Mans (2002) hypothesized that the two domains of savignin interact with each other, giving a globular form in the absence of thrombin. Binding of the C-terminal domain of the inhibitor to the fibrinogen-binding site of thrombin leads to the dissociation of the domains. This would yield an extended conformation that would allow binding of the N-terminal residues of the N-terminal domain to thrombin’s active site. To test this hypothesis, both theoretical and experimental approaches were employed to determine the molecular dimensions of uncomplexed savignin. In the theoretical approach, the hydrodynamic radius (Rh) of the extended form of savignin was calculated from the crystal structure data of the thrombin-ornithodorin complex, and found to be 2.319 nm. With the same programme, based on the crystal structure data for bikunin, a protein in which both domains are closely associated, the Rh value for the compact form of savignin was estimated as 1.96 nm. Using the equation that relates Rh to molecular mass, a value of 1.84 nm was calculated for savignin (12 430 Da). In the experimental approach, the SEC of salivary gland extracts, using lysozyme (Rh = 1.99 nm) as standards and chrymotrypsinogen (Rh = 2.31nm) indicated that uncomplexed savignin exists in both the globular and extended conformations. However, the majority of inhibitory activity was associcated with the extended form. Heat stability assays as well as SDS-PAGE experiments indicated the possible existence of the compact form of savignin. Generation of adequate amounts of savignin will allow further structural studies, to determine the structure of savignin in the uncomplexed form and in complex with thrombin. Expression of full length savignin and the separate N- and C-domains will facilitate further kinetic analysis. In the recombinant production of savignin, various factors were investigated: cell-lines, transformation efficiency, induction times, purification strategy and protease cleavage of expressed fusion protein. Even though large quantities of expressed fusion protein were obtained, cleavage of the target protein and its separation from the fusion partner by enzymatic means presented a major hurdle. Expression of Nsav was not observed and is most likely as a result of misfolding of the recombinant form. Due to the probable non-specific cleavage of the fusion protein, switching the prokaryotic expression system to other expression systems, like yeast- or baculovirus-insect cell-expression systems, is warranted. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

In this study the structural characteristics and antibacterial activities of two peptides derived from the carboxy-terminal of a tick defensin were investigated. Two defensin isoforms (OsDef1 and OsDef2) were previously identified in the midgut of the tick, Ornithodoros savignyi. Both OsDef1 and OsDef2 were found to be active against Gram-positive bacteria but showed no antibacterial activity against Gram-negative bacteria. OsDef2 was found to be slightly more active than OsDef1 and was, therefore, used as the template for the design of smaller antimicrobial peptides. Two peptide analogues were synthesised using the carboxy-terminal sequence of OsDef2 and differed in that in the one peptide the cysteine residues were present (Os) and in the other the cysteine residues were omitted (Os-C). Structurally, Os contained more α-helical properties than Os-C, whereas Os-C was more β-sheeted when prepared in 25 mM SDS. Both Os and Os-C showed no antibacterial activity when tested in Luria-Bertani broth or Mueller-Hinton broth indicating that the activities of Os and Os-C were influenced by the presence of broth salts and proteins. When tested in sodium phosphate buffer, both Os and Os-C exhibited Gram-positive and Gram-negative antibacterial activity. Os was slightly more active than Os-C against 3 of the 4 tested strains, with minimum bactericidal concentrations (MBCs) ranging from 0.94 μg/ml to 3.75 μg/ml. Os retained bactericidal activity against both Staphylococcus aureus and Escherichia coli when tested in the presence of 100 mM NaCl or 30% human serum. Os-C retained activity against E. coli in the presence of NaCl but became inactive in 30% human serum against both bacterial strains. At the MBCs, Os exhibited faster killing kinetics than Os-C killing both Bacillus subtilis and E. coli within 5 min, whereas Os-C took up to 120 min and 60 min, respectively. SYTOX Green permeabilization assays showed that both Os and Os-C caused permeabilization of E. coli membranes after 30 min exposure. At high peptide concentrations, both Os and Os-C were shown to interact with plasmid DNA. Both Os and Os-C exhibited no cytotoxic effects against SC-1 and Caco-2 cell lines, even at peptide concentrations 32 times higher than the highest MBC. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Biochemistry / unrestricted

