Lipid A’s Structure Mediates Neisseria gonorrhoeae Fitness during Experimental Infection of Mice and Men

Abstract

Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments.IMPORTANCE Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A’s structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.

Phosphoethanolamine Decoration of Neisseria gonorrhoeae Lipid A Plays a Dual Immunostimulatory and Protective Role during Experimental Genital Tract Infection

“…The presence of a lipid A PEA moiety also increases N. gonorrhoeae resistance to complement-mediated killing (15,16) but does not affect the serum resistance of N. meningitidis. We recently demonstrated that an lptA mutant of N. gonorrhoeae strain FA1090 was attenuated during experimental urethral infection of male subjects and cervicovaginal infection of female mice (17), which strongly supports the protective role of this lipid A modification during infection.…”

“…Human embryonic kidney (HEK) cells expressing the TLR4-MD2-CD14 receptor complex and mouse embryonic fibroblasts (MEFs) from C3H/HeN (wild-type TLR4) mice carrying the NF-B-inducing secreted embryonic alkaline phosphatase (SEAP) reporter gene (InvivoGen, CA) were used to assess the NF-B-stimulatory property of purified LOS and whole bacteria according to the manufacturer's instructions. Briefly, LOS from wild-type or lptA mutant gonococci was purified as previously described (17) and added to 5 ϫ 10 4 HEK TLR4 cells or MEFs at concentrations of 0.1, 1, 10, or 100 ng/ml in 96-well plates and incubated for 20 h at 37°C (total reaction volume, 200 l/well). For whole bacteria, a multiplicity of infection (MOI) of 1 was used.…”

“…In our recent experiments with human subjects and female mice, competitive infections were performed between mutant bacteria and the wild-type parent strain (17), and therefore, the effect of the PEA lipid A decoration on host responses could not be measured. Here we examined the impact of lipid A PEA decoration on induction of a proinflammatory response through TLR4 using human and murine tissue culture cell lines and on the host inflammatory response to infection as assessed by noncompetitive murine infection.…”

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage

“…Recent analysis of experimental infections with mixtures of wild-type N. gonorrhoeae and isogenic mutants that lacked lptA in both mice and men showed that lptA expression conferred a significant increase in the ability of the bacteria to survive in mammalian hosts (40). Modification of lipid A with PEA has been found to increase the resistance of N. gonorrhoeae to complement killing in normal human serum by increasing the binding of the complement regulatory protein C4b-binding protein and increasing resistance to endogenous cationic antimicrobial peptides (41,42).…”

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

“…An update of the nomenclature for all Neisserial lipid A PEA transferase enzymes will be announced on PubMLST (pubmlst.org/)]. Mutants of NmEptA increase bacterial sensitivity to CAMPs (12), resulting in attenuation in mice and human models of infection (13). EptA catalyzes the transfer of PEA from phosphatidylethanolamine (PE), via a PEA-enzyme intermediate, to the 1 and 4′ headgroups of the lipid A of lipooligosaccharide (SI Appendix, Scheme S1).…”