Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages.
Thesis (PhD)--Stellenbosch University, 2023.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
2022
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613783280582656 |
|---|---|
| access_status_str | Open Access |
| author | Okugbeni, Naomi |
| author2 | Kinnear, Craig |
| author_browse | Kinnear, Craig Okugbeni, Naomi |
| author_facet | Kinnear, Craig Okugbeni, Naomi |
| author_sort | Okugbeni, Naomi |
| collection | Thesis |
| description | Thesis (PhD)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125158 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:37.777Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125158 Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. Okugbeni, Naomi Kinnear, Craig Loos, Ben Johnson, Glynis Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Autophagic vacuoles Mycobacterium tuberculosis Macrophages Medical microscopy Western immunoblotting Thesis (PhD)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Autophagy, a cellular process that degrades unwanted proteins, organelles and intracellular pathogens is involved in host defence against Mycobacterium tuberculosis (M. tb), the pathogen that causes tuberculosis (TB). Tuberculosis, an ancient bacterial disease, remains one of the leading causes of deaths worldwide killing approximately 1.4 million individuals yearly. To alleviate this burden, more research is focusing on understanding the bacterial pathogenesis and host’s response with the aim of developing host-directed anti-TB therapies. Upon infection, M. tb is initially encapsulated in a phagosome for impending lysosomal degradation. Evidence has, however, shown that, during the course of infection, M. tb is able to damage this phagosome and translocate to the cytosol, triggering autophagy. Likewise, the exogenous stimulation of autophagy has been shown to facilitate M. tb killing. Despite these findings, knowledge gaps such as the time-dependent differences in basal autophagy activity during M. tb infection that could explain patterns of M. tb pathogenesis have not been fully elucidated. The overarching aim of our study was to track the activity of the autophagy machinery in uninfected and M. tb-infected macrophages over a period of 72 hours. Western blotting and immunofluorescence imaging of different autophagy markers in THP-1 and RAW 264.7 macrophages were used to accomplish this. The turnover of bacterial colony-forming units was also enumerated in these M. tb-infected macrophages at 4, 24, 48 and 72 hours post-infection. Lastly, some preliminary experiments to characterize the bacterial niche at selected time points were performed. We found that THP-1 and RAW 264.7 macrophages exhibit different time and infection-dependent patterns in autophagy turnover. Immunofluorescence microscopy analysis of bacteria-containing macrophages and intracellular bacterial enumeration identified 48 hours as the time point with the highest autophagy turnover. We further showed that usage of LC3B and p62, as well as a combined measurement of microscopic puncta count and relative cell volume were more accurate measures of autophagy activity. Higher resolution imaging of intracellular M. tb provided by Correlative Light and Electron Microscopy (CLEM) visually provided evidence of the different compartments occupied by M. tb at the start of infection and at 48 hours. Identification of 48 hours as the time point with highest intracellular M. tb turnover suggests that most cytosolic translocation occurs at this time point and should be a consideration when investigating autophagy in M. tb infection. Our study also highlights the potential value of LC3B and p62 as biomarkers for TB prognosis. With research investigating autophagy for host-directed anti-TB therapy, our research provides more clarity on the involvement of autophagy in M. tb clearance and identifies potential avenues for drug-targeting. In all, this study provided a comprehensive documentation of autophagy turnover across two cell lines and a time period of 72 hours in uninfected and M. tb-infected macrophages. This provides a strong background with valuable recommendations for future autophagy research. AFRIKAANSE OPSOMMING: Outophagie, 'n sellulêre proses wat ongewenste proteïene, organelle en intrasellulêre patogene afbreek, is betrokke by die gasheerverdediging teen Mycobacterium tuberculosis (M. tb), die patogeen wat tuberkulose (TB) veroorsaak. Tuberkulose, 'n ou bakterieёle siekte, bly steeds een van die grootste oorsake van sterftes wêreldwyd wat jaarliks ongeveer 1,4 miljoen mense doodmaak. Om hierdie las te verlig, fokus meer navorsing op die verstaan van die bakterieёle patogenese en die gasheer se reaksie met die doel om gasheergerigte anti-tuberkulose-terapieë te ontwikkel. By infeksie word M. tb aanvanklik ingekapsel in 'n fagosoom om lisosomale afbraak te bewerkstellig. Bewyse het egter getoon dat M. tb tydens die infeksie hierdie fagosoom kan beskadig en na die sitosol kan oorgaan, wat outofagie veroorsaak. Net so is bewys dat die eksogene stimulering van outofagie die dood van M. tb vergemaklik. Ten spyte van hierdie bevindings, is gapings soos die tydsafhanklike verskille in basale outofagie-aktiwiteit tydens M. tb-infeksie wat patrone van M. tb-patogenese kan verklaar, nie volledig toegelig nie. Die oorhoofse doel van ons studie was om die aktiwiteit van die outofagie-masjinerie in nie-geïnfekteerde en M. tb-geïnfekteerde makrofage oor 'n tydperk van 72 uur op te spoor. Western blotting en immunofluoressensiebeelding van verskillende outofagiemerkers in THP-1 en RAW 264.7 makrofage is gebruik om dit te bewerkstellig. Die omset van bakterieёle-kolonievormende eenhede is ook in hierdie M. tb-geïnfekteerde makrofage opgesom op 4, 24, 48 en 72 uur na infeksie. Laastens is 'n paar voorlopige eksperimente gedoen om die bakterieёle-nis op geselekteerde tydspunte te ondersoek. Ons het gevind dat THP-1 en RAW 264.7 makrofage verskillende tyd- en infeksieafhanklike patrone in outofagie-omset toon. Immunofluoressensiemikroskopie-analise van bakteriebevattende makrofage en intrasellulêre bakteriële opsomming identifiseer 48 uur as die tydstip met die hoogste outofagie-omset. Ons het verder getoon dat die gebruik van LC3B en p62, sowel as 'n gekombineerde meting van mikroskopiese puncta -telling en relatiewe selvolume, meer akkurate maatstawwe van outofagiese aktiwiteit was. Hoër resolusie beeldvorming van intrasellulêre M. tb wat deur Correlative Light and Electron Microscopy (CLEM) verskaf is, het visueel bewys gelewer van die verskillende kompartemente wat M. tb beset het aan die begin van die infeksie en na 48 uur. Identifikasie van 48 uur as die tydstip met die hoogste intrasellulêre omset van M. tb dui daarop dat die meeste sitosoliese translokasie op hierdie tydstip plaasvind en dit moet in ag geneem word by die ondersoek na outofagie by M. tb -infeksie. Ons studie beklemtoon ook die potensiële waarde van LC3B en p62 as biomerkers vir TB -prognose. Met navorsing wat outofagie ondersoek vir gasheergerigte anti-tuberkulose-terapie, bied ons studie meer duidelikheid oor die betrokkenheid van outofagie by M. tb-opruiming en identifiseer potensieële moontlikhede vir dwelmgerigtheid. In totaal het hierdie studie 'n omvattende dokumentasie verskaf van die omset van outofagie oor twee sellyne en 'n tydperk van 72 uur in nie-geïnfekteerde en M. tb-geïnfekteerde makrofage. Dit bied 'n sterk agtergrond met waardevolle aanbevelings vir toekomstige outofagie -navorsing om TB -las in makrofage te verstaan en te verlig. Doctorate 2022-01-12T11:47:44Z 2022-04-29T12:56:33Z 2022-07-13T03:00:08Z 2022-01 Thesis http://hdl.handle.net/10019.1/125158 en_ZA xxiv, 212 pages : illustrations xxiv, 212 pages : illustrations application/pdf |
| spellingShingle | Autophagic vacuoles Mycobacterium tuberculosis Macrophages Medical microscopy Western immunoblotting Okugbeni, Naomi Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. |
| title | Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. |
| title_full | Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. |
| title_fullStr | Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. |
| title_full_unstemmed | Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. |
| title_short | Tracking autophagy progression in mycobacterium tuberculosis-infected macrophages. |
| title_sort | tracking autophagy progression in mycobacterium tuberculosis infected macrophages |
| topic | Autophagic vacuoles Mycobacterium tuberculosis Macrophages Medical microscopy Western immunoblotting |
| url | http://hdl.handle.net/10019.1/125158 |
| work_keys_str_mv | AT okugbeninaomi trackingautophagyprogressioninmycobacteriumtuberculosisinfectedmacrophages |