BackgroundTriclosan is a widely used antimicrobial compound and emerging environmental contaminant. Although the role of the gut microbiome in health and disease is increasingly well established, the interaction between environmental contaminants and host microbiome is largely unexplored, with unknown consequences for host health. This study examined the effects of low, environmentally relevant levels of triclosan exposure on the fish gut microbiome. Developing fathead minnows (Pimephales promelas) were exposed to two low levels of triclosan over a 7-day exposure. Fish gastrointestinal tracts from exposed and control fish were harvested at four time points: immediately preceding and following the 7-day exposure and after 1 and 2 weeks of depuration.ResultsA total of 103 fish gut bacterial communities were characterized by high-throughput sequencing and analysis of the V3-V4 region of the 16S rRNA gene. By measures of both alpha and beta diversity, gut microbial communities were significantly differentiated by exposure history immediately following triclosan exposure. After 2 weeks of depuration, these differences disappear. Independent of exposure history, communities were also significantly structured by time. This first detailed census of the fathead minnow gut microbiome shows a bacterial community that is similar in composition to those of zebrafish and other freshwater fish. Among the triclosan-resilient members of this host-associated community are taxa associated with denitrification in wastewater treatment, taxa potentially able to degrade triclosan, and taxa from an unstudied host-associated candidate division.ConclusionsThe fathead minnow gut microbiome is rapidly and significantly altered by exposure to low, environmentally relevant levels of triclosan, yet largely recovers from this short-term perturbation over an equivalently brief time span. These results suggest that even low-level environmental exposure to a common antimicrobial compound can induce significant short-term changes to the gut microbiome, followed by restoration, demonstrating both the sensitivity and resilience of the gut flora to challenges by environmental toxicants. This short-term disruption in a developing organism may have important long-term consequences for host health. The identification of multiple taxa not often reported in the fish gut suggests that microbial nitrogen metabolism in the fish gut may be more complex than previously appreciated.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0069-6) contains supplementary material, which is available to authorized users.

Summary Many intracellular pathogens exploit host secretory trafficking to support their intracellular cycle, but knowledge of these pathogenic processes is limited. The bacterium Brucella abortus uses a Type IV secretion system (VirB T4SS) to generate a replication-permissive Brucella-containing vacuole (rBCV) derived from the host endoplasmic reticulum (ER), a process that requires host early secretory trafficking. Here we show that the VirB T4SS effector BspB contributes to rBCV biogenesis and Brucella replication by interacting with the Conserved Oligomeric Golgi (COG) tethering complex, a major coordinator of Golgi vesicular trafficking, thus remodeling Golgi membrane traffic and redirecting Golgi-derived vesicles to the BCV. Altogether, these findings demonstrate that Brucella modulates COG-dependent trafficking via delivery of a T4SS effector to promote rBCV biogenesis and intracellular proliferation, providing mechanistic insight into how bacterial exploitation of host secretory functions promotes pathogenesis.

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.

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.