Browsing by Author "Baatjies, Lucinda"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemInvestigation of the expression and immunogenicity of Mycobacterium tuberculosis Rv1460(Stellenbosch : Stellenbosch University, 2021, 2021-03) Baatjies, Lucinda; Williams, Monique Joy; Loxton, Andre Gareth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: A challenge that has plagued humanity for centuries is the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. In 2018, over 9.6 million individuals fell ill with the disease and 1.5 million lost their lives. Not all infected individuals get sick, 95% remain healthy (asymptomatic) and are said to be latently infected. Only 3-10% of infected individuals develop tuberculosis (TB). The shortcomings of current immunodiagnostics include the failure to detect progression from latent infection to active tuberculosis disease, and the inability to monitor treatment efficacy. This highlights the need for new tuberculosis biomarkers. These biomarkers should be highly sensitive and specific diagnosing TB infection, specifically distinguishing between latent infection and active disease. One strategy to identify Mtb antigens with diagnostic potential is to identify genes that are specifically induced during infection or in specific disease stages. Despite the importance of iron homeostasis during TB infection, the utility of bacterial antigens expressed during the iron limitation experienced by Mtb within the host remains largely unexplored. The Rv1460 gene encodes a transcriptional regulator of the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 mobilization of sulphur (SUF) operon, which encodes the primary iron-sulphur biogenesis system in Mtb. The SUF operon is induced during iron limitation and intracellular growth of Mtb, pointing to its importance during infection. This study therefore aimed to investigate the level of the Rv1460 protein during intracellular growth of Mtb, and to gain insight into the regulation involved in determining these levels. Furthermore, since Rv1460 levels are predicted to be elevated during intracellular growth and infection, we hypothesize that it is immunogenic and may induce an immune response in individuals infected with Mtb that has diagnostic potential. To facilitate rapid monitoring of Rv1460 expression at the single cell level during intracellular growth we sought to develop a fluorescent reporter for monitoring its expression. Two fluorescent reporter vectors were constructed by cloning two Rv1460 promoter regions (123 bp and 211 bp) upstream of a promoterless mCherry gene and transforming Mtb H37Rv with the resulting vector constructs. The expression of the mCherry protein had no effect on the growth of the fluorescent strains when compared to the wild type (wt) control. Florescence per cell was determined either by dividing fluorescence determined in a plate reader by colony forming units per well i.e., Relative fluorescent units (RFU)/Colony forming units (CFU) or by flow cytometry. The RFU/CFU measurements showed more than a 10x increase between day 4 and 10 for H37Rv attB::pMV306 (control strain), H37Rv attB::pMV306_123mCherry and H37Rv attB::pMV306_211mCherry (fluorescent reporter strains), while values remained constant between day 10 and 14. Flow cytometry showed no increase in fluorescence between day 4, 10 and 14 for the two reporter strains. The high background fluorescence observed in the control strain for the RFU/CFU measurements suggests that the increase observed between day 4 and 10 was due to autofluorescence of the cells or background fluorescence in the media. An increase in the Rv1460 transcript was observed between day 4 and 10, while between day 10 and 14 the transcript levels decreased. The mCherry transcript was significantly lower than the Rv1460 transcript and the ratio of the two transcripts did not remain constant in either strain over time. This suggests that the mCherry reporters did not accurately reflect changes in Rv1460 transcript levels over time. Monitoring fluorescence in the reporter strains during intracellular growth suggests that Rv1460 expression is induced in a subset of bacteria within macrophages, and this population increases over the course of a 72-hour infection. Rv1460 protein does not seem to be highly immunogenic under the conditions tested. Higher levels of Interleukin (IL)-8 (p = 0.08) and Matrix metallopeptidase (MMP)-9 (p = 0.00) was measured in the active TB group when compared to the Quantiferon (QFN) negative (neg) group for the short-term stimulation (12 hours) with Rv1460 suggestive of an effector or memory response. Short-term stimulation for all stimulation conditions showed significantly lower levels of Regulated and normal T cell expressed and secreted (RANTES) in the active TB group when compared to QFN positive (pos) and QFN neg groups. This cytokine selectively attracts monocyte/macrophages in the airways and strongly induces migration of T cells with the memory phenotype.