The in vivo identification of trypanosome proteins interacting with the Glossina midgut proteolytic lectin (GPL)

Abstract

Protein-protein interactions mediate in the transformation of parasites during the course of infection. The Glossina proteolytic lectin (Gpl) is associated with the transformation of the bloodstream trypomastigotes into the procyclic forms within the midgut of the tsetse. This allows the trypanosomes to adapt in their new invertebrate insect host because the trypanosomes encounter a hostile environment and infection is established when the trypanosomes are able to survive and differentiate. This study was undertaken to identify the trypanosome protein(s) that act as interacting partner(s) of the Glossina midgut proteolytic lectin (Gpl) leading to the proliferation of the African trypanosomes. This study was performed in-vivo using the GAL4 Yeast- Two-Hybrid system. The complete Open Reading Frame of the proteolytic lectin gene from Glossinafuscipesfuscipes was used as bait to fish for gene(s) present in a cDNA-AD fusion expression library constructed in vivo from the bloodstream forms of Trypanosoma brucei brucei. False positive clones were eliminated by selection of clones transcribing three reporter genes i.e. ADE2, HIS3, LacZ genes and by segregation analysis. Recombinant plasm ids from positive colonies were identified using differential blue/white color screening. Characterization of positive blue yeast colonies (ADE2+/HIS3+/LacZ+) by PCR using AD-insert screening amplimers yielded no results showinga ~-organization of the vector during a positive protein-protein interaction. Restriction digestion with Nde I and BamH Idropped inserts of approximately 800 bps while the empty vectors were of different and unexpected sizes. The putative positive library clones were identified, sequenced and analyzed by bioinformatics. Nucleotide and deduced amino acid sequence analysis showed that the cDNA insert corresponded to a truncated Gpl gene, a recombination that resulted as an outcomeof toxicity after a strong positive-positive interaction. PCR and restriction digestion characterization of a white yeast colony (ADE2+/HIS3+/LacZ-) yielded a cDNA insert of approximately 800 bps. The empty vector from restriction digestion was of the correct size. The nucleotide sequence of the cDNA insert showed a 97% identity with a hypothetical Trypanosoma brucei/AJ234097 gene and a partial identity (5%) with a serine-rich protein from Shizosaccharomyces pombe (PIRlT39903/T39903). When interrogated in the Trypanosoma brucei Gene Index (TGI) (PIRlT39903/T39903) from TIGR. In the NCBI database this gene corresponded to Trypanosoma brucei TREU 927 hypothetical protein (Tb09.21 1.4290) partial mRNA. Understanding the Trypanosome protein(s) interacting with Gpl will increase insight into the Tsetse/Trypanosome relationship which is an area of science hampered by lack of information. This will lead to the development of an effective parasite transmission blocking mechanism as a method of controlling African trypanosomosis. The resultsobtained from this research clearly show the toxic outcome of this complex interaction apd the need for further characterization of Gpl to provide more insight into its role and functions in vivo on trypanosome differentiation.