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eSymposia | Autophagy: Mechanisms and Disease


Characterization of autophagic flux over time in Mycobacterium tuberculosis-infected macrophages


Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

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Characterization of autophagic flux over time in Mycobacterium tuberculosis-infected macrophages Naomi Okugbeni1, André du Toit2, Glynis Johnson1, Ben Loos2, and Craig Kinnear1 1Dept. of Biomedical Sciences 2Dept. of Physiological Sciences, Stellenbosch University Tuberculosis (TB), is a respiratory disease that caused ~10 million cases in 2018. It is the leading cause of death by a single infectious agent- Mycobacterium tuberculosis (M. tb) (1). Autophagy is known to be involved in the killing of M.tb (2). Upon inhalation of M.tb, macrophages in the lungs engulf bacteria for phagosomal degradation. M.tb, however, damages the phagosome and escapes degradation (3). The phagosome and bacteria are then targeted for autophagic degradation but how this progresses has not been fully elucidated. We described the infection time course by quantifying autophagic flux of LC3B and p62 in M. tb-infected and uninfected macrophages using confocal microscopy, Western blotting, and bacterial enumeration in the absence and presence of Bafilomycin A1. Microscopy data revealed the variation in autophagic flux at 4, 24, 48, and 72 hours post-infection. Coupled with Western blot data, we described the basal autophagic flux in uninfected macrophages and M. tb-infected macrophages. Lastly, we correlated changes in autophagic flux to bacteria survival and showed that autophagic killing of M. tb is highest at 48 hours post-infection. These data characterized the progression of autophagy in M. tb-infected macrophages and highlighted protein markers useful for infection monitoring. This is an improvement on single time-point experiments and revealed the dynamic character of autophagic progression in M. tb-infected macrophages that may contribute to TB drug development and disease monitoring. References 1. WHO | Tuberculosis [Internet]. WHO. 2018 [cited 2018 Mar 24]. Available from: http://www.who.int/mediacentre/factsheets/fs104/en/ 2. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy Is a Defense Mechanism Inhibiting BCG and Mycobacterium tuberculosis Survival in Infected Macrophages. Cell. 2004 Dec 17;119(6):753–66. 3. van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell. 2007 Jun 29;129(7):1287–98. This work was funded by the National Research Foundation, South Africa and the South African Medical Research Council.

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