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Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads.
Thesis (MSc)--Stellenbosch University, 2023.
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| Language: | en_ZA en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867614140281913344 |
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| access_status_str | Open Access |
| author | Ymele Soko, Vivette |
| author2 | Mavumengwana, Vuyo |
| author_browse | Mavumengwana, Vuyo Ymele Soko, Vivette |
| author_facet | Mavumengwana, Vuyo Ymele Soko, Vivette |
| author_sort | Ymele Soko, Vivette |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/127358 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA en_ZA |
| last_indexed | 2026-06-10T12:47:18.472Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/127358 Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. Ymele Soko, Vivette Mavumengwana, Vuyo Loxton, Andre G. Smith, Liezel Allie, Nasiema Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Mycobacterium tuberculosis Apoptosis Necrosis -- Microbiology Tuberculosis -- Treatment Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Apoptosis is a natural immune protective mechanism that allows the host to clean up nonfunctional cells. Not surprisingly, blocking apoptosis is presented as one of the strategies Mycobacterium tuberculosis (M.tb) uses to avoid host immune defence. In the absence of apoptosis, infected cells are taken captive to support the growth of their invader and eventually progress to an uncontrolled death (necrosis), which in the case of M.tb infection is called caseous necrosis and is characteristic of pathology observed in tuberculosis patients. In this project, host-directed therapy (HDT) was exploited and involved using pro-apoptotic drugs to induce and restore the normal process of controlled cell death (apoptosis) in infected macrophages. The advantage of this strategy is that, unlike current tuberculosis (TB) therapy which is not specific to certain forms of bacteria. Including dormant and multi-resistant M.tb, HDT could affect all forms of bacteria because it does not directly target the pathogen. This project used Mycobacterium smegmatis mc2 155 (M. smegmatis)-infected macrophages as a model to assess the efficacy of selected apoptotic inducer (API) drugs in controlling the intracellular growth of M. smegmatis. The model has been structured so that its product can be cultured on agar plates to evaluate the growth of bacteria representative of the inhibition or growth of intracellular bacteria. Preliminary tests were performed to evaluate the antimicrobial susceptibility of mycobacterium against the selected (API) drugs. Amongst the 12 drugs that were assessed, cepharanthine, CP31398 dihydrochloride hydrate, marinopyrrole A, and nutlin-3a showed activity against M. tuberculosis and M. smegmatis mc2 155. These four drugs have a minimum inhibitory concentration (MIC) of 3.1 - 6.2 µg/mL, 6.2 – 12.5 µg/mL, 25 – 50 µg/mL and 50–100 µg/mL, respectively, against M. smegmatis mc2 155. Also, when tested against M.tb H37Rv, the MIC value of these drugs was one-fold lower than their respective MIC values observed on M. smegmatis. Furthermore, the minimum bactericidal concentration (MBC) test revealed that cepharanthine or CP-31398 dihydrochloride hydrate could kill M. smegmatis at 12.5 µg/mL or 25 µg/mL, respectively. Ex-vivo treatment of M. smegmatis-infected macrophages with tolerated concentrations of API drugs indicated there was some limitations in this host model due to spontaneous inhibition of M. smegmatis growth in THP-1 macrophages. Nonetheless, it was possible to test the efficacy of drugs intended for HDT using the difference in the relative survival of intracellular bacteria as indicators. Cepharanthine and CP-31398 dihydrochloride hydrate inhibited the intracellular growth of the bacteria after 12hrs treatments at a concentration representing half of their MIC value or one-quarter of their MBC value. Higher concentrations of these drugs, for instance, 6.2 µg/mL of cepharanthine; or 9.2 µg/mL of CP-31398 dihydrochloride hydrate, resulted in the absence of growth of intracellular bacteria after only 6 hrs treatment. Multi-cytokines analysis on the cell culture supernatants sampled during the treatment of macrophages indicated that mild expression of IFN-gamma, TNF-alpha, IL-1beta, IL-5, combined with the inhibition of IL-6 and IL-22 was required to eliminate intracellular M. smegmatis mc2 155. However, high levels of TNF-alpha, IL-1beta, IL-6, and IL-22 coupled with the absence of expression of IFN-gamma and IL-5 correlated with a high burden of intracellular bacteria. AFRIKAANS OPSOMMING: Apoptose is 'n natuurlike immuun beskermings meganisme wat die gasheer toelaat om niefunksionele selle uit ie sisteem te verwyder. Dit is nie verbasend dat