Effect of Lipidation on the Localization and Activity of a Lysozyme Inhibitor in Neisseria gonorrhoeae.

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme.IMPORTANCE Neisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme.