Can miRNA Indicate Risk of Illness after Continuous Exposure to M. tuberculosis?

Simple SummaryTuberculosis is the leading cause of mortality from a single infectious agent and is among the top 10 causes of death worldwide. Despite that, few studies focus on regulatory elements such as small non-coding RNAs in tuberculosis. This pilot work applied Next Generation Sequencing techniques to evaluate the global miRNA expression profile of patients with active tuberculosis; their respective healthy physicians, who are at constant risk of infection; and a second group of healthy controls. In addition, we observed miRNA–gene interactions affected by exposure to the bacteria. Our findings indicate a list of miRNAs that could be used as potential biomarkers to improve treatment strategies at early stages. We also observed modified pathways related to the immune response due to differential miRNA expression profiles. Finally, we alert and encourage the development of new strategies to avoid long-term exposure of healthy physicians, considering how closely related their miRNA profile was to tuberculosis patients using current safety protocols.AbstractThe role of regulatory elements such as small ncRNAs and their mechanisms are poorly understood in infectious diseases. Tuberculosis is one of the oldest infectious diseases of humans and it is still a challenge to prevent and treat. Control of the infection, as well as its diagnosis, are still complex and current treatments used are linked to several side effects. This study aimed to identify possible biomarkers for tuberculosis by applying NGS techniques to obtain global miRNA expression profiles from 22 blood samples of infected patients with tuberculosis (n = 9), their respective healthy physicians (n = 6) and external healthy individuals as controls (n = 7). Samples were run through a pipeline consisting of differential expression, target genes, gene set enrichment and miRNA–gene network analyses. We observed 153 altered miRNAs, among which only three DEmiRNAs (hsa-let-7g-5p, hsa-miR-486-3p and hsa-miR-4732-5p) were found between the investigated patients and their respective physicians. These DEmiRNAs are suggested to play an important role in granuloma regulation and their immune physiopathology. Our results indicate that miRNAs may be involved in immune modulation by regulating gene expression in cells of the immune system. Our findings encourage the application of miRNAs as potential biomarkers for tuberculosis.     

Comparative solution properties of cyclocopolymers having cationic,anionic, zwitterionic and zwitterionic/anionic backbones of similar degree of polymerization

A new class of ionic copolymers containing amine and phosphonate functionalities has been prepared for the first time. N,N-Diallyl-3-(Diethylphosphonato)-methylammonium chloride underwent cyclocopolymerization with SO₂ to give a cationic polyelectrolyte (CPE) in very good yields. Likewise, ethyl 3-(N,N-diallylammonio)methanephosphonate/SO₂ cyclopolymerization afforded the corresponding polyzwitterion-ester (PZE), which on neutralization with NaOH gave the anionic polyelectrolyte (APE). Both CPE and PZE on hydrolysis of the phosphonate ester functionalities gave the same polyzwitterionic acid (PZA). The pH-responsive PZA on treatment with excess NaOH was converted to dianionic polyelectrolyte (DAPE) which on treatment with 1 equivalent HCl afforded the zwitterionic-anionic polyelectrolyte (ZAPE). Solution properties of the CPE, PZE, DAPE, APE and ZAPE having similar degree of polymerization have been studied in some detail. The apparent basicity constants of the amine group in APE have been determined. An aqueous two-phase system of polyethylene glycol-DAPE-H₂O (0.4 N NaCl) has been constructed for its potential application in selective separation of toxic metal ions since polyphosphonates are expected to be effective chelators.     

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Functions in A Calcium Dependent Manner

Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca²⁺ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca²⁺ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca²⁺ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca²⁺ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca²⁺ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca²⁺ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.     

Neutrophils in Tuberculosis: Cell Biology,Cellular Networking and Multitasking in Host Defense

Neutrophils readily infiltrate infection foci, phagocytose and usually destroy microbes. In tuberculosis (TB), a chronic pulmonary infection caused by Mycobacterium tuberculosis (Mtb), neutrophils harbor bacilli, are abundant in tissue lesions, and their abundances in blood correlate with poor disease outcomes in patients. The biology of these innate immune cells in TB is complex. Neutrophils have been assigned host-beneficial as well as deleterious roles. The short lifespan of neutrophils purified from blood poses challenges to cell biology studies, leaving intracellular biological processes and the precise consequences of Mtb–neutrophil interactions ill-defined. The phenotypic heterogeneity of neutrophils, and their propensity to engage in cellular cross-talk and to exert various functions during homeostasis and disease, have recently been reported, and such observations are newly emerging in TB. Here, we review the interactions of neutrophils with Mtb, including subcellular events and cell fate upon infection, and summarize the cross-talks between neutrophils and lung-residing and -recruited cells. We highlight the roles of neutrophils in TB pathophysiology, discussing recent findings from distinct models of pulmonary TB, and emphasize technical advances that could facilitate the discovery of novel neutrophil-related disease mechanisms and enrich our knowledge of TB pathogenesis.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

