Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei expression library by polymerase chain reaction

Abstract

Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.

Keywords: Nagana; PCR; bioinformatics; drug targets; expression library; proteins.

Figure 1

Pwsa for the determination of RHSP 4 (Tb927.1.120). One of the clones matched RHSP4 when BLAST searched in the ntdb at NCBI with Score = 1081 bits (585), Expect = 0.0, Identities = 982/1168 (84%), Gaps = 66/1168 (6%) Strand = Plus/Minus.

Figure 2

Pwsa for the determination of PDE1α. One of the clones matched T. brucei TREU927 PDE1α when BLAST searched in the ptdb at NCBI with Score = 24.6 bits (52), Expect = 0.007, Identities = 12/27 (44%), Positives = 16/27 (59%), Gaps = 0/27 (0%).

Figure 3

Pwsa for the determination of VSGPx (emb|cad90550.1|). One of the clones matched T. b. brucei VSGPx when BLAST searched in the ptdb at NCBI with Score = 22.3 bits (46), Expect = 0.009, Identities = 9/22 (41%), Positives = 13/22 (59%), Gaps = 0/22 (0%).

Figure 4

Pwsa for the determination of VSGq (Tb11.v4.0036). One of the clones matched T. brucei VSGq when BLAST searched in the ptdb at NCBI with Score = 22.3 bits (46), Expect = 0.009, Identities = 9/28 (32%), Positives = 15/28 (54%), Gaps = 0/28 (0%).

Figure 5

Pwsa for the determination of CBS (Tb11.02.5400). One of the clones matched T. brucei TREU927 CBS when BLAST searched in the ptdb at NCBI with Score = 23.9 bits (50), Expect = 0.006, Identities = 11/40 (28%), Positives = 20/40 (50%), Gaps = 1/40 (3%).

Figure 6

Pwsa for the determination of αMT (Tb10.70.0260). One of the clones matched T. brucei TREU927 αMT when BLAST searched in the ptdb at NCBI with Score = 24.3 bits (51), Expect = 0.008, Identities = 11/29 (38%), Positives = 13/29 (45%), Gaps = 0/29 (0%).

Figure 7

Pwsa for the determination of SKTMP (Tb11.02.0290). One of the clones matched T. brucei TREU927 SKTMP when BLAST searched in the ptdb at NCBI with Score = 23.5 bits (49), Expect = 0.005, Identities = 8/22 (36%), Positives = 13/22 (59%), Gaps = 0/22 (0%).

Figure 8

Pwsa for the determination of HP I (Tb927.7.4460). One of the clones matched T. brucei TREU927 HP I when BLAST searched in the ptdb at NCBI with Score = 22.3 bits (46), Expect = 0.026, Identities = 12/37 (32%), Positives = 19/37 (51%), Gaps = 0/37 (0%).

Figure 9

Pwsa for the determination of HP II (Tb927.6.620). One of the clones matched T. brucei TREU927 HP II when BLAST searched in the ptdb at NCBI with Score = 24.3 bits (51), Expect = 0.010, Identities = 10/15 (67%), Positives = 12/15 (80%), Gaps = 0/15 (0%).

Figure 10

Pwsa for the determination of HP III (Tb927.3.1420). One of the clones matched T. brucei TREU927 HP III when BLAST searched in the ptdb at NCBI with Score = 23.5 bits (49), Expect= 0.012, Identities = 12/25 (48%), Positives = 14/25 (56%), Gaps = 0/25 (0%).

Figure 11

Pwsa for the determination of HP IV (Tb927.5.2770). One of the clones matched T. b. brucei strain 927/4 GUTAT10.1 HP VI when BLAST searched in the ptdb at NCBI with Score = 22.7 bits (47), Expect = 0.005, Identities = 13/27 (48%), Positives = 17/27 (63%), Gaps = 3/27 (11%).