Exploration of virulence and immune evasion functions of the candidate vaccine antigen SpyAD in the globally disseminated M1T1 group A <i>Streptococcus</i> strain

Group A <i>Streptococcus</i> (GAS; <i>S. pyogenes</i>) is a significant human pathogen responsible for a wide range of diseases, including invasive conditions with high mortality rates, for which no FDA-approved vaccine exists. The membrane-bound protein <i><u>S</u>. <u>py</u>ogenes</i> <u>a</u>dhesion and <u>d</u>ivision protein (SpyAD) is highly conserved among all sequenced GAS isolates and has emerged as a promising vaccine antigen, but its precise role in GAS pathogenesis remains unclear. Here, we investigate the contribution of SpyAD in establishing and maintaining infection in M1T1 GAS, a persistent and globally disseminated strain that is the most frequently isolated serotype in both invasive and noninvasive infections. We found that SpyAD is critical for GAS invasion of immortalized human pharyngeal and vaginal epithelial cells, as loss of SpyAD significantly impaired bacterial invasion. SpyAD deficiency also resulted in impaired biofilm formation. These features suggest that SpyAD facilitates bacterial persistence and contributes to GAS virulence. In addition, SpyAD enhanced GAS resistance to neutrophil-mediated killing, potentially through phagocytosis-dependent mechanisms. <i>In vivo</i> experiments demonstrated that SpyAD deficiency leads to decreased bacterial burden and prolonged survival in a murine model of systemic infection, as well as reduced colonization of the murine vaginal mucosa, further highlighting its role in GAS virulence. These findings underscore the importance of SpyAD as a factor in GAS pathogenesis in the M1T1 strain and support its potential as a target for vaccine development.IMPORTANCEGroup A <i>Streptococcus</i> (GAS) infections remain prevalent worldwide, with recent surges in severe invasive cases causing high morbidity and mortality. To effectively combat these invasive infections, a more thorough understanding of GAS pathogenesis-particularly mechanisms of immune invasion and evasion-is critical. In this study, we address this need by elucidating the role of the candidate GAS vaccine antigen SpyAD in these key processes in the highly prevalent M1T1 GAS strain. SpyAD enables GAS to evade neutrophil killing and facilitates epithelial cell invasion, likely helping the bacterium circumvent immune defenses. Moreover, SpyAD is important for systemic infection and vaginal mucosal infection <i>in vivo</i>. Vaccination targeting SpyAD may generate opsonophagocytic antibodies to improve GAS clearance and fortify protection against invasive disease.