Faculty of Natural and Agricultural Sciences
Department of Biochemistry
Selected Highlights from Research Findings
The research focus of the HIV research group remains the development of non-toxic treatments and novel methodologies for measuring disease progression. For the former aim, novel metallo-drugs (containing Au, Pt and Pd) and natural product extracts are screened for anti-HIV activity and for the latter, bio-fluid metabonomics is conducted. This past year several new drugs have been screened and found to have promising inhibitory activity against HIV enzymes. Data on the mechanism of action of the drugs have also been collected. Several members of the research group (staff and students) have received prestigious research awards (e.g NRF/NSTF, L'oreal, Oppenheimer Trust) and the principal investigator participated in sabbatical research with collaborators at the University of California San Francisco. The HIV research group upgraded the Flow Cytometer by adding an additional laser which now expands the instrument's capabilities and also acquired a real time electronic censing instrument (RT-CES). Both of these activities was made possible by a generous grant from the Technology Innovation Agency (TIA).
Contact person: Prof D Meyer.
2- DE profiling of proteins in hemolymph of unchallenged Ornithodoros savignyi ticks versus ticks challenged with heat-killed yeast revealed 5 proteins to be differentially expressed. These proteins could not be identified by de novo sequencing using MS/MS. In an attempt to investigate hemolymph proteins that recognize and bind to yeast cells, hemolymph was incubated with yeast cells. Elution of the bound proteins followed by SDS-PAGE analysis revealed three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples. The yeast-binding proteins could also not be identified by de novo sequencing using tandem mass spectrometry (MS/MS).
Using bacterial affinity beads, two high molecular mass proteins have been identified in tick hemolymph. These proteins are novel as no homologs could be identified by de novo sequencing. A degenerate primer obtained from a de novo sequence for one of the proteins led to the cloning of an unrelated hemocyte protein (savicalin). Bioinformatical analysis indicated that savicalin belongs to the lipocalin family.
Further characterization of the novel bacterial/ yeast binding proteins and savicalin identified in this study may contribute to the development of novel anti-infective agents and potential targets for anti-tick vaccine design.
Contact person: Dr ARM Gaspar.
The Welcome Trust funded project concerning anti-tick feeding vaccine development ended during 2010. The project started in 2005, involving research groups from 8 countries and resulted in encouraging outputs to pursue in the coming years. Our research group could identify 3 potential antigens as vaccine candidates, inter alia by iRNA. These antigens were from Boophilus microplus (cattle tick): Protein elongation factor 1 alpha, boophilin G2 (a presumed thrombin and trypsin inhibitor) and a membrane bound Tissue Factor Pathway Inhibitor (TFPI)-like Kunitz protein. The latter 2 antigens are implicated as blood coagulation inhibitors, vital for successful tick feeding. The antigens were expressed in sufficient quantities for vaccine trials by GenScript (USA) after several own attempts with a few different expression systems.
The research culminated in 2010 with a huge vaccination trial in collaboration with UPBRC at Onderstepoort Veterinary Faculty. A total of 16 calves were used. Vaccinated animals were challenged with B. microplus larvae. Three experimental parameters applied to gauge the success of vaccination: the reduction of the numbers of female ticks successfully feeding, the reduction in their average engorgement weight and the reduction in their egg laying ability. Encouraging results were achieved but these need confirmation before further development.
Contact person: Prof AWH Neitz.
The Discovery project is aimed at providing a publicly available informatics resource where comprehensive information on the parasite and host proteins are stored, together with the results from relevant 3rd-party investigations as well as results from our own high-throughput analysis. The comprehensive data included in the resource is aimed as wide as possible, including protein, gene-ontology, orthology, metabolic, structural, expression, interactome and chemoinformatics information. This is combined with a data-mining interface for researchers to perform the selection of putative drug target protein and lead compounds according to their specific highly-flexible criteria. The system is not designed to be a repository, but a resource to perform advanced data mining, filtering and selection according to biologically-relevant criteria. The resource is unique in integrating comparative genomics, protein annotations and chemoinformatics in a single system.
Protein information includes data from the human, mosquito and the various malaria genome projects. Chemical information is from PDB, KEGG and DrugBank. Information includes basic annotations, motifs, domains, binding sites, structural features, orthology information, ontology terms, protein-protein interactions, protein-ligand interactions, pathogen-host interactions and comparative genomics information. Chemical information includes protein interactions and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties. The researcher accessing the resource is then able to perform advanced searching and filtering of proteins and chemical compounds according to possible interactions and the different types of properties described in the database. This may be initiated either from the protein or chemical compound as starting point. Additional work currently being performed includes the development of more accurate statistical scoring methods for the predictions, a literature mining component as well as the inclusion of additional chemical data sources.
Contact person: Prof F Joubert.
