Year: 2013
Type: dissertation
Abstract: Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and \ncauses high morbidity and mortality, despite the widespread availability of effective \nantibiotics against most strains of Mycobacterium tuberculosis, which is the \ncausative agent of TB. One of the primary reasons that hinder TB control is that \nmany cases of active disease go undetected or are discovered late. This is, in large \npart, due to the relative insensitivity and limited specificity, amongst other \nlimitations, of the current TB diagnostics tests. Moreover, M. tuberculosis \ninfection can be asymptomatic and latent, or cause active disease. Therefore, an \nideal or effective TB diagnostic needs to distinguish between these two states. The \naim of this study was to develop novel diagnostic reagents for M. tuberculosis \nusing phage displayed peptides and nucleic acid aptamers with a view to discerning \nlatent from active TB. \nUsing a linear (X12) and constrained (CX7C) phage display libraries, five rounds of \nselection (biopanning) were performed. Ten phage displayed peptides that bind to \nthe mycobacteria surface were selected. These phage clones were identified using \nboth random clone picking and high throughput (HTP) sequencing. A phage clone \ndisplaying the CPLHARLPC peptide was identified by HTP sequencing as the most \nenriched, representing 82.49% of the selected CX7C phage population. Further \ncharacterization showed that it bound better to different mycobacteria species, \nincluding M. tuberculosis, than the unselected phage library. Moreover, using \nsurface plasmon resonance (SPR) technology, the chemically synthesised \nCPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate \nand not non-mycobacteria lysates. \nIn addition, using the systematic evolution of ligands by exponential enrichment \n(SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were \nselected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five \naptamers were identified after five rounds of selection. Two of these aptamers, \nGH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and \nKD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the \nEsxA homologue. \nTaken together, these findings suggest that a combination of phage display, SELEX \nand HTP sequencing can be a useful tool for the identification of specific detection \nreagents that can bind to mycobacteria and its associated targets. These reagents \ncould be exploited to develop alternative molecular probes for TB diagnostics.
Author: Nqobile Angel Cebile. Ngubane
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1859734804