Magister Scientiae - MSc / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). TB mainly affects lungs of patients but other parts of the body can also be affected. It kills approximately 2 million people annually. HIV/AIDS and drug resistance make TB difficult to control. Mtb CarD protein forms a physiological complex with Ribonucleic Acid Polymerase (RNAP). This complex causes Mtb to undergo dormancy rendering it difficult to control using current antibiotics. CarD and a size-reduced subunit β1 (denoted β1m for “minimized”) of Thermus thermophilus RNAP, in which the central domain has been replaced by a Gly-Gly linker, were produced and purified using affinity nickel nitrilotriaceticacid and glutathione-Stransferase (GST) affinity chromatography techniques respectively. CarD N terminal domain (CarDN) was generated from CarD by inserting a stop codon by site directed mutagenesis. CarD was stabilised by adding 5 % (v/v) glycerol to PBS pH 7.4 ensuring protein stability of up to 67 days rather than 2 days without glycerol. CarDN was stable in PBS pH 7.4 without addition of glycerol. This suggests that the CarD C terminal domain may be responsible for CarD instability. To further purify the proteins both anion exchange and gel permeation chromatography techniques were used. CarD and CarDN degrade immediately after anion exchange potentially because of the high ion concentration which partially unfolds the protein making it prone to proteolytic cleavage. GST-pull down assays were used to demonstrate complex formation between RNAP β1m and both CarD and CarDN confirming that complex formation is dependent on the N-terminal domain of CarD.

Overuse of conventional antibiotics has led to increased multidrug resistant micro-organisms. Therefore, development of alternative drugs with new mechanisms of action in the control of resistant micro-organisms is urgently needed. Defensins, one of the larger groups of naturally occurring antimicrobial peptides (AMPs), found in a variety of species, may serve as templates for the development of novel therapeutic agents. The work completed in this study is based on an antimicrobial peptide (AMP), Os, derived from the C-terminus of a tick Ornithodoros savignyi defensin isoform 2 (OsDef2). OsDef2 was found to be active against Gram-positive bacteria only, whereas Os, showed bactericidal activity towards both Gram-positive and Gram-negative bacteria. In this study a series of synthetic shorter peptides, based on the sequence of Os, was utilised in order to determine whether shorter peptides would retain their antibacterial activity and selectivity. Initial screening indicated that only two fragments, Os(3-12) and Os(11-22), were active towards the tested Gram-negative and Gram-positive bacteria. The minimum bactericidal concentrations (MBCs) of the two fragments were determined and ranged from 30 μg/ml to 120 μg/ml. The MBCs of the parent peptide, Os (1.88 to 15 μg/ml), was considerably lower than that of Os(3-12) and Os(11-22). As previously observed for Os, neither of the peptides showed cytotoxic effects towards eukaryotic cells. The amidated analogue of one of the active peptides, Os(11-22)NH2, was further evaluated in terms of its secondary structure, antibacterial and antioxidant activities as well as cytotoxicity. Amidation increased the activity of Os(11-22) 16 fold towards B. subtilis (MBC of 1.88 μg/ml) and 32 fold towards both Escherichia coli and Pseudomonas aeruginosa (MBC of 3.75 μg/ml), whereas a 2 fold decrease in activity was observed against Staphylococcus aureus (MBC of 60 μg/ml). Circular dichroism data showed that amidation altered the secondary structure of Os(11-22) from α-helical to mostly random coiled. In the presence of 30% serum the activity of Os(11-22)NH2 unexpectedly increased 8 fold against S. aureus (MBC of 7.5 μg/ml), but decreased 32 fold against E. coli (MBC of 120 μg/ml). The activity of Os(11-22)NH2 in 100 mM NaCl decreased 4 fold against E. coli (MBC of 15 μg/ml), but was completely lost (MBC >120 μg/ml) against S. aureus. The kinetics of bactericidal activity indicated that Os(11-22)NH2 killed B. subtilis and E. coli within 30 min and 120 min, respectively, whereas Os killed both bacteria within 5 min. Even at high concentrations Os(Os(11-22)NH2 was non-toxic towards human erythrocytes and SC-1 cells, moreover an increase in SC-1 cell number was observed at 120 μg/ml. The peptide showed strong antioxidant activity and was found to be 4 fold more active than glutathione (GSH), however Os was 3.4 fold more antioxidative than Os(11-22)NH2. Os(11-22)NH2 can be considered a dual functional peptide, since it possesses both antibacterial and antioxidant activity. The amidated peptide has the potential for use against the damaging effects of oxidative stress associated with infectious diseases and recovery of chronic wounds. Further investigation into structure-function properties of Os(11-22)NH2 is necessary. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Biochemistry / Unrestricted