Development of novel reagents for tuberculosis detection.
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Date
2013
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Abstract
Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and
causes high morbidity and mortality, despite the widespread availability of effective
antibiotics against most strains of Mycobacterium tuberculosis, which is the
causative agent of TB. One of the primary reasons that hinder TB control is that
many cases of active disease go undetected or are discovered late. This is, in large
part, due to the relative insensitivity and limited specificity, amongst other
limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis
infection can be asymptomatic and latent, or cause active disease. Therefore, an
ideal or effective TB diagnostic needs to distinguish between these two states. The
aim of this study was to develop novel diagnostic reagents for M. tuberculosis
using phage displayed peptides and nucleic acid aptamers with a view to discerning
latent from active TB.
Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of
selection (biopanning) were performed. Ten phage displayed peptides that bind to
the mycobacteria surface were selected. These phage clones were identified using
both random clone picking and high throughput (HTP) sequencing. A phage clone
displaying the CPLHARLPC peptide was identified by HTP sequencing as the most
enriched, representing 82.49% of the selected CX7C phage population. Further
characterization showed that it bound better to different mycobacteria species,
including M. tuberculosis, than the unselected phage library. Moreover, using
surface plasmon resonance (SPR) technology, the chemically synthesised
CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate
and not non-mycobacteria lysates.
In addition, using the systematic evolution of ligands by exponential enrichment
(SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were
selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five
aptamers were identified after five rounds of selection. Two of these aptamers,
GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and
KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the
EsxA homologue.
Taken together, these findings suggest that a combination of phage display, SELEX
and HTP sequencing can be a useful tool for the identification of specific detection
reagents that can bind to mycobacteria and its associated targets. These reagents
could be exploited to develop alternative molecular probes for TB diagnostics.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.
Keywords
Tuberculosis--Epidemiology., Mycobacterium tuberculosis., Theses--Medical microbiology.