Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses.

Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC's ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC's response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

A Survey of Multigenic Protein-Altering Variant Frequency in Familial Exudative Vitreo-Retinopathy (FEVR) Patients by Targeted Sequencing of Seven FEVR-Linked Genes.

While Inherited Retinal Diseases (IRDs) are typically considered rare diseases, Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie Disease (ND) are more rare than retinitis pigmentosa. We wanted to determine if multigenic protein-altering variants are common in FEVR subjects within a set of FEVR-related genes. The potential occurrence of protein-altering variants in two different genes has been documented in a very small percentage of patients, but potential multigenic contributions to FEVR remain unclear. Genes involved in these orphan pediatric retinal diseases are not universally included in available IRD targeted-sequencing panels, and cost is also a factor limiting multigenic-sequence-based testing for these rare conditions. To provide an accurate solution at lower cost, we developed a targeted-sequencing protocol that includes seven genes involved in Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie disease. Seventy-six DNA samples from persons refered to clinic with possible FEVR and some close relatives were sequenced using a novel Oakland-ERI orphan pediatric retinal disease panel (version 2) providing 900 times average read coverage. The seven genes involved in FEVR/ND were: NDP (ChrX), CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11). A total of 33 variants were found that alter protein sequence, with the following relative distribution: LRP5 13/33 (40%), FZD4 9/33 (27%), ZNF408 6/33 (18%), (KIF11 3/33 (9%), NDP 1/33 (3%), CTNNB1 1/33 (3%). Most protein-altering variants, 85%, were found in three genes: FZD4, LRP5, and ZNF408. Four previously known pathogenic variants were detected in five families and two unrelated individuals. Two novel, likely pathogenic variants were detected in one family (FZD4: Cys450ter), and a likely pathogenic frame shift termination variant was detected in one unrelated individual (LRP5: Ala919CysfsTer67). The average number of genes with protein-altering variants was greater in subjects with confirmed FEVR (1.46, n = 30) compared to subjects confirmed unaffected by FEVR (0.95, n = 20), (p = 0.009). Thirty-four percent of persons sequenced had digenic and trigenic protein-altering variants within this set of FEVR genes, which was much greater than expected in the general population (3.6%), as derived from GnomAD data. While the potential contributions to FEVR are not known for most of the variants in a multigenic context, the high multigenic frequency suggests that potential multigenic contributions to FEVR severity warrant future investigation. The targeted-sequencing format developed will support such exploration by reducing the testing cost to $250 (US) for seven genes and facilitating greater access to genetic testing for families with this very rare inherited retinal disease.

Systemic Mastocytosis: A Rare Cause of Diarrhea.

Mastocytosis is a spectrum of neoplastic, clonal cell disorders that are characterized by mast cell hyperplasia and accumulation. Disease and clinical presentation can vary depending on the extent of spread, ranging from skin-limited cutaneous mastocytosis to systemic mastocytosis that can mimic other disease processes. Symptoms may include pruritus, flushing, hypotension, headaches, abdominal pain, nausea, vomiting, and diarrhea. Although gastrointestinal (GI) symptoms are present in a majority of patients with systemic disease, the actual percentage of gut mast cell infiltration remains unknown. Here we describe a case of diarrhea secondary to GI involvement of systemic mastocytosis. A 55-year-old woman with a known history of systemic mastocytosis and medical noncompliance complained of persistent chronic diarrhea for one year. She was evaluated for other causes of diarrhea but all additional testing was unrevealing. She ultimately underwent upper endoscopy and colonoscopy in which biopsy and histologic analysis confirmed the presence of mastocyte infiltration. She was restarted on her medical therapy and her symptoms resolved. In conclusion, systemic mastocytosis is an uncommon cause of chronic diarrhea. However, in select patients, it is important to obtain a thorough medical history and exclude other potential causes.

Master Sculptor at Work: Enteropathogenic Escherichia coli Infection Uniquely Modifies Mitochondrial Proteolysis during Its Control of Human Cell Death.

