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.

猪链球菌（Streptococcus suis）是一种重要的人畜共患病原菌，其在宿主扁桃体的定植被认为是人类与动物感染的重要传染源，然而猪链球菌争夺稳定扁桃体定植微环境的机制仍不明晰。重排热点蛋白（Rearrangement hotspot, Rhs）的特征为含有YD肽重复序列，可折叠形成包裹C端毒素（C-terminal toxin, CT）结构域的大型β-笼状结构（β-cage structure）。在革兰氏阴性菌（Gram-negative bacteria）中，Rhs蛋白已被证实为Ⅵ型分泌系统（type VI secretion system, T6SS）分泌的多态性效应因子（polymorphic effectors）。与之相对，在革兰氏阳性菌（Gram-positive bacteria）中出现的其远缘同源物——即壁关联蛋白A（wall-associated protein A, WapA）——的转运机制仍未被完全阐明。本研究报道了分子量为350 kDa的WapA-CT1，该蛋白可通过调控扁桃体菌群促进猪链球菌定植，其与由两个上游基因编码的类SecF蛋白（SecF-like protein）以及SrtB分选酶（sortase）相关联。未折叠的WapA-CT1可在其N端Sec信号基序（Sec-signal motif）与同源类SecF蛋白的引导下，通过经典Sec分泌通路（Sec pathway）跨细胞膜转运。裂解实验（cleavage assay）鉴定出该蛋白包含四个结构片段：N端NCWB片段、两个中间Rhs结构域（Rhs domains），二者可折叠形成两侧经Lid/RAC结构域（Lid/RAC plugs）封堵的β-桶状结构（β-barrel structure），以及C端PreT-CT毒素结构域（PreT-CT toxin domain）。SrtB可在该裂解事件发生前后与NCWB区域结合，并在毒性效应因子介导的细菌间拮抗作用（interbacterial antagonism）中发挥关键作用。目前的数据尚不足以证实SrtB可催化NCWB区域生成细胞壁锚定基序（cell wall anchoring motif），但二者的相互作用或许可助力Rhs1与Rhs2桶状结构穿过肽聚糖层（peptidoglycan layer），并释放其包裹的C端毒素。该研究成果凸显了Rhs/WapA型多态性毒素（Rhs/WapA polymorphic toxins）转运机制的多样性，以及其在细菌感染过程中与宿主菌群竞争最优定植微环境的重要作用。