Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers

<p>Abstract</p> <p>Background</p> <p><it>Haemophilus ducreyi</it>, the causative agent of the sexually transmitted disease chancroid, contains a <it>flp </it>(fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. <it>H. ducreyi </it>mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A <it>tadA </it>mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a <it>flp1flp2 </it>mutant is virulent in rabbits. Whether a <it>flp </it>deletion mutant would cause disease in humans is not clear.</p> <p>Results</p> <p>We constructed 35000HPΔ<it>flp1-3</it>, a deletion mutant that lacks expression of all three Flp proteins but has an intact <it>tad </it>secretion system. 35000HPΔ<it>flp1-3 </it>was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with <it>flp1-3 </it>in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔ<it>flp1-3 </it>(ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (<it>P </it>= 0.52). Mutant papules were significantly smaller (mean, 11.2 mm²) than were parent papules (21.8 mm²) 24 h after inoculation (<it>P </it>= 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (<it>P </it>= 0.001).</p> <p>Conclusion</p> <p>These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of <it>flp1-3 </it>is also necessary for <it>H. ducreyi </it>to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo.</p

Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection

BACKGROUND: Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies. RESULTS: We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively. CONCLUSIONS: Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization

Haemophilus ducreyi SapA Contributes to Cathelicidin Resistance and Virulence in Humans▿

Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's genome contains homologs of genes sensitive to antimicrobial peptides (sap operon) in nontypeable Haemophilus influenzae. In this study, we characterized the sap-containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi-infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HPsapA, and compared the percent survival of wild-type 35000HP and 35000HPsapA exposed to several human APs, including α-defensins, β-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HPsapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HPsapA after exposure to LL-37, which was complemented by introducing sapA in trans. Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared mutant strain 35000HPsapA with strain 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HPsapA was significantly lower than that of sites inoculated with 35000HP (33.3% versus 66.7%; P = 0.007). Together, these data establish that SapA acts as a virulence factor and as one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans

35000HPΔPEAT is fully virulent in vivo.

<p>^a Volunteers 441 and 442 were inoculated in the first iteration. Volunteers 444, 445, and 446 were inoculated in the second iteration. Volunteer 447 was inoculated in the third iteration. Volunteers 451 and 453 were inoculated in the fourth iteration.</p><p>^b M, Male; F, Female</p><p>^c P, 35000HP (parent); M, 35000HPΔPEAT (mutant)</p><p>^d Mutant-inoculated sites received estimated delivered doses of 56, 112, or 224 CFU.</p><p>^e Mutant-inoculated sites received estimated delivered doses of 40, 80, or 159 CFU.</p><p>35000HPΔPEAT is fully virulent in vivo.</p

<i>H</i>. <i>ducreyi</i> PEA transferases confer resistance to α- and β-defensins.

<p>35000HP, 35000HPΔPEAT and 35000HPΔPEAT/pPEAT were tested for resistance to the (A) α-defensin HD-5 (B) β-defensin HBD-3, and (C) human cathelicidin LL-37. Asterisks indicate statistically significant differences from 35000HP (<i>P</i> < 0.05). Complementation with pPEAT restored parental levels of susceptibility to defensins. Data represent average ± standard error of six independent replicates, and statistical significance was determined by Student’s t-test.</p

Bacterial strains and plasmids used in study.

<p>^a StrepR, resistance to streptomycin; Cm^R, resistance to chloramphenicol; AmpR, resistance to ampicillin; KanR, resistance to kanamycin; SpecR, resistance to spectinomycin.</p><p>Bacterial strains and plasmids used in study.</p

Putative <i>H</i>. <i>ducreyi</i> PEA transferases.

<p>Putative <i>H</i>. <i>ducreyi</i> PEA transferases.</p

LptA contributes to modification of lipid A with PEA.

<p>Negative-ion MALDI-MS spectra of <i>O</i>-LOS from (A) 35000HP<i>ΔptdB</i>, (B) 35000HP<i>ΔptdA</i>, (C) 35000HP<i>ΔlptA</i>, and (D) 35000HP. The Fig shows zoomed images from representative spectra for each strain. The <i>O</i>-deacylated monophosphorylated lipid A (MPLA) was observed at <i>m/z</i> 951.46 or 951.45, this structure plus the addition of PEA was observed at <i>m/z</i> 1074.46 or 1074.47. The MPLA plus PEA was not observed in the 35000HP<i>ΔlptA</i> samples.</p