Abstact

Type VII secretion systems (T7SSb) are widespread in Bacillota and mediate both interbacterial antagonism and host interactions through the export of diverse effector proteins, including polymorphic LXG toxins. Despite extensive variation in toxin repertoires, T7SSb systems universally secrete the small WXG100 protein EsxA, suggesting a conserved role in secretion. However, the relationship between EsxA and LXG toxin export remains unresolved. Here, we define the hierarchy and mechanistic basis of substrate export by the T7SSb of Streptococcus intermedius. We show that EsxA is required for the secretion of all LXG toxins, whereas EsxA export occurs independently of these effectors, establishing a unidirectional dependency. This requirement is not mediated by direct interaction, as EsxA does not associate with LXG toxin complexes. Instead, structural and mutational analyses reveal that EsxA forms a homodimer containing a bipartite export motif that is essential for both its own secretion and for LXG toxin export. Consistent with this, LXG toxins possess analogous composite export motifs, indicating that these substrates are independently recognized by the secretion machinery. We further show that while all four ATPase domains of the EssC secretion ATPase are required for system activity, only the Walker motifs of the D1 domain are necessary for substrate translocation. Finally, we demonstrate that EsxA export depends on EssC compatibility, indicating that EssC contributes to substrate recognition. Together, these findings establish EsxA as a conserved T7SSb substrate whose export is a prerequisite for LXG toxin secretion and define domain-specific requirements of EssC that underlie substrate recognition and translocation.