Abstract
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.
Highlights
Neisseria gonorrhoeae is a strict human pathogen that has caused the sexually transmitted disease gonorrhea for thousands of years (Sparling et al, 1990)
Since it is highly sensitive to killing by normal human serum (NHS), which is mediated by an antibody- and classical complement pathway (CCP)-dependent mechanism (Shafer et al, 1982), we reasoned that the killing capacity of NHS would be sufficient to use it as a selective pressure when screening the transposon library
A randomly picked Himar I mutant was used as a control in the NHS bactericidal assay and it was as NHS-susceptible as parent strain F62 indicating that the possession of the transposon per se was not responsible to the elevated NHSresistance displayed by WMS 100
Summary
Neisseria gonorrhoeae is a strict human pathogen that has caused the sexually transmitted disease gonorrhea for thousands of years (Sparling et al, 1990). Gonococci have developed a number of mechanisms to escape both innate and adaptive immune responses of the human host, which likely explains why immunity to re-infection does not occur. We are interested in the genetic basis for how gonococci can evade antimicrobial agents that it encounters during infection of mucosal surfaces or when growing in the bloodstream In this respect, our previous work showed that the MtrC-MtrD-MtrE efflux pump can export host-derived antimicrobial agents such as cationic antimicrobial peptides (CAPs; Shafer et al, 1998), as well as certain classical antibiotics (Veal et al, 2002). In addition to energy-dependent efflux, gonococcal resistance to CAPs has been linked to the decoration of lipid A by phosphoethanolamine (PEA; Lewis et al, 2009), which likely interferes with the ability of CAPs to bind to negatively charged groups on the bacterial surface
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