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Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress
Thesis (MSc)--Stellenbosch University, 2022.
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867614114503720960 |
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| access_status_str | Open Access |
| author | Cook, Emma Nancy |
| author2 | Kenyon, Colin |
| author_browse | Cook, Emma Nancy Kenyon, Colin |
| author_facet | Kenyon, Colin Cook, Emma Nancy |
| author_sort | Cook, Emma Nancy |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125061 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:53.692Z |
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| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125061 Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress Cook, Emma Nancy Kenyon, Colin Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Mycobacterium tuberculosis -- Pathogenesis -- South Africa Antitubercular agents -- South Africa Drug resistance in microorganisms -- South Africa UCTD Thesis (MSc)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Despite the availability of multiple antituberculosis agents, thoroughly tested treatment regimens, and a vaccine, Tuberculosis is still one of the leading causes of death by a single pathogen worldwide, especially in developing countries, such as South Africa. Mycobacterium tuberculosis, the causative agent of Tuberculosis, has previously been reported to have infected one-third of the global population. However, an area of increasing concern regarding Tuberculosis is the rise in antibiotic-resistant isolates observed over the years since antituberculosis compounds were first discovered. Of the antibiotic-resistant clinical isolates reported in 2017, 82% were resistant to multiple antibiotics, and 8.2% were identified as totally drug-resistant; as a direct consequence, none of the available antibiotics would be effective in treating patients infected with such strains. These antibiotic-resistant strains require more complicated treatment regimens, and over time more cases of totally drug resistant M. tuberculosis are recognized. The rise in antibiotic-resistant M. tuberculosis, in conjunction with the slow discovery and development of new antituberculosis agents, emphasizes the need for more research into the antibiotic resistance acquisition mechanisms in M. tuberculosis. Research into the innate adaptive immune response system found in most prokaryotes and archaea, the CRISPR-Cas system, has in recent years revealed several interesting developments. Research into the CRISPR-Cas system has paralleled the field of RNA interference, in that RNA interference was initially thought to function only in immunity; however, further investigation revealed its involvement in gene regulation. Similarly, the CRISPR-Cas system has been shown to be involved in gene regulation, DNA repair, and even genome evolution, over and above the two most well-known functions, plasmid immunity and protection against bacteriophages. Research into the innate CRISPR-Cas system in M. tuberculosis is a relatively new field of research; however, the preliminary findings suggest that M. tuberculosis harbors one of the most complex CRISPR-Cas systems researched to date. This observation is somewhat counterintuitive considering that horizontal gene transfer in M. tuberculosis is of a historic nature and the niche environment in which M. tuberculosis has evolved to thrive, the alveolar macrophage, results in M. tuberculosis having minimal to no contact with bacteriophages. Therefore, the need for an innate immune response system such as the CRISPR-Cas system is low, which prompts the question, is the CRISPR-Cas system in M. tuberculosis simply a remnant of the evolutionary process, or is this system exploited for other cellular processes. Moreover, some of the nine CRISPR-associated Cas genes have been associated with drug resistance, gene regulation, and differential survival under environmental stress conditions when expressed in Mycobacterium smegmatis; although, the preliminary studies conducted thus far have published incongruent findings. Subsequently, despite the potential that the CRISPR-Cas system may be involved in other cellular processes, no studies have investigated the expression of the Cas operon to date. Therefore, this study aimed to investigate the relative transcription of the nine CRISPR-associated Cas genes in antibiotic exposed samples. In order to achieve this aim, the secondary aim of this study was the design and optimization of a qPCR assay, which in addition to its employment in this study could be used in future studies to further investigate the transcription of the CRISPR-associated Cas genes in response to other environmental stressors as