Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development.
Drug-induced liver injury (DILI) accounts for 20-40% of all instances of clinical hepatic failure and is a common reason for withdrawal of an approved drug or discontinuation of a potentially new drug from clinical/nonclinical development. Numerous individual risk factors contribute to the susceptibility to human DILI and its severity that are either compound-and/or patient-specific. Compound-specific primary mechanisms linked to DILI include: cytotoxicity, reactive metabolite formation, inhibition of bile salt export pump (BSEP), and mitochondrial dysfunction. Since BSEP is an energydependent protein responsible for the efflux of bile acids from hepatocytes, it was hypothesized that humans exposed to drugs that impair both mitochondrial energetics and BSEP functional activity are more sensitive to more severe manifestations of DILI than drugs that only have a single liability factor. As annotated in the United States National Center for Toxicological Research Liver Toxicity Knowledge Base (NCTR-LTKB), the inhibitory properties of 24 Most-DILI-, 28 Less-DILI-, and 20 No-DILIconcern drugs were investigated. Drug potency for inhibiting BSEP or mitochondrial activity was generally correlated across human DILI concern categories. However, drugs with dual potency as mitochondrial and BSEP inhibitors were highly associated with more severe human DILI, more restrictive product safety labeling related to liver injury, and appear more sensitive to the drug exposure (Cmax) where more restrictive labeling occurs. Conclusion: These data affirm that severe manifestations of human DILI are multifactorial, highly associated with combinations of drug potency specifically related to known mechanisms of DILI (like mitochondrial and BSEP inhibition), and, along with patient-specific factors, lead to differences in the severity and exposure thresholds associated with clinical DILI. (HEPATOLOGY 2014;60:1015-1022
Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.
Mitochondrial dysfunction has been implicated as an important factor in the development of idiosyncratic organ toxicity. An ability to predict mitochondrial dysfunction early in the drug development process enables the deselection of those drug candidates with potential safety liabilities, allowing resources to be focused on those compounds with the highest chance of success to the market. A database of greater than 2000 compounds was analyzed to identify structural and physicochemical features associated with the uncoupling of oxidative phosphorylation (herein defined as an increase in basal respiration). Many toxicophores associated with potent uncoupling activity were identified, and these could be divided into two main mechanistic classes, protonophores and redox cyclers. For the protonophores, potent uncoupling activity was often promoted by high lipophilicity and apparent stabilization of the anionic charge resulting from deprotonation of the protonophore. The potency of redox cyclers did not appear to be prone to variations in lipophilicity. Only 11 toxicophores were of sufficient predictive performance that they could be incorporated into a structural-alert model. Each alert was associated with one of three confidence levels (high, medium, and low) depending upon the lipophilicity-activity profile of the structural class. The final model identified over 68% of those compounds with potent uncoupling activity and with a value for specificity above 99%. We discuss the advantages and limitations of this approach and conclude that although structural alert methodology is useful for identifying toxicophores associated with mitochondrial dysfunction, they are not a replacement for the mitochondrial dysfunction assays in early screening paradigms.
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