Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.     

Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.     

Study on the interaction between anti‐tuberculosis drug ethambutol and bovine serum albumin: multispectroscopic and cyclic voltammetric approaches

The binding of bovine serum albumin (BSA) to ethambutol (EMB) was investigated using spectroscopic methods, viz., fluorescence, Fourier transform infrared (FTIR), ultraviolet (UV)/vis absorption and cyclic voltammetry techniques. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of serum albumin by EMB is static, which was also confirmed by lifetime measurements. The number of binding sites, n, and binding constant, K, were obtained at various temperatures. The distance, r, between EMB and the protein was evaluated according to the Förster energy transfer theory. Based on displacement experiments using site probes, viz., warfarin, ibuprofen and digitoxin, the site of binding of EMB in BSA was proposed to be Sudlow's site I. The effect of EMB on the conformation of BSA was analyzed by using synchronous fluorescence spectra (SFS) and 3D fluorescence spectra. The results of fluorescence, UV/vis absorption and FTIR spectra showed that the conformation of BSA was changed in the presence of EMB. The thermodynamic parameters including enthalpy change (ΔH⁰), entropy change (ΔS⁰) and free energy change (ΔG⁰) for BSA–EMB were calculated according to the van't Hoff equation and are discussed.     

The mycobacterial cell wall: structure, biosynthesis and sites of drug action

Structural analysis has been successfully implemented recently to obtain valuable information on the mycobacterial cell wall components, many of which have formed the basis for biosynthesis and functional studies towards developing better drugs and possible vaccines. The highly complex and well organized structure unique to mycobacteria, represents the best target for novel antimycobacterial agents. Until recently, our knowledge of the enzymes responsible for the biogenesis of the cell wall components was almost negligible. The pathways are now being elucidated in several laboratories. Highlights of this review include significant advances in the structure and biochemistry of the major cell wall components and potenital targets for generation of new drugs.     

Inhibition of Mycobacterium tuberculosis InhA: Design,synthesis and evaluation of new di-triclosan derivatives

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC₅₀ of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC₉₉ of 12.9 µM against M. bovis.     

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.     

Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan (AG)--peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several antitubercular drugs. For instance, ethambutol (EMB) targets AG biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB, as well as the single Emb from Corynebacterium glutamicum. Here, we present for the first time an experimental analysis of the membrane topology of Emb. The domain organization clearly positions highly conserved loop regions, like the recognized glycosyltransferase C motif and the hydrophilic C-terminus towards the periplasmic side of the cell. Moreover, the assignment and orientation of hydrophobic segments identified a loop region, which might dip into the membrane and could possibly line a transportation channel for the emerging substrate. Site-directed mutations introduced into plasmid-encoded Cg-emb were analyzed in a C. glutamicumDeltaemb strain for their AG glycosyl composition and linkage analysis. Mutations analyzed did not perturb galactan synthesis; however, D297A produced a dramatically reduced arabinan content and prevented growth, indicating an inactive Emb. A second D298A mutation also drastically reduced arabinan content; however, growth of the corresponding mutant was not altered, indicating a certain tolerance of this mutation in terms of Emb function. A W659L-P667A-Q674E triple mutation in the chain length regulation motif (Pro-motif) resulted in a reduced arabinose deposition in AG but retained all arabinofuranosyl linkages. Taken together, the data clearly define important residues of Emb involved in arabinan domain formation and, for the first time, shed new light on the topology of this important enzyme.     

乙胺丁醇与异烟肼协同对耻垢分枝杆菌生长的影响研究（英文）

【目的】异烟肼(isoniazid, INH)和乙胺丁醇(ethambutol, EMB)是治疗结核病(tuberculosis,TB)的2种主要一线药物,虽然这2种药在临床上的联合使用有效地遏制了结核病的蔓延,但是它们之间潜在的协同作用机制却十分不清楚。【方法】本项目以耻垢分枝杆菌为模式菌株,首先,克隆Ms0606基因并以异丙基β-D-1-硫代吡喃半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导蛋白质的表达,再采用等温滴定量热法(isothermal titration calorimetry, ITC)证实Ms0606蛋白与EMB存在相互作用,进一步通过凝胶迁移实验(electrophoretic mobility shift assay, EMSA)证明EMB对Ms0606的DNA结合活性的影响,然后检测基因Ms0606超表达菌株在INH及INH+EMB处理下细菌的生长曲线。【结果】最终纯化得到纯度较高的Ms0606蛋白,EMB与Ms0606以1:1的比例结合,并且EMB能特异性地增强Ms0606的DNA结合活性,在非致死剂量...     

结核分枝杆菌乙胺丁醇耐药机制的研究进展

耐多药结核病的出现和流行对结核病的防控造成了严重威胁。乙胺丁醇(Ethambutol, EMB)是一线抗结核药物,常与异烟肼、利福平等联合应用,还可用于耐药结核病的治疗。但近年来EMB耐药形势严峻,我国复治结核病患者中EMB耐药率已达17.2%,并呈上升趋势;耐多药结核病患者中,EMB耐药率约为51.3%~66.7%,情况不容乐观。明确EMB耐药的产生机制对于有效防控EMB耐药率的上升、充分发挥EMB的作用十分重要,因此本文对结核分枝杆菌EMB的耐药现状、EMB的作用机制及其耐药产生机制方面的研究进展进行了综述。     

Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosis

A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations.