Browsing by Author "Arries, Jesmine S."
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- ItemThe identification of novel proteins involved in iron-sulphur cluster biogenesis in mycobacteria(Stellenbosch : Stellenbosch University, 2016-03) Arries, Jesmine S.; Williams, Monique J.; Sampson, Samantha L.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human GeneticsENGLISH ABSTRACT : Mycobacterium tuberculosis remains one of the world’s deadliest pathogens, and although decades of research have been dedicated to the discovery and development of anti-tuberculosis agents and vaccines, tuberculosis (TB) remains an ongoing global public health threat. Given the emergence of drug resistance and the poor treatment outcomes associated with this disease, it is imperative that we improve our understanding of the pathogenesis of M. tuberculosis in order to develop new anti-TB drugs and strategies for shortening drug treatment. Iron-sulphur (Fe-S) clusters are ubiquitous cofactors that are required for the maturation of various proteins, many of which are involved in essential biological processes. Multiprotein complexes are required for the in vivo assembly of Fe-S clusters, and the SUF system, encoded by the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465- Rv1466 operon in M. tuberculosis, is thought to be the major Fe-S cluster biogenesis machinery in this organism. This process is poorly understood in mycobacteria, and it is currently unclear if proteins outside of this operon are involved in Fe-S cluster biogenesis. In this study, we sought to identify novel proteins involved in Fe-S cluster assembly in mycobacteria using various approaches. Initially we optimised blue-native PAGE for the analysis of mycobacterial cytoplasmic protein complexes, and demonstrated that protein complexes could be identified by immunodetection of BN-PAGE and subsequent gel fraction analysis by mass spectrometry. However, due to the complexity of the cytoplasmic fraction and poor resolution of this technique, we were unable to use it to identify complexes containing the Fe-S cluster biogenesis machinery. In a second approach, we set out to generate an antibody against Csd, an Fe-S cluster assembly protein, to facilitate immunodectection of the Fe-S cluster biogenesis machinery. However, heterologous expression of the M. tuberculosis Csd in Escherichia coli proved unsuccessful due to insolubility of the recombinant protein. In an attempt to achieve soluble expression of Csd, a 6xHis-tag fusion was expressed in the closely related organism Mycobacterium smegmatis. Soluble expression of the fusion protein was optimised and confirmed by immunodetection. We then employed affinity enrichment using nickel-charged magnetic beads and mass spectrometry to identify potential interacting partners for Csd. Strains expressing a 6xHis- tag SufC fusion and a 6xHis-tag mCherry fusion were also included for comparison. Utilising this method, we were able to demonstrate affinity enrichment for Fe-S cluster assembly proteins, and Fe-S cluster-containing enzymes, as well as identify potential novel interacting partners. This work has established the methodology for identifying novel protein-protein interactions in mycobacteria, and lays the foundation for elucidating the process of Fe-S cluster assembly in mycobacteria.