Measurement of Autophagy Activity Reveals Time-Dependent, Bacteria-Specific Turnover during Mycobacterium tuberculosis Infection

Naomi Okugbeni; André du Toit; Victoria Cole-Holman; Glynis Johnson; Ben Loos; Craig Kinnear

doi:10.3390/pathogens12010024

Measurement of Autophagy Activity Reveals Time-Dependent, Bacteria-Specific Turnover during Mycobacterium tuberculosis Infection

Pathogens. 2022 Dec 23;12(1):24. doi: 10.3390/pathogens12010024.

Authors

Naomi Okugbeni^{1

2}, André du Toit³, Victoria Cole-Holman², Glynis Johnson¹, Ben Loos³, Craig Kinnear^{1

2}

Affiliations

¹ DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, US/SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa.
² South African Medical Research Council Genomics Centre, Tygerberg 7505, South Africa.
³ Neuro Research Group, Department of Physiological Sciences, Faculty of Sciences, Stellenbosch University, Stellenbosch 7602, South Africa.

Abstract

The intracellular pathogen, Mycobacterium tuberculosis (M. tb) uses various mechanisms to evade its killing. One of such is phagosomal damage and cytosolic translocation which is then targeted by the host's bactericidal autophagy pathway. It is suggested that cytosolic translocation of M. tb is time-dependent, occurring at later time points of 48 to 72 h post-infection. It is, however, not known whether increased autophagic targeting correlates with these time points of infection. We investigated the time-dependent profile of autophagy activity through the course of M. tb infection in mammalian macrophages. Autophagy activity was inferred by the turnover measurement of autophagy markers and M. tb bacilli in THP-1 and RAW 264.7 macrophages. Over a period of 4 to 72 h, we observed highest autophagy turnover at 48 h of infection in M. tb-containing cells. This was evident by the highest turnover levels of p62 and intracellular M. tb. This supports observations of phagosomal damage mostly occurring at this time point and reveal the correlation of increased autophagy activity. The findings support the preservation of autophagy activity despite M. tb infection while also highlighting time-dependent differences in M. tb-infected macrophages. Future studies may explore time-dependent exogenous autophagy targeting towards host-directed anti-tuberculosis therapy.

Keywords: LC3B; RAW 264.7; THP-1; autophagic flux; autophagy turnover; biomarker; immunofluorescence microscopy; p62; tuberculosis; western blot.

Grants and funding

The South African government through the National Research Foundation of South Africa (NRF) (Grant number: CSUR13091742463) and the South African Medical Research Council (SAMRC)through its baseline funding supported this work. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. NO is supported through funding by the National Research Foundation of South Africa through its Professional Development Programme (PDP). Funders were not involved in the writing of the manuscript or the decision to submit it for publication. The authors were not paid by a pharmaceutical company or other agency to write this article. The corresponding author (CK) had full access to all the data in the study and the final responsibility for the decision to submit for publication.