Mycobacterial RNase E-associated proteins

RNase E and its complex with other proteins ('degradosome') play an important role in RNA processing and decay in Escherichia coli and in many other bacteria. To identify the proteins which can potentially interact with this enzyme in mycobacteria, Mycobacterium tuberculosis H37Rv RNase E was cloned and expressed as a 6HisFLAG-tagged fusion protein. Analysis of the mycobacterial RNase E overexpressed and purified from M. bovis BCG revealed the presence of GroEL and two other copurified proteins, products of the Mb1721 (inorganic polyphosphate/ATP-NAD kinase) and Mb0825c (acetyltransferase) genes. Identical copies of these two genes can be found in M. tuberculosis H37Rv.     

Mycobacteria use their surface-exposed glycolipids to infect human macrophages through a receptor-dependent process

Two subfamilies of the polar glycopeptidolipids (GPLs) located on the surface of Mycobacterium smegmatis, along with unknown phospholipids, were recently shown to participate in the nonopsonic phagocytosis of mycobacteria by human macrophages (Villeneuve, C., G. Etienne, V. Abadie, H. Montrozier, C. Bordier, F. Laval, M. Daffe, I. Maridonneau-Parini, and C. Astarie-Dequeker. 2003. Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids. J. Biol. Chem. 278: 51291-51300). As demonstrated herein, a phospholipid mixture that derived from the methanol-insoluble fraction inhibited the phagocytosis of M. smegmatis. Inhibition was essentially attributable to phosphatidylinositol mannosides (PIMs), namely PIM2 and PIM6, because the purified phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol were inactive. This was further confirmed using purified PIM2 and PIM6 from M. bovis BCG that decreased by half the internalization of M. smegmatis. Both compounds also inhibited the uptake of M. tuberculosis and M. avium but had no effect on the internalization of zymosan used as a control particle of the phagocytic process. When coated on latex beads, PIM2 and polar GPL (GPL III) favored the particle entry through complement receptor 3. GPL III, but not PIM2, also directed particle entry through the mannose receptor. Therefore, surface-exposed mycobacterial PIM and polar GPL participate in the receptor-dependent internalization of mycobacteria in human macrophages.     

Degradation process of Mycobacterium leprae cells in infected tissue examined by the freeze-substitution method in electron microscopy

Mycobacterium leprae cells (strain Thai-53) harvested from infected mouse foot pads were examined by electron microscopy using the freeze-substitution technique. The population of M. leprae cells from the infected tissue consisted of a large number of degraded cells and a few normal cells. These thin sectioned cell profiles could be categorized into four groups depending on the alteration of the membrane structures, and the degradation process is considered to occur in stages, namely from stages 1 to 3. These are the normal cells with an asymmetrical membrane, a seemingly normal cell but with a symmetrical membrane (stage 1), a cell possessing contracted and highly concentrated cytoplasm with a membrane (stage 2), and a cell that has lost its membrane (stage 3). The peptidoglycan layer was found to remain intact in these cell groups.     

Coenzyme Q1-catalyzed luminol chemiluminescent assay for rapid antimicrobial susceptibility testing of Mycobacterium bovis

Coenzyme Q1 is herein proposed as the best catalyst among coenzymes Q and vitamins K for quinone-catalyzed luminol chemiluminescent assays applied to rapid determination of viability or rapid antimicrobial susceptibility tests of Mycobacterium bovis. Luminol chemiluminescence intensity (LCI) was determined 10 min after the incubation of M. bovis with coenzyme Q1, and was proportional to CFU (colony-forming unit)/ml in the range of 9,000 to 2,250,000. LCI depended on the the production of the superoxide anion (O2-) rather than H2O2 during a 10-min incubation of M. bovis with coenzyme Q1, as superoxide dismutase reduced LCI more effectively than catalase. The minimal inhibitory concentrations (MICs) of 10 kinds of antituberculous agents estimated on the basis of decrease in LCI after one or two days' cultivation were in good agreement with MICs determined by turbidity analysis, which requires upwards of 1 week to complete.     

Crossreactivity of SLE autoantibodies with 70 kDa heat shock proteins of Mycobacterium tuberculosis

Heat shock proteins (hsp) may be involved in the initiation and perpetuation of autoimmune diseases. In order to investigate the possible role of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody production in SLE, the immuno-crossreactivity of SLE autoantibodies with Mycobacterium tuberculosis sonic extract and hsp-70 kDa was investigated. These proteins showed significant binding with Protein A-Sepharose isolated SLE IgG. Western blotting of hsp-70 with SLE IgG showed strong recognition, suggesting possible involvement of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody induction in SLE.     

Direct application of AvaII PCR restriction fragment length polymorphism analysis (AvaII PRA) targeting 644 bp heat shock protein 65 (hsp65) gene to sputum samples

To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS 6110 PCR. Although this method showed a sensitivity slightly lower than IS 6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS 6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.     

Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.     

Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system

The importance of non-tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention. Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC) and to compare biocide susceptibility of planktonic and biofilm organisms. Scanning electron microscopy was also carried out to observe biofilm morphology. With the exception of Sporicidin and Virkon the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values. The MBEC assay system was seen to produce multiple and reproducible biofilms of M. phlei and to be a useful tool for susceptibility studies.     

分枝杆菌TZh51菌株的分离鉴定及其生物修复污染土壤的特性

[目的]为获得降解芘的微生物菌株,并用其生物修复被多环芳烃污染的土壤.[方法]芘降解菌的分离采用平板升华法.根据表型观察、生理生化特性和16S rDNA的序列同源性分析,对菌株进行分类学鉴定.通过活菌计数、HPLC测定多环芳烃的残留量,研究菌株在固体、液体无机盐培养基以及在污染土壤中降解多环芳烃(polycyclic aromatic hydrocarbons,PAHs)的能力.[结果]分离到4株能降解芘的菌株TZh51、TZh52、TG42和TG52.实验结果表明,TZh51降解PAHs的能力强于其余3株菌.TZh51被鉴定为分枝杆菌属(Mycobacterium sp.),但与已发表的分枝杆菌菌株M11为不同的种.TZh51接种在芘膜的固体无机盐培养基上,测定获得最大芘降解量的条件是培养温度为3512和芘膜厚度为130 ng/mm2.在芘浓度为50、100 mg/L的液体无机盐培养基中培养,6天时TZh51的芘降解率分别达到91.9%、71.8%,10天时菌体数量分别达到最大值为2.0、6.0×108cfu/mL;TZh51降解芘的效果强于M11.在种植作物的处理中,到第6周时TZh51的菌体数量达到每克干土含7.2×108个菌落数,到第8周时菲、荧蒽和芘的降解率分别达到91.4%、86.9%和85.8%;[结论]TZh51具有很强降解PAHs的能力;另外,TZh51与作物联合生物修复污染土壤的效果明显.     

Mycobacterium malmoense Isolated from Soil

Mycobacterium malmoense was isolated from a soil sample, and biological, biochemical, antigenic, and genetic characteristics of the isolate were described. This is the first report of isolation of this organism in Japan.