Molecular and cellular characterization of the intracellular phenotype of Mycobacterium avium

Mycobacterium avium cause disseminated disease in immunocompromised people such as AIDS patients. Subsequent to crossing the intestinal epithelium, M. avium thrive within vacuoles in macrophages. The bacteria exhibit a different, more invasive, phenotype after being in macrophages compared to M. avium from laboratory conditions. We hypothesized that this intracellular phenotype contributes to disease in a variety of ways, such as influencing apoptosis of the macrophage. We further studied this intracellular phenotype, analyzing the molecular and cellular details. Using microscopy, we found that a portion of M. avium survive after the macrophage becomes apoptotic. From apoptotic bodies, some bacteria were observed escaping the vacuole and macrophage to the extra-cellular space and others were seen invading the macrophage ingesting the apoptotic body. We also found that macrophages infected by M. avium undergo autophagy, and that bacteria from autophagic macrophages were viable. After developing an in vitro system that elicits the intracellular phenotype, we determined that macrophages infected by bacteria exhibiting the intracellular phenotype undergo early-onset cell-death frequently compared to macrophages infected by bacteria exposed to laboratory conditions. With the use of real time PCR and microarray analysis, several genes, including genes of the twin-arginine translocase system, were shown to be upregulated by bacteria with the intracellular phenotype, while others were downregulated. The twin-arginine translocase system of M. avium was further characterized, and a tatb antisense strain was found to enter and survive in macrophages more efficiently than the wildtype strain, but was more sensitive to β-lactam antibiotics. Finally, we analyzed four transposon mutants of M. avium that were impaired in their ability to be taken up by macrophages to determine if the vacuole in which they reside, an aspect partially controlled by the bacteria, and therefore a part of the intracellular behavior of M. avium, differs from the vacuole containing wildtype bacteria. Some of these mutants were found in vacuoles with marked difference compared to the wildtype-containing vacuoles, others were similar to the wildtype. This work has increased our understanding of the intracellular phenotype of M. avium and how it may contribute to aspects of disease such as cell-to-cell spread

A rapid, low pH, nutrient stress, assay to determine the bactericidal activity of compounds against non-replicating Mycobacterium tuberculosis.

There is an urgent need for new anti-tubercular agents which can lead to a shortened treatment time by targeting persistent or non-replicating bacilli. In order to assess compound activity against non-replicating Mycobacterium tuberculosis, we developed a method to detect the bactericidal activity of novel compounds within 7 days. Our method uses incubation at low pH in order to induce a non-replicating state. We used a strain of M. tuberculosis expressing luciferase; we first confirmed the linear relationship between luminescence and viable bacteria (determined by colony forming units) under our assay conditions. We optimized the assay parameters in 96-well plates in order to achieve a reproducible assay. Our final assay used M. tuberculosis in phosphate-citrate buffer, pH 4.5 exposed to compounds for 7 days; viable bacteria were determined by luminescence. We recorded the minimum bactericidal concentration at pH 4.5 (MBC4.5) representing >2 logs of kill. We confirmed the utility of the assay with control compounds. The ionophores monensin, niclosamide, and carbonyl cyanide 3-chlorophenylhydrazone and the anti-tubercular drugs pretomanid and rifampicin were active, while several other drugs such as isoniazid, ethambutol, and linezolid were not

Synthesis and Evaluation of the 2-Aminothiazoles as Anti-Tubercular Agents.

The 2-aminothiazole series has anti-bacterial activity against the important global pathogen Mycobacterium tuberculosis. We explored the nature of the activity by designing and synthesizing a large number of analogs and testing these for activity against M. tuberculosis, as well as eukaryotic cells. We determined that the C-2 position of the thiazole can accommodate a range of lipophilic substitutions, while both the C-4 position and the thiazole core are sensitive to change. The series has good activity against M. tuberculosis growth with sub-micromolar minimum inhibitory concentrations being achieved. A representative analog was selective for mycobacterial species over other bacteria and was rapidly bactericidal against replicating M. tuberculosis. The mode of action does not appear to involve iron chelation. We conclude that this series has potential for further development as novel anti-tubercular agents

The representative aminothiazole 20 possesses bactericidal activity.

<p><i>M</i>. <i>tuberculosis</i> was inoculated to a starting of OD₅₉₀ of 0.1 in medium containing compound 20. CFU/mL was enumerated at indicated time points by serial dilution onto solid medium. The limit of detection was 20. Note that the lines for 0.625, 1.25 and 2.5 overlap.</p

Chemical synthesis of analogs comprised of thiazole core replacement.

<p>Reagents:: (i) 4-(t-butyl)PhCOCl, EtOH, 1 h; (ii) EtOH, reflux, 16 h; (iii) H₂SO₄, r.t., 16 h; (iv) EDC, CH₂Cl₂, r.t., 16 h.</p

Chemical synthesis of C-4 ketone and carboxamide analogs.

<p>Reagents: (i) EDC.HCl, HOBt, NCH₃(OCH₃), DIPEA, CH₂Cl₂, 16 h; (ii) CH₃MgBr, THF, -78°C–r.t., 2 h; (iii) 1-adamantanoyl chloride, Et₃N, THF, r.t., 1 h.</p

Chemical synthesis of pyrazole-based analogs.

<p>Reagents: (i) KO^<i>t</i>Bu, BuOH, reflux, 3 h; (ii) RCOCl, THF, r.t., 1 h; (iii) RNCO, THF, 1 h.</p