Streptococcus suis exports WapA polymorphic toxins to compete with host microbiota for an optimal colonization

Streptococcus suis is an important zoonotic pathogen, and its colonization in host tonsil is believed to be a vital source of infection for humans and animals, while the mechanism of S. suis competing for a stable tonsil niche is unknown. Rearrangement hotspot (Rhs) proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin (CT) domain. In Gram-negative bacteria, Rhs proteins have been identified as polymorphic effectors exported by type VI secretion system. In contrast, the delivery mechanism of their distantly related homologues emerged in Gram-positive bacteria, referred to as wall-associated protein A (WapA), remains incompletely understood. Here we reported the 350-kDa WapA-CT1, facilitating the S. suis colonization by manipulating tonsil microbiota, is linked with a SecF-like protein and a SrtB sortase encoded by two upstream genes. The unfolded WapA-CT1 was translocated across cell membrane via the canonical Sec pathway under the guidance of its N-terminal Sec-signal motif and cognate SecF-like protein. A cleavage assay identified four fragments within it: the N-terminal NCWB fragment, two middle Rhs domains (Rhs1&2) that may fold as a β-barrel structure enclosed by Lid/RAC plugs on each side, and a C-terminal PreT-CT toxin domain. SrtB interacts with the NCWB region before or after this cleavage, and plays vital roles for the interbacterial antagonism mediated by the toxic effectors. The current data is insufficient to confirm that SrtB catalyzes NCWB to produce a cell wall anchoring motif, while their interaction may facilitate the Rhs1&2-barrel to traverse the peptidoglycan layer and deliver the encapsulated C-terminal toxins. This discovery underscores the diversity of mechanisms delivering Rhs/WapA polymorphic toxins, and their roles competing with host microbiota for an optimal colonization during bacterial infection.