Bio-oligomers as antibacterial agents and strategies for bacterial detection
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Date
28/11/2014Author
Kasturiarachchi, Jagath Chandana
Metadata
Abstract
In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides
and aptamers, as therapeutic and diagnostic entities.
Therapeutic Bio-Oligomers;
A series of peptoid analogs have been designed and synthesised using solid phase
synthesis. These peptoids have been subjected to biological evaluation to determine
structure-activity relationships that define their antimicrobial activity. In total 13
peptoids were synthesised. Out of 13 different peptoids, only one peptoid called
Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal
activity. TOP kills bacteria under non-dividing and dividing conditions. The
Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli
and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus
(MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The
highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM.
The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the
10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for
TOP as 100. This TI is well above previously reported peptidomimetics TI of around
20. TOP demonstrates selective bacterial killing in co-culture systems and
intracellular bacterial killing activity.
Diagnostic Bio-Oligomers;
In the second part of my thesis, I investigated aptamer and peptide-based molecular
probes to detect MRSA. As well as screening aptamers and peptide probes against
whole MRSA, I over-expressed and purified PBP2A protein. This purified protein
was used as a target for aptamer and peptide probes to detect MRSA.
Two different aptamer libraries were initially screened for utility. In-vitro conditions
for SELEX were optimised. Biopanning with a phage derived peptides was also
performed. Target sequences for both methods were identified and chemically
synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and
confocal imaging showed no specificity for MRSA detection with either method. The
Bio-Oligomers and the in-vitro selection methodology require further refinement to
improve diagnostic utility.