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.

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2

“…Also in this group were the predicted PEtn transferases PtdA and PtdB from Haemophilus ducreyi. The acceptor molecule targeted by these H. ducreyi PEtn transferases has not been identified, but these proteins were not required for the addition of PEtn to lipid A (24). There are no reports of PEtn decoration of cell components other than LPS in P. multocida, and this bacterium does not produce OPG.…”

“…PEtn is also incorporated into OPG, located in the periplasm of many bacteria, including E. coli (22). The addition of PEtn to key positions on bacterial surface structures confers resistance to polymyxins and host innate immune antimicrobials, including cathelicidins and alpha and beta defensins (21,24,40). The most common surface structure to be decorated with PEtn in Gramnegative bacteria is the LPS structure, where it can be attached to lipid A, to Kdo, to Hep within the inner core, or to the outer core galactose (10,38,(41)(42)(43).…”

Massive analysis of 64,628 bacterial genomes to decipher water reservoir and origin of mobile colistin resistance genes: is there another role for these enzymes?

“…Because polymyxins share chemical similarities with cationic antimicrobial peptides (cAMPs) secreted by vertebrates and invertebrates, it is possible that decoration of lipid A of those bacteria with ethanolamine constitutes an immune defense against those metabolites. There is evidence that expression of PET in Haemophilus ducreyi is associated with polymyxin resistance as well as resistance to human defensins 44 . Although this hypothesis may explain another role of these enzymes against innate immunity from eukaryotic cells, this could not explain the huge prevalence of those enzymes in water environments.…”

Cross-resistance to human cationic antimicrobial peptides and to polymyxins mediated by the plasmid-encoded MCR-1?

“…Cross-resistance between several human CAMPs and polymyxins has rarely been evidenced in Enterobacteriaceae [7,8]. In addition, resistance to polymyxins and to human CAMPs has been associated with production of a phosphoethanolamine transferase in the Gram-negative species Haemophilus ducreyi and Campylobacter jejuni [9,10]. Therefore, our working hypothesis was that a crossed resistance to CAMPs and polymyxins might be observed among MCR-1-producing E. coli.…”