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In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system.

ABSTRACT: Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.

SUBMITTER: Marchesini MI

PROVIDER: S-EPMC3139020 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system.

Marchesini María Inés MI Herrmann Claudia K CK Salcedo Suzana P SP Gorvel Jean-Pierre JP Comerci Diego J DJ

Cellular microbiology 20110624 8

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cell ...[more]

PMID: 21707904

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Project description: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.ImportanceMany intracellular bacterial pathogens encode specialized secretion systems that deliver effector proteins into host cells to mediate the multiple stages of their intracellular cycles. Because these intracellular events occur sequentially, classical genetic approaches cannot address the late roles that these apparatuses play, as secretion-deficient mutants cannot proceed past their initial defect. Here we have designed a functionally controllable VirB type IV secretion system (T4SS) in the bacterial pathogen Brucella abortus to decipher its temporal requirements during the bacterium's intracellular cycle in macrophages. By controlling production of the VirB11 ATPase, which energizes the T4SS, we show not only that this apparatus is required early to generate the Brucella replicative organelle but also that it contributes to completion of the bacterium's cycle and bacterial egress. Our findings expand upon the pathogenic functions of the Brucella VirB T4SS and illustrate targeting of secretion ATPases as a useful strategy to manipulate the activity of bacterial secretion systems.

Dataset Information

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system.

Publications

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system.

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