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The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages
Thesis (MSc)--Stellenbosch University, 2021.
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867613979238465536 |
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| access_status_str | Open Access |
| author | Mkhonza, Mbali Nondumiso |
| author2 | Baker, Bienyameen |
| author_browse | Baker, Bienyameen Mkhonza, Mbali Nondumiso |
| author_facet | Baker, Bienyameen Mkhonza, Mbali Nondumiso |
| author_sort | Mkhonza, Mbali Nondumiso |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/110433 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:44.746Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/110433 The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages Mkhonza, Mbali Nondumiso Baker, Bienyameen Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics. Mycobacterium smegmatis Mycobacterium tuberculosis Gene expression Macrophages UCTD Thesis (MSc)--Stellenbosch University, 2021. ENGLISH ABSTRACT: Tuberculosis (TB) is a communicable disease caused by the virulent mycobacterial strain, Mycobacterium tuberculosis (M. tb). With an estimated 1.3 million deaths, of HIV uninfected individuals, and 10 million infected people in 2019, the World Health Organisation deemed TB a global epidemic and implemented the End TB Strategy to eradicate the disease by 2035. Since the current treatment regimen makes use of antibiotics, the increasing number of multi-drug resistant M. tb strains has resulted in a need for research into alternative options. One approach that could be of interest is host directed therapy which aims to identify and support the mechanism of action used by the host during its immune response to infection. Previously generated Ampliseq data identified CMPK2, which is suspected of playing a role in the differentiation of monocytic cells into macrophages, and RSAD2, associated with antiviral response, as being among the top upregulated genes following mycobacterial infection. Prior research on these genes had focused on their role during viral infections and thus, we aimed to better understand their involvement in the killing of mycobacteria in macrophages. During the present study, in vitro analysis of cultured THP-1 cells following differentiation into macrophages and infection with Mycobacterium smegmatis (non-pathogenic strain) and Mycobacterium bovis BCG (facultatively pathogenic strain) was performed. Thereafter, siRNA mediated knockdown in M. smegmatis was conducted to determine the effect that decreased expression of these genes had on the survival of the mycobacteria. For the vector-based knock up of RSAD2 and CMPK2, M. bovis BCG infected cells were treated with gene specific vector. Mycobacterial survival (via colony forming units) and relative gene expression (via qPCR) was then assessed. Gene expression results showed a significant difference (p=0.00030) between infected only and those treated with RSAD2 siRNA at 12 hours post M. smegmatis-infection, indicating that knockdown was achieved when using specific siRNA. Similarly, we showed that CMPK2 levels measured at 12 hours post M. smegmatis infection also achieved a significant decrease in the gene levels, p<0.0001. However, the mycobacterial survival results under these treatment conditions did not indicate a significant change following gene specific knockdown of CMPK2 or RSAD2, suggesting that the change in mRNA levels did not translate to an overall difference in the killing of the mycobacteria. Next, when a vector-based approach was used to knock-up CMPK2 and RSAD2, the results indicated no significant change in M. bovis BCG survival at 24 hours post infection. However, at 48 hours post M. bovis BCG infection, a significant decrease in the mycobacterial survival was observed following RSAD2 and CMPK2 vector knock up, p = 0.0003 and p= 0.0317 respectively. In conclusion, the present study demonstrated that successful siRNA-mediated knockdown of CMPK2 and RSAD2 in M. smegmatis could be achieved in infected cells on an mRNA level. Analysis of M. bovis BCG survival following knock-up with the specific gene vectors indicated a significant change at 48 hours post infection. AFRIKAANSE OPSOMMING: Tuberkulose (TB) is 'n oordraagbare siekte wat deur die bacterium Mycobacterium tuberculosis (M. tb) veroorsaak word. Met 'n geskatte 1,3 miljoen sterftes, van MIV-onbesmette individue, en tien miljoen geïnfekteerde mense in 2019, beskou die Wêreldgesondheidsorganisasie TB as 'n wêreldwye epidemie en het die End TB-strategie geïmplementeer om die siekte teen 2035 uit te roei. Aangesien die huidige behandelingsregime gebruik maak van antibiotika, is die toenemende aantal multi-middelweerstandige M. tb het gelei tot die behoefte aan navorsing oor alternatiewe opsies. 