Browsing by Author "Pietersen, Ray-Dean Donovan"
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- ItemEmploying omics to investigate the macrophage response to infection with pathogenic and non-pathogenic mycobacteria(Stellenbosch : Stellenbosch University, 2021-10) Pietersen, Ray-Dean Donovan; Baker, Bienyameen; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis kills more than a million people each year and its lengthy treatment is complicated by drug resistant variants of Mycobacterium tuberculosis. Pathogenic mycobacteria have the ability to survive inside its human hosts and use the host’s own molecules to subvert host defences and avoid normal macrophage functions that normally contain and kill invading bacteria. New drug and drug targets as well as treatment options are continuously being sought to eradicate tuberculosis. One possible means of treatment is to target host molecules through the use of host directed therapeutics to stop or disrupt the pathogenic mycobacteria from using host molecules for its intracellular survival. Host-directed therapeutics (HDTs) have the advantage of not being subjected to drug resistance. HDTs are synthetically produced and can act to supress, through the use of siRNA (short interfering RNA), or activate, through the use of saRNA (short activating RNA), the expression of genes. Changing the expression level of a specific host gene can lead to the intracellular killing of pathogenic mycobacteria. Two separate studies using siRNA demonstrated that by enhancing the process of autophagy, which can overcome the blocking effect on phagosome maturation, resulted in decreased intracellular growth of the mycobacteria. Previous research indicates that the culturing of mycobacteria in the presence of detergents like Tween-80 disrupts the mycobacterial capsule and results in a different host response. This has an important implication in that mycobacteria proliferate and spread in the absence of detergent inside humans and mimicking this detergent-free state in the macrophage infection model, might elucidate drug targets that can be overlooked otherwise. To investigate the effect of Tween-80 on mycobacteria, a metabolomics study was performed to compare mycobacteria cultured in the presence and absence of Tween-80. Metabolome variations were seen for both Mtb H37Rv and R179 cultured in the presence of Tween-80. Overall, various metabolites were up-regulated but significantly more for H37Rv as compared to R179. Tween-80 provided an additional carbon source that caused a shift in the central carbon metabolism and hence a different metabolic profile. These results gave strong evidence to support the exclusion of detergent from mycobacterial cultures that will be used for macrophage infections. The effect of detergent was also tested on the response of macrophages by infecting mouse bone marrow-derived macrophages (BMDMs) with R179 grown in the presence and absence of Tween-80 and analysing the macrophage transcriptome through high throughput (RNA-Seq). The transcriptome profile for the BMDMs was different between infection with R179 cultured with and without Tween-80. Furthermore, while an overall down-regulation of genes were observed for infection with mycobacteria grown in Tween-80, the detergent-free cultured Mtb provoked a largely up-regulation of genes as well as a strong proinflammatory response. This demonstrated that it is important to exclude the use of detergents in the culturing of mycobacteria that will be used for infection studies. The current project aimed to identify differentially expressed genes in host cells between infection with pathogenic (Mtb H37Rv and Beijing clinical isolate R179) and non-pathogenic (M. smegmatis and M. bovis BCG) mycobacteria. The virulence level differ between these species. To ascertain if virulence could have an impact on the macrophage response, BMDMs were infected with Beijing Mtb clinical isolates R5527 (hyper-virulent) or R1507 (hypo-virulent). The macrophage responses were compared through transcriptomics profiles and showed differences for infection between hypo-and hyper-virulent Mtb. Prominent differences were revealed in the cytokine and chemokine levels induced between the hypo- and hyper-virulent Mtb, including pro-inflammatory cytokines IL- 1β, IL-6, IL-12β and the chemokine RANTES (CCL5). It could therefore be expected to find differentially expressed host genes in the comparison between the 4 mycobacterial species. The approach for identifying molecules that are potentially involved in the intracellular survival of mycobacteria, was to compare the macrophage response to infection between non-pathogenic mycobacteria (M. smegmatis and M. bovis BCG) that are normally killed inside macrophages and pathogenic mycobacteria (Mtb H37Rv and Beijing clinical isolate R179) that normally survive inside macrophages. The aim was to identify macrophage molecules that are differentially expressed between infection with pathogenic and non-pathogenic mycobacteria. Mouse bone marrow-derived macrophages were infected with mycobacteria that were cultured in the absence of detergent. RNA-Seq was used to determine the expression levels of genes by measuring mRNA transcripts. The focus was placed on genes that were differentially expressed when macrophages were infected with pathogenic mycobacteria and having a fold change of 2 or more. These genes were shortlisted and validated through qPCR. Pathway analysis (using STRING) revealed that genes having an antiviral function and induced by interferon signalling featured strongly among the differentially expressed genes. This list included mouse Mx genes that do not code for functional proteins. This lead to the study shifting to a human macrophage cell-based model to test the selected genes for their potential role in the intracellular survival of mycobacteria. Selected genes included MX1, MX2, IFITM1, IFITM3, OAS1, OAS2 and OAS3. BCG-infection could be grouped together with the response under infection with the pathogenic strains and was therefore used an indicator for infection with the pathogenic species. Gene expression levels were knocked down or up through the use of siRNA or non-viral vectors respectively to test if any of the genes of interest play a role in the intracellular survival of mycobacteria. Knocking down MX2 in M. smegmatis-infected THP-1 macrophages caused increased intracellular bacterial growth. The knock-down of IFITM1 and IFITM3 in M. smegmatis infected macrophages resulted in the same trend of increased intracellular growth. The same type of observation was made in a related published study for the knocking down of OAS genes for which it was concluded that these differentially expressed genes with an antiviral role also seem to have a protective role during mycobacterial infections. In contrast, knocking up the expression of MX1 and OAS3 in BCG-infected macrophages caused a significant reduction in the number of intracellular mycobacteria. The simultaneous treatment of BCG-infected macrophages with MX1 and OAS3 vectors resulted in a highly significant decrease in CFU at 48 hours post-infection as compared to the untreated control. This result is therefore in line with previously published genes that have an antiviral role in host cells and also being protective against mycobacteria. MX1 and OAS3 could be promising candidates if explored as targets in host-directed therapies against tuberculosis.