Specialized shuttle proteins recognize and address type IX secretion effectors

Members of the phylum Bacteroidota utilize the type IX secretion system (T9SS) to transport a diverse array of substrates either in the environment or onto their surface via a dedicated transport apparatus. In many species like Flavobacterium johnsoniae , the T9SS supports the secretion of specialized adhesins like SprB that promote gliding motility. T9SS substrates feature a N-terminal Sec-dependent signal peptide, enabling export to the periplasm, and a conserved C-terminal domain (CTD) that is recognized by the T9SS for translocation across the outer membrane. Following translocation, T9SS substrates are engaged by a shuttle protein, which ensures their final localization. CTDs are classified into two distinct sequence families, Type A and Type B. While Type A CTDs, which are recognized by the PorV shuttle protein, have been well characterized, the role of Type B CTDs remain less explored. The genome of F. johnsoniae encodes 12 substrates with Type B CTDs, most of which are genetically linked to genes encoding PorP/SprF-like proteins. PorP/SprF are thought to act as specialized shuttle proteins essential for the secretion of their cognate Type B substrates. In this study, we identified the shuttle protein responsible for the secretion of three orphan Type B CTDs. Our findings reveal that two conserved motifs within Type B CTDs are necessary but not sufficient for their specific recognition by cognate shuttle proteins. Moreover, we demonstrate that CTDs serve a dual purpose: they contain both a secretion signal, sufficient for recognition and secretion of substrates by the T9SS, and a targeting signal, which directs substrates to their final localization.