Functional characterization of the gonococcal polyphosphate pseudo-capsule

Abstract

Neisseria gonorrhoeae is an exclusively human pathogen able to evade the host immune system through multiple mechanisms. Gonococci accumulate a large portion of phosphate moieties as polyphosphate (polyP) on the exterior of the cell. Although its polyanionic nature has suggested that it may form a protective shield on the cell surface, its role remains controversial. Taking advantage of a recombinant His-tagged polyP-binding protein, the presence of a polyP pseudo-capsule in gonococcus was demonstrated. Interestingly, the polyP pseudo-capsule was found to be present in specific strains only. To investigate its putative role in host immune evasion mechanisms, such as resistance to serum bactericidal activity, antimicrobial peptides and phagocytosis, the enzymes involved in polyP metabolism were genetically deleted, generating mutants with altered polyP external content. The mutants with lower polyP content on their surface compared to the wild-type strains, became sensitive to complement-mediated killing in presence of normal human serum. Conversely, naturally serum sensitive strains that did not display a significant polyP pseudo-capsule became resistant to complement in the presence of exogenous polyP. The presence of polyP pseudo-capsule was also critical in the protection from antibacterial activity of cationic antimicrobial peptide, such as cathelicidin LL-37. Results showed that the minimum bactericidal concentration was lower in strains lacking polyP than in those harboring the pseudo-capsule. Data referring to phagocytic killing resistance, assessed by using neutrophil-like cells, showed a significant decrease in viability of mutants lacking polyP on their cell surface in comparison to the wild-type strain. The addition of exogenous polyP overturned the killing phenotype of sensitive strains suggesting that gonococcus could exploit environmental polyP to survive to complement-mediated, cathelicidin and intracellular killing. Taken together, data presented here indicate an essential role of the polyP pseudo-capsule in the gonococcal pathogenesis, opening new perspective on gonococcal biology and more effective treatments. Innate immunity plays a key role in the clearance of Neisseria gonorrhoeae. However, the complex molecular mechanisms evolved by N. gonorrhoeae to evade the host immune system are still an open-ended question. Moreover, the antibiotic resistance crisis highlights its public health relevance thriving for a deeper understanding of gonococcal pathogenesis to advance our chance to defeat gonococcal infections. Polyphosphate (polyP) is an anionic polymer that N. gonorrhoeae accumulates both internally and externally to the bacterial cell but its role is still controversial. Here, we demonstrate the presence of a surface exposed pseudo-capsule, made of polyP that shields N. gonorrhoeae from strategies put in place by the innate immune response, including complement-mediated bactericidal activity of normal human serum, antibacterial activity of cationic antimicrobial peptides, such as cathelicidin LL-37 and the phagocyte-dependent intracellular killing. Our findings highlight an additional strategy used by N. gonorrhoeae to counteract the host immune system and pave the way to novel approaches to tackle gonococcal infections.