the expression of fgbA, which is required for virulence (26).…”

Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers

“…All 18 genes or operons known to be required for the full virulence of H. ducreyi in humans were expressed in vivo (13)(14)(15)(16)(17)46). However, H. ducreyi harvested from pustules only had relatively increased expression of hgbA and genes belonging to the flp-tad and lspB-lspA2 operons (Table 3).…”

“…Biopsy specimens of endpoint pustules infected with 35000HP were obtained from four healthy adult volunteers who participated in csrA and relA spoT mutant-parent trials (15,16). Demographics of the volunteers, inoculation doses, and duration of infection are described in Table 1.…”

“…The stringent-response mediators (p)ppGpp and DksA are partially required for pustule formation in humans and have more profound effects on gene expression in stationary-phase cells than in mid-log-phase cells (14,15). Similarly, the RNA-binding protein CsrA is partially required for virulence in humans and has a more profound effect on resistance to oxidative stress and macrophage killing in stationary-phase cells than in mid-log cells (16).…”

“…Of those systems, the H. ducreyi genome contains homologs of the envelope stress response regulators CpxRA and RpoE, the stringent-response regulators (p)ppGpp and DksA, and the general posttranscriptional regulators Hfq and CsrA (11)(12)(13)(14)(15)(16). Although CpxR is dispensable for human infection, the activation of CpxR by the deletion of cpxA renders H. ducreyi avirulent in humans (17,18).…”