Botteaux, A. | bioRxiv Science

bioRxiv2024

Conservation of C4BP-binding Sequence Patterns in Streptococcus pyogenes M and Enn Proteins

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP-binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP, and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to identification of amino acids that are crucial for C4BP-binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most populated among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

microbiology↗

bioRxiv2024

The Streptococcus pyogenes M protein is involved in phenotypic resistance to phage A25 infection in presence of human serum.

Streptococcus pyogenes is responsible for mild to life-threatening infections. Bacteriophages, or phages, and their virulence genes play a key role in the emergence and expansion of epidemics. However, relatively little is known about the biology of S. pyogenes phages, particularly in biologically relevant environments. During infection, S. pyogenes conceals from the host immune system through the binding of human serum proteins. This evasion is mediated by surface proteins, such as the M protein which is a major virulence determinant of S. pyogenes. Here, we demonstrate that human serum proteins also confer phenotypic resistance to phage A25 infection by impeding phage adsorption. We have found that, although not directly involved in phage A25 infection, the M protein is involved in this inhibition through the binding of both IgG and albumin, especially in absence of bound fatty acids. These findings highlight the importance of studying phages within a physiological context, specifically in the environmental conditions in which they will be used. Author summaryThe issues of antimicrobial resistance and resurgence of life-threatening infection, like the recent cases of invasive S. pyogenes infections, are prompting the scientific community to use phages as a complementary therapy. Phages are often characterized in laboratory conditions which are very different from the infection site. During human infection, Streptococcus pyogenes uses serum proteins to protect against the immune system. Our data illustrate how the human host environment also modulates phage susceptibility of S. pyogenes. We found that human serum transiently protects a M25 strain against infection by the lytic phage A25. This protective effect is mediated in part by the M protein, a major virulence determinant and the target of current vaccines. This new function for the M protein highlights the need to characterize bacteria-phage interactions in a more physiological context to increase the chances of success of phage therapy.

microbiology↗