The Achilles' Heel Targeted by TB47 Is Protected in Select Mycobacteria.
Buruli ulcer, the third largest mycobacterial disease, and tuberculosis, an ancient infectious disease which today claims more human life than any other bacterial infection are both caused by Mycobacteria. TB47, a new antibiotic candidate, inhibits these two mycobacteria by varying degrees. Here we report how the differences in the target of TB47 in them account for their differential susceptibilities to TB47. It is potentially highly potent against leprosy, the second largest mycobacterial disease.
- Mycobacteria usually grow very slowly, for example, it takes 3 to 6 weeks for Mycobacterium tuberculosis and ~3 months for Mycobacterium ulcerans to form a visible colony on agar containing enriched media. Traditional methods that depend on enumerating colony forming units (CFU) of bacteria to evaluate efficacy of a drug would significantly prolong the duration of preclinical studies required for development of antimycobacterial compounds/treatments. Autoluminescent reporter mycobacteria were constructed using the luxCDABE operon, which can emit light without the addition of exogenous substrates. Relative light units (RLU) counts correlated well with CFUs of mycobacteria. Use of this technology enabled rapid (3 seconds) and serial real-time monitoring of mycobacteria in the same samples in vitro and even in the same small live animals for evaluation of drug activity. Therefore, this approach can drastically reduce the time, effort, animals and resources necessary for assessments that require monitoring of mycobacteria growth. This method is highly efficient in active compound discovery both in vitro and in vivo.
- Existing treatments for mycobacterial infections are lengthy or losing effectiveness due to resistance/toxicity. New powerful drugs with new mechanisms of action could address the current need for novel regimens. TB47 is a new agent with activity against M. tuberculosis, the bacteria that causes tuberculosis. Using various in vitro,in macrophage and in vivo approaches, we found that TB47 is bacteriostatic against M. tuberculosis.
- We also assessed the activity of TB47 against M. ulcerans, the causative agent of Buruli Ulcer, using in vitro assays and an established mouse model mainly due to an international collaboration with Australia where Buruli Ulcer is still a problem. Buruli ulcer is a disease that manifests as skin ulcer resulting from destruction of skin and dermal tissue in response to a toxin secreted by M. ulcerans.In the mouse model of Buruli Ulcer, the footpad is used to assess pathology and response to treatment. When using TB47 to treat the autoluminescent M. ulcerans reporter strain infected mice, we were surprised to find that only several days, RLUs from the footpads reached to the background reading though only 3 mice used at the very beginning. Using this model, we further found that TB47 was bactericidal against M. ulcerans and prevented footpad swelling and pathology that are characteristic in untreated mice, as can be seen in the figure below.
- For uncovering mechanism of action of TB47, We spent more than one year to screen the spontaneous TB47-resistant M. tuberculosis mutants and found lots of colonies appeared on TB47 containing plates, but none of them was really resistant to TB47. M. ulcerans and Mycobacterium marinum are closely related in evolution. We selected TB47-resistant M. marinum mutants. However, we failed for > 10 times in the case of selection of TB47-resistant M. ulcerans mutants at the beginning. We postulated that it could be super sensitive to TB47. We then used as low as 0.02 μg/mL TB47 and succeeded in getting the resistant mutants. We identified mutations by sequencing the whole genomes of the mutants. A common mutation was a single nucleotide mutation resulting in an amino acid replacement in the gene that encodes for QrcB, a protein of the electron transport chain. We also hypothesize that CycAB protein complex confers protection from the poisoning effect of TB47 of the Qrc complex, as shown below.
- We propose that QrcB is the Achilles’ Heel of mycobacteria that is targeted by TB47. Different species of mycobacteria exhibit varying levels of sensitivity to TB47. We hypothesize that molecular differences in the QrcB orthologs in different mycobacteria determine the level of inhibition by TB47. Likely equally important is how robust the CydAB complex is in each mycobacterium. For example, M. smegmatis genome encodes a robust CydAB complex and this potentially protects this mycobacteria from TB47. On the other hand, lack of active CydAB in M. ulcerans could potentially explain why TB47 is bactericidal against it. It is very interesting that no CydAB ortholog in M. leprae, the causative agent of leprosy, which indicates that TB47 may be supercidal to M. leprae. This indicates that screening/evaluation of anti-TB drugs using M. tuberculosis-based models are very important, as different mycobacteria may have different pathway activities (just like this study) and different enzymes. For example, the first-line anit-TB drug pyrazinamide is not active against most of mycobacteria including M. bovis which contains only one single nucleotide polymorphism in pncA gene compared to that from M. tuberculosis. However, if a drug/compound is active against one pathway of one mycobacterium, it may also affect the same pathway in another mycobacterium and so this agent may have additive/synergistic activity in combination.
- If we can find an agent to disrupt protector(s) such as CydAB complex and combine it with TB47, it is possible that the combination will exhibit synergy in bactericidal activity against mycobacteria. This is a case supporting that proteins encoded by nonessential genes can be potential drug targets and novel drug discovery strategy based on synthetic lethality would greatly strengthen the preclinical development of drugs to treat mycobacterial infection.
- This post has been written by Tianyu Zhang, Yang Liu and Yamin Gao.