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.

Transcriptome Profiling of Trypanosoma brucei Development in the Tsetse Fly Vector Glossina morsitans

“…Tsetse flies were infected with pleomorphic T . brucei brucei RUMP 503 parasites and following verification of the infection status by microscopic examination, trypanosomes were isolated from midgut, proventriculus and salivary gland tissues [16]. Total RNA was processed for sequencing on the Illumina platform and the resulting reads were aligned to the T .…”

“…brucei brucei RUMP 503 and we used the Glossina morsitans morsitans colony maintained in the insectary at Yale University for in vivo experiments. All manipulations, including tsetse infections, tissue dissections and RNA isolations were done as previously described [16]. Flies were dissected after a minimum of 40 days post infection and 72 h after their last blood meal, and infected salivary gland, proventriculus and midgut tissues were collected from the same flies.…”

Adult blood-feeding tsetse flies, trypanosomes, microbiota and the fluctuating environment in sub-Saharan Africa

“…In the presence of trypanosomes, tsetse flies will modify the expression of several of their genes. In response to this differential gene regulation, trypanosomes regulate the expression of their own genes for their survival (Savage et al, 2012). For example, T. b. gambiense and T. b. rhodesiense express genes associated with reactions against stress (Simo et al, 2010), indicating that trypanosomes are exposed to environmental stress within the tsetse fly midgut.…”

Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly

“…Transcripts were analyzed using complementary DNA templates normalized against T. brucei α-tubulin prepared from trypanosomeinfected tsetse midgut, cardia, and salivary glands, as well as the MCF parasites isolated from tsetse saliva, and BSF trypanosomes were experimentally raised in rodent blood using previously described methods. 4 Transcripts corresponding to TbLPP2, TbLPP3, and TbLPP2-related protein were the most abundant in salivary glands, followed by BSF and cardia samples ( Figure 2A and Supplemental Figure 2). We could not detect transcripts of the TbLPP1 gene in any of the tissue samples tested using our assay conditions (Figure 2A).…”

“…2 A tight control of gene expression occurs during tsetse colonization by T. brucei, where unique transcripts from parasites infecting midgut, cardia, and salivary glands have been reported. 3,4 Lipid phosphate phosphatases (LPPs) are members of the phosphatidic acid phosphatase superfamily. 5 LPP enzymes catalyze the dephosphorylation of phosphorous lipids, important in a broad range of physiological functions including cell migration, proliferation, and differentiation.…”