Enteropathogenic Escherichia coli (EPEC) causes severe diarrheal disease and is present globally. EPEC virulence requires a bacterial type III secretion system to inject >20 effector proteins into human intestinal cells. Three effectors travel to mitochondria and modulate apoptosis; however, the mechanisms by which effectors control apoptosis from within mitochondria are unknown. To identify and quantify global changes in mitochondrial proteolysis during infection, we applied the mitochondrial terminal proteomics technique mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS). MS-TAILS identified 1,695 amino N-terminal peptides from 1,060 unique proteins and 390 N-terminal peptides from 215 mitochondrial proteins at a false discovery rate of 0.01. Infection modified 230 cellular and 40 mitochondrial proteins, generating 27 cleaved mitochondrial neo-N termini, demonstrating altered proteolytic processing within mitochondria. To distinguish proteolytic events specific to EPEC from those of canonical apoptosis, we compared mitochondrial changes during infection with those reported from chemically induced apoptosis. During infection, fewer than half of all mitochondrial cleavages were previously described for canonical apoptosis, and we identified nine mitochondrial proteolytic sites not previously reported, including several in proteins with an annotated role in apoptosis, although none occurred at canonical Asp-Glu-Val-Asp (DEVD) sites associated with caspase cleavage. The identification and quantification of novel neo-N termini evidences the involvement of noncaspase human or EPEC protease(s) resulting from mitochondrial-targeting effectors that modulate cell death upon infection. All proteomics data are available via ProteomeXchange with identifier PXD016994IMPORTANCE To our knowledge, this is the first study of the mitochondrial proteome or N-terminome during bacterial infection. Identified cleavage sites that had not been previously reported in the mitochondrial N-terminome and that were not generated in canonical apoptosis revealed a pathogen-specific strategy to control human cell apoptosis. These data inform new mechanisms of virulence factors targeting mitochondria and apoptosis during infection and highlight how enteropathogenic Escherichia coli (EPEC) manipulates human cell death pathways during infection, including candidate substrates of an EPEC protease within mitochondria. This understanding informs the development of new antivirulence strategies against the many human pathogens that target mitochondria during infection. Therefore, mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS) is useful for studying other pathogens targeting human cell compartments.

A Unified Strategy for the Syntheses of the Isoquinolinium Alkaloids Berberine, Coptisine, and Jatrorrhizine.

Total syntheses of the antibacterial alkaloids berberine, coptisine, and jatrorrhizine have been achieved in four steps through a unified route. The key step of this strategy is an efficient intramolecular Friedel-Crafts alkoxyalkylation which, following oxidation, establishes the isoquinolinium core of these natural products. Herein, the design and development of this synthetic strategy, which has enabled the shortest and most efficient syntheses of these alkaloids reported to date, is described.

Thermodynamic signatures and cluster properties of self-assembly in systems with competing interactions.

Colloidal particles, amphiphiles and functionalized nanoparticles are examples of systems that frequently exhibit short-range attraction coupled with long-range repulsion. We vary the ratio of attraction and repulsion in a simple isotropic model with competing interactions, using molecular simulations, and observe significant differences in the properties of the self-assembled clusters that form. We report conditions that lead to the self-assembly of clusters of a preferred size, accompanied by a change in the slope of the pressure with respect to density, similar to micelles formed by amphiphilic molecules. We also report conditions where repulsion dominates, clusters of a preferred size form and the pressure vs. density slope is unaffected by self-assembly. We investigate cluster structure by calculating the size distributions, free colloid density, cluster shape and density profiles. The system dynamics are characterized by cluster life-times. We do not find qualitative differences in structure or dynamics of the clusters, regardless the pressure behavior. Therefore, thermodynamic and structural quantities are required to classify the different clustering characteristics that are observable in systems with competing interactions. Our results have implications in terms of development of design principles for stable cluster self-assembly.

Assembly, structure, function and regulation of type III secretion systems.

Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.

Quantitative Mass Spectrometry Identifies Novel Host Binding Partners for Pathogenic Escherichia coli Type III Secretion System Effectors.

Enteropathogenic and enterohemorrhagic Escherichia coli cause enteric diseases resulting in significant morbidity and mortality worldwide. These pathogens remain extracellular and translocate a set of type III secreted effector proteins into host cells to promote bacterial virulence. Effectors manipulate host cell pathways to facilitate infection by interacting with a variety of host targets, yet the binding partners and mechanism of action of many effectors remain elusive. We performed a mass spectrometry screen to identify host targets for a library of effectors. We found five known effector targets and discovered four novel interactions. Interestingly, we identified multiple effectors that interacted with the microtubule associated protein, ensconsin. Using co-immunoprecipitations, we confirmed that NleB1 and EspL interacted with ensconsin in a region that corresponded to its microtubule binding domain. Ensconsin is an essential cofactor of kinesin-1 that is required for intracellular trafficking, and we demonstrated that intracellular trafficking was severely disrupted during wild type EPEC infections but not during infections with ΔnleB1 or ΔespL mutants. Our findings demonstrate the efficacy of quantitative proteomics for identifying effector-host protein interactions and suggest that vesicular trafficking is a crucial cellular process that may be targeted by NleB1 and EspL through their interaction with ensconsin.

Determination of the critical micelle concentration in simulations of surfactant systems.

