Date Published: July 5, 2018
Publisher: Public Library of Science
Author(s): Stephanie A. Ragland, Marίa V. Humbert, Myron Christodoulides, Alison K. Criss, Christoph Tang.
The bacterial pathogen Neisseria gonorrhoeae (Gc) infects mucosal sites rich in antimicrobial proteins, including the bacterial cell wall-degrading enzyme lysozyme. Certain Gram-negative bacteria produce protein inhibitors that bind to and inhibit lysozyme. Here, we identify Ng_1063 as a new inhibitor of lysozyme in Gc, and we define its functions in light of a second, recently identified lysozyme 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, which produce lysozyme. 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.
Neisseria gonorrhoeae (Gc) is a Gram-negative diplococcus and the causative agent of the sexually transmitted infection gonorrhea. The World Health Organization (WHO) estimates 78 million cases of gonorrhea occur each year, with over 800,000 cases reported annually in the United States [1–3]. The lack of a protective vaccine, widespread prevalence of antibiotic-resistant Gc, and treatment failures with last line therapeutics have prompted the United States Centers for Disease Control to label antibiotic-resistant Gc as an urgent threat to public health [1, 4–6]. Likewise, the WHO lists the control and elimination of Gc infection as a high priority . Dissecting Gc pathogenesis and virulence is critical for the development of novel therapeutics and vaccines.
The success of Gc as a human pathogen requires that it defends itself against antimicrobial components found at mucosal surfaces. In this work, we identified Ng_1063 as a new functional homolog of the MliC-type lysozyme inhibitors in Gc. We then interrogated its functions alongside another, recently characterized lysozyme inhibitor, Ng_1981 . We found that Ng_1063 and Ng_1981 interact with lysozyme in the physiological context of the bacterium, and both proteins’ expression was increased upon exposure to lysozyme. Gc lacking both ng1063 and ng1981 was markedly reduced in survival after exposure to human lysozyme and lysozyme-containing human secretions, and this effect was greater than either single mutant alone. Ng_1063 was exposed on the surface of Gc, while Ng_1981 was not; however, a fraction of Ng_1981 protein was released into the extracellular milieu. Based on these results, we conclude that Gc produces two distinct inhibitors of lysozyme that together confer full resistance to this abundant antimicrobial defense protein.