Bacteroidales T6SS minor Hcp subunits form heteromers recognising effectors

The type VI secretion system (T6SS) is a macromolecular protein complex found in Gram-negative bacteria that mediates intercellular antagonism and gut Bacteroidales use this system to increase their competitiveness. Furthermore, T6SS is a recognized gut colonization factor that influences gut biodiversity. Bacteroidales T6SSs are divergent from their well-characterized Pseudomonadota counterparts. One conserved feature in many Bacteroidales T6SSs is the presence of multiple Hcp proteins with low sequence similarity, encoded within the secretion system’s locus and adjacent to variable effector cassettes. Here we provide evidence that in the Bacteroides fragilis T6SS, these Hcp subunits, similar to other protein export systems function as high and low abundance major and minor subunits. First, we showed that these minor Hcp proteins form a network of interactions. Biochemical characterization of one of the minor Hcp complexes formed between Hcp1 and Hcp2 showed that they form hetero-hexamers with variable stoichiometry. Furthermore, we showed that this complex recognizes the secreted effector Bte1. Our results suggest that these minor subunits function as recognition particles for effectors to mediate secretion and that this is a conserved feature in Bacteroidales T6SS. The functionality of these Hcp-effector modules is encoded in a high-synteny region within T6SS loci, and bioinformatic analyses imply that this arrangement participates in establishing effector variability. Exploiting this feature could pave the way for characterizing numerous Bacteroidales effectors with unknown functions through copurification with their cognate Hcps, unlocking new insights into bacterial interactions and into mechanisms of gut biodiversity establishment.