Alternative methods for determining the critical micelle concentration (cmc) are investigated using canonical and grand canonical Monte Carlo simulations of a lattice surfactant model. A common measure of the cmc is the "free" (unassociated) surfactant concentration in the presence of micellar aggregates. Many prior simulations of micellizing systems have observed a decrease in the free surfactant concentration with overall surfactant loading for both ionic and nonionic surfactants, contrary to theoretical expectations from mass-action models of aggregation. In the present study, we investigate a simple lattice nonionic surfactant model in implicit solvent, for which highly reproducible simulations are possible in both the canonical (NVT) and grand canonical (µVT) ensembles. We confirm the previously observed decrease of free surfactant concentration at higher overall loadings and propose an algorithm for the precise calculation of the excluded volume and effective concentration of unassociated surfactant molecules in the accessible volume of the solution. We find that the cmc can be obtained by correcting the free surfactant concentration for volume exclusion effects resulting from the presence of micellar aggregates. We also develop an improved method for determination of the cmc based on the maximum in curvature for the osmotic pressure curve determined from µVT simulations. Excellent agreement in cmc and other micellar properties between NVT and µVT simulations of different system sizes is observed. The methodological developments in this work are broadly applicable to simulations of aggregating systems using any type of surfactant model (atomistic/coarse grained) or solvent description (explicit/implicit).

Steered molecular dynamics study of inhibitor binding in the internal binding site in dehaloperoxidase-hemoglobin.

The binding free energy of 4-bromophenol (4-BP), an inhibitor that binds in the internal binding site in dehaloperoxidase-hemoglobin (DHP) was calculated using Molecular Dynamics (MD) methods combined with pulling or umbrella sampling. The effects of systematic changes in the pulling speed, pulling force constant and restraint force constant on the calculated potential of mean force (PMF) are presented in this study. The PMFs calculated using steered molecular dynamics (SMD) were validated by umbrella sampling (US) in the strongly restrained regime. A series of restraint force constants ranging from 1000 down to 5 kJ/(mol nm(2)) were used in SMD simulations. This range was validated using US, however noting that weaker restraints give rise to a broader sampling of configurations. This comparison was further tested by a pulling simulation conducted without any restraints, which was observed to have a value closest to the experimentally measured free energy for binding of 4-BP to DHP based on ultraviolet-visible (UV-vis) and resonance Raman spectroscopies. The protein-inhibitor system is well suited for fundamental study of free energy calculations because the DHP protein is relatively small and the inhibitor is quite rigid. Simulation configuration structures are compared to the X-ray crystallography structures of the binding site of 4-BP in the distal pocket above the heme.

Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways.

Enteric bacterial pathogens commonly use a type III secretion system (T3SS) to successfully infect intestinal epithelial cells and survive and proliferate in the host. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC; EHEC) colonize the human intestinal mucosa, form characteristic histological lesions on the infected epithelium and require the T3SS for full virulence. T3SS effectors injected into host cells subvert cellular pathways to execute a variety of functions within infected host cells. The EPEC and EHEC effectors that subvert innate immune pathways--specifically those involved in phagocytosis, host cell survival, apoptotic cell death and inflammatory signalling--are all required to cause disease. These processes are reviewed within, with a focus on recent work that has provided insights into the functions and host cell targets of these effectors.

Probing the Statistical Validity of the Ductile-to-Brittle Transition in Metallic Nanowires Using GPU Computing.

We perform a large-scale statistical analysis (>2000 independent simulations) of the elongation and rupture of gold nanowires, probing the validity and scope of the recently proposed ductile-to-brittle transition that occurs with increasing nanowire length [Wu et al. Nano Lett. 2012, 12, 910-914]. To facilitate a high-throughput simulation approach, we implement the second-moment approximation to the tight-binding (TB-SMA) potential within HOOMD-Blue, a molecular dynamics package which runs on massively parallel graphics processing units (GPUs). In a statistical sense, we find that the nanowires obey the ductile-to-brittle model quite well; however, we observe several unexpected features from the simulations that build on our understanding of the ductile-to-brittle transition. First, occasional failure behavior is observed that qualitatively differs from that predicted by the model prediction; this is attributed to stochastic thermal motion of the Au atoms and occurs at temperatures as low as 10 K. In addition, we also find that the ductile-to-brittle model, which was developed using classical dislocation theory, holds for nanowires as small as 3 nm in diameter. Finally, we demonstrate that the nanowire critical length is higher at 298 K relative to 10 K, a result that is not predicted by the ductile-to-brittle model. These results offer practical design strategies for adjusting nanowire failure and structure and also demonstrate that GPU computing is an excellent tool for studies requiring a large number of independent trajectories in order to fully characterize a system's behavior.

Critical care: assessing blood pressure, circulation and intravascular volume.

All nurses should possess core critical assessment skills in order to appropriately assess critically ill patients. It is anticipated that the possession of these core skills will enable nurses to appropriately assess and identify those patients who are at risk of deterioration. By comprehensively assessing individual patients and identifying problems early, nurses can initiate specific interventions that may stabilize and improve patient outcomes and help prevent unnecessary intensive care unit admission.