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Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response
Thesis (PhD)--Stellenbosch University, 2021.
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2022
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| _version_ | 1867613769606103040 |
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| access_status_str | Open Access |
| author | Obasa, Zimvo |
| author2 | Sampson, Samantha |
| author_browse | Obasa, Zimvo Sampson, Samantha |
| author_facet | Sampson, Samantha Obasa, Zimvo |
| author_sort | Obasa, Zimvo |
| collection | Thesis |
| description | Thesis (PhD)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125056 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:24.431Z |
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| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
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| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125056 Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response Obasa, Zimvo Sampson, Samantha Mouton, Jomien Smith, Liezel Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Mycobacterium tuberculosis Persisters Macrophages Host-parasite relationships UCTD Thesis (PhD)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Approximately one-third of the world’s population is asymptomatically infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Despite the relatively effective anti-TB drugs, the pathogen is adept at persisting within its host. It is believed that this is because of a subpopulation of mycobacteria called persisters. Persisters are defined as a small, viable, but non-replicating (VBNR) drug-tolerant population. As a result of this population, lengthy antibiotic treatment may be required to completely eradicate the infection, with consequent treatment failure and drug resistance. Importantly, persisters also represent a reservoir of latent infection, which may progress to active disease when host immunity is compromised. There is limited understanding of how the host environment influences persister formation, and vice versa. This limited understanding is mostly attributed to the lack of tools to identify, isolate, and characterise VBNR mycobacteria from a heterogeneous population. To overcome this, we exploited a dual-fluorescence replication reporter plasmid expressing a constitutively expressed green fluorescent protein and an inducible red fluorescent protein. We applied this tool in both macrophage and mouse infection models. In this study, we aimed to i) assess the transcriptional profiles of macrophages containing either VBNR (persister) or actively replicating (AR) M. tuberculosis populations and ii) establish a tractable murine model that can be used to study M. tuberculosis persister populations in the context of the host, in conjunction with our replication reporter system. Understanding these phenotypes has important implications for how we approach TB prevention and treatment. To achieve the first aim, RAW264.7 murine macrophages were infected with the M. tuberculosis H37Rv::pTiGc reporter strain. After 4 days, macrophages were sorted into those containing VBNR or AR bacteria (as identified by the dual fluorescent reporter system) and uninfected macrophages using BD FACSJazzTM cell sorter. Following sorting, ribonucleic acid (RNA) from these populations was extracted and sequenced. To achieve the second aim, BALB/c mice were infected via an intravenous injection with 1X106 CFU of the M.tb H37Rv:: pTiGc reporter strain and infection was allowed to proceed for up to 51 days, depending on the validation step. A series of steps were followed to validate the use of this model. Specifically, we wanted to a) Confirm that the compound used to induce the replication reporter protein (theophylline) does not affect the host, pathogen or disease progression, b) Demonstrate theophylline induction of the replication reporter in vivo, c) Determine regulation of TurboFP635 induction in vivo following withdrawal of theophylline and d) Determine theophylline induction in vivo at early and later time points. At designated time points, infected mice were euthanized, lungs and spleens were harvested and homogenised. Infected cells from lung and spleen tissues were prepared and analysed using flow cytometry imaging, histopathology and serial dilution plating to determine bacterial organ burden. We were able to identify and sort macrophages containing AR and VBNR bacteria using our dual-fluorescent replication reporter system. Macrophages containing VBNR bacteria were strictly selected