Tsetse fly tolerance to T. brucei infection: transcriptome analysis of trypanosome-associated changes in the tsetse fly salivary gland

Abstract

Background: For their transmission, African trypanosomes rely on their blood feeding insect vector, the tsetse fly (Glossina sp.). The ingested Trypanosoma brucei parasites have to overcome a series of barriers in the tsetse fly alimentary tract to finally develop into the infective metacyclic forms in the salivary glands that are transmitted to a mammalian host by the tsetse bite. The parasite population in the salivary gland is dense with a significant number of trypanosomes tightly attached to the epithelial cells. Our current knowledge on the impact of the infection on the salivary gland functioning is very limited. Therefore, this study aimed to gain a deeper insight into the global gene expression changes in the salivary glands of Glossina morsitans morsitans in response to an infection with the T. brucei parasite. A detailed whole transcriptome comparison of midgut-infected tsetse with and without a mature salivary gland infection was performed to study the impact of a trypanosome infection on different aspects of the salivary gland functioning and the mechanisms that are induced in this tissue to tolerate the infection i.e. to control the negative impact of the parasite presence. Moreover, a transcriptome comparison with age-matched uninfected flies was done to see whether gene expression in the salivary glands is already affected by a trypanosome infection in the tsetse midgut.

Results: By a RNA-sequencing (RNA-seq) approach we compared the whole transcriptomes of flies with a T. brucei salivary gland/midgut infection versus flies with only a midgut infection or versus non-infected flies, all with the same age and feeding history. More than 7500 salivary gland transcripts were detected from which a core group of 1214 differentially expressed genes (768 up- and 446 down-regulated) were shared between the two transcriptional comparisons. Gene Ontology enrichment analysis and detailed gene expression comparisons showed a diverse impact at the gene transcript level. Increased expression was observed for transcripts encoding for proteins involved in immunity (like several genes of the Imd-signaling pathway, serine proteases, serpins and thioester-containing proteins), detoxification of reactive species, cell death, cytoskeleton organization, cell junction and repair. Decreased expression was observed for transcripts encoding the major secreted proteins such as 5'-nucleotidases, adenosine deaminases and the nucleic acid binding proteins Tsals. Moreover, expression of some gene categories in the salivary glands were found to be already affected by a trypanosome midgut infection, before the parasite reaches the salivary glands.

Conclusions: This study reveals that the T. brucei population in the tsetse salivary gland has a negative impact on its functioning and on the integrity of the gland epithelium. Our RNA-seq data suggest induction of a strong local tissue response in order to control the epithelial cell damage, the ROS intoxication of the cellular environment and the parasite infection, resulting in the fly tolerance to the infection. The modified expression of some gene categories in the tsetse salivary glands by a trypanosome infection at the midgut level indicate a putative anticipatory response in the salivary glands, before the parasite reaches this tissue.

Keywords: RNA-seq; Salivary gland; Tolerance; Trypanosoma brucei; Tsetse fly.

Fig. 1

Heat map showing the Euclidean distances between the samples as calculated from the regularized log transformation. MG: midgut; SG: salivary glands. The trypanosome infection status of the respective tissue is indicated by – or +

Fig. 2

Differentially expressed transcripts between the experimental conditions. Red and blue triangles indicate significance at a 10% adjusted p-value, upregulation and downregulation respectively. Grey triangles indicate transcripts that showed no change. MG: midgut; SG: salivary glands. The trypanosome infection status of the respective tissue is indicated by – or +

Fig. 3

a Gene Ontology enrichment analysis for biological process of up-regulated transcripts. The transcripts included had an expression value higher than 2 fold change in both comparisons. The GO terms were slimmed prior to the analysis and were considered significant for a false discovery rate of 0.01. A total of 290 transcripts were used in the analysis from which 217 had a GO term annotation. The number of transcripts with a GO term is indicated in the corresponding pie slice. b Classification of downregulated transcripts using Gene Ontology. The transcripts included had an expression value lower than 2 fold change in both comparisons. A total of 91 transcripts were used in the classification from which 56 had a GO annotation. The GO terms were slimmed prior to the analysis. The number of transcripts with a GO term is indicated in the corresponding pie slice

Fig. 4

a-j Graphical representation of transcripts expression in different functional category. The heat maps were obtained by plotting the mean of normalized read counts (scaled by row) in the three infection conditions. Colors display z-scores from −1 (low expression: dark blue) to 1 (high expression: red) for normalized gene expression values. MG: midgut; SG: salivary glands. The trypanosome infection status of the respective tissue is indicated by – or +. FC: Fold Change; NDE: not differentially expressed

Fig. 5

Overview of the major gene transcript impact of T. brucei infection in the salivary glands. Only the transcripts that showed a ≥ 2 fold differential expression are presented in the figure. Red: increased expression; Blue: decreased expression