'n Benadering wat van belowend kan wees, is gasheergerigte terapie wat die meganisme van die gasheer se immuunrespons om infeksie te identifiseer en vernietig ondersteun. Ampliseq data wat in ‘n vorige studie gegenereer is, het CMPK2 geïdentifiseer, wat vermoedelik ‘n rol speel in die differensiasie van monosiet tot makrofaag, asook RSAD2, wat verbind is met antivirale reaksie, as een van die top gereguleerde gene na infeksie met M.tb. Vorige navorsing van die gene het gefokus op hulle rol tydens virale infeksies en daarom het ons studie gepoog om hul betrokkenheid by die dood van M. tb in makrofage beter te verstaan. In hierdie studie is in vitro analise van THP1-selle gedoen na differensiasie in makrofage en infeksie met Mycobacterium smegmatis (nie-patogeniese bakterium) en Mycobacterium bovis BCG (fakultatiewe patogeen). Daarna is siRNA-gemedieerde uitklop in M. smegmatis uitgevoer om die effek te bepaal wat die verminderde uitdrukking van hierdie gene op die oorlewing van die mikobakterieë het. Vir die vektorgebaseerde uitklop van RSAD2 en CMPK2 is M. bovis BCG- geïnfekteerde selle behandel met geen-spesifieke vektore. Mikobakteriese oorlewing (dmv kolonievormende eenhede) en relatiewe geenuitdrukking (dmv qPCR) is vervolgens bepaal. Resultate van gene-uitdrukking het 'n beduidende verskil (p = 0.0030) getoon tussen M. smegmatis geïnfekteerde selle en geïnfekteerde selle wat 12 uur na infeksie met RSAD2 siRNA behandel is, wat daarop dui dat die uitklop bereik is tydens die gebruik van spesifieke siRNA. Ons het ook gewys dat CMPK2-vlakke gemeet 12 uur na M. smegmatis-infeksie ook 'n beduidende afname in die geenvlakke behaal het, p<0.0001. Die mikobakteriese oorlewingsresultate onder hierdie behandelingstoestande dui egter nie op 'n beduidende verandering na geen-spesifieke afbreek van CMPK2 of RSAD2 nie, wat daarop dui dat die verandering in mRNA-vlakke nie 'n algemene verskil in die doodmaak van die mikobakterieë is nie. 'n Vektor-gebaseerde benadering is gebruik is om CMPK2 en RSAD2 op te reguleer waarvan die resultate geen beduidende verandering in M. bovis BCG-oorlewing 24 uur na infeksie aangedui nie. 48 uur na M. bovis BCG-infeksie is daar egter 'n beduidende afname in die mikobakteriese oorlewing waargeneem na onderskeidelik RSAD2- en CMPK2-vektore, p = 0.0003 en p = 0.0317. Die huidige studie het getoon dat suksesvolle siRNA-gemedieerde uitklop van CMPK2 en RSAD2 in M. smegmatis geïnfekteerde selle op 'n mRNA-vlak bereik kon word. Analise van M. bovis BCG-oorlewing na opknapping met die spesifieke geen vektore dui op 'n beduidende verandering 48 uur na infeksie. Masters 2021-04-30T09:29:11Z 2021-04-30T09:29:11Z 2021-03 Thesis http://hdl.handle.net/10019.1/110433 en_ZA Stellenbosch University 94 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacterium smegmatis Mycobacterium tuberculosis Gene expression Macrophages UCTD Mkhonza, Mbali Nondumiso The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages |
| title | The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages |
| title_full | The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages |
| title_fullStr | The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages |
| title_full_unstemmed | The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages |
| title_short | The role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages |
| title_sort | role of cmpk2 and rsad2 in the killing of mycobacteria in macrophages |
| topic | Mycobacterium smegmatis Mycobacterium tuberculosis Gene expression Macrophages UCTD |
| url | http://hdl.handle.net/10019.1/110433 |
| work_keys_str_mv | AT mkhonzambalinondumiso theroleofcmpk2andrsad2inthekillingofmycobacteriainmacrophages AT mkhonzambalinondumiso roleofcmpk2andrsad2inthekillingofmycobacteriainmacrophages |