based on high GFP, high TurboFP635 selection, whereas macrophages containing AR bacteria were selected based on a high GFP, low TurboFP635 signal. More importantly, we successfully interrogated the transcriptional profiles of macrophages containing differentially replicating bacteria and the gene ontologies associated with these phenotypes following RNA-seq. Based on the RNA-seq data, we observed that macrophages containing AR bacteria exhibit an overexpression of transcriptional signatures associated with cell proliferation, whereas macrophages containing VBNR bacteria exhibit an overexpression of transcriptional signatures associated bacterial growth restriction. In the mouse study, we were able to i) identify and isolate GFP+ cells to assess TurboFP365 expression in vivo, ii) to determine the optimal theophylline dose and treatment duration required for maximal TurboFP635 expression in mice, iii) confirm that theophylline does not affect the disease progression, iv) determine that TurboFP635 signal is reduced if no theophylline is administered, v) TurboFP635 can be induced in in vivo at early and later time points, and vi) we were able to show that the reporter plasmid was retained at later time points and was still functional. This study is the first to successfully interrogate the macrophage immune response to differentially replicating bacteria using the replication reporter system. More importantly, this study was the first of its kind to validate and demonstrate the use of the dual-fluorescent replication reporter system for the identification of M. tuberculosis persister in specific in vivo niches, coupled with cell sorting and imaging flow cytometry. This system allows for tracking the presence of live bacteria, as well as monitoring bacterial replication. These findings provide an avenue for future studies to i) elucidate the physiological state of M. tuberculosis persisters and ii) simultaneously interrogate both the pathogen and host transcriptional signatures both in vitro and in vivo, and iii) to interrogate the effect of drug treatment and/or vaccination on persister populations and their role in latent TB infection as this may have important implications for TB prevention and treatment. AFRIKAANSE OPSOMMING: Ongeveer 'n derde van die wêreld se bevolking is asimptomaties besmet met Mycobacterium tuberculosis, die veroorsakende middel van tuberkulose (TB). Ten spyte van die relatief effektiewe anti-TB-middels, is die patogeen bedrewe om binne sy gasheer voort te gaan. Daar word geglo dat dit is as gevolg van 'n subpopulasie van mycobacteria wat persisters genome word. Persisters word gedefinieer as 'n klein, lewensvatbare, maar nie-repliserende (VBNR) dwelm-verdraagsame populasie. As gevolg van hierdie populasie kan lang antibiotiese behandeling nodig wees om die infeksie heeltemal uit te wis, met gevolglike mislukking van die behandeling en weerstand teen geneesmiddels. Belangrik is dat persisters ook 'n reservoir van latente infeksie verteenwoordig, wat tot aktiewe siektes kan oorgaan as gasheerimmuniteit in die gedrang kom. Daar is 'n beperkte begrip van hoe die gasheer omgewing die vorming van persister beïnvloed, en omgekeerd. Hierdie beperkte begrip word meestal toegeskryf aan die gebrek aan gereedskap om VBNR-mycobacteria uit 'n heterogene populasie te identifiseer, te isoleer en te karakteriseer. Om dit te oorkom, het ons 'n dubbel-fluorescentie replikasie reporter plasmied uitgebuit wat 'n konstitutief uitgedrukte groen fluorescerende proteïen en 'n induseerbare rooi fluorescerende proteïen uitdruk. Ons het hierdie instrument toegepas in eide makrofage- en muis infeksie modelle. In hierdie studie het ons ten doel gehad om i) die transkripsie profiele van makrofage te evalueer wat of VBNR (persister) of aktief replikerende (AR) M. tuberculosis populasies bevat en ii) 'n opspoorbare muis model te bepaal wat gebruik kan word om M. tuberculosis persister populasies te bestudeer in die konteks van die gasheer, in samewerking met ons replikasie reporter stelsel. Om hierdie fenotipes te verstaan, het belangrike implikasies vir hoe ons TB - voorkoming en behandeling benader. Om die eerste doel te bereik, is RAW264.7 muriene makrofage besmet met die M. tuberculosis H37Rv :: pTiGc reporter strein. Na 4 dae is makrofage gesorteer in dié wat VBNR- of AR - bakterieë bevat en soos geïdentifiseer met die dubbel-fluorescerende replikasie reporter stelsel oninfekteerde makrofage met behulp van BD FACSJazzTM -selsorteerder. Na sortering is ribonukleïensuur (RNA) uit hierdie populasies onttrek en opeenvolgend ge-sequence. Om die tweede doel te bereik, is BALB/c -muise besmet via 'n binneaarse inspuiting met 1x106 CFU van die M.tb H37Rv :: pTiGc -reporter strain en infeksie was toegelaat om tot tot 51 dae te duur, afhangende van die validerings stap. 