well as validate mutation studies conducted on the genes within the Cas operon. This study began with the generation of a reliable experimental sample set consisting of M. tuberculosis samples exposed for 24 hours to the MIC and ½ MIC of RIF and INH at mid-log phase, which were then processed for RNA extraction. Additionally, this study validated and optimized a reliable RNA extraction method to extract RNA of suitable quality and quantity for downstream qPCR analysis from 10 ml of M. tuberculosis culture. Finally, the most significant observation made from the relative transcription conducted in this study was the ubiquitous expression of all nine of the CRISPR-associated Cas genes under all the conditions examined. Unlike the comparable CRISPR-Cas system in Escherichia coli, which is repressed under control conditions despite continuous interaction with bacteriophages and horizontal gene transfer events. In conclusion, although this study is a preliminary study, it has established a foundation upon which future studies can be developed. Firstly, this study successfully designed and optimized a qPCR assay specifically for the CRISPR-associated Cas genes in M. tuberculosis, which can be utilized for future relative quantification studies, as well as adapted for absolute quantification. Due to the conserved nature of the CRISPR-associated Cas genes in Mycobacterium sp., this assay could also be employed to investigate transcription levels in other strains. Secondly, this study was the first to examine the CRISPR-associated Cas genes in M. tuberculosis using qPCR, thus, establishing the baseline transcription levels of the CRISPR-associated Cas genes in two common laboratory strains, H37Rv and CDC1551, and presented the first available data on the transcription of these genes in response to antibiotic stress. Therefore, building upon the qPCR assay, baseline transcription levels, and preliminary observations regarding the expression of the CRISPR-associated Cas genes in response to antibiotic stress, future studies should aim to elucidate the complex atypical features of the CRISPR-Cas system in M. tuberculosis. AFRIKAANSE OPSOMMING: Ten spyte van die beskikbaarheid van veelvuldige antituberkulose-middels, deeglik getoetsde behandelingsregimes en 'n entstof, is Tuberkulose steeds een van die hoofoorsake van dood deur 'n enkele patogeen wêreldwyd, veral in ontwikkelende lande, soos Suid-Afrika. Mycobacterium tuberculosis, die veroorsakende middel van Tuberkulose, is vroeër berig dat dit een derde van die wêreldbevolking besmet het. 'n Area van toenemende kommer met betrekking tot Tuberkulose is die toename in antibiotika-weerstandige isolate wat oor die jare waargeneem is sedert antituberkuloseverbindings die eerste keer ontdek is. Van die antibiotika-weerstandige kliniese isolate wat in 2017 aangemeld is, was 82% weerstandbiedend teen veelvuldige antibiotika, en 8,2% is as heeltemal middelweerstandig geïdentifiseer; as 'n direkte gevolg, sal geen van die beskikbare antibiotika effektief wees in die behandeling van pasiënte wat met sulke stamme besmet is nie. Hierdie antibiotika-weerstandige stamme vereis meer ingewikkelde behandelingsregimes, en met verloop van tyd word meer gevalle van totaal middelweerstandige M. tuberculosis herken. Die toename in antibiotika-weerstandige M. tuberculosis, tesame met die stadige ontdekking en ontwikkeling van nuwe antituberkulose-middels, beklemtoon die behoefte aan meer navorsing oor die verkrygingsmeganismes vir antibiotika-weerstandigheid in M. tuberculosis. Navorsing na die aangebore aanpasbare immuunresponsstelsel wat in die meeste prokariote en archaea voorkom, die CRISPR-Cas-stelsel, het die afgelope jare verskeie interessante ontwikkelings aan die lig gebring. Navorsing oor die CRISPR-Cas-stelsel het parallel geloop met die veld van RNAinmenging, deurdat daar aanvanklik gedink is dat RNA-inmenging slegs in immuniteit funksioneer; verdere ondersoek het egter ook betrokkenheid by geenregulering aan die lig gebring. Net so is getoon dat die CRISPR-Cas-stelsel betrokke is by geenregulering, DNA-herstel, en selfs genoom-evolusie, bo en behalwe die twee bekendste funksies, plasmied-immuniteit en beskerming teen bakteriofage. Navorsing oor die aangebore CRISPR-Cas-stelsel in M. tuberculosis is 'n relatief nuwe navorsingsveld; die voorlopige bevindinge dui egter daarop dat M. tuberculosis een van die mees komplekse CRISPR-Cas-stelsels huisves wat tot dusver nagevors is. Hierdie waarneming is ietwat teen-intuïtief as in ag geneem word dat horisontale geenoordrag in M. tuberculosis van 'n historiese aard is en die nis-omgewing waarin M. tuberculosis ontwikkel het om te floreer, die alveolêre makrofaag, lei daartoe dat M. tuberculosis minimale tot geen kontak met bakteriofage het nie. Daarom