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1.  Proteomic Selection of Immunodiagnostic Antigens for Trypanosoma congolense 
Animal African Trypanosomosis (AAT) presents a severe problem for agricultural development in sub-Saharan Africa. It is caused by several trypanosome species and current means of diagnosis are expensive and impractical for field use. Our aim was to discover antigens for the detection of antibodies to Trypanosoma congolense, one of the main causative agents of AAT. We took a proteomic approach to identify potential immunodiagnostic parasite protein antigens. One hundred and thirteen proteins were identified which were selectively recognized by infected cattle sera. These were assessed for likelihood of recombinant protein expression in E. coli and fifteen were successfully expressed and assessed for their immunodiagnostic potential by ELISA using pooled pre- and post-infection cattle sera. Three proteins, members of the invariant surface glycoprotein (ISG) family, performed favorably and were then assessed using individual cattle sera. One antigen, Tc38630, evaluated blind with 77 randomized cattle sera in an ELISA assay gave sensitivity and specificity performances of 87.2% and 97.4%, respectively. Cattle immunoreactivity to this antigen diminished significantly following drug-cure, a feature helpful for monitoring the efficacy of drug treatment.
Author Summary
Animal African Trypanosomosis (AAT) is a set of diseases whereby animals are infected with single-cell parasites that replicate in their bloodstream. The disease in cattle results in weight-loss and death, and AAT is a significant veterinary problem for sub-Saharan Africa. One of the principal trypanosome species responsible for AAT in cattle is Trypanosoma congolense and, although there are drug-treatments for these infections, current diagnostic methods are impractical for field use. Our aim was to discover protein molecules from the parasite to which infected animals make antibodies, to then make these proteins in bacteria and to subsequently demonstrate that they can be used to detect antibodies in cattle serum, thus diagnosing AAT. To discover the diagnostic proteins, we dissolved parasites in a detergent solution and applied them to beads coated with antibodies from infected cattle and to beads coated with antibodies from un-infected cattle. We then compared the proteins bound to each and selected those proteins that were at least 100-fold enriched by the infected cattle antibodies. We refined this list, according to practical and performance considerations, and settled on one protein, called Tc38630. Testing Tc38630 with cattle sera showed that it can detect about nine out of ten AAT infections.
doi:10.1371/journal.pntd.0002936
PMCID: PMC4055490  PMID: 24922510
2.  Crystal structure of Leishmania major ADP ribosylation factor-like 1 and a classification of related GTPase family members in this Kinetoplastid 
ADP ribosylation factor-like (ARL) proteins are small GTPases that undergo conformational changes upon nucleotide binding, and which regulate the affinity of ARLs for binding other proteins, lipids or membranes. There is a paucity of structural data on this family of proteins in the Kinetoplastida, despite studies implicating them in key events related to vesicular transport and regulation of microtubule dependent processes. The crystal structure of Leishmania major ARL1 in complex with GDP has been determined to 2.1 Å resolution and reveals a high degree of structural conservation with human ADP ribosylation factor 1 (ARF1). Putative L. major and Trypanosoma brucei ARF/ARL family members have been classified based on structural considerations, amino acid sequence conservation combined with functional data on Kinetoplastid and human orthologues. This classification may guide future studies designed to elucidate the function of specific family members.
doi:10.1016/j.molbiopara.2010.08.002
PMCID: PMC3065712  PMID: 20801163
ADP ribosylation factor-like; GTPase; Leishmania; protein structure
3.  The architecture of Trypanosoma brucei tubulin-binding cofactor B and implications for function 
The Febs Journal  2013;280(14):3270-3280.
Tubulin-binding cofactor (TBC)-B is implicated in the presentation of α-tubulin ready to polymerize, and at the correct levels to form microtubules. Bioinformatics analyses, including secondary structure prediction, CD, and crystallography, were combined to characterize the molecular architecture of Trypanosoma brucei TBC-B. An efficient recombinant expression system was prepared, material-purified, and characterized by CD. Extensive crystallization screening, allied with the use of limited proteolysis, led to structures of the N-terminal ubiquitin-like and C-terminal cytoskeleton-associated protein with glycine-rich segment domains at 2.35-Å and 1.6-Å resolution, respectively. These are compact globular domains that appear to be linked by a flexible segment. The ubiquitin-like domain contains two lysines that are spatially conserved with residues known to participate in ubiquitinylation, and so may represent a module that, through covalent attachment, regulates the signalling and/or protein degradation associated with the control of microtubule assembly, catastrophe, or function. The TBC-B C-terminal cytoskeleton-associated protein with glycine-rich segment domain, a known tubulin-binding structure, is the only such domain encoded by the T. brucei genome. Interestingly, in the crystal structure, the peptide-binding groove of this domain forms intermolecular contacts with the C-terminus of a symmetry-related molecule, an association that may mimic interactions with the C-terminus of α-tubulin or other physiologically relevant partners. The interaction of TBC-B with the α-tubulin C-terminus may, in particular, protect from post-translational modifications, or simply assist in the shepherding of the protein into polymerization.
doi:10.1111/febs.12308
PMCID: PMC3806363  PMID: 23627368
CAP-Gly domain; CD; crystallography; tubulin-binding; ubiquitin-like

Results 1-3 (3)