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

Abstract

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.

Fig 1. Deletion of two putative PEA transferase genes in H. ducreyi increases susceptibility to HBD-3.

(A) 35000HPΔlptA ptdA, (B) 35000HPΔlptA ptdB, and (C) 35000HPΔptdA ptdB were compared with 35000HP for resistance to the β-defensin HBD-3. All three mutants lacking two putative PEA transferase genes were significantly more sensitive than 35000HP to HBD-3, indicated by asterisks (P < 0.05). Data represent average ± standard error of 3–4 independent assays, and statistical significance was determined by Student’s t-test.

Fig 2. H. ducreyi PEA transferases confer resistance to α- and β-defensins.

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 (P < 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.

Fig 3. H. ducreyi PEA transferases do not confer resistance to human serum.

35000HP, 35000HPΔPEAT and FX517 were examined for resistance to human serum. There was no significant difference in sensitivity to serum between 35000HP and 35000HPΔPEAT; FX517, a ΔdsrA mutant that served as a control for serum sensitivity, was significantly more sensitive to serum than 35000HP, indicated by asterisk (P < 0.05). Data represent average ± standard error of six independent assays, and statistical significance was determined by Student’s t-test.

Fig 4. H. ducreyi putative PEA transferases contribute to modification of lipid A with PEA.

Negative-ion MALDI-MS spectra of O-LOS from (A) 35000HP/pLSSK, (B) 35000HPΔPEAT/pLSSK, and (C) 35000HPΔPEAT/pPEAT. The compositions of the glycoforms are described in Table 5. Masses labeled in bold were only observed in the parent strain and the corresponding PEAT complemented strain. The asterisk in the glycoform nomenclature designates the number of PEA groups present on the O-LOS.

Fig 5. LptA contributes to modification of lipid A with PEA.

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