Homology Modeling of Wild-type,D516V,and H526L Mycobacterium Tuberculosis RNA Polymerase and Their Molecular Docking Study with Inhibitors

Abstract

Rifamicyns (Rifs) are antibiotic widely used for the treatment of tuberculosis (TB); nevertheless, their efficacy has been limited by a high percentage of mutations, principally in the rpoB gene. In this work, the first three-dimensional molecular model of the hypothetical structures for the wild-type and D516V and H526L mutants of Mycobacterium tuberculosis (mtRNAP) were elucidated by a homology modeling method. In addition, the orientations and binding affinities of some Rifs with those new structures were investigated. Our findings could be helpful for the design of new more potent rifamycin analogs.     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

Rifamycins: an insight into biological activity based on structural investigations

The structures of two compounds of the antibiotic rifamycin series have been studied by X-ray methods: rifamycin B, C₃₉H₄₉NO₁₄, which is active on bacterial RNA polymerase, and rifamycin Y, C₃₉H₄₇NO₁₅, which is devoid of any antibacterial activity. In both cases their p-iodoanilide derivative has been used and the final crystal structure data are now given.A comparative study of the structures of rifamycin B and rifamycin Y, and of the structures of two other ansa-compounds with similar activity on bacterial RNA polymerase, tolypomycin Y and streptovaricin C, together with the analysis of the activity ratios of a series of semisynthetic rifamycins, makes it possible to suggest that the following constitutional and conformational features are common to all the ansa-compounds with anti-bacterial activity: (i) a naphthoquinone or naphthohydroquinone nucleus with free oxygen functions, O(1) and O(2), on C(1) and C(8) respectively: (ii) an amide nitrogen attached to C(2); (iii) a 17-membered chain bridging the chromophoric group; (iv) two free hydroxyl groups attached to atoms C(21) and C(23) of the ansa-bridge and in the same relative positions with respect to each other and with respect to O(1) and O(2). However, the constitution and the conformation of the remaining part of the ansa-bridge can be rather different in different compounds.     

Dxr is essential in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>and fosmidomycin resistance is due to a lack of uptake

   

Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme [1], mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration.     

Importance of determining viability ofMycobacterium leprae inside macrophages—anin vitro method using uracil

It has been demonstrated thatMycobacterium leprae, are caPable of taking uP uracil and incorPorating it into trichloroacetic acid-insoluble materials, both as free susPension of bacteria, as well as when they are inside the macrophages, a host cell for theirin vivo survival. Same amount of bacteria show better incorPoration inside macroPhages than as free bacterial susPension. Both tyPes of incorPoration are inhibited by rifamPicin an antileProsy drug and an RNA synthesis inhibitor. Thus uracil uPtake byMycobacterium lePrae inside macroPhages has been used for standardising a raPidin vitro viability assay for the leProsy causing bacteria.     

RifP; a membrane protein involved in rifamycin export in <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis>

The rifamycin gene cluster in Amycolatopsis mediterranei includes the gene rifP, whose role in antibiotic production has not yet been established. In this work, the rifP gene was silenced and the results indicated that it codes for a protein to export rifamycin, avoiding its accumulation inside the cell. An antisense cassette was constructed by inserting the rifP gene in an antisense orientation downstream from the modified ermE^* promoter, and upstream of the Tasd terminator (aspartate semialdehyde dehydrogenase of A. lactamdurans). Partial silencing of the rifP gene by the use of the antisense cassette, cloned in the plasmid pUAMAE5, resulted in a 70% decrease in the extracellular rifamycin B. A protein of 53 kDa was absent in the membrane fraction of the silenced strain. This is the same size of the expected product from the rifP gene. The 2D structure analysis indicated it belongs to a Drug:H⁺ antiporter family which includes a wide number of membrane transport proteins.     

Few amino acid positions in <Emphasis Type="Italic">rpoB</Emphasis> are associated with most of the rifampin resistance in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mutations in rpoB, the gene encoding the β subunit of DNA-dependent RNA polymerase, are associated with rifampin resistance in Mycobacterium tuberculosis. Several studies have been conducted where minimum inhibitory concentration (MIC, which is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited) of rifampin has been measured and partial DNA sequences have been determined for rpoB in different isolates of M. tuberculosis. However, no model has been constructed to predict rifampin resistance based on sequence information alone. Such a model might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates.     

Modification of Rifamycin Polyketide Backbone Leads to Improved Drug Activity against Rifampicin-resistant Mycobacterium tuberculosis

Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.     

