Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause ...of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.
Summary Background Ipilimumab is an approved treatment for patients with advanced melanoma. We aimed to assess ipilimumab as adjuvant therapy for patients with completely resected stage III melanoma ...at high risk of recurrence. Methods We did a double-blind, phase 3 trial in patients with stage III cutaneous melanoma (excluding lymph node metastasis ≤1 mm or in-transit metastasis) with adequate resection of lymph nodes (ie, the primary cutaneous melanoma must have been completely excised with adequate surgical margins) who had not received previous systemic therapy for melanoma from 91 hospitals located in 19 countries. Patients were randomly assigned (1:1), centrally by an interactive voice response system, to receive intravenous infusions of 10 mg/kg ipilimumab or placebo every 3 weeks for four doses, then every 3 months for up to 3 years. Using a minimisation technique, randomisation was stratified by disease stage and geographical region. The primary endpoint was recurrence-free survival, assessed by an independent review committee, and analysed by intention to treat. Enrollment is complete but the study is ongoing for follow-up for analysis of secondary endpoints. This trial is registered with EudraCT, number 2007-001974-10, and ClinicalTrials.gov , number NCT00636168. Findings Between July 10, 2008, and Aug 1, 2011, 951 patients were randomly assigned to ipilimumab (n=475) or placebo (n=476), all of whom were included in the intention-to-treat analyses. At a median follow-up of 2·74 years (IQR 2·28–3·22), there were 528 recurrence-free survival events (234 in the ipilimumab group vs 294 in the placebo group). Median recurrence-free survival was 26·1 months (95% CI 19·3–39·3) in the ipilimumab group versus 17·1 months (95% CI 13·4–21·6) in the placebo group (hazard ratio 0·75; 95% CI 0·64–0·90; p=0·0013); 3-year recurrence-free survival was 46·5% (95% CI 41·5–51·3) in the ipilimumab group versus 34·8% (30·1–39·5) in the placebo group. The most common grade 3–4 immune-related adverse events in the ipilimumab group were gastrointestinal (75 16% vs four <1% in the placebo group), hepatic (50 11% vs one <1%), and endocrine (40 8% vs none). Adverse events led to discontinuation of treatment in 245 (52%) of 471 patients who started ipilimumab (182 39% during the initial treatment period of four doses). Five patients (1%) died due to drug-related adverse events. Five (1%) participants died because of drug-related adverse events in the ipilimumab group; three patients died because of colitis (two with gastrointestinal perforation), one patient because of myocarditis, and one patient because of multiorgan failure with Guillain-Barré syndrome. Interpretation Adjuvant ipilimumab significantly improved recurrence-free survival for patients with completely resected high-risk stage III melanoma. The adverse event profile was consistent with that observed in advanced melanoma, but at higher incidences in particular for endocrinopathies. The risk–benefit ratio of adjuvant ipilimumab at this dose and schedule requires additional assessment based on distant metastasis-free survival and overall survival endpoints to define its definitive value. Funding Bristol-Myers Squibb.
Human papillomavirus 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique among viruses, uptake occurs ...asynchronously over a protracted period of time, with half-times between 9 and 12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPGs). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage, respectively, of the L2 N terminus contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications accounted for only 30 to 50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid.
HPVs are the main cause of anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicate that perhaps a unique niche for initial infection that could allow for rapid infection exists. In addition, our results may help to develop novel, preventive antiviral measures.
Intestinal pathogenic Escherichia coli are a major cause of worldwide morbidity and mortality. Currently seven intestinal pathovars are recognized causing a wide range of intestinal disorders that ...are sometimes associated with severe and even lethal complications. The arsenal of virulence factors is used to subvert cellular functions of the host thereby enhancing adaptation, virulence and pathogenicity. Virulence factor profiles are largely the result of the acquisition of mobile genetic elements such as prophages and pathogenicity islands. A group of highly adapted intestinal pathogenic E. coli that are characterized by the induction of 'attaching-and-effacing (A/E) lesions' have acquired a decisive pathogenicity island, the 'locus of enterocyte effacement - LEE' by horizontal gene transfer. This review focuses on recent advances in our understanding of A/E E. coli. It highlights novel functions of effector proteins, addresses the LEE flanking regions where additional genetic elements such as the LifA/Efa1 region have been identified, and points to implications for diagnostics and therapy due to the putative interconversion of A/E E. coli during infection.
Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of ...the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.
Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH ...oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.
Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that ...the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.
Type IV pili are ubiquitous bacterial motors that power surface motility. In peritrichously piliated species, it is unclear how multiple pili are coordinated to generate movement with directional ...persistence. Here we use a combined theoretical and experimental approach to test the hypothesis that multiple pili of Neisseria gonorrhoeae are coordinated through a tug-of-war. Based on force-dependent unbinding rates and pilus retraction speeds measured at the level of single pili, we build a tug-of-war model. Whereas the one-dimensional model robustly predicts persistent movement, the two-dimensional model requires a mechanism of directional memory provided by re-elongation of fully retracted pili and pilus bundling. Experimentally, we confirm memory in the form of bursts of pilus retractions. Bursts are seen even with bundling suppressed, indicating re-elongation from stable core complexes as the key mechanism of directional memory. Directional memory increases the surface range explored by motile bacteria and likely facilitates surface colonization.
Key points
We recently found that feeding healthy mice a diet with reduced levels of branched-chain amino acids (BCAAs), which are associated with insulin resistance in both humans and rodents, ...modestly improves glucose tolerance and slows fat mass gain.
In the present study, we show that a reduced BCAA diet promotes rapid fat mass loss without calorie restriction in obese mice.
Selective reduction of dietary BCAAs also restores glucose tolerance and insulin sensitivity to obese mice, even as they continue to consume a high‐fat, high‐sugar diet.
A low BCAA diet transiently induces FGF21 (fibroblast growth factor 21) and increases energy expenditure.
We suggest that dietary protein quality (i.e. the precise macronutrient composition of dietary protein) may impact the effectiveness of weight loss diets.
Obesity and diabetes are increasing problems around the world, and although even moderate weight loss can improve metabolic health, reduced calorie diets are notoriously difficult to sustain. Branched‐chain amino acids (BCAAs; leucine, isoleucine and valine) are elevated in the blood of obese, insulin‐resistant humans and rodents. We recently demonstrated that specifically reducing dietary levels of BCAAs has beneficial effects on the metabolic health of young, growing mice, improving glucose tolerance and modestly slowing fat mass gain. In the present study, we examine the hypothesis that reducing dietary BCAAs will promote weight loss, reduce adiposity, and improve blood glucose control in diet‐induced obese mice with pre‐existing metabolic syndrome. We find that specifically reducing dietary BCAAs rapidly reverses diet‐induced obesity and improves glucoregulatory control in diet‐induced obese mice. Most dramatically, mice eating an otherwise unhealthy high‐calorie, high‐sugar Western diet with reduced levels of BCAAs lost weight and fat mass rapidly until regaining a normal weight. Importantly, this normalization of weight was mediated not by caloric restriction or increased activity, but by increased energy expenditure, and was accompanied by a transient induction of the energy balance regulating hormone FGF21 (fibroblast growth factor 21). Consumption of a Western diet reduced in BCAAs was also accompanied by a dramatic improvement in glucose tolerance and insulin resistance. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance.
Key points
We recently found that feeding healthy mice a diet with reduced levels of branched-chain amino acids (BCAAs), which are associated with insulin resistance in both humans and rodents, modestly improves glucose tolerance and slows fat mass gain.
In the present study, we show that a reduced BCAA diet promotes rapid fat mass loss without calorie restriction in obese mice.
Selective reduction of dietary BCAAs also restores glucose tolerance and insulin sensitivity to obese mice, even as they continue to consume a high‐fat, high‐sugar diet.
A low BCAA diet transiently induces FGF21 (fibroblast growth factor 21) and increases energy expenditure.
We suggest that dietary protein quality (i.e. the precise macronutrient composition of dietary protein) may impact the effectiveness of weight loss diets.
Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% ...of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).
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