Structural and Biochemical Characterization of Compounds Inhibiting Mycobacterium tuberculosis Pantothenate Kinase

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.     

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Covalent Modification Cycles through the Spatial Prism

Covalent modification cycles are basic units and building blocks of posttranslational modification and cellular signal transduction. We systematically explore different spatial aspects of signal transduction in covalent modification cycles by starting with a basic temporal cycle as a reference and focusing on steady-state signal transduction. We consider, in turn, the effect of diffusion on spatial signal transduction, spatial analogs of ultrasensitive behavior, and the interplay between enzyme localization and substrate diffusion. Our analysis reveals the need to explicitly account for kinetics and diffusional transport (and localization) of enzymes, substrates, and complexes. It demonstrates a complex and subtle interplay between spatial heterogeneity, diffusion, and localization. Overall, examining the spatial dimension of covalent modification reveals that 1), there are important differences between spatial and temporal signal transduction even in this cycle; and 2), spatial aspects may play a substantial role in affecting and distorting information transfer in modules/networks that are usually studied in purely temporal terms. This has important implications for the systematic understanding of signaling in covalent modification cycles, pathways, and networks in multiple cellular contexts.     

Interactions of allelic variance of PNPLA3 with nongenetic factors in predicting nonalcoholic steatohepatitis and nonhepatic complications of severe obesity

Objective : Allelic variation (rs738409C→G) in adiponutrin (patatin‐like phospholipase domain‐containing protein 3, PNPLA3) has been associated with hepatic steatosis and liver fibrosis. The physiologic impact of the PNPLA3 G allele may be exacerbated in patients with severe obesity. In this study, we investigated the interactions of PNPLA3 rs738409 with a broad panel of metabolic and histologic characteristics of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) in patients with medically complicated obesity. Design and Methods : Consecutive patients undergoing bariatric surgery were selected for a prospective study. They underwent extensive laboratory and histologic (liver biopsy) assessment, as well as evaluation of rs738409 polymorphism by TaqMan assay. Results : Only 12 (8.3%) of the 144 patients had normal liver histology, with 72 (50%) NASH, of whom 15 (10.4% of total patients) had fibrosis stage 2‐3. PNPLA3 GG genotype correlated positively (P < 0.05) with serum levels of alanine aminotransferase (ALT), asparate aminotransferase (AST), glucose, fibrinogen, and insulin‐dependent diabetes mellitus, homeostasis model assessment—insulin resistance, and presence of NASH. Multivariate analysis indicated that PNPLA3 rs738409 G versus C allele remained an (independent) risk factor for NASH, in addition to CK‐18 >145 IU/l, glucose >100 mg/dl, and C‐reactive protein (CRP) >0.8 mg/dl. The probability of NASH increased from 9% (no risk factor) to 82% if all four risk factors were present. Conclusions : In this cohort of patients with medically complicated obesity, PNPLA3 rs738409 G allelic expression is associated with hepatic (NASH) and nonhepatic complications of obesity, such as insulin resistance. These novel findings may be related to a greater impact of PNPLA3 variant in magnitude and scope in patients with severe obesity than in less obese populations. Further studies are needed to characterize the nature of these associations.     

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin‐induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label‐free LC‐MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO‐P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal‐regulated kinase (ERK) were identified to be key mediators of pro‐ and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO‐P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.     

How Do Urban Indian Private Practitioners Diagnose and Treat Tuberculosis? A Cross-Sectional Study in Chennai

Setting

Private practitioners are frequently the first point of healthcare contact for patients with tuberculosis (TB) in India. Inappropriate TB management practices among private practitioners may contribute to delayed TB diagnosis and generate drug resistance. However, these practices are not well understood. We evaluated diagnostic and treatment practices for active TB and benchmarked practices against International Standards for TB Care (ISTC) among private medical practitioners in Chennai.

Design

A cross-sectional survey of 228 practitioners practicing in the private sector from January 2014 to February 2015 in Chennai city who saw at least one TB patient in the previous year. Practitioners were randomly selected from both the general community and a list of practitioners who referred patients to a public-private mix program for TB treatment in Chennai. Practitioners were interviewed using standardized questionnaires.

Results

Among 228 private practitioners, a median of 12 (IQR 4–28) patients with TB were seen per year. Of 10 ISTC standards evaluated, the median of standards adhered to was 4.0 (IQR 3.0–6.0). Chest physicians reported greater median ISTC adherence than other MD and MS practitioners (score 7.0 vs. 4.0, P<0.001), or MBBS practitioners (score 7.0 vs. 4.0, P<0.001). Only 52% of all practitioners sent >5% of patients with cough for TB testing, 83% used smear microscopy for diagnosis, 33% monitored treatment response, and 22% notified TB cases to authorities. Of 228 practitioners, 68 reported referring all patients with new pulmonary TB for treatment, while 160 listed 27 different regimens; 78% (125/160) prescribed a regimen classified as consistent with ISTC. Appropriate treatment practices differed significantly between chest physicians and other MD and MS practitioners (54% vs. 87%, P<0.001).

Conclusion

TB management practices in India’s urban private sector are heterogeneous and often suboptimal. Private providers must be better engaged to improve diagnostic capacity and decrease TB transmission in the community.     

Binding Cooperativity Matters: A GM1-Like Ganglioside-Cholera Toxin B Subunit Binding Study Using a Nanocube-Based Lipid Bilayer Array

Protein-glycan recognition is often mediated by multivalent binding. These multivalent bindings can be further complicated by cooperative interactions between glycans and individual glycan binding subunits. Here we have demonstrated a nanocube-based lipid bilayer array capable of quantitatively elucidating binding dissociation constants, maximum binding capacity, and binding cooperativity in a high-throughput format. Taking cholera toxin B subunit (CTB) as a model cooperativity system, we studied both GM₁ and GM₁-like gangliosides binding to CTB. We confirmed the previously observed CTB-GM₁ positive cooperativity. Surprisingly, we demonstrated fucosyl-GM₁ has approximately 7 times higher CTB binding capacity than GM₁. In order to explain this phenomenon, we hypothesized that the reduced binding cooperativity of fucosyl-GM₁ caused the increased binding capacity. This was unintuitive, as GM₁ exhibited higher binding avidity (16 times lower dissociation constant). We confirmed the hypothesis using a theoretical stepwise binding model of CTB. Moreover, by taking a mixture of fucosyl-GM₁ and GM₂, we observed the mild binding avidity fucosyl-GM₁ activated GM₂ receptors enhancing the binding capacity of the lipid bilayer surface. This was unexpected as GM₂ receptors have negligible binding avidity in pure GM₂ bilayers. These unexpected discoveries demonstrate the importance of binding cooperativity in multivalent binding mechanisms. Thus, quantitative analysis of multivalent protein-glycan interactions in heterogeneous glycan systems is of critical importance. Our user-friendly, robust, and high-throughput nanocube-based lipid bilayer array offers an attractive method for dissecting these complex mechanisms.