Identification of Genes Involved in Biogenesis of Outer Membrane Vesicles (OMVs) in Serovar Typhi.

Outer membrane vesicles (OMVs) are nano-sized proteoliposomes discharged from the cell envelope of Gram-negative bacteria. OMVs normally contain toxins, enzymes and other factors, and are used as vehicles in a process that has been considered a generalized, evolutionarily conserved delivery system among bacteria. Furthermore, OMVs can be used in biotechnological applications that require delivery of biomolecules, such as vaccines, remarking the importance of their study. Although it is known that serovar Typhi ( Typhi), the etiological agent of typhoid fever in humans, delivers toxins (e.g., HlyE) via OMVs, there are no reports identifying genetic determinants of the OMV biogenesis in this serovar. In the present work, and with the aim to identify genes participating in OMV biogenesis in Typhi, we screened 15,000 random insertion mutants for increased HlyE secretion. We found 9 Typhi genes (generically called genes) determining an increased HlyE secretion that were also involved in OMV biogenesis. The genes corresponded to , and (envelope stability), and (LPS synthesis), (), (synthesis and remodeling of peptidoglycan), (stress sensor serine endopeptidase) and (global transcriptional regulator). We found that Typhi Δ mutants were prone to secrete periplasmic, functional proteins with a relatively good envelope integrity. In addition, we showed that zzz genes participate in OMV biogenesis, modulating different properties such as OMV size distribution, OMV yield and OMV protein cargo.