Research Article: SliC is a surface-displayed lipoprotein that is required for the anti-lysozyme strategy during Neisseria gonorrhoeae infection

Date Published: July 5, 2018

Publisher: Public Library of Science

Author(s): Ryszard A. Zielke, Adriana Le Van, Benjamin I. Baarda, Marco F. Herrera, Christopher J. Acosta, Ann E. Jerse, Aleksandra E. Sikora, Christoph Tang.

http://doi.org/10.1371/journal.ppat.1007081

Abstract

Lysozymes are nearly omnipresent as the first line of immune defense against microbes in animals. They exert bactericidal action through antimicrobial peptide activity and peptidoglycan hydrolysis. Gram-negative bacteria developed several weapons to battle lysozymes, including inhibitors of c-type lysozymes in the MliC/PliC family and the Neisseria adhesin complex protein (ACP). Until the recent discovery of ACP, no proteinaceous lysozyme inhibitors were reported for the genus Neisseria, including the important human pathogen N. gonorrhoeae. Here, we describe a previously unrecognized gonococcal virulence mechanism involving a protein encoded by the open reading frame ngo1063 that acts to counteract c-type Iysozyme and provides a competitive advantage in the murine model of gonorrhea. We named this protein SliC as a surface-exposed lysozyme inhibitor of c-type lysozyme. SliC displays low overall primary sequence similarity to the MliC/PliC inhibitors, but we demonstrate that it has a parallel inhibitory mechanism. Our studies provide the first evidence that bacterial proteinaceous lysozyme inhibitors protect against host lysozyme during infection based on lack of attenuation of the ΔsliC mutant in lysozyme knock-out mice, and that the conserved residues involved in lysozyme inhibition, S83 and K103, are functionally indispensable during infection in wild type mice. Recombinant SliC completely abrogated the lytic activity of human and chicken c-type lysozymes, showing a preference towards human lysozyme with an IC50 of 1.85 μM and calculated KD value of 9.2 ± 1.9 μM. In contrast, mutated SliC bearing S83A and K103A substitutions failed to protect fluorescein-labeled cell-wall from lysozyme-mediated hydrolysis. Further, we present data revealing that SliC is a surface-displayed lipoprotein released in membrane vesicles that is expressed throughout all phases of growth, in conditions relevant to different niches of the human host, and during experimental infection of the murine genital tract. SliC is also highly conserved and expressed by diverse gonococcal isolates as well as N. meningitidis, N. lactamica, and N. weaveri. This study is the first to highlight the importance of an anti-lysozyme strategy to escape the innate immune response during N. gonorrhoeae infection.

Partial Text

The first line of host immune defense against bacterial pathogens in plants and in invertebrate and vertebrate animals involves degradation of peptidoglycan through innate immune system components such as lysozymes [1–3]. Peptidoglycan (murein) is the major structural component of the bacterial cell envelope that provides resistance against turgor pressure and prevents cell death due to lysis. Peptidoglycan forms a giant three-dimensional network built of linear glycan strands of alternating β-(1,4) linked N-acetylmuramic acid and N-acetylglucosamine sugars that are cross-linked by short peptides [4]. Lysozymes are powerful host weapons exerting bacterial killing by hydrolytic action on the glycosidic bond linking the sugars, which breaks peptidoglycan [3, 5]. Three main classes of lysozymes have been distinguished within the animal kingdom including c- (conventional or chicken), g- (goose), and i- (invertebrate) type lysozyme [2, 3]. The mammalian lysozyme-like gene family consists of lysozyme c, lactalbumin and calcium-binding lysozyme [5]. Recent studies, however, showed the presence of eight additional diverse types of lysozyme-like genes in the genome of the common ancestor of all extant mammals, and ten lysozyme-like sequences distributed over five chromosomes in humans [6]. Therefore, it is not surprising that bacteria have evolved sophisticated mechanisms to escape killing via murein hydrolysis by chemical modifications of the peptidoglycan backbone and by synthesis of proteinaceous lysozyme inhibitors [7, 8]. The latter anti-lysozyme strategy targets i-, g-, or c-type lysozymes and encompasses at least five inhibitor families distributed exclusively in Gram-negative bacteria and predominantly in Proteobacteria [8]. Among the inhibitors of c-type lysozyme are Ivy (Inhibitor of vertebrate lysozyme) and Ivy-like proteins, the MliC/PliC family (membrane-bound or periplasmic lysozyme inhibitor of c-type lysozyme, respectively), and the NeisseriaAdhesin Complex Protein, ACP [8, 9].

To establish infection, microbes must evade a combination of host antimicrobial peptides and enzymes, including lysozymes, which are abundantly secreted by the epithelium and produced within professional phagocytes [32]. Described for the first time by Alexander Fleming in 1922 as a substance with the ability to “lyse” bacteria, lysozyme additionally acts as an antimicrobial peptide by interacting directly with cell membranes via its positively charged amino acids [33]. Pathogenic and commensal bacteria have developed many methods to evade lysozyme; however, until the recently reported ACP [9], no proteinaceous lysozyme inhibitor has been described for the genus Neisseria. ACP is a surface-exposed meningococcal adhesin that induces cross-strain bactericidal antibodies [30]. Despite low primary sequence homology, the apo crystal structure of N. meningitidis ACP resembles an eight-stranded antiparallel ß-barrel similar in the overall fold to those of MliC/PliC proteins [9, 11, 12, 19]. In vitro assays have demonstrated that ACP proteins act as inhibitors of HEWL and HL, contributing to lysozyme tolerance in the presence of the membrane-permeabilizing agent lactoferrin [9, 30]. Nevertheless, the importance of lysozyme inhibition for host colonization has not been addressed in Neisseria and investigations in other bacterial species are also sparse [13, 14].

 

Source:

http://doi.org/10.1371/journal.ppat.1007081