is die behoefte aan 'n aangebore immuunresponsstelsel soos die CRISPR-Cas-stelsel laag, wat die vraag laat ontstaan, is die CRISPR-Cas-stelsel in M. tuberculosis bloot 'n oorblyfsel van die evolusionêre proses, of word hierdie stelsel uitgebuit vir ander sellulêre prosesse. Boonop is sommige van die nege CRISPR-geassosieerde Cas-gene geassosieer met geneesmiddelweerstand, geenregulering en differensiële oorlewing onder omgewingstrestoestande wanneer dit in Mycobacterium smegmatis uitgedruk word; alhoewel, die voorlopige studies wat tot dusver uitgevoer is, het inkongruente bevindings gepubliseer. Ten spyte van die potensiaal dat die CRISPR-Cas-stelsel by ander sellulêre prosesse betrokke kan wees, het geen studies tot op hede die uitdrukking van die Cas operon ondersoek nie. Daarom was hierdie studie daarop gemik om die relatiewe transkripsie van die nege CRISPRgeassosieerde Cas-gene in antibiotika blootgestelde monsters te ondersoek. Om hierdie doel te bereik, was die sekondêre doel van hierdie studie die ontwerp en optimalisering van 'n qPCR-toets wat, benewens die gebruik daarvan in hierdie studie, gebruik kan word in toekomstige studies om die transkripsie van die CRISPR-geassosieerde Cas-gene verder te ondersoek in reaksie op ander omgewingstressors, asook mutasiestudies wat op die gene binne die Cas operon uitgevoer is, te bekragtig. Hierdie studie het begin met die ontwikkeling van 'n betroubare eksperimentele monsterstel bestaande uit M. tuberculosis monsters wat vir 24 uur blootgestel is aan die MIC en ½ MIC van RIF en INH in middel-log fase, wat dan very RNA ekstraksie verwerk is. Daarbenewens het hierdie studie 'n betroubare RNA-ekstraksiemetode bekragtig en geoptimaliseer om RNA van geskikte kwaliteit en kwantiteit vir stroomafwaartse qPCR-analise uit 10 ml M. tuberculosis-kultuur te onttrek. Ten slotte, die mees betekenisvolle waarneming gemaak van die relatiewe transkripsie wat in hierdie studie uitgevoer is, was die alomteenwoordige uitdrukking van al nege van die CRISPRgeassosieerde Cas-gene onder al die toestande wat ondersoek is. Anders as die vergelykbare CRISPRCas-stelsel in Escherichia coli, wat onder beheertoestande onderdruk word ten spyte van voortdurende interaksie met bakteriofage en horisontale geenoordraggebeurtenisse. Ten slotte, alhoewel hierdie studie 'n voorlopige studie is, het dit 'n grondslag gevestig waarop toekomstige studies ontwikkel kan word. Eerstens het hierdie studie suksesvol 'n qPCR-toets ontwerp en geoptimaliseer spesifiek vir die CRISPR-geassosieerde Cas-gene in M. tuberculosis, wat gebruik kan word vir toekomstige relatiewe kwantifiseringstudies, sowel as aangepas vir absolute kwantifisering. As gevolg van die bewaarde aard van die CRISPR-geassosieerde Cas-gene in Mycobacterium sp., kan hierdie toets ook aangewend word om transkripsievlakke in ander stamme te ondersoek. Tweedens was hierdie studie die eerste om die CRISPR-geassosieerde Cas-gene in M. tuberculosis met behulp van qPCR te ondersoek, en sodoende die basislyn transkripsievlakke van die CRISPR-geassosieerde Cas-gene in twee algemene laboratoriumstamme, H37Rv en CDC1551, vas te stel en die eerste beskikbare data oor die transkripsie van hierdie gene in reaksie op antibiotiese stres. Daarom moet toekomstige studies daarop gemik wees om die komplekse atipiese kenmerke van die CRISPR-Cas-stelsel in M. tuberculosis. Masters 2022-03-01T13:57:29Z 2022-04-29T12:52:18Z 2023-03-18T03:00:13Z 2022-03 Thesis http://hdl.handle.net/10019.1/125061 en_ZA Stellenbosch University xxxiv, 246 pages : illustrations application/pdf application/vnd.ms-excel Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterium tuberculosis -- Pathogenesis -- South Africa Antitubercular agents -- South Africa Drug resistance in microorganisms -- South Africa UCTD Cook, Emma Nancy Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress |
| title | Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress |
| title_full | Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress |
| title_fullStr | Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress |
| title_full_unstemmed | Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress |
| title_short | Investigating the expression of the CRISPR-associated Cas genes in Mycobacterium tuberculosis under antibiotic stress |
| title_sort | investigating the expression of the crispr associated cas genes in mycobacterium tuberculosis under antibiotic stress |
| topic | Mycobacterium tuberculosis -- Pathogenesis -- South Africa Antitubercular agents -- South Africa Drug resistance in microorganisms -- South Africa UCTD |
| url | http://hdl.handle.net/10019.1/125061 |
| work_keys_str_mv | AT cookemmanancy investigatingtheexpressionofthecrisprassociatedcasgenesinmycobacteriumtuberculosisunderantibioticstress |