Transcript expression analysis of putative Trypanosoma brucei GPI-anchored surface proteins during development in the tsetse and mammalian hosts

Abstract

Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of unknown genes encoding predicted T. brucei surface proteins during the complete developmental cycle. This knowledge may form the foundation for the development of future novel transmission blocking strategies against metacyclic parasites.

Figure 1. Expression profiling of three known genes from experimental cDNA templates.

As experimental controls alpha-tubulin, procyclin and BARP gene expression was analyzed A) alpha-tubulin, B) procyclin, C) BARP PCR amplified from infected tsetse salivary glands (lane 1), proventriculus (lane 2), midguts (lane 3), and bloodstream form (lane 4) cDNAs.

Figure 2. Validation of semi-quantitative RT-PCR analysis.

Fold-change expression was measured by qRT-PCR analysis for randomly selected trypanosome genes in tsetse SG, PV, and MG tissues, relative to alpha-tubulin expression (bar graph). The values obtained by the semi-quantitative fold-change analysis for the same genes are shown as numerical data below each graph. Tb927.10.5700/5710 are two related genes amplified by the same primers. Asterisks (*) denote statistical significance (p≤0.05), diamonds (♦) denote non-significant trend p≤0.10.

Figure 3. Tissue specificity of trypanosome gene expression.

Percentage of trypanosome transcripts corresponding to putative probable GPI-anchored proteins detected by semi-quantitative RT-PCR by expression profile classification.