Mini-binders targeting Streptococcus equi ssp. zooepidemicus M-like protein inhibit the bacterial adhesion and exert protective effects in vivo.

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is one of the most common pathogens causing streptococcal disease in pigs in China and has been identified as a zoonotic pathogen, and thus poses great threat to the health of humans and pigs. The M-like protein (SzM) is the primary virulence factor of SEZ. Monoclonal antibodies (mAbs) targeting SzM have been demonstrated to provide effective protection against SEZ infection, but their preparation is cumbersome. Here, we designed mini-binders targeting SzM from scratch based on the RFdiffusion approach. Four potential binders were obtained in a short period of time, among which binder 3 showed the highest binding affinity to SzM protein. In vitro adhesion inhibition analysis demonstrated that binder 3 significantly suppressed the adhesion of SEZ to fibrinogen and HEp-2 cells. In vivo experiments showed that binder 3 treatment improved the survival rate (80 %) of mice infected with a 100-fold lethal dose of SEZ and significantly reduced the organ bacterial load. Our study provides new insights into the rapid development of stable anti-SEZ agents, which are expected to be ideal alternatives to mAbs.