Three small partner proteins facilitate the type VII-dependent secretion of an antibacterial nuclease

The type VIIb secretion system (T7SSb) is a multisubunit protein export machine found in Gram-positive Bacillota which plays a key role in interbacterial competition. The T7SSb secretes a variety of toxic effector proteins targeting closely related strains; however, the mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by the Staphylococcus aureus T7SSb, which forms a pre-secretion complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins of unknown function: EsxB, EsxC, and EsxD. Here, we show that these three proteins bind to EsaD and function as EsaD export factors and we report preliminary structural information for a complete T7SSb substrate pre-secretion complex. Cryo-electron microscopy of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers an elongated conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export. IMPORTANCE: Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.