Permeabilization of the mycobacterial envelope for protein cytolocalization studies by immunofluorescence microscopy

BACKGROUND: The establishment of the cellular localization of proteins in M. tuberculosis will provide of valuable information for the identification of new drug/vaccine/diagnostic targets. Cytolocalization by inmunofluorescence microscopy has been limited in mycobacteria because to difficulties in effectively permeabilize it. RESULTS: A treatment combining lysozyme with triton X-100 was found to be an effective permeabilization method of the mycobacterial envelope. CONCLUSION: A rapid and simple permeabilization protocol has been successfully assessed in pure cultures of both Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. This method can be successful used in the cytolocalization of proteins by immunolabeling

Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: Implications for drug discovery

International audienceBackground: Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise in bacterial drug resistance in recent years has led to the need of new drugs and drug regimens. Model systems are regularly used to speed-up the drug discovery process and circumvent biosafety issues associated with manipulating M. tuberculosis. These include the use of strains such as Mycobacterium smegmatis and Mycobacterium marinum that can be handled in biosafety level 2 facilities, making high-throughput screening feasible. However, each of these model species have their own limitations. Results: We report and describe the first complete genome sequence of Mycobacterium aurum ATCC23366, an environmental mycobacterium that can also grow in the gut of humans and animals as part of the microbiota. This species shows a comparable resistance profile to that of M. tuberculosis for several anti-TB drugs. The aims of this study were to (i) determine the drug resistance profile of a recently proposed model species, Mycobacterium aurum, strain ATCC23366, for anti-TB drug discovery as well as Mycobacterium smegmatis and Mycobacterium marinum (ii) sequence and annotate the complete genome sequence of this species obtained using Pacific Bioscience technology (iii) perform comparative genomics analyses of the various surrogate strains with M. tuberculosis (iv) discuss how the choice of the surrogate model used for drug screening can affect the drug discovery process. Conclusions: We describe the complete genome sequence of M. aurum, a surrogate model for anti-tuberculosis drug discovery. Most of the genes already reported to be associated with drug resistance are shared between all the surrogate strains and M. tuberculosis. We consider that M. aurum might be used in high-throughput screening for tuberculosis drug discovery. We also highly recommend the use of different model species during the drug discovery screening process

Identification of DNA binding motifs of the Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system.

International audienceThe Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 genes, by using gene fusions, electrophoretic mobility shift assays and DNase I footprinting experiments. A consensus sequence for PhoP binding was deduced. It consists of two direct repeats, DR1/DR2, associated with a third repeat, DR3, important in some cases for PhoP binding to DR1/DR2 but located at a variable distance from these direct repeats. DR1/DR2 and DR3 consensus sequences were used to screen the whole-genome sequence for other putative binding sites potentially corresponding to genes directly regulated by PhoP. The identified 87 genes, encoding transcription regulators, and proteins involved in secondary metabolites biosynthesis, transport and catabolism are proposed to belong to the PhoP regulon. A consensus sequence derived from the analysis of PhoP binding to four gene promoter regions is proposed. We show for the first time the involvement of a third direct repeat motif in this binding reaction. The consensus sequence was instrumented to study the global regulation mediated by PhoP in M. tuberculosis. This analysis leads to the identification of several genes that are potentially regulated by this key player

Autofluorescence of Mycobacteria as a Tool for Detection of Mycobacterium tuberculosis▿

The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for Mycobacterium tuberculosis. Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of Mycobacterium tuberculosis was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that <106 bacilli ml−1 of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 μl of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries

DNase I footprinting assay of the <i>pks2</i> and <i>msl3</i> promoter regions with PhoP-P.

<p>Radiolabeled PCR fragments corresponding to (A) pks2 (−136 to +62), (B) msl3 (−312 to −79), (C) fadD21(−194 to +7) and (D) lipF (−639 to −450) were used as DNA targets. Various amounts of PhoP-P were used with pks2 and msl3 (lane 1: 0; lane 2: 1.8 pmol; lane 3: 3.6 pmol; lane 4: 7.2 pmol and lane 5: 14.4 pmol) and were incubated with 0.2 pmol of DNA before DNase 1 digestion. For fadD21 and lipF different amount of PhoP-P were used ((lane 1: 0; lane 2: 1.8 pmol; lane 3: 18 pmol; lane 4: 180 pmol and lane 5: 900 pmol) and were incubated with 1 pmol of DNA before DNase I digestion. Lane 6: A+G Maxam and Gilbert reaction. Protected regions are indicated by a line with colored regions. The blue, orange and red segments correspond to the DR1, DR2 and DR3 sites, respectively.</p

Beta-galactosidase activity in <i>M. smegmatis</i> expressing <i>lacZ</i> under the control of the promoter regions of the <i>M. tuberculosis</i> H37Rv <i>lipF</i>, <i>pks2</i>, <i>msl3</i> and <i>fadD21</i> genes.

<p>The <i>lipF</i>, <i>pks2</i>, <i>msl3</i> and <i>fadD21</i> promoter-<i>lacZ</i> fusions carried by the pJEM15 plasmid were introduced into <i>M. smegmatis</i>. The positive control is <i>M. smegmatis</i> transformed with pJEM31(PAN) containing <i>lacZ</i> under the control of a mobile genetic element normalized against <i>M. smegmatis</i> transformed with pJEM15 empty vector. The results shown are the mean of two independent experiments.</p

Identification of the DR1–3 sites on the DNA promoters recognized byPhoP.

<p>For each DNA sequence shown to bind PhoP-P, sequences with a complete or partial match to the DR1, DR2 and DR3 sites were identified (underlined). The degree of conservation of the consensus sequence was calculated and is shown as the ratio of matching nucleotides to the consensus: % identity.</p

Electrophoretic mobility assays with the lipF, pks2, msl3 and fadD21 promoter fragments and PhoP-P.

<p>2A. The large DNA fragments initially selected — lipFa (222 bp), pks2 (148 bp), msl3a (235 bp), and fadD21 (210 bp) — were incubated with PhoP-P in the presence of poly dI-dC at 10 µg/ml and run on a native polyacrylamide gel (8%), in 0.5× TBE buffer. Complete binding was assessed on the basis of the amount of shifted material for each fragment in the presence of 4 µM of PhoP-P (protein/DNA ratio: 100/1), except for lipFa, for which 8 µM PhoP-P was required (protein/DNA ratio: 200/1). 2B. Smaller DNA fragments — lipFa1 (68 bp), pks2a (59 bp), pks2b (40 bp), pks2d (104 bp), msl3a1 (45 bp), msl3a2 (83 pb), fadD21a (55 bp) and fadD21b (79 bp) — were designed and incubated with PhoP-P in the presence of poly dI-dC at 10 µg/ml. Protect40 (40 bp) was tested in all mobility shift assays, as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042876#pone.0042876-Gupta1" target="_blank">[26]</a>. 2C. Protect40 (40 bp) fragments with an altered sequence were used: Pho1 (DR1 altered sequence), Pho2 (DR2 altered sequence) and Pho3 (DR1 and DR2 altered sequences). Wild-type Protect40 (40 bp) was tested as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042876#pone.0042876-Gupta1" target="_blank">[26]</a>.</p

List of DNA fragments used in this study.

b<p>Fragments used for electrophoretic mobility shift assays.</p