Postreplication Roles of the Brucella VirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase

Abstract

Brucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, the Brucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodeled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by the Brucella VirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of the Brucella intracellular cycle. To address this issue, we have generated a B. abortus strain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of the virB11 gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression of virB11 prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in the Brucella intracellular cycle.

Brucella abortus Senses the Intracellular Environment through the BvrR/BvrS Two-Component System, Which Allows B. abortus To Adapt to Its Replicative Niche

“…It would be interesting in the future to monitor the state of BvrR phosphorylation at late stages of the intracellular life cycle, when the bacteria have moved from the ER to autophagosome-like compartments and are ready to exit the cell (38). In fact, a VirB requirement in postreplication events has recently been demonstrated (39). Thus, it is likely that under these circumstances, bacteria again increase their competence for intracellular survival and would then activate the virulence circuit formed by BvrR/BvrS, VjbR, and VirB to initiate another cycle of replication within host cells.…”

A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication

“…For fluorescence microscopy purposes, all strains were modified to express the fluorescent protein DsRed m via integration of the miniTn7K- dsRed at the attTn 7 locus by electroporation of pUC18T-miniTn7K- dsRed (Smith et al, 2016) with the helper plasmid pUC18T-Tn7 tnp , as described previously(Myeni et al, 2013). Electroporants were selected on TSA plates containing 30 μg/ml of kanamycin, and correct insertion of mini Tn7K-dsRed was confirmed using PCR primers RC603 and RC604 (Table S1).…”

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum

“…However, both these effectors play only modulatory roles as they are not essential for rBCV expansion. Moreover, there is evidence that T4SS inactivation in bacteria residing already within an rBCV has limited influence on the rate of Brucella replication (Smith et al, 2016). Although we cannot rule out the activity of yet unidentified T4SS-independent factors, these data may suggest that, following initial entry into the ER lumen, bacteria may not need to actively induce membrane acquisition in order to expand the rBCV.…”