Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages

Salmonella enterica serovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonella virulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity). Salmonella lacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonella gene-encoded type III secretion systems (T3SSs)-Salmonella pathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonella virulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.