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.

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“…We observed a significant increase in cellular, extracellular, and cell surface polyP in response to INH. Compared to log-phase cells, stationary phase Mtb have more polyP and are more resistant to killing by INH [27, 53], and capsular polyP accumulation protects N. gonorrhoeae from antibiotics [23, 24]. Combining these observations, one possibility is that Mtb increase polyP in response to INH as a protective measure.…”

“…In addition to intracellular polyP, bacteria also have extracellular polyP. The pathogenic bacterium Neisseria gonorrhoeae has polyP in its capsule, and the polyP potentiates resistance to antimicrobials [23, 24]. We observed that both Mtb and M. smegmatis accumulate extracellular polyP [27].…”

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“…We observed a significant increase in cellular, extracellular, and cell surface polyP in response to INH. Compared to log-phase cells, stationary phase Mtb have more polyP and are more resistant to killing by INH (Herbert et al, 1996 ; Rijal et al, 2020 ), and capsular polyP accumulation protects N. gonorrhoeae from antibiotics (Mookherjee et al, 2013 ; Manca et al, 2023 ). Combining these observations, one possibility is that Mtb increase polyP in response to INH as a protective measure.…”

“…Pathogenic bacteria lacking PPK or having reduced PPK levels exhibit defects in stress response, quorum sensing, growth, survival, and virulence (Rao et al, 1998 ; Rashid and Kornberg, 2000 ; Rashid et al, 2000 ; Chavez et al, 2004 ; Sureka et al, 2009 ; Zhang et al, 2010 ; Mookherjee et al, 2013 ; Manca et al, 2023 ). For instance, intracellular polyP is necessary for the survival of Mtb in host cells (Chavez et al, 2004 ; Sureka et al, 2007 ; Chuang et al, 2013 ; Singh et al, 2013 ), and deletion of PPK1 in M. smegmatis attenuates the survival of ingested M. smegmatis in human macrophages (Rijal et al, 2020 ).…”

“…In addition to intracellular polyP, bacteria also have extracellular polyP. The pathogenic bacterium Neisseria gonorrhoeae has polyP in its capsule, and the polyP potentiates resistance to antimicrobials (Mookherjee et al, 2013 ; Manca et al, 2023 ). We observed that both Mtb and M. smegmatis accumulate extracellular polyP (Rijal et al, 2020 ).…”