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

Erin P Smith,Robert Child,Cheryl N Miller,Jennifer A Cundiff,Jean Celli,Howard A Shuman

doi:10.1128/mbio.01730-16

Abstract

ABSTRACTBrucella 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.

Highlights

Intracellular Gram-negative bacterial pathogens have the capacity to subvert host cell functions and generate or reach compartmentalized niches that provide them with survival, persistence, and proliferation abilities essential to their virulence
To assess the role of the VirB T4SS in postreplication stages of the Brucella intracellular cycle, we reasoned that enabling VirB T4SS activity to allow rBCV biogenesis and disabling its function to test its role in bacterial replication within rBCVs, autophagic BCVs (aBCVs) formation, and bacterial egress would circumvent the early trafficking blockage that characterizes constitutively defective virB mutant strains in which single or multiple virB genes are inactivated (2, 10, 15, 18)
We sought to generate a B. abortus strain in which VirB T4SS activity is conditional by constructing an in-frame deletion mutant in an essential virB gene and complementing it with a conditionally expressed, single chromosomal copy inserted at a secondary locus

Summary

Introduction

Intracellular Gram-negative bacterial pathogens have the capacity to subvert host cell functions and generate or reach compartmentalized niches that provide them with survival, persistence, and proliferation abilities essential to their virulence. The ability of Brucella spp. to cause disease depends on their intracellular cycle within host phagocytes, such as macrophages or dendritic cells (8, 9), in which the bacterium resides in a membrane-bound compartment called the Brucella-containing vacuole (BCV) (10) Upon their initial formation following phagocytic uptake, BCVs undergo maturation events along the endocytic pathway to become an endosomal. We have designed and generated strains of Brucella abortus in which expression of an essential virB gene depends upon the presence of the inducer anhydrotetracycline (ATc) and can be induced or repressed in a temporal manner to control functionality of the VirB T4SS Applying this approach to the VirB11 ATPase, we show that VirB T4SS expression within the eBCV is required early for optimal rBCV biogenesis and is necessary for aBCV formation and bacterial egress, uncovering novel roles of this secretion machinery in postreplication stages of the Brucella intracellular cycle

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