Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis

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

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

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

<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

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

Haemophilus ducreyi resists killing by antimicrobial peptides encountered during human infection, including cathelicidin LL-37, α-defensins, and β-defensins. In this study, we examined the role of the proton motive force-dependent multiple transferable resistance (MTR) transporter in antimicrobial peptide resistance in H. ducreyi. We found a proton motive force-dependent effect on H. ducreyi's resistance to LL-37 and β-defensin HBD-3, but not α-defensin HNP-2. Deletion of the membrane fusion protein MtrC rendered H. ducreyi more sensitive to LL-37 and human β-defensins but had relatively little effect on α-defensin resistance. The mtrC mutant 35000HPmtrC exhibited phenotypic changes in outer membrane protein profiles, colony morphology, and serum sensitivity, which were restored to wild type by trans-complementation with mtrC. Similar phenotypes were reported in a cpxA mutant; activation of the two-component CpxRA regulator was confirmed by showing transcriptional effects on CpxRA-regulated genes in 35000HPmtrC. A cpxR mutant had wild-type levels of antimicrobial peptide resistance; a cpxA mutation had little effect on defensin resistance but led to increased sensitivity to LL-37. 35000HPmtrC was more sensitive than the cpxA mutant to LL-37, indicating that MTR contributed to LL-37 resistance independent of the CpxRA regulon. The CpxRA regulon did not affect proton motive force-dependent antimicrobial peptide resistance; however, 35000HPmtrC had lost proton motive force-dependent peptide resistance, suggesting that the MTR transporter promotes proton motive force-dependent resistance to LL-37 and human β-defensins. This is the first report of a β-defensin resistance mechanism in H. ducreyi and shows that LL-37 resistance in H. ducreyi is multifactorial