Expression profiling of Trypanosoma congolense genes during development in the tsetse fly vector Glossina morsitans morsitans

Abstract

Background: The tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense's developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies.

Methods: In this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370).

Results: Consistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse's cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 'GARP', 'iii' and 'i' subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse's cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans' proboscis.

Conclusion: Collectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse's proboscis. We discuss our results in terms of their relevance to disease transmission processes.

Keywords: Gene expression analysis; Glossina morsitans morsitans; Trypanosoma congolense; Tsetse cardia; Tsetse proboscis and confocal microscopy.

Fig. 1

A Volcano plot showing differentially expressed genes of T. congolense parasites in the cardia relative to those in the probosces of G. morsitans. Only transcripts with at least 30 reads mapped and at least five RPKM (Reads Per Kilobase Million) by CLC-Genomics and EdgeR analysis [36, 99] in either library (C-parasites or PB-parasites) were considered. Red dots indicate DE genes with an FC of ≥ 2 (log₂ = 1) and false detection rate (FDR) corrected P-value of < 0.05 between cardia and proboscis parasites. The x-axis displays magnitude of fold-changes and y-axis the statistical significance (-log₁₀ of P-value). Points having FC of < 2 (log₂ < 1) on an FDR corrected P-value of < 0.05 are shown in black, and indicate genes with non-significance change between different developmental states

Fig. 2

Differential expression of genes in metabolic pathways. Heat maps showing the expression profiles consisting of respective RPKMs clustered using Euclidean distance calculation and ward.D clustering methods. a KEGG and TrypanoCYC enriched metabolic pathway. b-f Genes that function in the oxidative phosphorylation pathway, antioxidant (defense), pentose phosphate pathway, lipid metabolism, and glycolytic pathway respectively

Fig. 3

Volcano plot showing DE genes between cardia and proboscis parasites encoding proteins putatively associated with trypanosome differentiation. The red dots indicate points-of-interest with a fold-change of ≥ 2 (log₂ = 1) and False Detection Rate (FDR) corrected P < 0.05 between cardia and proboscis parasites. Black dots represent genes with no significance change in expression level

Fig. 4

Heatmaps of expression of T. congolense genes encoding putative cell surface proteins. a GPI anchored proteins. Prediction of the putative GPI-anchored cell surface proteins was determined by FragAnchor [44], PredGPI [43] and BigPI [45]. b Transmembrane proteins. Trans-membrane helices was predicted using TMHMM [46]. The expression profiles consist of respective log₂ transformed RPKM clustered using Euclidean distance calculation and ward.D clustering methods. ¹Fam13/16 (VSGs), ²Fam50 (Brucei alanine-rich protein), ³Fam51 (Expression site-associated gene 4), ⁴Fam54 (Amino acid transporter), ⁵Fam56 (ABC transporter), ⁶Fam57 (Folate transporter, ESAG10), ⁷Fam60 (Membrane transporter protein), ⁸Fam61 (Nucleoside transporter), ⁹Fam67 (Cysteine peptidase), ¹⁰Fam12 (Procyclin-like), ¹¹Fam14 (Transferrin receptor-like, PAG-like)

Fig. 5

Expression analysis of selected CSP genes. a Expression levels of T. congolense gapdh from infected tsetse’s midgut, cardia and proboscis organs as well as bloodstream form parasites purified from infected mice blood. Abbreviations: BSF, bloodstream form; MG, tsetse midgut parasites; card, cardia parasites; PB, proboscis parasites. b Stage-regulated gene expression profiles for genes that putatively encode GPI-anchored and transmembrane proteins, normalized to gapdh levels as shown in A. * indicates transmembrane protein encoding genes. c Expression analysis of two GPI-anchored protein encoding genes via RT-qPCR. d Localization of T. congolense TcIL3000_0_02370 protein in parasites residing in the midgut, cardia and proboscis organs examined by immunofluorescent staining and confocal laser microscopy. Red indicates immunofluorescence staining of TcIL3000_0_02370, and blue indicates the DAPI staining of nucleus and kinetoplast