Functional and Structural Characterisation of Spy0136, a Novel Group A Streptococcal Virulence Factor

Abstract

Streptococcus pyogenes (Group A Streptococcus, GAS) is a leading human pathogen associated with a broad range of diseases and over half a million deaths annually all over the world. GAS generates numerous virulence factors contributing to the bacteria's pathogenesis. However, the function of many of these factors has yet to be identified, and more studies to gain a better understanding of host and bacterial factors are required for developing therapeutic and prevention strategies. The GAS whole-genome study has recently provided new opportunities to identify novel virulence factors by discovering several open reading frames in the GAS genome. This study aimed to investigate the functional characterisation of one of these newly discovered genes annotated as spy0136. It is a conserved gene across all major emm types of GAS, including the most prevalent invasive strains and flanks the fibrinogen-binding, collagen-binding, T antigen (FCT) region of the GAS genome. Through producing the recombinant form of Spy0136, the structural properties of this protein and its contribution to GAS virulence were evaluated using several in-vitro and in-vivo assays. Spy0136 was found to bind C1s, C1r, C3, C5 complement proteins, and fibrinogen, suggesting a potential involvement of this protein in immune evasion mechanisms. This binding was shown to be glycan-dependent for C1s, C3, and C5. Functional analysis of Spy0136 in host immune evasion revealed that the protein inhibits complement-mediated haemolysis of erythrocytes. Moreover, activation of all three pathways of complement and the formation of C5b-9 on the GAS surface were inhibited in the presence of Spy0136. All these observations suggest that Spy0136 is a novel GAS immune evasion factor that interferes with the human complement system. Additionally, Spy0136-mediated protection was observed in whole blood killing assays, and in-vivo analysis of the wild-type and spy0136-knockout strains of GAS in a wax worm infection model showed attenuated virulence of the mutant strain than the wild-type and confirmed the role of Spy0136 in GAS virulence. This study provides novel insights into the mechanisms that GAS employs to fight the complement system and interact with its human host. This information could help to develop new preventive and therapeutic approaches against GAS.