A subset of type 4 secretion system effectors of <i>Brucella</i> spp. associates to outer membrane vesicles

The establishment of a replicative niche in the hostile environment of the host presents an enormous challenge for pathogens. Intracellular pathogens such as <i>Brucella</i> spp., the Gram-negative causative agents of Brucellosis, must subvert diverse host functions to ensure survival and replication. One of the key adaptations to achieve this is the translocation of effector proteins into host cells via its type 4 secretion system (T4SS). But effector identification in <i>Brucella</i> is particularly challenging, as previously identified effectors lack a conserved translocation signal, exhibit variable requirements for translocation, and in some cases appear to be translocated in a T4SS-independent manner. Here, we demonstrate that a subset of previously described T4SS effector proteins associates with outer membrane vesicles (OMVs) in different <i>Brucella</i> species. Most of these effector proteins encode predicted signal peptides for periplasmic export or transmembrane domains. Among them, BspC and VceA carry functional signal peptides that direct their export into the periplasm in a Sec-dependent manner. From the periplasm, these proteins are subsequently secreted into the extracellular milieu, likely via the formation and release of OMVs. Our findings provide new insights into protein secretion by <i>Brucella</i>, suggesting that OMVs may represent an alternative secretion pathway to the T4SS.