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.Haemophilus ducreyi is a gram-negative coccobacillus and the causative agent of the sexually transmitted genital ulcer disease (GUD) chancroid (1,8). Globally, chancroid is a significant sexually transmitted disease, with more than 6 million cases reported in 1997 (60). In the United States, several outbreaks were reported between 1980 and 2000 (24, 36, 51), but since then the number of cases has greatly diminished, and today the soft chancres characteristic of H. ducreyi infection occur only in isolated cases that are typically associated with the sex trade industry (57). Chancroid is endemic in some developing countries in Africa, Asia, and South America, where it accounts for almost half of all GUD cases, although these numbers could be higher as H. ducreyi cases remain poorly documented (55,57). GUD is a recognized cofactor for human immunodeficiency virus acquisition and transmission (25,37), and a better understanding of H. ducreyi pathogenesis is necessary to allow a rational approach to the identification of vaccine candidates that could be used to prevent chancroid.H. ducreyi is a strict human pathogen, and it is likely that during the different stages of ulcer production (i.e., progression of a papule into a pustule followed by frank ulceration [1,8,50]), this pathogen controls gene expression to enhance its growth and to evade the host immune system. Information about regulatory networks that might control the expression of H. ducreyi virulence factors is very limited at present. Although nucleotide sequence analysis of the H. ducreyi 35000HP genome (GenBank accession no. NC002940) revealed the presence of several genes encoding predicted proteins with homology to known bacterial regulators...

Deletion of mtrC in Haemophilus ducreyi Increases Sensitivity to Human Antimicrobial Peptides and Activates the CpxRA Regulon

“…5B, double asterisks). 35000HP⌬cpxR, in which the Cpx regulon is constitutively inactive (29), showed no significant difference from 35000HP in survival of LL-37 at concentrations ranging from 0.2 to 20 g/ml (data not shown). Together, these data suggest that, although activation of CpxRA causes increased sensitivity to LL-37, the MTR transporter itself independently contributes to LL-37 resistance.…”

“…We tested this hypothesis by examining mRNA expression levels of selected Cpx-regulated genes, including dsrA, ompP2B, and two additional genes known to be either upregulated (HD0281) or downregulated (HD1155) by activation of CpxRA (28). HD0281 encodes the putative major fimbrial subunit FimA, and HD1155 encodes LspB, which is required for secretion of the antiphagocytic factors LspA1 and LspA2 (29,52). qRT-PCR analysis showed that, relative to 35000HP, the mtrC mutant had increased transcriptional levels of fimA and ompP2B and decreased mRNA levels of lspB and dsrA (Fig.…”

“…For example, the secreted proteins LspA1 and LspA2 are key to resisting phagocytosis, and the outer membrane protein (OMP) DsrA confers resistance to killing by human serum (17,24). Although little is known about how H. ducreyi regulates virulence factors, DsrA and LspA2, along with the secretory protein LspB, are controlled by the CpxRA two-component regulator (29,48). H. ducreyi lacking the sensor kinase component gene cpxA is attenuated in a human model of infection, possibly because of dysregulation of important virulence determinants (48).…”

Characterization of the CpxRA Regulon in Haemophilus ducreyi

“…The earliest documented example of gene regulation in H. ducreyi involved a heme acquisition system (18), and in a later study, mutant analysis indicated that inactivation of the lspA1 gene would cause increased expression of the LspA2 protein (61). The use of DNA microarray analysis and real-time reverse transcriptase (RT) PCR showed that several hundred H. ducreyi genes are differentially regulated in the presence of fetal calf serum (FCS), and subsequent experiments revealed that the H. ducreyi CpxR response regulator is involved in negatively controlling expression of the lspB-lspA2 operon (35). Two-component signal transduction systems (TCSs) typically comprise a membrane-bound sensor (histidine protein) kinase and a cytoplasmic response regulator (for reviews, see references 38, 65, and 69).…”

“…The demonstration of a functional CpxR ortholog in H. ducreyi (35), together with the presence of a closely linked open reading frame (ORF) encoding a predicted at 4°C, after which they were incubated in primary antibody for 3 to 4 h at room temperature or overnight at 4°C. Membranes were then incubated for 1 h at room temperature in a 1:20,000 dilution of either goat anti-mouse IgG-horseradish peroxidase (IgG-HRP) or goat anti-rabbit IgG-HRP (Bio-Rad, Hercules, CA).…”