Identification of biosignatures associated with Mycobacterium tuberculosis

Simple item page
dc.contributor.advisorMouton, Jomienen_ZA
dc.contributor.advisorSampson, Samanthaen_ZA
dc.contributor.authorDikhoba, Lesedi Brighten_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2021-08-25T14:00:16Zen_ZA
dc.date.accessioned2022-02-22T10:16:50Zen_ZA
dc.date.available2022-08-31T03:00:15Zen_ZA
dc.date.issued2021-12en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2021.en_ZA
dc.description.abstractENGLISH ABSTRACT: Latent tuberculosis (TB) is thought to be caused by a subpopulation of Mycobacterium tuberculosis, called persisters. Persisters are defined as a small, viable, but non-replicating (VBNR), reversibly drug-tolerant, but not genetically resistant, bacterial subpopulation. It has been shown that M. tuberculosis exhibits heterogeneous replication rates following macrophage infection, consisting of actively replicating (AR) M. tuberculosis and VBNR persister M. tuberculosis. However, our understanding of how M. tuberculosis manipulates the physiology of macrophages to establish a persistent TB infection within the host is very limited. Therefore, a detailed understanding of how M. tuberculosis persisters manipulate macrophage responses to establish TB infection is essential. In this study, we aimed to use a combination of fluorescence dilution (FD), fluorophore-conjugated monoclonal antibody staining and flow cytometry to identify whether a specific macrophage phenotype is associated with either AR or VBNR M. tuberculosis::pTiGc H37Rv. We first infected THP-1 macrophages with M. tuberculosis::pTiGc H37Rv and used colony forming units (c.f.u) plating to assess the inoculum, uptake and survival of M. tuberculosis::pTiGc for 120 h after the infection. Secondly, we applied the FD reporter system and flow cytometry to assess M. tuberculosis::pTiGc replication dynamics upon THP-1 macrophage infection over 120 h. Lastly, we exploited the use of FD technology in combination with fluorophore-conjugated primary antibody staining to monitor the macrophage polarisation of THP-1 macrophages infected with AR M. tuberculosis::pTiGc and THP-1 macrophages infected VBNR M. tuberculosis::pTiGc. Using c.f.u plating, we observed a 9.88 ± 0.83% mean percentage uptake of M. tuberculosis::pTiGc by THP-1 macrophages. C.f.u counts of intracellular M. tuberculosis::pTiGc following a 120 h infection time course revealed stable intracellular bacterial numbers. Using the FD reporter system, at earlier time points (0 h and 24 h) of THP-1 macrophage infection with M.tuberculosis::pTiGc, a relatively homogeneous population was observed, similar to thatobserved in in vitro-cultured M. tuberculosis::pTiGc. However, as the infection progressed (72 h and 120 h post infection), a heterogeneous population was observed, with a clear AR intracellular subpopulation and a more slowly replicating or halted intracellular subpopulation (VBNR). At 72 h post infection, we observed a proportion of 0.3 ± 0.01% VBNR persister-like M. tuberculosis::pTiGc and at 120 h post infection a proportion of 1.3 ± 0.35%. This demonstrated an increase in persister percentages as infection progressed. Using the FD reporter system in combination with fluorophore-conjugated primary antibody staining, we observed that the antibody selected to represent M1 polarisation, anti-CD274, positively stained THP-1 macrophages containing VBNR M. tuberculosis::pTiGc (expressing high GFP and high TurboFP635 intensity) and the antibodies selected to represent M2 polarisation, anti-CD206 and anti-ARG-1, positively stained THP-1 macrophages containing AR M. tuberculosis::pTiGc (expressing high GFP, low TurboFP635 intensity). These findings indicated THP-1 macrophages containing VBNR M. tuberculosis::pTiGc have polarised into a pro-inflammatory, M1-like state, suggesting that VBNR M. tuberculosis::pTiGc evade recognition by intracellular immune receptors to establish a latent TB infection. In contrast, THP-1 macrophages containing AR M. tuberculosis::pTiGc have polarised into an anti-inflammatory, M2-like state, suggesting that AR M. tuberculosis::pTiGc overcome host defence by reprogramming macrophage polarisation in order to proliferate. These findings support that the FD reporter in combination with cell sorting would allow isolation of the THP-1 macrophages containing VBNR M. tuberculosis::pTiGc and THP-1 macrophages containing AR M. tuberculosis::pTiGc for transcriptomic analysis to probe into the mechanisms M. tuberculosis use to establish differential macrophage infection.