Lines of Evidence for Horizontal Gene Transfer of a Phenazine Producing Operon into Multiple Bacterial Species

Phenazines are secondary metabolites with broad-spectrum antibiotic activity against bacteria, fungi, and eukaryotes. In pseudomonad species, a conserved seven-gene phenazine operon (phzABCDEFG) is required for the conversion of chorismic acid to the broad-spectrum antibiotic phenazine-1-carboxylate. Previous analyses of genes involved in phenazine production from nonpseudomonad species uncovered a high degree of sequence similarity to pseudomonad homologues. The analyses undertaken in this study wished to eluciadate the evolutionary history of genes involved in the production of phenazines. Furthermore, I wanted to determine if the phenazine operon has been transferred through horizontal gene transfer. Analyses of GC content, codon usage patterns, frequency of 3:1 dinucleotides, sequence similarities, and phylogenetic reconstructions were undertaken to map the evolutionary history of phenazine genes from multiple bacterial species. Patchy phyletic distribution, high sequence similarities, and phylogenetic evidence infer that pseudomonad, Streptomyces cinnamonensis, Pantoea agglomerans, Burkholderia cepacia, Pectobacterium atrosepticum, Brevibacterium linens, and Mycobacterium abscessus species all contain a phenazine operon which has most likely been transferred among these species through horizontal gene transfer. The acquisition of an antibiotic-associated operon is significant, as it may increase the relative fitness of the recipient species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthetic Biology in Action: Developing a Drug Against MDR-TB

The amalgamation of the research efforts of biologists, chemists and geneticists led by scientists at the Department of Zoology, University of Delhi has resulted in the development of a novel rifamycin derivative; 24-desmethylrifampicin, which is highly effective against multi-drug resistant (MDR) strains of Mycobacterium tuberculosis. The production of rifamycin analogue was facilitated by genetic-synthetic strategies that have opened an interdisciplinary route for the development of more such rifamycin analogues aiming at a better therapeutic potential. The results of this painstaking effort of nearly 25 years of a team of students and scientists led by Professor Rup Lal have been recently published in the Journal of Biological Chemistry (www.jbc.org/content/289/30/21142.long). This strategy can now find applications for developing newer rifamycin analogues that can be harnessed to overcome the problem of MDR, extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis.     

New C25 carbamate rifamycin derivatives are resistant to inactivation by ADP-ribosyl transferases

A novel series of 3-morpholino rifamycins in which the C25 acetate group was replaced by a carbamate group were prepared and found to exhibit significantly improved antimicrobial activity than rifampin against Mycobacterium smegmatis. Further characterization of such compounds suggests that relatively large groups attached to the rifamycin core via a C25 carbamate linkage prevent inactivation via ribosylation of the C23 alcohol as catalyzed by the endogenous rifampin ADP-ribosyl transferase of M. smegmatis. SAR studies of the C25 carbamate rifamycin series against M. smegmatis and other bacteria are reported.     

Streptovaricins inhibit Focus Formation by MSV (MLV) Complex

We recently reported that the streptovaricins inhibit the reaction by which DNA is transcribed from the RNA template resident in murine leukaemia virions (MLV)¹. The reports^{2, 3} which first indicated that this DNA polymerase is present in oncogenic RNA viruses have been confirmed and extended^4–8. The enzyme provides a mechanism whereby an RNA virus can insert stable genetic information into a host chromosome. Gallo and co-workers described an RNA dependent DNA polymerase in lymphoblastic leukaemic cells which was inhibited by N-demethylrifampicin⁹ and this antibiotic, together with a number of other rifamycin derivatives, also inhibited the oncogenic viral DNA polymerase¹⁰. Like the streptovaricins, the rifamycins are ansa macrolide antibiotics (Fig. 1).     

Target Range of Zwittermicin A, an Aminopolyol Antibiotic from Bacillus cereus

Zwittermicin A is a novel antibiotic produced by Bacillus cereus UW85, which suppresses certain plant diseases in the laboratory and in the field. We developed a rapid method for large-scale purification of zwittermicin A and then studied the in vitro activity of zwittermicin A against bacteria, fungi, and protists. Zwittermicin A was highly active against the Oomycetes and their relatives, the algal protists, and had moderate activity against diverse Gram-negative bacteria and certain Gram-positive bacteria as well as against a wide range of plant pathogenic fungi. Zwittermicin A was more active against bacteria and fungi at pH 7–8 than at pH 5–6. When zwittermicin A was combined with kanosamine, another antibiotic produced by B. cereus, the two acted synergistically against Escherichia coli and additively against Phytophthora medicaginis, an Oomycete. The results indicate that there are diverse potential applications of this new class of antibiotic. Received: 1 December 1997 / Accepted: 9 January 1998     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。