blokkering van apoptose een van die metodes (virulente faktore) is wat deur Mycobacterium tuberculosis (M.tb) gebruik word om sy gasheer se immuun verdediging te vermy nie. In die afwesigheid van apoptose, word M.tb besmette selle gevange geneem om die groei van M.tb te bevorder en sodoende onbeheerde sel dood (nekrose) te vorder, wat in die geval van M.tb-infeksie kaseuse nekrose genoem. Kaseuse nekrose is ‘n kenmerkend kenmerk in pasiënte met tuberkulose. In hierdie projek is gasheergerigte terapie (HDT) voorgestel, waar pro-apoptotiese middels gebruik word om die normale proses van beheerde seldood (apoptose) in geïnfekteerde makrofage te induseer apoptosis te herstel. Die voordeel van hierdie strategie is dat, anders as huidige tuberkulose (TB) terapie wat nie spesifiek is vir dormant en multi-weerstandig bakterieë nie, kan HDT alle vorme van bakterieë beïnvloed omdat dit nie die patogeen direk teiken nie. Hierdie projek het Mycobacterium smegmatis mc2 155 (M. smegmatis)-geïnfekteerde makrofage as 'n model gebruik om die doeltreffendheid van geselekteerde apoptotiese induseerder (API) middels in die beheer van die intrasellulêre groei van M. smegmatis te bepaal. Die model is so gestruktureer dat intrasellulêre bakterie op agarplate gekweek is wat verteenwoordigend is van die inhibisie of groei van intrasellulêre bakterieë. Voorlopige toetse is uitgevoer om die antimikrobiese vatbaarheid van mykobakterie teen die geselekteerde (API) middels te evalueer. Onder die 12 middels wat geassesseer is, het kefarantien, CP-31398 dihidrochloriedhidraat, marinopirrool A en nutlin-3a aktiwiteit teen M.tb H37Rv en M. smegmatis mc2 155 getoon. Hierdie vier middels het 'n minimum inhiberende konsentrasie (MIC) van onderskeidelik 3,1 - 6,2 µg/ml, 6,2 – 12,5 µg/ml, 25 – 50 µg/mL en 50-100 µg/ml teen M. smegmatis mc2 155 getoon. Die MIC waardes teen M.tb H37Rv was een konsentrasie laer as hul onderskeie MIC-waardes teen M. smegmatis. Ook het kefarantien en CP-31398 dihidrochloriedhidraat 'n minimum bakteriedodende konsentrasie (MBC) van 12.5 µg/mL en 25 µg/ml onderskeidelik teen M. smegmatis getoon. Ex-vivo behandeling van M. smegmatis-geïnfekteerde makrofage met draaglike konsentrasies van API-middels, het aangedui dat hierdie model kon werk ten spyte van die beperking van spontane inhibisie van M. smegmatis groei in THP-1 makrofage wat in die model gerapporteer is. Die verskil in die relatiewe oorlewing van intrasellulêre bakterieë is gebruik as aanwysers vir die doeltreffendheid van die behandeling. Cepharanthine en CP-31398 dihidrochloriedhidraat het die intrasellulêre groei van die bakterieë na 12 uur behandeling geïnhibeer met ‘n konsentrasie wat die helfte van hul MIC waarde of een kwart van hul MBC waarde verteenwoordig. Hoër konsentrasies van hierdie middels, byvoorbeeld 6.2 µg/mL kefarantien; of 9.2 µg/ml CP-31398 dihidrochloriedhidraat het gelei tot die afwesigheid van groei van intrasellulêre bakterieë na slegs 6 uur behandeling. Multi-sitokiene analise op die selkultuur-supernatante wat tydens die behandeling van makrofage gemonster is, het aangedui dat ligte uitdrukking van IFN-gamma, TNF-alfa, IL-1beta, IL-5, gekombineer met die inhibisie van IL-6 en IL-22 was nodig om intrasellulêre M. smegmatis mc2 155 uit te skakel. Hoë vlakke van TNF-alfa, IL-1beta, IL-6 en IL-22 tesame met die afwesigheid van uitdrukking van IFNgamma en IL-5 het egter gekorreleer met 'n hoë las van intrasellulêre bakterieë. Masters 2023-02-28T06:21:45Z 2023-05-18T07:17:59Z 2023-02-28T06:21:45Z 2023-05-18T07:17:59Z 2023-03 Thesis http://hdl.handle.net/10019.1/127358 en_ZA en_ZA Stellenbosch University application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterium tuberculosis Apoptosis Necrosis -- Microbiology Tuberculosis -- Treatment Ymele Soko, Vivette Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. |
| title | Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. |
| title_full | Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. |
| title_fullStr | Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. |
| title_full_unstemmed | Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. |
| title_short | Evaluation of mycobacterium smegmatis infected d THP-1 macrophages as a model to assess host directed therapy: using apoptotic agents as repurposed anti-tuberculosis drug leads. |
| title_sort | evaluation of mycobacterium smegmatis infected d thp 1 macrophages as a model to assess host directed therapy using apoptotic agents as repurposed anti tuberculosis drug leads |
| topic | Mycobacterium tuberculosis Apoptosis Necrosis -- Microbiology Tuberculosis -- Treatment |
| url | http://hdl.handle.net/10019.1/127358 |
| work_keys_str_mv | AT ymelesokovivette evaluationofmycobacteriumsmegmatisinfecteddthp1macrophagesasamodeltoassesshostdirectedtherapyusingapoptoticagentsasrepurposedantituberculosisdrugleads |