'n Reeks stappe is gevolg om die gebruik van hierdie model te bekragtig. Ons wou spesifiek a) bevestig dat die verbinding wat gebruik word om die replikasie reporter proteïen (theophylline) beïnvloed nie die gasheer, patogeen of siekte progressie nie, b) demonstreer theophylline induksie van die replikasie reporter in vivo c) die regulering van TurboFP635 bepaal induksie in vivo na onttrekking van theophylline en d) bepaal theophylline induksie in vivo op vroeë en latere tyd punte. Op die aangewese tyds punt is besmette muise genoodsaak, longe en milt geoes en gehomogeniseer. Besmette selle uit longen miltweefsels is voorberei en geanaliseer met behulp van vloei -sitometrie -beeldvorming, histopatologie en reeks verdunning plating om die bakteriese orgaan-las te bepaal. Ons kon makrofage wat AR- en VBNR-bakterieë bevat, identifiseer en sorteer met behulp van ons dubbel-fluorescerende replikasie-reporter stelsel. Nog belangriker, ons het die transkripsie profiele van makrofage met suksesvol differensiele bakterieë en die geen ontologieë wat met hierdie fenotipes verband hou, gevolg na aanleiding van RNA-seq. Op grond van die RNA-seq data, het ons opgemerk dat makrofage wat AR-bakterieë bevat, 'n ooruitdrukking toon van transkripsionele handtekeninge wat verband hou met sel proliferasie, terwyl makrofage wat VBNR-bakterieë bevat, 'n ooruitdrukking toon van transkripsionele seine wat verband hou met bakteriële groei beperking. In die muis studie kon ons i) GFP+ selle identifiseer en isoleer om TurboFP365 -uitdrukking in vivo te bepaal, ii) om die optimale theophylline dosis en behandelings duur te bepaal wat nodig is vir die maksimum TurboFP635 -uitdrukking in muise, iii) bevestig dat theophylline geen invloed op die siekte progressie het nie, iv) bepaal dat die TurboFP635 -sein verminder word as geen theophylline toegedien word nie, v) TurboFP635 kan in vivo geïnduseer word op vroeë en latere tyd punte, en vi) ons kon wys dat die reporter plasmied op latere tyds punte behou word en nog steeds funksioneel was. Hierdie studie is die eerste om die immuunrespons van makrofage op verskillende bakterieë met behulp van die replikasie -reporter stelsel suksesvol te gebruik. Nog meer belangriker, hierdie studie was die eerste in sy soort om die gebruik van die dubbel-fluorescerende replikasie reporter stelsel te bevestig en te demonstreer vir die identifisering van M. tuberculosis persister in spesifieke in vivo nisse, tesame met sel sortering en beelding van vloei sitometrie. Hierdie sisteem ondersteun die monitering van lewendige bakterieë, sowel as bakterieële replikasie. Hierdie bevindinge bied 'n uitweg vir toekomstige studies om i) die fisiologiese toestand van M. tuberculosis persisters toe te lig en ii) gelyktydig die patogeensowel as die gasheer transkripsie -sein te ondervra, beide in vitro en in vivo, en iii) om die effek van geneesmiddel behandeling en /of inenting op persister populasie en hul rol in latente TB - infeksie te ondervra, aangesien dit belangrike implikasies kan hê vir die voorkoming en behandeling van TB. Doctoral 2022-02-08T11:33:45Z 2022-04-29T12:52:05Z 2022-02-08T11:33:45Z 2021-10 Thesis http://hdl.handle.net/10019.1/125056 en_ZA xx, 173 pages : illustrations application/pdf |
| spellingShingle | Mycobacterium tuberculosis Persisters Macrophages Host-parasite relationships UCTD Obasa, Zimvo Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response |
| title | Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response |
| title_full | Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response |
| title_fullStr | Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response |
| title_full_unstemmed | Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response |
| title_short | Mycobacterium tuberculosis persisters at the host-pathogen interface: development and application of tools for identification and elucidating impact on host response |
| title_sort | mycobacterium tuberculosis persisters at the host pathogen interface development and application of tools for identification and elucidating impact on host response |
| topic | Mycobacterium tuberculosis Persisters Macrophages Host-parasite relationships UCTD |
| url | http://hdl.handle.net/10019.1/125056 |
| work_keys_str_mv | AT obasazimvo mycobacteriumtuberculosispersistersatthehostpathogeninterfacedevelopmentandapplicationoftoolsforidentificationandelucidatingimpactonhostresponse |