Brucella abortus, the intracellular causative agent of brucellosis, relies on type IV secretion system (T4SS) effector-mediated modulation of host cell functions to establish a replicative niche, the ...Brucella-containing vacuole (BCV). Brucella exploits the host's endocytic, secretory, and autophagic pathways to modulate the nature and function of its vacuole from an endocytic BCV (eBCV) to an endoplasmic reticulum (ER)-derived replicative BCV (rBCV) to an autophagic egress BCV (aBCV). A role for the host ER-associated degradation pathway (ERAD) in the B. abortus intracellular cycle was recently uncovered, as it is enhanced by the T4SS effector BspL to control the timing of aBCV-mediated egress. Here, we show that the T4SS effector BspA also interferes with ERAD, yet to promote B. abortus intracellular proliferation. BspA was required for B. abortus replication in bone marrow-derived macrophages and interacts with membrane-associated RING-CH-type finger 6 (MARCH6), a host E3 ubiquitin ligase involved in ERAD. Pharmacological inhibition of ERAD and small interfering RNA (siRNA) depletion of MARCH6 did not affect the replication of wild-type B. abortus but rescued the replication defect of a
deletion mutant, while depletion of the ERAD component UbxD8 affected replication of B. abortus and rescued the replication defect of the
mutant. BspA affected the degradation of ERAD substrates and destabilized the MARCH6 E3 ligase complex. Taken together, these findings indicate that BspA inhibits the host ERAD pathway via targeting of MARCH6 to promote B. abortus intracellular growth. Our data reveal that targeting ERAD components by type IV effectors emerges as a multifaceted theme in Brucella pathogenesis.
Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The ...bacterium Brucella abortus generates a host endoplasmic reticulum‐derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system‐mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans‐Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase‐activating Protein (GAP) ACAP1 to dysregulate Arf6‐/Rab8a‐dependent transport within the recycling endosome, which resulted in accretion of TGN‐associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.
SYNOPSIS
Host membrane transport remodeling via secreted effector proteins promotes proliferation of Brucella abortus bacteria within host cell vacuoles. Here, the effector BspF delivered by the Type IV secretion system is found to facilitate secretory vesicle rerouting by modulating Arf6/Rab8a GTPase signaling.
BspF interferes with host cell retrograde membrane transport through the tubular recycling endosome.
BspF binding to GTPase‐activating protein ACAP1 decreases Arf6/Rab8a‐dependent retrograde transport to the trans‐Golgi network (TGN).
BspF modulation of retrograde transport facilitates Syntaxin‐6‐dependent recruitment of TGN‐associated vesicles to Brucella‐containing vacuoles, and facilitates bacterial growth.
Brucella abortus effector protein BspF binding to host GTPase‐activating protein ACAP1 facilitates intracellular bacterial growth by decreasing retrograde vesicular transport to the trans‐Golgi network.
Intracellular bacterial pathogens cause many infectious diseases of public health importance, where they modulate cellular functions during their lifecycle to promote essential virulence traits such ...as proliferation, dissemination, or persistence. Despite their significance, the underlying mechanisms of these virulence strategies are rarely understood, which limits our knowledge of bacterial pathogenesis. Species of the genus Brucella are highly infectious bacteria that cause the worldwide zoonotic disease, brucellosis, and their proliferation within phagocytes depends on the expression of a Type IV Secretion System (T4SS) that delivers effector proteins into the host cell and mediates biogenesis of the replicative Brucella-containing vacuole (rBCV). Determining the functions of these individual T4SS effector proteins is central to our understanding of how Brucella generates its intracellular niche to establish a successful infection. Several T4SS effector proteins have been identified, however their functions and role during Brucella infection remains largely unknown. Here, we describe the function of four T4SS effector proteins, BspB, RicA, BspA and BspF, and how they contribute to rBCV biogenesis or intravacuolar replication of the bacterium by targeting secretory compartments. BspB and RicA engage Rab2 functions in an antagonistic manner to balance endoplasmic reticulum (ER)-to-Golgi apparatus vesicular transport to support rBCV biogenesis. BspA promotes bacterial replication in rBCVs by inhibiting ER associated degradation (ERAD) through interactions with the E3 ubiquitin ligase, MARCH6, thereby interfering with E3 ubiquitin complex function. BspF interacts with the Arf6 GTPase activating protein (GAP) ACAP1, resulting in blockage of Arf6/Rab8a-dependent retrograde transport at the recycling endosome (RE) to promote intravacuolar replication. BspF contains a Gcn5-related N-acetyltransferase domain which is important for its interaction with ACAP1 and inhibition of retrograde transport. This work provides some of the first functional insight into how multiple T4SS effector proteins target distinct secretory membrane compartments to contribute to Brucella’s infectious cycle.
Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the ...function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium
, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative
-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in
replication. Defects in rBCV biogenesis and
replication caused by deletion of
were dependent on the host GTPase Rab2a and suppressed by the deletion of
, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and
intracellular replication were abolished upon deletion of both
and
, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the
intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA's functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs
replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors.
Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium
delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.
ABSTRACT Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. ...While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors. IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.
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