结核分枝杆菌RD1区重组11 kD蛋白的克隆表达及效力

目的在大肠杆菌中表达结核分枝杆菌RD1区11kD蛋白,并检测表达产物的效力。方法构建11kD蛋白编码基因原核表达载体,转化大肠杆菌BL21(DE3)IPTG诱导表达,离子层析柱分离纯化重组蛋白。以结核分枝杆菌致敏豚鼠进行皮肤变态反应(DTH)测定。结果结核分枝杆菌11kD蛋白在大肠杆菌中以可溶形式表达,表达蛋白占总菌体蛋白的33%以上,纯化后纯度达95%以上,并可诱导结核分枝杆菌致敏豚鼠产生迟发型超敏反应,5μg/ml11kD蛋白的效力与50IU/ml TB-PPD相当。结论重组11kD蛋白已在大肠杆菌中成功表达,并有望成为结核病皮试鉴别诊断用新试剂。     

Determination of the bound conformation of a competitive nanomolar inhibitor of mycobacterium tuberculosis type II dehydroquinase by NMR spectroscopy

The synergy between tuberculosis and the AIDS epidemic, along with the surge of multidrug-resistant isolates of M. tuberculosis, has reaffirmed tuberculosis as a primary public health threat. It is therefore necessary to discover new, safe, and more efficient antibiotics against this disease. On the other hand, mapping the dynamic interactions of inhibitors of a target protein can provide information for the development of more potent inhibitors and consequently, more potent potential drugs. In this context, the conformational binding of our previously reported nanomolar inhibitor of M. tuberculosis type II dehydroquinase, the 3-nitrophenyl derivative 1, was studied using saturation transfer difference (STD) and transferred NOESY experiments. These studies have shown that in the bound state, one conformation of those present in solution of the competitive nanomolar inhibitor 3-nitrophenyl derivative 1 is selected. In the bound conformation, the aromatic ring is slightly shifted from coplanarity, with the double bond and the nitro group of 1 oriented towards the double bond side.     

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1β in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.     

应用等位基因特异性扩增法快速检测结核分支杆菌对利福平的耐药性

目的　建立一套等位基因特异性扩增 (ASA)的检测体系 ,用于结核分支杆菌对利福平耐药性的快速检测。方法　根据结核分支杆菌野生型基因设计ASA特异性引物 ,ASAPCR扩增 39株结核杆菌利福平耐药株的rpoB基因 ,经琼脂糖凝胶电泳检测 ,进而推断利福平的耐药性 ;同时进行DNA序列测定以验证ASA法的检测结果 ,并分析上海地区结核杆菌rpoB基因的突变特点。结果　在 39株利福平耐药株中共检出 36株 ,敏感性为92 3% ;与DNA测序结果的符合率为 87.2 % ,并鉴定了 11种不同类型的 4 1种突变形式 ,其中包括 9种点突变和 2种基因缺失。结论　用等位基因特异性扩增法分析结核分支杆菌的rpoB基因突变具有较高的特异性与敏感性。该法快速、简便、可靠 ,可应用于临床利福平耐药性的检测。     

Studies of the Binding of Modest Modulators of the Human Enzyme,Sirtuin 6, by STD NMR

Pyrazinamide (PZA), an essential constituent of short‐course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite the structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 by different mechanisms and through different binding sites, as suggested by saturation transfer difference (STD) NMR. Available experimental evidence, such as that derived from crystal structures and kinetic experiments, has been of only limited utility in elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For instance, crystallographic structural analysis of sirtuin binding sites does not help us understand important differences in binding affinities among sirtuins or capture details of such dynamic process. Hence, STD NMR was utilized throughout this study. Our results not only agreed with the binding kinetics experiments but also gave a qualitative insight into the binding process. The data presented herein suggested some details about the geometry of the binding epitopes of the ligands in solution with the apo‐ and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates might be possible. These derivatives might be accessible by employing the malleability of this scaffold to assist in the identification by STD NMR of the motifs that interact with the apo‐ and holoenzymes in solution.     

Inhibition of Citrinin Production in Penicillium citrinum Cultures by Neem [Azadirachta indica A. Juss (Meliaceae)]

The efficacy of different concentrations of aqueous neem leaf extract (3.12 to 50 mg/mL) on growth and citrinin production in three isolates of Penicillium citrinum was investigated under laboratory conditions. Mycotoxin production by the isolates was suppressed, depending on the concentration of the plant extract added to culture media at the time of spore inoculation. Citrinin production in fungal mycelia grown for 21 days in culture media containing 3.12 mg/mL of the aqueous extract of neem leaf was inhibited by approximately 80% in three isolates of P. citrinum. High-performance liquid chromatography was performed to confirm the spectrophotometric results. Vegetative growth was assessed, but neem extract failed to inhibit it. Neem leaf extract showed inhibition of toxin production without retardation in fungal mycelia growth.     

