The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an “obesogenic” bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.

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.

Summary The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.

Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors. IMPORTANCE Brucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion.

ABSTRACTThere are a number of genetic tools available for studyingFrancisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems forF. tularensisbased on the Tet repressor, TetR. For the inducible system, atetoperator sequence was cloned into a modifiedF. tularensis groESLpromoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,luxCDABE, was cloned under the hybridFrancisellatetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association withtetO.Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml−1. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists ofFTRpregulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with theluxreporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploringF. tularensisprotein function.

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.

Pantothenate, commonly referred to as vitamin B 5 , is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ⌬panG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ⌬panG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

bRecent literature has reported increased accuracy of Trichomonas vaginalis transcription-mediated amplification (TMA)-based analyte-specific reagent (ASR) testing in female populations. A retrospective investigation assessed 7,277 female first-void urine, cervical, or vaginal specimens submitted from a high-prevalence sexually transmitted infection (STI) community to characterize prevalence of disease etiologies. The most common STI phenotype reflected detection of solely T. vaginalis (54.2% of all health care encounters that resulted in STI detection). In females with detectable T. vaginalis, codetection of Chlamydia trachomatis and Neisseria gonorrhoeae occurred in 7.8% and 2.7% of health care encounters, respectively. The mean age of women with detectable T. vaginalis (30.6) was significantly higher than those for women with C. trachomatis or N. gonorrhoeae (22.3 and 21.6, respectively; P < 0.0001). T. vaginalis was the predominant sexually transmitted agent in women over the age of 20 (P < 0.0002). C. trachomatis was the most commonly detected agent in females under the age of 21, particularly from cervical specimens. However, first-void urine detection rates for T. vaginalis and C. trachomatis within this age demographic demonstrated no difference (P ‫؍‬ 0.92). While overall and cervical specimen-derived detection of T. vaginalis within African American majority geographical locales outweighed that within majority Caucasian geographical regions (P < 0.004), this difference was not noted with first-void urine screening (P ‫؍‬ 0.54). Health care professionals can consider TMA-based T. vaginalis screening for a wide age range of patients; incorporation of first-void urine specimens into screening algorithms can potentiate novel insight into the epidemiology of trichomoniasis.