Abstract
Neisseria gonorrhoeae (Gc, gonococcus) is a Gram-negative diplococcus and obligate human pathogen. Gc is the causative agent gonorrhea, a major global health problem. Gc colonization of mucosal surfaces leads to the abundant recruitment of neutrophils. Both the mucosa and neutrophils produce various defenses to neutralize microbes, yet Gc can survive in the presence of these defenses. One antimicrobial protein common to both the mucosa and neutrophils is lysozyme. In addition to targeted hydrolysis of bacterial cell wall peptidoglycan (PG), lysozyme can also kill bacteria through ionic interactions leading to membrane instability. Besides its antimicrobial activities, lysozyme hydrolysis and degradation of PG facilitates the release of immunomodulatory PG. Thus, lysozyme resistance in bacteria serves to limit both the antimicrobial and immunomodulatory activities of lysozyme. However, it is unclear how Gc resists lysozyme or how this resistance contributes to virulence.
Our first observation for how Gc resists killing by lysozyme came from a serendipitous study investigating how two PG turnover proteins in Gc affect bacterial survival in the presence of neutrophils. During periods of cell wall remodeling, PG must be released from the intact cell wall in a process termed PG turnover. The lytic transglycosylases LtgA and LtgD are part of the PG turnover machinery in Gc, and both of these enzymes’ activities result in the extracellular release of immunomodulatory PG monomers. We found that ΔltgAΔltgD mutant Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild-type. Addition of the immunomodulatory PG monomers that are released by LtgA and LtgD failed to alter ΔltgAΔltgD mutant survival in the presence of neutrophils, pointing to a mechanism other than PG monomer release for LtgA- and LtgD-mediated survival. We found two reasons to explain decreased survival of the ΔltgAΔltgD mutant. First, ΔltgAΔltgD mutant Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD mutant Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude from this study that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule-localized antimicrobial proteins. Notably, these observations are the first to implicate a possible link between bacterial degradation by lysozyme to increased neutrophil activation.
Because some Gram-negative bacteria produce protein inhibitors that bind to and occlude the active site of lysozyme, we next investigated the possibility of Gc protein inhibitors of lysozyme. Here, we identified Ng_1063 as a new inhibitor of lysozyme in Gc, and defined its functions in light of a second, recently identified inhibitor, Ng_1981. In silico analyses indicated that Ng_1063 bears sequence and structural homology to MliC-type inhibitors of lysozyme. Recombinant Ng_1063 inhibited lysozyme-mediated killing of a susceptible mutant of Gc and the lysozyme-sensitive bacterium Micrococcus luteus. This inhibitory activity was dependent on serine 83 and lysine 103 of Ng_1063, which are predicted to interact with lysozyme’s active site residues. Lysozyme co-immunoprecipitated with Ng_1063 and Ng_1981 from intact Gc. Ng_1063 and Ng_1981 protein levels were also increased in Gc exposed to lysozyme. Gc lacking both ng1063 and ng1981 was significantly more sensitive to killing by lysozyme than wild-type or single mutant bacteria. When exposed to human tears or saliva, in which lysozyme is abundant, survival of Δ1981Δ1063 Gc was significantly reduced compared to wild-type, and survival was restored upon addition of recombinant Ng_1981. Δ1981Δ1063 mutant Gc survival was additionally reduced in the presence of human neutrophils. We found that while Ng_1063 was exposed on the surface of Gc, Ng_1981 was both in an intracellular pool and extracellularly released from the bacteria, suggesting that Gc employs these two proteins at multiple spatial barriers to fully neutralize lysozyme activity. Together, these findings identify Ng_1063 and Ng_1981 as critical components for Gc defense against lysozyme. These proteins may be attractive targets for antimicrobial therapy aimed to render Gc susceptible to host defenses and/or for vaccine development, both of which are urgently needed against drug-resistant gonorrhea.
Lysozyme-sensitive Gram-positive bacteria can activate macrophages, dependent on lysozyme and the PG-sensor NOD2. We hypothesize that the lysozyme-sensitive ΔltgAΔltgD mutant Gc may be similarly activating neutrophils. However, it is unknown how lysozyme or NOD2 functions to sense PG in neutrophils. Therefore, we began by testing what types of PG are immunomodulatory in neutrophils. Neutrophils exhibit increased markers of activation when exposed to insoluble, polymeric PG compared with soluble, monomeric PG. Activation by insoluble, polymeric PG was dependent on phagocytosis. Moreover, we found that pre-treatment of polymeric PG with lysozyme, which degrades PG into monomers, ablated neutrophil activation. We propose that soluble, monomeric PG has limited access to the neutrophil cytosol where NOD2 resides, while insoluble, polymeric PG, as occurs in an intact bacterium, is phagocytosed and processed intracellularly by lysozyme, preceding NOD2 activation. Future studies are aimed at testing the requirement for lysozyme and NOD2 in neutrophil responses to PG or lysozyme-sensitive Gc.
