Using fluorescene to understand Mycobacterial Heterogeneity at a Single Cell Level

Parbhoo, Trisha (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Tuberculosis remains a major worldwide health threat. Among those infected there is risk of Mycobacterium tuberculosis, the causative agent of tuberculosis, developing into an asymptomatic dormant state. Cell-to-cell phenotypic variation is known to contribute to the establishment of diverse colonization states to evade host immune responses. There are major knowledge gaps regarding dormant or viable but non-culturable (VBNC) bacteria as they are difficult to isolate and heterogeneous populations are not reflected by colony forming unit plating. This has led to the application of single-cell techniques, such as flow cytometry, which offers a rapid, high-throughput tool to analyse the physiological and biochemical characteristics of bacteria at a single cell level in M. tuberculosis. Here we discussed various applications of flow cytometry for further understanding the physiological nature of bacterial systems. We aim to provide a better understanding of these physiological states of mycobacteria, and this thesis describes efforts to develop tools towards this aim. To identify and enumerate live and dead Mycobacterium smegmatis within a heterogeneous population we optimised the LIVE/DEAD BacLight Bacterial Viability and counting kit, exploiting flow cytometry. M. smegmatis was quantified by means of standardization beads, and the kit shows promise for developing a rapid, culture-free counting method for mycobacteria, ultimately replacing colony forming unit (CFU) plating. Efforts were initiated for applying this method to bacteria exposed to various anti-tuberculosis antibiotics, as well as for bacteria spiked into artificial sputum, which will require further investigation. In addition, flow cytometry was applied together with a recently developed Fluorescence Dilution (FD) reporter system, to enable measurement of differentially replicating mycobacteria in macrophage infection models. Previous studies have reported M. smegmatis to be rapidly killed upon uptake into macrophages. In contrast, results from our lab using a M. smegmatis macrophage infection model uncovered an unexpected sub-population of apparently dividing M. smegmatis. The nature of this population was explored using a fluorescently labelled anti-tuberculosis cell-surface binding antibody in combination with FD, for determination of whether the replicating population was intra- or extracellular of the macrophage. However, further investigation will need to be performed to determine the location of this population. The findings of this study suggest the feasibility of a real-time tool to distinguish and enumerate live and dead cells within a heterogeneous mycobacterial population. Additionally, FD in combination with other markers offers a promising technique for studying population-wide adaptation to environmental stress, or even to anti-tuberculosis drugs.

AFRIKAANSE OPSOMMING: Tuberkulose bly 'n wêreldwye gesondheidsbedreiging. Tussen diegene wat met Mycobacterium turbuculosis besmet is, die veroorsakende agent van tuberkulose, is daar 'n verdere risiko dat die bakterium in 'n asimptomatiese dormante staat kan ontwikkel. Sel-tot-sel fenotipiese variasies is bekend daarvoor om by te dra tot die vestiging van diverse kolonisasie toestande om die gasheer se immuunreaksie te ontduik. Daar is groot kennisgapings tussen dormante, ook bekend as lewensvatbaar maar nie-kultuurbare bakterieë, omdat dit moeilik is om hulle te isoleer en heterogene bevolkings word nie weerspieël deur enkele kolonievormende eenheide nie. Dit het gelei tot die toepassing van enkelseltegnieke, soos vloeisitometrie, wat 'n vinnige, hoë-deurset instrument is om die fisiologiese en biochemiese eienskappe van bakterieë op 'n enkelselvlak te analiseer. Hier bespreek ons verskeie toepassings van vloeisitometrie vir verdere begrip van die fisiologiese aard van die bakteriële stelsel. Ons doel is om 'n beter begrip van hierdie fisiologiese toestande van mikrobakterieë te voorsien. Hierdie tesis beskryf pogings om gereedskap te ontwikkel vir hierdie doel. Om lewende en dooie Mycobacterium smegmatis te identifiseer binne 'n heterogene bevolking, het ons die LIVE/DEAD BacLight Bakteriese Lewensvaatbaarheid protokol in parallel met vloeisitometrie gebruik. M. smegmatis was gekwantifiseer by wyse van gestandariseerde krale, en die tegniek wys belowende resultate vir die ontwikkeling van ‘n vinnige, kultuur-vrye metode om mikrobakterieë te tel, in plaas van om kolonievormende eenhede te plaat. Pogings is geïnisieer vir die toepassing van hierdie metode op bakterieë wat blootgestel is aan verskeie anti-tuberkulose antibiotika, asook bakterieë in kunsmatige speeksel, wat verdere ondersoek vereis. Daarbenewens is vloeisitometrie saam met die onlangse ontwikkelde Fluoresensie Verwatering (FV) rapporteerstelsel gebruik, om ons in staat te stel om lesings van differensiële repliserende mikrobakterieë in 'n makrofaag infeksiemodel vas te stel. Vorige studies het aangedui dat M. smegmatis vinnig doodgemaak is tydens opname in makrofage. In teenstelling hiermee, het die resultate van ons labaratorium se M. smegmatis macrofaag infeksiemodel, 'n onverwagte subbevolking van repliserende M. smegmatis blootgestel. Die aard van die bevolking was ondersoek met behulp van 'n fluoreserend gemerkte anti-tuberkulose seloppervlakbindende teenliggaam in kombinasie met FV, wat gebruik was om te bepaal of die repliserende bevolking intra- of ekstrasellulêr van die makrofaag is. Daar sal egter verdere ondersoek uitgevoer moet word om die ligging van hierdie bevolking te bepaal. Die bevindinge van hierdie studie dui op die lewensvatbaarheid van 'n ware tyd instrument wat tussen lewende en dooie selle kan onderskei in 'n heterogene mikobakteriële bevolking. Daarbenewens bied die gebruik van FV, in kombinasie met ander merkers, 'n belowende tegniek te wees vir die bestudering van bevolking-wye aanpassing op omgewingstres, of op anti-tuberkulose dwelms.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101451
This item appears in the following collections: