In‐Cell NMR Spectroscopy of Intrinsically Disordered Proteins

This review summarizes the results of in‐cell Nuclear Magnetic Resonance, NMR, spectroscopic investigations of the eukaryotic and prokaryotic intrinsically disordered proteins, IDPs: α‐synuclein, prokaryotic ubiquitin‐like protein, Pup, tubulin‐related neuronal protein, Tau, phenylalanyl‐glycyl‐repeat‐rich nucleoporins, FG Nups, and the negative regulator of flagellin synthesis, FlgM. The results show that the cellular behavior of IDPs may differ significantly from that observed in the test tube.     

5-Chloro-2-thiophenyl-1,2,3-triazolylmethyldihydroquinolines as dual inhibitors of Mycobacterium tuberculosis and influenza virus: Synthesis and evaluation

This study describes synthesis and evaluation of novel 5-Chloro-2-thiophenyl-1,2,3-triazolylmethyldihydroquinolines 7a-o as dual inhibitors of Mycobacterium tuberculosis and influenza virus. Huisgen’s [3+2] dipolar cycloaddition of 6-(azidomethyl)-5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline 5 with various alkynes 6a-o using sodium ascorbate and copper sulphate gave new dihydroquinoline-1,2,3-triazoles 7a-o in good to excellent yields. The new compounds were evaluated for in vitro antimycobacterial against M. tuberculosis H37Rv (Mtb) and antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1). Among the fifteen new analogs, compounds 7a (MIC: 3.12 µg/mL), 7j and 7k (MIC: 6.25 µg/mL) were identified as potent antitubercular agents. The virus-inhibiting activity of all the fifteen compounds was found to be moderate, and among them the compound 7l, bearing thiophene moiety appeared the most active with good selectivity index (IC₅₀ = 19.5 µg/mL; SI = 15). The results presented here will help developing newer dual inhibitors of tuberculosis and influenza virus.     

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

结核分枝杆菌多表位诊断抗原的制备

将结核分枝杆菌(Mycobacterium tuberculosis,Mtb)多个B细胞预测表位串联表达(命名为B102),并初步评价其作为诊断抗原的血清学诊断价值。将MtbPstS1、ESAT6、CFP10、Ag85B、Ag85A及PPE54等6个蛋白的11个B细胞预测表位串联,加入合适的连接臂后全基因合成;将多表位片段插入带有TRX标签的表达质粒中,在大肠杆菌BL21(DE3)中诱导表达,并利用Ni~(2+)-Chelating亲和层析和DEAE阴离子交换层析纯化目的蛋白;利用Western blotting (WB)技术对目的蛋白抗原性进行鉴定,并建立Mtb抗体检测竞争法ELISA技术,初步评价此方法对阴阳血清样本的鉴别能力。目的蛋白以包涵体形式存在,其表达量约占菌体总蛋白的31.25%,经纯化及复性后蛋白B102可溶性存在,浓度为3.124mg/mL,纯度为96.71%;WB实验表明目的蛋白能与Mtb阳性血清相应抗体发生反应。对60份Mtb阳性血清及60份Mtb阴性血清进行检测得出其灵敏度为90.00%,特异性为93.33%,阳性预测值为93.10%,阴性预测值为90.32%,符合率为91.67%,McNemer检验的结果提示与"金标准"诊断结果无差异,Kappa=0.833,提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的Mtb多表位诊断抗原,作为诊断抗原可以应用于Mtb的血清学检测中。     

Dispersal patterns in a medium‐density Irish badger population: Implications for understanding the dynamics of tuberculosis transmission

European badgers (Meles meles) are group‐living mustelids implicated in the spread of bovine tuberculosis (TB) to cattle and act as a wildlife reservoir for the disease. In badgers, only a minority of individuals disperse from their natal social group. However, dispersal may be extremely important for the spread of TB, as dispersers could act as hubs for disease transmission. We monitored a population of 139 wild badgers over 7 years in a medium‐density population (1.8 individuals/km²). GPS tracking collars were applied to 80 different individuals. Of these, we identified 25 dispersers, 14 of which were wearing collars as they dispersed. This allowed us to record the process of dispersal in much greater detail than ever before. We show that dispersal is an extremely complex process, and measurements of straight‐line distance between old and new social groups can severely underestimate how far dispersers travel. Assumptions of straight‐line travel can also underestimate direct and indirect interactions and the potential for disease transmission. For example, one female disperser which eventually settled 1.5 km from her natal territory traveled 308 km and passed through 22 different territories during dispersal. Knowledge of badgers' ranging behavior during dispersal is crucial to understanding the dynamics of TB transmission, and for designing appropriate interventions, such as vaccination.     

Tuberculosis Exacerbates HIV-1 Infection through IL-10/STAT3-Dependent Tunneling Nanotube Formation in Macrophages

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Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.     

Mycobacterial PE_PGRS proteins contain calcium-binding motifs with parallel beta-roll folds

The PE_PGRS family of proteins unique to mycobacteria is demonstrated to contain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel beta-roll or parallel beta-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE PGRS proteins in the light of macrophage-pathogen interaction and pathogenesis is presented.     

Exosomes in tuberculosis: Still terra incognita?

Today, diagnosis, vaccination, and treatment of tuberculosis (TB) remain major clinical challenges. Therefore, an introduction of new diagnostic measures and biomarkers is necessary to improve infection control. The ideal biomarker for TB infection can be defined as a host or pathogen-derived biomolecule, which is potent for identifying infection and determining its clinical stage. Exosomes, defined as cell-derived nanovesicles released into biological fluids, are involved in cell–cell communication and immune modulation. These vesicles have emerged as a new platform for improving the clinical diagnosis and prognosis of different infectious diseases and cancers. The role of these nanovehicles, as alternative biomarkers for the improvement of TB diagnosis and treatment, has been demonstrated in a significant body of literature. In this review, we summarized recent progress in the clinical application of exosome-based biomarkers in TB infection.