Role of Neuraminidase A of S. pneumoniae on platelet-bacteria-interaction

Introduction Streptococcus pneumoniae as an opportunistic human pathogen is a major causative agent of severe community-acquired pneumonia. Pneumococci express pneumolysin (Ply), a cholesterol dependent cytolysin, which forms pores in membranes of eukaryotic cells including platelets. We have shown that pore formation results in diminished platelet function, representing a new pathomechanism during pneumococcal pneumonia. Furthermore, we demonstrated efficient neutralization of Ply with pharmaceutical human IgG (IVIG, Privigen). Besides Ply, S. pneumoniae expresses neuraminidase A (NanA), an enzyme that cleaves specifically sialic acid residues from eukaryotic glycoproteins. This increases binding of Ply to desialylated surface-associated platelet glycoproteins. Here, we aim to decipher how NanA activity affects platelet damage caused by Ply expressing and Ply-deficient strains and the inhibition of these effects by IVIG.

Method Blood was obtained from healthy human donors and isolated platelets were incubated with S. pneumoniae D39 and TIGR4 wild-type strains and their isogenic mutants ∆nanA, ∆ply, ∆cps for 2h and analysed by flow cytometry. Desialylation was evaluated by Erythrina Cristagalli Lectin staining. Pneumolysin binding and membrane permeability was determined by staining with monoclonal anti-ply antibody and -anti-human β-Tubulin antibody. In neutralization experiments, samples were supplemented with human polyvalent immunoglobulin preparations (IVIG; IgG-enriched Privigen®​) [5 mg/mL].

Results Compared to wild-type D39 and TIGR4, desialylation of the platelet surface was reduced after incubation with D39∆nanA and TIGR4∆nanA (MOI (ratio bacteria to platelets) 0.1; D39: 101,147 vs. 70,202, p=0.0078; TIGR4: 214,786 vs. 69,943 p<0.0001). Ply binding (1,159 vs. 357, p=0.0062) and platelet damage (β-Tubulin expression: 1,989 vs. 470, p=0.0054) were nominally reduced when platelets were incubated with TIGR4∆nanA mutants compared to wild-types. Similar trends were observed with the D39 equivalents, but failed to reach significance (Ply binding: 369 vs. 259, p=0.0648; β-Tubulin expression: 655 vs. 410, p=0.0641). Desialylation of platelets was decreased when IVIG was added for D39 (44,567 vs. 20,892, p<0.0001) and TIGR4 (95,089 vs. 20,951, p=0.0002). Additionally, IVIG reduced accessibility of β-Tubulin even after infection at MOI 1 (D39: 24,548 vs. 632, p=0.0078; TIGR4: 49,132 vs. 1,237, p=0.0078).

Conclusion NanA and Ply are important virulence factors of S. pneumoniae and profoundly influence the interplay between platelets and this pathogen. The deficiency of NanA in S. pneumoniae reduces Ply binding to the platelet surface, hence also weakening its destructive effect on platelets. Therapeutic doses of human polyvalent immunoglobulin preparations can prevent the effects of both, NanA and Ply. Therefore, human IgG preparations represent promising candidates for therapeutical intervention during severe S. pneumoniae pneumonia to prevent complications like ARDS.

 

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Conflict of Interest

No conflict of interest to be disclosed.

Publikationsverlauf

Artikel online veröffentlicht:
20. Februar 2023