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J Biol Chem. 2003 Feb 28;278(9):7310-9. Epub 2002 Dec 06.
Tracking the putative biosynthetic precursors of oxygenated mycolates of Mycobacterium tuberculosis. Structural analysis of fatty acids of a mutant strain deviod of methoxy- and ketomycolates.

Dinadayala P, Laval F, Raynaud C, Lemassu A, Laneelle MA, Laneelle G, Daffe M.

Departement de Mecanismes Moleculaires des Infections Mycobacteriennes, Institut de Pharmacologie et Biologie Structurale du CNRS et de l'Universite Paul Sabatier (UMR 5089), 205 route de Narbonne, Toulouse 31077 cedex 04, France.

Disruption of the mma4 gene (renamed hma) of Mycobacterium tuberculosis has yielded a mutant strain defective in the synthesis of both keto- and methoxymycolates, with an altered cell-wall permeability to small molecules and a decreased virulence in the mouse model of infection (Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Laneelle, M. A., Yu, K., Quemard, A., Smith, I., and Daffe, M. (2000) Mol. Microbiol. 36, 630-637). Assuming that the mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural analysis of mycolates from the hma-inactivated strain was performed using a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, proton NMR spectroscopy, and chemical degradation techniques. These consisted most exclusively of alpha-mycolates, composed of equal amounts of C(76)-C(82) dicyclopropanated (alpha(1)) and of C(77)-C(79) monoethylenic monocyclopropanated (alpha(2)) mycolates, the double bond being located at the "distal" position. In addition, small amounts of cis-epoxymycolates, structurally related to alpha(2)-mycolates, was produced by the mutant strain. Complementation of the hma-inactivated mutant with the wild-type gene resulted in the disappearance of the newly identified mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene in Mycobacterium smegmatis led to the lowering of diethylenic alpha mycolates of the recipient strain and the production of keto- and hydroxymycolates. These data indicate that long-chain ethylenic compounds may be the precursors of the oxygenated mycolates of M. tuberculosis. Because the lack of production of several methyltransferases involved in the biosynthesis of mycolates is known to decrease the virulence of the tubercle bacillus, the identification of the substrates of these enzymes should help in the design of inhibitors of the growth of M. tuberculosis.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12473649&dopt=Abstract

Dis Colon Rectum. 2002 Dec;45(12):1685-8.
Nested polymerase chain reaction in the diagnosis of negative Ziehl-Neelsen stained Mycobacterium tuberculosis fistula-in-ano: report of four cases.

Shan YS, Yan JJ, Sy ED, Jin YT, Lee JC.

Department of Surgery, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.

PURPOSE: Mycobacterium is one of the causes of granulomatous diseases within the anorectal region. Early diagnosis of infection is important before the use of antituberculosis chemotherapy. Clinical diagnosis is usually dependent on microscopic detection using Ziehl-Neelsen stain and mycobacterial culture, but the sensitivity and specificity of these two methods are low. In this study nested polymerase chain reaction was used to detect mycobacterial infection in anal fistulas. METHODS: Paraffin-embedded specimens from three patients and discharge from one patient were used. DNA extraction was performed using phenol/chloroform techniques. IS6110-based nested polymerase chain reaction, yielding a 259-bp amplicon, for the diagnosis of infection was done to facilitate treatment. RESULTS: Four cases of suspected fistulas-in-ano presented with persistent fistula or unhealed wound. Histopathologic examination revealed granulomatous inflammation with failed microscopic detection of acid-fast bacilli using Ziehl-Neelsen stain. Nested polymerase chain reaction confirmed the presence of in all cases. The anal lesions healed rapidly following a course of antituberculosis therapy. CONCLUSION: Molecular diagnosis of fistula-in-ano by nested polymerase chain reaction is useful for clinically highly suspected infection despite a negative Ziehl-Neelsen stain.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12473896&dopt=Abstract

Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):17037-42. Epub 2002 Dec 13.
Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach.

Mougous JD, Leavell MD, Senaratne RH, Leigh CD, Williams SJ, Riley LW, Leary JA, Bertozzi CR.

Howard Hughes Medical Institute, Department of Molecular and Cell Biology, Department of Chemistry, and School of Public Health, University of California, Berkeley, CA 94720, USA.

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO 4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12482950&dopt=Abstract

J Hepatobiliary Pancreat Surg. 2002;9(4):515-8.
Pancreatic tuberculosis.

Sanabe N, Ikematsu Y, Nishiwaki Y, Kida H, Murohisa G, Ozawa T, Hasegawa S, Okawada T, Toritsuka T, Waki S.

Department of Surgery, Hamamatsu Medical Center, 328 Tomitsuka, Hamamatsu 432-8580, Japan.

A 63-year-old Japanese man visited our institute with fever of unknown origin. Findings on preoperative imaging modalities were consistent with pancreatic carcinoma, but a positive tuberculin skin test indicated tuberculosis infection. Negative results for MycobacteriumDNA polymerase chain reaction from sputum and bone-marrow aspiration biopsy specimens ruled out pulmonary and miliary tuberculosis, respectively. Positron emission tomography (PET) with 2-[fluorine-18]-fluoro-2-deoxy- d-glucose (FDG) showed multiple labeled spots within the pancreas body and chest. Distal pancreatectomy was performed with a diagnosis of suspected pancreatic carcinoma, but the histological and microbiological diagnosis was Mycobacterium infection. A rare case of pancreatic tuberculosis evaluated by FDG PET is reported and discussed herein.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12483276&dopt=Abstract

nms.ac.jp

Buruli ulcer, caused by Mycobacterium ulcerans, is emerging as the third most common mycobacterial disease after leprosy and tuberculosis in some tropical regions. Although a toxin of the polyketide family is central to the pathogenesis of the disease, there are still several parameters that need clarification. Among them and of crucial interest are the curative drug treatment and the test for early detection of the disease. In this study, we used mouse monoclonal antibodies, raised against synthetic sugars of the terminal trisaccharide of M. leprae PGL-1, to detect the immunoreactivity of this antigen in tissue infected with M. ulcerans. Thirty specimens of skin tissue from Buruli ulcer patients (3 plaques, 10 nodules, 1 ulcerated nodule, 7 deep ulcer beds and 9 ulcers in healing) were obtained from Ghana. Eighty-three percent of the submitted cases were compatible with the lesions of Buruli ulcer. AFB were positive in 33% of plaques, 40% of nodules, 44% of actives ulcers and 22% of the ulcer in healing stage. Immunohistochemically, phenolic glycolipid-1 (PGL-1) was detected in all AFB-positive cases. This observation implies that Mycobacterium ulcerans may express an M. leprae PGL-1-like substance and should tentatively emulate research to further characterize such a substance. The search for an early diagnostic tool for the Buruli disease may benefit from such investigations.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12483968&dopt=Abstract

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