title: 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.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Trombley
authors.affiliation: Buck Institute for Research on Aging, Novato, CA, United States of America.
authors.forename: Deborah M B
authors.initials: DM
authors.lastname: Post
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Sherri D
authors.initials: SD
authors.lastname: Rinker
authors.affiliation: Buck Institute for Research on Aging, Novato, CA, United States of America.
authors.forename: Lorri M
authors.initials: LM
authors.lastname: Reinders
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Kate R
authors.initials: KR
authors.lastname: Fortney
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Beth W
authors.initials: BW
authors.lastname: Zwickl
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Janowicz
authors.affiliation: Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN, United States of America; Department of Biostatistics, Richard M. Fairbanks School of Public Health, Indianapolis, IN, United States of America.
authors.forename: Fitsum M
authors.initials: FM
authors.lastname: Baye
authors.affiliation: Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN, United States of America; Department of Biostatistics, Richard M. Fairbanks School of Public Health, Indianapolis, IN, United States of America.
authors.forename: Barry P
authors.initials: BP
authors.lastname: Katz
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America; Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States of America; Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, United States of America; Center for Immunobiology, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Spinola
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States of America.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Bauer
mesh_terms: D000284:Administration, Oral; D000328:Adult; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002602:Chancroid / Q000188:drug therapy / Q000382:microbiology / Q000473:pathology; D002939:Ciprofloxacin / Q000627:therapeutic use; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004982:Ethanolaminephosphotransferase / Q000235:genetics* / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D006191:Haemophilus ducreyi / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D064368:Healthy Volunteers; D006801:Humans; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D009154:Mutation; D011485:Protein Binding; D055672:Static Electricity; D023084:alpha-Defensins / Q000494:pharmacology; D023083:beta-Defensins / Q000494:pharmacology; D054804:Cathelicidins
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D004983:Ethanolamines; D008050:Lipid A; D023084:alpha-Defensins; D023083:beta-Defensins; D002939:Ciprofloxacin; C005448:phosphorylethanolamine; D004982:Ethanolaminephosphotransferase; D054804:Cathelicidins