Differential protein expression throughout the life cycle of Trypanosoma congolense, a major parasite of cattle in Africa

Abstract

Trypanosoma congolense is an important pathogen of livestock in Africa. To study protein expression throughout the T. congolense life cycle, we used culture-derived parasites of each of the three main insect stages and bloodstream stage parasites isolated from infected mice, to perform differential protein expression analysis. Three complete biological replicates of all four life cycle stages were produced from T. congolense IL3000, a cloned parasite that is amenable to culture of major life cycle stages in vitro. Cellular proteins from each life cycle stage were trypsin digested and the resulting peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ). The peptides were then analyzed by tandem mass spectrometry (MS/MS). This method was used to identify and relatively quantify proteins from the different life cycle stages in the same experiment. A search of the Wellcome Trust's Sanger Institute's semi-annotated T. congolense database was performed using the MS/MS fragmentation data to identify the corresponding source proteins. A total of 2088 unique protein sequences were identified, representing 23% of the ∼9000 proteins predicted for the T. congolense proteome. The 1291 most confidently identified proteins were prioritized for further study. Of these, 784 yielded annotated hits while 501 were described as "hypothetical proteins". Six proteins showed no significant sequence similarity to any known proteins (from any species) and thus represent new, previously uncharacterized T. congolense proteins. Of particular interest among the remainder are several membrane molecules that showed drastic differential expression, including, not surprisingly, the well-studied variant surface glycoproteins (VSGs), invariant surface glycoproteins (ISGs) 65 and 75, congolense epimastigote specific protein (CESP), the surface protease GP63, an amino acid transporter, a pteridine transporter and a haptoglobin-hemoglobin receptor. Several of these surface disposed proteins are of functional interest as they are necessary for survival of the parasites.

Graphical abstract

Fig. 1

(A) The iTRAQ reagent is an isobaric tag consisting of a charged reporter group, a peptide-reactive group and a neutral balance portion. The reporter group ranges in mass from m/z 114 to 117, whereas the balance group ranges in mass from 28 to 31 Da, such that the combined mass remains constant (145 Da) for each of the four reagents. The figure was taken with permission from Ref. . (B) A simplified schematic overview of the iTRAQ sample preparation and experiment. Proteins from each life cycle stage were digested to tryptic peptides and labeled with different isobaric tags in parallel prior to mixing and simultaneous MS analysis. The figure was modified with permission from Ref. .

Fig. 2

Example of a mass spectrum for a single iTRAQ labeled peptide. (A) Precursor ion scan (MS) of a peptide labeled with the iTRAQ reagent. The differentially labeled forms are indistinguishable and contribute to the same peak. (B) Product ion scan (MS/MS) of the fragmented iTRAQ-labeled peptide. The b and y ions are indicative of the peptide sequence. (C) Enlarged region of the product ion spectrum, with the iTRAQ reporter ions indicating the relative levels of peptide in the four original samples. The figure was compiled from spectra 16.1.1.3838.2 of replicate number one. The source protein is β-tubulin.

Fig. 3

Venn diagram showing the distribution of unique protein hits among the 3 biological replicates of the iTRAQ experiments.

Fig. 4

Immunoblots on T. congolense cell lysates using various monoclonal antibodies. The inserted iTRAQ expression data shown represent the average of all reliable replicates (in some cases data with a p-value > 0.05 were included, averages that have some uncertain data included are marked with an asterisk) and normalized so that the expression level in PCF always equals 1. (A) Glutamic acid/alanine rich protein. (B) Major lysosomal membrane protein, p67. (C) Glycerol-3 phosphate dehydrogenase. (D) β tubulin. (E) Flagellar calcium binding protein. (F) Congolense epimastigote specific protein. The multiple or smeared bands observed in panels A, B and F are possibly due to glycosylation or other post-translational modifications of the proteins.