Detection of Mycobacterial DNA in Human Bone Marrow.

Bone marrow is a cell-rich tissue of the reticuloendothelial system essential in the homeostasis and accurate functioning of hematopoiesis and of the immune system; moreover, it is also rich in lipids because it contains marrow adipocytes. This work aimed to evaluate the detection of mycobacterial DNA in human bone marrow as a tool to understand the complex pathology caused by the main pathogen Mycobacterium tuberculosis (Mtb). Formalin-fixed paraffin-embedded human bone marrow samples were studied using both conventional PCR + hybridization and in situ PCR to figure out the cell distribution of the targeted DNA. Samples were retrospectively collected from HIV+ patients with microbiologically proved mycobacterial infection and from subjects without evidence of infection. Mycobacterium avium (Mav) as well as Mtb DNA was detected in both settings, including tissues with and without granulomas. We detected DNA from both mycobacterial species, using in situ PCR, inside bone marrow macrophages. Other cell types, including adipocytes, showed positive signals only for Mtb DNA. This result suggested, for the first time, that marrow adipocytes could constitute an ideal reservoir for the persistence of Mtb, allowing the bacilli to establish long-lasting latent infection within a suitable lipid environment. This fact might differentiate pathogenic behavior of non-specialized pathogens such as Mav from that of specialized pathogens such as Mtb.

Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

Transcriptional analysis of and resistance level conferred by the aminoglycoside acetyltransferase gene aac(2')-Id from Mycobacterium smegmatis.

OBJECTIVES: To analyse the correlation between the expression levels of the aac(2')-Id gene from Mycobacterium smegmatis mc(2)155 and the resistance levels to aminoglycosides conferred by the encoded aminoglycoside 2'-N-acetyltransferase [AAC(2')-Id]. METHODS: Expression levels were studied using a transductional fusion with the lacZ gene. The promoter region was characterized by primer extension analysis and ribonuclease protection assay. The aac(2')-Id gene was placed under the control of different mycobacterial promoters; deletions of the promoter region were done. Each of the plasmids was introduced in M. smegmatis mc(2)155 and the MICs were determined by resazurin assay. RESULTS: The aac(2')-Id gene is transcribed from two promoters: P1 (weaker) and P2 (stronger) located 38 and 1 nt upstream of the start codon, respectively. P2 promoter (producing a leaderless mRNA) was confirmed by producing deletions in the aac(2')-Id promoter and analysing the ability of the re-constructed genes to confer resistance to aminoglycosides. The expression levels (in terms of beta-galactosidase units) varied during the phase of growth of cultures, reaching high levels during the early exponential and the stationary phase and reduced levels during entry into stationary phase. Both the levels of expression and the MICs were more elevated at lower temperatures. Cloning the gene under the control of other strong mycobacterial promoters also resulted in higher MIC values. CONCLUSIONS: In M. smegmatis mc(2)155, the aminoglycoside resistance levels conferred by the AAC(2')-Id enzyme directly rely on the strength of the promoter driving transcription of the aac(2')-Id gene.

Transcriptional analysis of Mycobacterium fortuitum cultures upon hydrogen peroxide treatment using the novel standard rrnA-P1.

BACKGROUND: The ability of an intracellular pathogen to establish infection depends on the capacity of the organism to survive and replicate inside the host. Mycobacterium fortuitum is a bacteria that contains genes involved in the detoxification of the oxygen reactive species such as those produced by the host during the infection. In this work, we investigate the effects of hydrogen peroxide on the transcription and expression of these genes by developing a real time quantitative PCR technique (qRT-PCR) using the ribosomal promoter region (rrnA-P1) as reference product for quantification of the mRNA levels. RESULTS: M. fortuitum cultures were treated with different hydrogen peroxide concentrations (0.02 to 20 mM) during several periods of time (30 to 120 minutes). The activity of the enzymes KatGII and SodA, and the transcription of corresponding genes were evaluated. The transcriptional regulator furAII gene was also studied. The ribosomal promoter region rrnA-P1 was validated as referential product under the stress conditions checked by qRT-PCR. Minor changes were observed under the conditions tested except when bacteria were incubated in the presence of 20 mM hydrogen peroxide. Under those conditions, the levels of transcription of the three genes under study increased at 30 minutes of treatment. The viability of the bacteria was not influenced under the conditions tested. CONCLUSION: In this work, we have quantified transcriptional responses to stress suggesting that, the opportunistic pathogen M. fortuitum is more resistant and differs in behaviour in the presence of hydrogen peroxide, when compared to the major pathogen Mycobacterium tuberculosis and the saprophyte Mycobacterium smegmatis. Besides, we demonstrate the mycobacterial non-coding region rrnA-P1 to be a suitable reference product in the analysis of qRT-PCR transcriptional data of M. fortuitum.

Analysis of the precursor rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard.

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons. Mycobacterium chelonae and M. fortuitum are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome. Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first rrnA promoter in both species, namely, rrnA P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when M. fortuitum infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its rrn operons is preferentially used under such conditions.