Exploring the Use of Medicinal Plants and Their Bioactive Derivatives as Alveolar NLRP3 Inflammasome Regulators during Mycobacterium tuberculosis Infection

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a successful intracellular pathogen that is responsible for the highest mortality rate among diseases caused by bacterial infections. During early interaction with the host innate cells, M. tuberculosis cell surface antigens interact with Toll like receptor 4 (TLR4) to activate the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) canonical, and non-canonical inflammasome pathways. NLRP3 inflammasome activation in the alveoli has been reported to contribute to the early inflammatory response that is needed for an effective anti-TB response through production of pro-inflammatory cytokines, including those of the Interleukin 1 (IL1) family. However, overstimulation of the alveolar NLRP3 inflammasomes can induce excessive inflammation that is pathological to the host. Several studies have explored the use of medicinal plants and/or their active derivatives to inhibit excessive stimulation of the inflammasomes and its associated factors, thus reducing immunopathological response in the host. This review describes the molecular mechanism of the NLRP3 inflammasome activation in the alveoli during M. tuberculosis infection. Furthermore, the mechanisms of inflammasome inhibition using medicinal plant and their derivatives will also be explored, thus offering a novel perspective on the alternative control strategies of M. tuberculosis-induced immunopathology.     

Preliminary Evaluation of Artemisinin–Cholesterol Conjugates as Potential Drugs for the Treatment of Intractable Forms of Malaria and Tuberculosis

To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin–cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened in vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC₅₀) against Pf drug‐sensitive NF54, and drug‐resistant K1 and W2 strains ranged from 0.03–2.6, 0.03–1.9, and 0.02–1.7 μm . Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony‐forming unit (CFU) assay after 24 h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3–38 % and 18–52 % at 10 and 80 μm , respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB in vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.     

Cyclopolymerization protocol for the synthesis of a poly(zwitterion‐alt‐sulfur dioxide) to investigate the polyzwitterion‐to‐poly(anion‐zwitterion) transition

The zwitterionic monomer, 3‐(N,N‐diallyl,N‐carboethoxymethylammonio)propanesulfonate, on cocyclopolymerization with sulfur dioxide in DMSO using azoisobutyronitrile as the initiator afforded the polyzwitterion (PZ) copolymer in excellent yields. The PZ on acidic hydrolysis of the ester groups led to the corresponding polyzwitterionic acid (PZA). The pH‐responsive PZA on treatment with sodium hydroxide gave the new poly(eletrolyte‐zwitterion) (PEZ). The solubility, viscosity behaviors, and solution properties of the salt‐tolerant PZ, PZA, and PEZ were studied in detail. Like common PZs, PZ was found to be insoluble in salt‐free but soluble in salt‐added water. The apparent basicity constants of the carboxyl group in PEZ have been determined. As the name implies, the PEZ possesses dual type of structural feature common to both conventional anionic polyelectrolytes and PZs, and its aqueous solution behavior is found to be similar to that observed for a typical alternating anionic‐zwitterionic copolymer. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Bis[3‐(diethoxyphosphoryl)propyl]diallylammonium chloride: Synthesis and use of its cyclopolymer as an antiscalant

A new symmetrically substituted cationic monomer bis[3‐(diethoxyphosphoryl)propyl]diallylammonium chloride has been synthesized and cyclopolymerized to give the corresponding cationic polyelectrolyte (+) (CPE) bearing two identical (diethoxyphosphoryl)propyl penedents on the pyrrolidinium repeating units. The hydrolysis of the phosphonate ester in (+) (CPE) gave a pH‐responsive cationic polyacid (+) (CPA) bearing the motifs of a tetrabasic acid. The (+) (CPA) under pH‐induced transformation was converted into a water‐insoluble polyzwitterion acid (±) (PZA) or water‐soluble polyzwitterion/monoanion (± ?) (PZMAN) or polyzwitterion/dianion (± =) (PZDAN) or polyzwitterion/trianion (± ≡) (PZTAN), all having identical degree of polymerization. The interesting solubility and viscosity behaviors of the polymers have been investigated in some detail. The apparent protonation constants of the anionic centers in (± ≡) (PZTAN) and its corresponding monomer (± ≡) (ZTAN) have been determined. Evaluation of antiscaling properties of the PZA using supersaturated solutions of CaSO₄ revealed ≈100% scale inhibition efficiency at a meager concentration of 10 ppm for a duration over 71 h at 40°C. The PZA has the potential to be an effective antiscalant in Reverse Osmosis plants. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40615.     

Inhibition Studies of Mycobacterium tuberculosis Salicylate Synthase (MbtI)

Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate‐utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate‐based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI‐catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3‐dihydroxybenzoate scaffold, proved to be low‐micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol‐pyruvyl side chain found in chorismate and isochorismate.     

Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System**

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 ( 1 ) and Tyrphostin AG538 ( 2 ). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.     

Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci

The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species. Linezolid was the most effective drug in inhibiting staphylococci in the biofilm, without an increase in the MIC, when compared to planktonic cells. None of the isolates were resistant to this drug.