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Kenners reken dat latente tuberkulose (TB) veroorsaak word deur n sub-populasie van Mycobacterium tuberculosis genaamd persisters. Persisters word gedefinieër as ‘n klein, lewensvatbare, maar nie-repliserende (VBNR) bakteriële populasie. Die sub-populasie selle is ook omkeerbaar antibiotika verdraend, maar nie geneties antibiotika weerstandig nie. Daar is gewys dat M. tuberculosis ‘n heterogeniese replikasie koers volg na makrofaag infeksies bestaande uit aktief repliserende (AR) asook VBNR persister M. tuberculosis. Ons verstandhouding oor hoe M. tuberculosis die makrofaag se fisiologie manipuleer om ‘n perister staat te vorm is egter beperk. Daarom is ‘n in diepte studie nodig wat die verstandhouding rondom hoe presies M. tuberculosis persisters makrofaag reaksies manipuleer noodsaaklik. Ons doel in die studie is om ‘n kombinasie van fluoreserende verdunnings (FD), fluorofoor-gehegde monoklonale teenliggaampies asook vloeisitometrie gebruik te maak om te bepaal of spesifieke makrofaag fenotipes met AR of VBNR M. tuberculosis::pTiGc H37Rv selle geassosieer word vir latente infeksies. Eerstens het ons THP-1 makrofage met M.tuberculosis::pTiGc H37Rv geïnfekteer en kolonie vormende eenheid (KVE) uitplaat tegnieke gebruik om die inset, opneem asook oorlewing van bakterieë binne THP-1 makrofage te bevestig. Tweedens het ons die FD rapporterende sisteem en vloesitometrie gebruik en M. tuberculosis::pTiGc replikasie tydens THP-1 makrofaag infeksies bestudeer na 120h. Laastens het ons die FD tegnologie gebruik in kombinasie met fluorofoor-gehegde primêre teenliggaampies om makrofaag polarisering van AR en VBNR M. tuberculosis::pTiGc geïnfekteerde THP-1 selle te monitor. Deur gebruik te maak van KVE uitplaat tegnieke het ons ‘n gemiddelde 9.88% ± 0.83 opneem persentasie waargeneem. KVE tellings van intrasellulêre M.tuberculosis::pTiGc na ‘n 120h infeksie periode het baie stabiele intrasellulêre getalle onthul. Die dubbele rapporterende sisteem het gewys dat die M. tuberculosis::pTiGc populasies heterogenies is tydens makrofaag infeksies. By vroeër tydspunte (0h en 24h) was die populasie redelik homogenies wat ooreenstem met in vitro M. tuberculosis::pTiGc kulture. Soos die infeksie vorder (72h en 120h) word die oppulasie meer heterogenies met ‘n AR intrasellulêre sub-populasie asook ‘n meer stadig of geen repliserende sub-populasie. Ons het onderskeidelik ‘n 0.3% ± 0.01 en 1.3% ± 0.35 VBNR proporsie selle gevind by 72h en 120h. Die het gedemonstreer dat soos die infeksie verloop verhoog die proporsie persister selle.. Ons het M1 teenliggaam, anti-CD274, positief gemerkde THP-1 makrofage wat VBNR M. tuberculosis::pTiGc (hoë GFP en hoë urboFP635 intensiteit) bevat opgemerk asook die gekose M2 teenliggaamp, anti-CD206 en anti-ARG-1, gemerkde THP-1 makrofage met AR M. tuberculosis::pTiGc (hoë GFP, lae TurboFP635 intensiteit). Die bevindings dui daarop dat THP-1 makrofage wat VBNR M. tuberculosis::pTiGc bevat gepolariseer word na ‘n pro-inflammatoriese, M1 tipe, staat en dat VBNR M. tuberculosis::pTiGc intrasellulêre immuun response reseptore ontwyk om ‘n latente infeksie te vestig. Kontrasterend tot die bogenoemde uitkoms is daar gevind dat THP-1 makrofage wat AR M. tuberculosis::pTiGc bevat gepolariseer het na ‘n anti-inflammatoriese, M2 tipe, staat wat daarop dui dat AR M. tuberculosis::pTiGc gasheer beskermings meganismes oorkom deur herprogrammering van makrofaag polarisering om verder te kan vermeerder. Die resultate verskaf waardevolle informasie rakende die toekomstige gebruik van die FD sisteem in kombinasie met sel sortering om THP-1 makrofage wat AR en VBNR M. tuberculosis::pTiGc suksesvol te isoleer vir transkriptomiese analisering om meer in diepte die meganisme te ondersoek wat M. tuberculosis gebruik om verskillende makrofaag infeksies the handhaaf.af_ZA
dc.description.versionMastersen_ZA
dc.embargo.terms2022-08-31en_ZA
dc.format.extentxxii, 118 pages : illustrationsen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/124212en_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMycobacterium tuberculosisen_ZA
dc.subject.lcshFluorescence dilutionen_ZA
dc.subject.lcshCytometryen_ZA
dc.subject.lcshMacrophagesen_ZA
dc.subject.lcshLatent virus diseasesen_ZA
dc.titleIdentification of biosignatures associated with Mycobacterium tuberculosisen_ZA
dc.typeThesisen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
dikhoba_identification_2021.pdf
Size:
8.16 MB
Format:
Adobe Portable Document Format
Description:
Download
License bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
license.txt
Size:
1.71 KB
Format:
Plain Text
Description:
Download
Collections
Masters Degrees (Molecular Biology and Human Genetics)