Three new web-based applications were finished or updated by my students last year: the database of horizontally transferred genomic islands identified in bacterial genomes (http://anjie.bi.up.ac.za/geidb/geidb-home.php), Gene Island Sniffer program for an automated detection of genomic islands in bacterial genomes (http://www.bi.up.ac.za/SeqWord/sniffer/index.html); and the MetaLingvo program for clustering and identification of environmental sequences generated in metagenomic projects (http://www.bi.up.ac.za/SeqWord/metalingvo/index.html). The new on-line tools were integrated into the SeqWord project web-portal and presented on the international conferences and in a publication.
The results achieved in this project were presented by my PhD student and myself on three international conferences: Cold Spring Harbor Laboratory Genome Informatics conference, Cambridge, UK, September 15-19, 2010 (poster); 9th European Conference on Computational Biology conference (ECCB), Ghent, Belgium, September 26-29, 2010 (2 posters); and Bio-IT conference 2010, Hanover, Germany, October 6-8 (round table discussion moderation and a poster). A workshop was hold in High Medical School of Hanover (Germany) by my PhD student and me to discuss a work plan of future collaboration with our partner team in Germany.
Publications:
(1) Beloqui A, Nechitaylo TY, López-Cortés N, Ghazi A, Guazzaroni ME, Polaina J, Strittmatter AW, Reva O, Waliczek A, Yakimov MM, Golyshina OV, Ferrer M, Golyshin PN. Diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species. Appl Environ Microbiol. 2010 Sep;76(17):5934-46. Epub 2010 Jul 9.
(2) Rückert C, Blom J, Chen X, Reva O, Borriss R. Genome sequence of B. amyloliquefaciens type strain DSM7(T) reveals differences to plant-associated B. amyloliquefaciens FZB42.
J Biotechnol. 2011 Jan 21. [Epub ahead of print].
(3) Gounder K, Brzuszkiewicz E, Liesegang H, Wolherr A, Daniel R, Gottschalk G, Reva O, Kumwenda B, Srivastava M, van Heerden E and Litthauer D. Hyperplasticity in the Thermus scotoductus SA01 genome: acquire, adapt or die. Submitted to BMC Genetics in 2011.
Contact person: Dr O Reva.
Crystals of the malarial Spermidine synthase protein were successfully grown with two different inhibitory compounds. X-ray diffraction data was collected from which the interaction of these compounds within the protein active site could be resolved at the atomic level. These results provided insights into the binding of compounds that were identified with in silico, dynamic pharmacophore-based screening and are important for further studies aimed at improving the ligand binding efficiency. These experiments were performed at The University of Lund and Max Lab in Lund, Sweden (two manuscripts in preparation)
Contact person: Prof L Birkholtz.
Crystals of the malarial Spermidine synthase protein were successfully grown with two different inhibitory compounds. X-ray diffraction data was collected from which the interaction of these compounds within the protein active site could be resolved at the atomic level. These results provided insights into the binding of compounds that were identified with in silico, dynamic pharmacophore-based screening and are important for further studies aimed at improving the ligand binding efficiency. These experiments were performed at The University of Lund and Max Lab in Lund, Sweden (two manuscripts in preparation)
Contact person: Prof AI Louw.
The MARTI-test for rapid TB diagnosis, currently being developed for the market, was tested to demonstrate its ability to detect extrapulmonary TB. Initial results confirmed this potential application, making it potentially the first evidence-based diagnostic test that can do this. The challenge now was to demonstrate that the test can be done with less sophisticated instrumentation and at higher sample throughput. For this purpose, basic knowledge was required to define the mycolic acid antigen that is recognised by human TB patient antibodies. By developing recombinant monoclonal antibodies against mycolic acids as well as using chemically synthetic mycolic acids, a structure-function relationship was defined and published. The monoclonal antibodies allowed the conceptualization of a Point of Care TB diagnostic device that was filed for patenting. Chemical synthesis of mycolic acids was improved with the collaboration of University of Bangor, UK and the method published
Contact person: Prof JA Verschoor.
Two MSc students presented their work at local conferences. Ms Malebe presented work on molecular markers for drought tolerance in tea (Camellia sinensis) at a South African Biochemistry and Informatics of Natural Products (SABINA) conference in Benoni, 4-9 October 2010. Mr Shane van Breda presented his research in a paper entitled "Differential extraction of caffeine and polyphenols from fresh tea leaves of Camellia sinensis by water" at Bela Bela, Warmbaths, 26-29 October 2010.
Several MSc and PhD students attended a project management workshop in Pretoria, 2-4 September 2010. Prof Apostolides presented a workshop on High Performance Liquid Chromatography and Bioinformatics for Natural Products at the University of Pretoria 30 August to 1 September 2010, attended by 15 persons. This will be repeated in the future at regular intervals.
Prof Apostolides was an invited speaker at the 3rd International Conference on Tea in Shizuoka, Japan, 26-29 October 2010 where he presented 2 papers and a keynote address
Contact person: Prof Z Apostolides.
Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry
Contact person: Dr M Beukes.
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