Bioinformatic discovery of type 11 secretion system (T11SS) cargo across the .

Type 11 secretion systems (T11SS) are broadly distributed amongst , with more than 3,000 T11SS family outer membrane proteins (OMPs) comprising ten major sequence similarity network clusters. Of these, only seven, all from animal-associated cluster 1, have been experimentally verified as secretins of cargo, including adhesins, haemophores and metal-binding proteins. To identify novel cargo of a more diverse set of T11SS, we identified gene families co-occurring in gene neighbourhoods with either cluster 1 or marine microbe-associated cluster 3 T11SS OMP genes. We developed bioinformatic controls to ensure that perceived co-occurrences are specific to T11SS, and not general to OMPs. We found that both cluster 1 and cluster 3 T11SS OMPs frequently co-occur with single-carbon metabolism and nucleotide synthesis pathways, but that only cluster 1 T11SS OMPs had significant co-occurrence with metal and haem pathways, as well as with mobile genetic islands, potentially indicating the diversified function of this cluster. Cluster 1 T11SS co-occurrences included 2,556 predicted cargo proteins, unified by the presence of a C-terminal -barrel domain, which fall into 141 predicted UniRef50 clusters and approximately ten different architectures: four similar to known cargo and six uncharacterized types. We experimentally demonstrate T11SS-dependent secretion of an uncharacterized cargo type with homology to plasmin-sensitive protein. Unexpectedly, genes encoding marine cluster 3 T11SS OMPs only rarely co-occurred with the C-terminal -barrel domain and instead frequently co-occurred with DUF1194-containing genes. Overall, our results show that with sufficiently large-scale and controlled genomic data, T11SS-dependent cargo proteins can be accurately predicted.