Mouse Anti-Mycobacterium Tuberculosis Hybridoma [C262L23]

Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2(-/-) mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation. IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyl-adenosine in the evolution of an environmental ancestor to M. tuberculosis. On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.

Long intergenic non-coding RNAs (lincRNAs) are long non-coding transcripts from the intergenic regions of annotated protein-coding genes. lincRNA cyclooxygenase 2 (Cox2) is an early-primary response gene regulated by the NF-kappa B signaling pathway in macrophages. It was found that lincRNACox2 was significantly increased in patients with the Mycobacterium tuberculosis (M. tuberculosis) H37Ra strain infection and macrophages, using reverse transcription-quantitative PCR (RT-qPCR). ELISA, western blotting and RT-qPCR results indicated that the inflammatory response factors tumor necrosis factor-alpha, interferon-gamma, interleukin-6, Cox2 and inducible nitric oxide synthase were significantly increased in H37Ra infected macrophages. In addition, the inflammatory regulating proteins NF-kappa B and Stat3 were significantly increased in H37Ra infected macrophages but decreased in lincRNACox2 knockdown macrophages infected with H37Ra. Moreover, the knockdown of lincRNACox2 increased the apoptotic rate of H37Ra infected macrophages and facilitated the proliferation of H37Ra. Collectively, the present results suggested that lincRNACox2 may be required for the activation of NF-kappa B and Stat3, in order to regulate inflammatory responses involved in resistance to M. tuberculosis infection.