Phosphoethanolamine substitution of lipid A and resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum.

The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide beta-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.

Phase variable changes in genes lgtA and lgtC within the lgtABCDE operon of Neisseria gonorrhoeae can modulate gonococcal susceptibility to normal human serum.

The alpha-chain of the core oligosaccharide of the lipo-oligosaccharide (LOS) produced by Neisseria gonorrhoeae can undergo reversible and rapid changes in structure due to phase-variable production of certain enzymes employed in the biosynthesis of the lacto-N-neotetraose structure. Five of these enzymes are encoded by the lgtABCDE operon, and polynucleotide tracts within three of these genes (lgtA, lgtC and lgtD) can be substrates for slipped-strand mispairing events that lead to nucleotide insertions or deletion events which result in variable production of their respective gene products. We now report that phase-variable synthesis of the lgtA and lgtC gene products in strain FA19 results in the production of elongated LOS alpha-chains and that the presence of these LOS species can result in gonococci being sensitive to the bacteriolytic action of serum-antibody and complement. Hence, phase variation within the lgtABCDE operon can significantly impact the ability of gonococci to subvert this important host defense system.

Lipooligosaccharide Structure is an Important Determinant in the Resistance of Neisseria Gonorrhoeae to Antimicrobial Agents of Innate Host Defense.

The strict human pathogen Neisseria gonorrhoeae has caused the sexually transmitted infection termed gonorrhea for thousands of years. Over the millennia, the gonococcus has likely evolved mechanisms to evade host defense systems that operate on the genital mucosal surfaces in both males and females. Past research has shown that the presence or modification of certain cell envelope structures can significantly impact levels of gonococcal susceptibility to host-derived antimicrobial compounds that bathe genital mucosal surfaces and participate in innate host defense against invading pathogens. In order to facilitate the identification of gonococcal genes that are important in determining levels of bacterial susceptibility to mediators of innate host defense, we used the Himar I mariner in vitro mutagenesis system to construct a transposon insertion library in strain F62. As proof of principle that this strategy would be suitable for this purpose, we screened the library for mutants expressing decreased susceptibility to the bacteriolytic action of normal human serum (NHS). We found that a transposon insertion in the lgtD gene, which encodes an N-acetylgalactosamine transferase involved in the extension of the α-chain of lipooligosaccharide (LOS), could confer decreased susceptibility of strain F62 to complement-mediated killing by NHS. By complementation and chemical analyses, we demonstrated both linkage of the transposon insertion to the NHS-resistance phenotype and chemical changes in LOS structure that resulted from loss of LgtD production. Further truncation of the LOS α-chain or loss of phosphoethanolamine (PEA) from the lipid A region of LOS also impacted levels of NHS-resistance. PEA decoration of lipid A also increased gonococcal resistance to the model cationic antimicrobial polymyxin B. Taken together, we conclude that the Himar I mariner in vitro mutagenesis procedure can facilitate studies on structures involved in gonococcal pathogenesis.

Genetic basis for nongroupable Neisseria meningitidis.

Nongroupable Neisseria meningitidis may constitute one-third or more of meningococcal isolates recovered from the nasopharynx of human carriers. The genetic basis for nongroupability was determined in isolates obtained from a population-based study in which 60 (30.9%) of 194 meningococcal isolates from asymptomatic carriers were not groupable. Forty-two percent of nongroupable isolates were related to serogroup Y ET-508/ST-23 clonal complex strains, the most common groupable carrier isolate from the study population. Nongroupable isolates were all rapidly killed by 10% normal human serum. The capsule loci of 6 of the ET-508/ST-23 complex strains and of 25 other genetically diverse nongroupable meningococci were studied in detail. Serogroup A or novel capsule biosynthesis genes were not found. Nongroupable isolates were genetically serogroup Y, B, or C isolates that did not express capsule but were related to groupable isolates found in the population (class I); capsule deficient because of insertion element-associated deletions of capsule biosynthesis genes (class II); or isolates that lacked all capsule genes and formed a distinct genetic cluster not associated with meningococcal disease (class III).