Browsing by Author "Mkhonza, Mbali Nondumiso"
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- ItemThe role of CMPK2 and RSAD2 in the killing of mycobacteria in macrophages(Stellenbosch : Stellenbosch University, 2021-03) Mkhonza, Mbali Nondumiso; Baker, Bienyameen; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.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.