Ticks are blood feeding ectoparasites that ingest large volumes of vertebrate blood. They are the most important arthropods that are capable of transmitting pathogens which cause disease in humans and domestic animals. Ticks are exposed to various microorganisms during feeding as well as in their habitat. They therefore must have a very good immune system to recognize and destroy these microorganisms. In the present study a micro-broth dilution assay was used to determine whether antifungal activity was present in different tick tissue extracts with or without challenge. The midguts gave the highest inhibition of yeast growth, followed by the salivary glands and then the hemolymph. This was seen with unchallenged tick tissue extracts, as well as tissue extracts collected after yeast challenge (2 hours). Thus all of the tick tissue extracts that was analyzed in this study had antifungal activity. Proteomics was used to determine whether proteins were differentially expressed in the hemolymph plasma, after a fungal challenge. 2DE was used since proteins are not only separated by molecular mass, but also by their charge. The proteins that were separated on the 2D-gels ranged between 17.5-76 kDa and not all proteins present on the 1D-gels (14-97 kDa) could be seen on the 2D-gels. Ticks were challenged for 2 hours to define the proteins that play a role in the short term innate immune response during a fungal infection. Various proteins were differentially expressed in the hemolymph samples that were collected 2 hours after ticks were injected with saline, â-1,3-glucan or yeast (or 72 hours). Injury and fungal challenge play a role in producing proteins that might play a role in the fungal response of the tick. Five spots that were statistically significant in the hemolymph collected 2 hours after ticks were injected with yeast cells were analyzed with MS/MS. No matches were found with MASCOT database searching or with EST searching. This can be due to the limited information that is available on the soft ticks, as only hard tick ESTs heve been published. It was also attempted to identify hemolymph proteins that might play a role in the recognition of fungi. Hemolymph was incubated with live Candida albicans cells and eluted with buffer. Three protein bands (97, 88 and 26 kDa) were found to be present whether ticks were challenged or unchallenged. These proteins were subjected to MS/MS analysis and database searching was performed revealing no matches to other known proteins. The antifungal response was found to be present in the soft tick O. savignyi and might play a vital function in the innate immune response during a fungal infection. These proteins may serve as lead molecules that could be used in the development of novel antifungal drugs, as well as in vaccine development. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Invertebrates do not possess an adaptive immune system, but rely on several mechanisms similar to the innate immune system of mammals. The synthesis and release of a host of potent antimicrobial proteins is an important component of this immune response. The antibacterial activity in the hemolymph of Ornithodoros savignyi is specific for Gram-positive bacteria, and the synthesis and release of the antibacterial factors need to be induced by challenging the ticks with heat-killed Gram-negative bacterial suspensions. The induction of the factors is very rapid, leading to a maximal response within one hour following bacterial challenge. The factors are stable at high temperatures, and were found to be protein in nature. By using reverse phase high performance liquid chromatography, four fractions exhibiting antibacterial activity were identified in the hemolymph of immune challenged ticks. Four antibacterial peptides were isolated from these fractions, and the mass analyses of the peptides indicate that there are at least two different antibacterial peptides present in the hemolymph. The N-terminal amino acid sequence of one of the peptides was determined, and the analysis showed that the peptide has high homology with defensin peptides isolated from other tick species. This led to the putative classification of the peptides as part of the invertebrate defensin family. The presence of lysozyme in O. savignyi was studied using molecular biological methods. Vertebrate and invertebrate lysozyme sequences were used to design a lysozyme-specific primer, which was used to amplify specific DNA products from whole tick cDNA using the polymerase chain reaction (PCR). The conditions for the amplification reaction were optimized, the products of the optimized reaction were cloned into a cloning vector and the nucleotide sequences of the products were determined. The nucleotide sequences were used for similarity searches of sequence databases to determine homology with sequences of known proteins. It is deduced the degenerate primer was not specific for lysozyme and did not playa significant role in the amplification of the PCR products. This method is thus not feasible for the investigation of the lysozyme of O. savignyi. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

Most studies on innate immunity in ticks have focused on the antimicrobial peptides from hemolymph, such as defensins and lysozyme, while less is known about bacterial recognition molecules, or antimicrobial mechanisms in other tissues. The current study attempted to identify novel antimicrobial mechanisms, with a focus on bacterial recognition by hemolymph proteins and antimicrobial activity in salivary gland extracts. Using bacteria as affinity beads, two high molecular mass molecules (Protein X and Protein Y) have been identified in tick hemolymph. These proteins are thought to interact with the bacterial surface via ionic interactions. Tandem mass spectrometry analysis followed by de novo sequencing indicated that these proteins are novel as no homologs could be identified from sequence databases. In an attempt to clone Protein X, using a degenerate primer obtained from a de novo sequence, an unrelated hemocyte protein was identified. This protein, named savicalin, was shown to belong to the lipocalin family based on bioinformatical analysis. Transcriptional profiling indicated that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. To date, this is the first tick lipocalin not derived from salivary glands. Interestingly, up-regulation of its mRNA transcript in response to bacterial challenge suggests that this protein could be involved in antimicrobial activity. Up-regulation after feeding also suggests a role in the post-feeding development of the tick. Two different approaches were used to purify the Gram-positive antibacterial activity from salivary gland extracts. The first attempt entailed a two-step separation approach. Tricine SDS-PAGE of the active fraction showed 3 components (~20, ~10 and ~7 kDa). BLAST searches using the N-terminal sequences of the latter proteins identified the ~20 kDa protein as savignin, while the other two proteins could not be matched. The second strategy included an ultrafiltration step (10 kDa cut-off) and MS-analysis of the active fraction in this case indicated the presence of various components with molecular masses ranging from 0.99 – 7.182 kDa, with 12 predominant components ranging from 0.99 - 4.448 kDa. Further tandem mass spectrometry analysis of the active fraction revealed the presence of three tick actin-derived fragments. This is of interest as actin fragments have been implicated in innate immunity of other invertebrates. In this study, synthetic peptides corresponding to one of the detected tick actin fragments as well as actin5C (detected in Drosophila hemolymph) were found not to inhibit the growth of Bacillus subtilis when tested up to a concentration of 100 ìg/ml. It is envisaged that future studies of immunoprotective molecules of the tick, O. savignyi, may contribute to the development of novel anti-infective agents and potential targets for anti-tick vaccine design. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted