# A subset of type 4 secretion system effectors of Brucella spp. associates to outer membrane vesicles

## TL;DR

Some Brucella proteins use outer membrane vesicles to leave the bacteria, possibly as an alternative to their main secretion system.

## Contribution

Identifies a subset of Brucella T4SS effectors that associate with outer membrane vesicles for secretion.

## Key findings

- A subset of T4SS effectors associates with outer membrane vesicles in Brucella species.
- BspC and VceA use signal peptides for periplasmic export and subsequent secretion via OMVs.
- OMVs may represent an alternative secretion pathway to the T4SS in Brucella.

## Abstract

The establishment of a replicative niche in the hostile environment of the host presents an enormous challenge for pathogens. Intracellular pathogens such as Brucella 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 Brucella 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 Brucella 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 Brucella, suggesting that OMVs may represent an alternative secretion pathway to the T4SS.

A subset of type 4 secretion system effectors of Brucella spp. can be exported to the periplasm and then secreted to the environment likely via the formation and release of outer membrane vesicles, which suggests that Brucella may use outer membrane vesicles as an alternative secretion pathway to the type 4 secretion system.