Insight into Structure-Function Relationships and Inhibition of the Fatty Acyl-AMP Ligase (FadD32) Orthologs from Mycobacteria

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is one of the targets of first-line antituberculous drugs. This pathway contains a number of potential targets, including some that have been identified only recently and have yet to be explored. One such target, FadD32, is required for activation of the long meromycolic chain and is essential for mycobacterial growth. We report here an in-depth biochemical, biophysical, and structural characterization of four FadD32 orthologs, including the very homologous enzymes from Mycobacterium tuberculosis and Mycobacterium marinum. Determination of the structures of two complexes with alkyl adenylate inhibitors has provided direct information, with unprecedented detail, about the active site of the enzyme and the associated hydrophobic tunnel, shedding new light on structure-function relationships and inhibition mechanisms by alkyl adenylates and diarylated coumarins. This work should pave the way for the rational design of inhibitors of FadD32, a highly promising drug target.     

Distribution and function of prophage phiRv1 and phiRv2 among Mycobacterium tuberculosis complex

Mycobacterium tuberculosis complex (MTBC) is notorious for causing diseases, such as tuberculosis. Tuberculosis caused by M. tuberculosis remains a global public health concern. Two prophages, phiRv1 and phiRv2, can be found among most MTBC genomes. However, no precise functions have been assigned for the two prophages. In this paper, to find out the function of these two prophages, the distribution and function of phiRv1 and phiRv2 in MTBC genomes were analyzed from multiple omics data. We found that complex insertion, deletion, and reorganization appeared on the locus of two prophages in MTBC genomes; some genes of the two prophages can be translated and are functional from proteomic data; the expression of other prophage genes, such as Rv1577c, Rv2650c, Rv2652c, Rv2659c, and Rv2658c, can vary with environmental stresses and might enhance the fitness of MTBC. These data will facilitate our in-depth understanding of their function.     

The epidemiology of mycobacterium leprae: Recent insight

Abstract Leprosy is still a health problem in many countries. Because the causative organism, Mycobacterium leprae cannot be cultured in vitro, it is virtually impossible to assess exposure, and the onset of infection and disease. As a consequence, the chain of infection, considered as the relationships between M. leprae , transmission and human host, is poorly understood. Here, we discuss a number of organism-, host- and environmental-related factors which may be incriminated in the dynamic process of the development of leprosy disease. The use of modern molecular and immunological tools has become a valuable addition to epidemiological research. Understanding of the epidemiology of leprosy is a prerequisite for effective control of the disease.     

ATG5 provides host protection acting as a switch in the atg8ylation cascade between autophagy and secretion

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Desensitization by histamine of H2 receptor-mediated adenylate cyclase activation in the human gastric cancer cell line HGT-1

Short-term treatment of cultured HGT-1 cells with histamine produced a time-dependent (half-life: 20 min) and homologous desensitization of histamine H2 receptor activity mediating cAMP generation in HGT-1 cells and gastric acid secretion in normal gastric mucosa. Histamine treatment resulted in loss of response of the adenylate cyclase to histamine in purified plasma membranes, but had no effect on basal, vasoactive intestinal peptide (VIP)- or NaF-stimulated enzyme activities. We propose that the desensitization of gastric histamine H2 receptor by histamine evidenced in cellular or subcellular preparations from HGT-1 cells could be involved in the physiological regulation and pharmacological control of gastric cell function in man.     

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.     

结核分枝杆菌硫醇乙酰基转移酶基因敲除株的构建及其生物学特性分析

为探索硫醇乙酰基转移酶(mycothiol acetyltransferase,MshD)在结核分枝杆菌中的生物学特性,本实验利用噬菌体为载体的同源重组技术,构建结核分枝杆菌mshD基因敲除株、mshD基因回补株,用实时定量聚合酶链反应(real time-quantitative polymerase chain reaction, RT-qPCR)对所构建的菌株进行验证。分别收集H37Ra野生株、mshD基因敲除株、mshD基因回补株对数生长期菌液各5 mL, 离心收集菌体并培养,以观察菌落形态、生物膜形成及生长曲线测定;用5 mmol/L H₂O₂、0.05% SDS,50 ℃热激及低氧条件下分别处理基因敲出菌株和野生菌株,将菌液进行10倍梯度稀释,培养4～6周后检测抗胁迫能力并计算存活率。结果显示, 与野生株H37Ra相比,mshD基因敲除株菌落褶皱减少且菌落偏小,生长趋势较为缓慢;生物膜形成所需时间增长且褶皱明显减少;抗逆能力下降,存活率略低于野生株和回补株。揭示了mshD基因对结核分枝杆菌的生长具有重要作用,为进一步揭示该基因的功能和作用机制奠定了基础。     

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.     

Reactive oxygen species‐mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium‐infected macrophages by activating regulated IRE1‐dependent decay pathway

Mycobacterium avium, a slow‐growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress‐mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1‐dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol‐requiring enzyme 1 (IRE1)/apoptosis signal‐regulating kinase 1 (ASK1)/c‐Jun N‐terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD‐associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium‐infected macrophages. Interestingly, M. avium‐induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4μ8c (RIDD blocker). Macrophages pretreated with N‐acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC‐pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin‐, 4μ8c‐, and NAC‐treated macrophages compared with the control. The data indicate that the ROS‐mediated ER stress response induces apoptosis of M. avium‐infected macrophages by activating IRE1α‐RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.