Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense

Abstract

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and 26 VSG EST clusters were found in the bloodstream library, 6 of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the Trypanosoma brucei and Trypanosoma cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml. The sequence data have been submitted to EMBL under the following accession numbers: FN263376-FN292969.

Fig. 1

Bar graph showing the abundances of the indicated EST clusters in each of the four regular (non-normalized) cDNA libraries. VSG, variant specific glycoprotein; ESAG, expression-site associated gene; CESP, congolense epimastigote-specific protein.

Fig. 2

A. Line diagram depicting the amino acid sequences of nascent T. congolense VSGs with the N-terminus on the left and C-terminus on the right. Numbers are approximate amino acid positions and vertical bars indicate conserved cysteines (redrawn from [37]). B. Bar graph showing the relative abundances of the 13 mVSG EST clusters containing 224 mVSG EST clones identified in the MCF EST library. Arrows indicate two previously identified T. congolense mVSGs [35]. C. Bar graph showing relative abundances of the 26 VSG EST clusters containing 184 VSG clones identified in the BSF EST library. Note that 6 of these 26 clusters encode mVSGs, including the most abundant cluster, which constitutes 62% of the 184 VSG clones (see text). IL3000 VSG was expressed by bloodstream T. congolense clone IL3000 used to generate the four developmental stages. Each of the bsfVSG groups 7 - 19 has only a single VSG EST.

Fig. 3

Bar graphs showing the relative abundances of ESTs encoding (A.) ribosomal proteins and (B.) the indicated protein families in each of the four regular libraries.

Fig. 3

Bar graphs showing the relative abundances of ESTs encoding (A.) ribosomal proteins and (B.) the indicated protein families in each of the four regular libraries.

Fig. 4

Bar graph showing the relative abundances of EST clusters encoding enzymes of the indicated metabolic pathways, transporters or flagellar proteins.

Fig. 5

Bar graph comparing β - and α-tubulin EST abundances in the indicated libraries.

Fig. 6

Relative abundances of the five most abundant “hypothetical” EST clusters in each of the indicated libraries, and their names and properties. The black square and open circle denote the same EST sequence in two different libraries.

Fig. 7

Relative abundances of the five most abundant “no hit” EST clusters in each of the indicated libraries, and their names and properties. The black square denotes the same EST sequence in two different libraries.