Leishmania infection induces a limited differential gene expression in the sand fly midgut

Abstract

Background: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages.

Results: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics.

Conclusion: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Keywords: Leishmania infantum; Lutzomyia longipalpis; Midgut; Parasite; RNA-Seq; Sand fly; Transcriptome; Transcriptomics; Vector.

Fig. 1

Overview of the transcriptome repertoire displaying the overall percentage of contigs (% of contigs) and abundance (%TPM) for all time points. The distribution of the mapped reads to the functional classification are highlighted

Fig. 2

Midgut sequencing overall analysis. a. Principal component analysis (PCA) describing the position of each midgut time point on the expression space. Expression space was generated based on the log₂ of TPMs using the 10,000 most highly expressed transcripts across libraries. The Eigenvalues and % variance for PC1 and PC3 were 6221.99 and 77.19% and 330.34 and 4.1%, respectively. b-h. Gene expression validation by nCounter (Nanostring). Linear regression analyses comparing the expression profiles of randomly chosen transcripts obtained with RNA-Seq and nCounter (Nanostring) techniques for the seven time points. All comparisons were statistically significant (p < 0.0001). R²: regression coefficient. n: number of transcripts. The color codes labeling each time point were as follow: Aqua (1d); Royal Blue (2d); Sea Green (4d); Sandy Brown (6d); Saddle Brown (8d); Red (12d); and Fuchsia (14d). The triangle and circle shapes represent Leishmania-infected and uninfected samples, respectively

Fig. 3

Analysis of differentially expressed (DE) midgut transcripts across time points. a. Number of DE transcripts up- and down-regulated in Leishmania infected over uninfected midguts at each time point. b. Venn diagrams depicting the number of DE transcripts unique and shared amongst the time points 1d through 6d. c. Venn diagrams depicting the number of DE transcripts unique and shared amongst time points 6d through 14d

Fig. 4

DE transcripts sorted by molecular functions. a-b. Heatmaps and cluster analyses depicting differences in the number of DE genes up-regulated (a) and down-regulated (b) by Leishmania infection belonging to different groups of molecular function. Legends: Cs: cytoskeleton; Detox: oxidative metabolism/detoxification; Extmat: extracellular matrix; Imm: immunity; Met: metabolism; Ne: nuclear export; Nr: nuclear regulation; Pe: protein export; Pm: protein modification; Prot: proteosome machinery; Ps: protein synthesis machinery; S: secreted protein; St: signal transduction; Storage: storage protein; Te: transposable element; Tf: transcription factor; Tm: transcription machinery; Tr: transporters and channels; Uk: unknown protein. c-d. Heatmaps and cluster analyses depicting differences in the number of DE genes up-regulated (c) and down-regulated (d) by Leishmania infection, belonging to different sorts of oxidative metabolism/detoxification molecular function. Legends: Dehyd: dehydrogenase; Glutat: glutathione s-transferase; P450: cytochrome P450; Oxidase: oxidase/peroxidase; Reduc: reductase; Abc: Transporter ABC superfamily; Thio: thioredoxin binding protein; Transf: sulfotransferase. e-f. Heatmaps and cluster analyses depicting differences in the number of DE genes up-regulated (e) and down-regulated (f) by Leishmania infection belonging to different sorts of metabolism molecular function, respectively. Legends: Aa: amino acid metabolism; Carb: carbohydrate metabolism; Energy: energy production; Int: intermediate metabolism; Lipd: lipid metabolism; Nuc: nucleotide metabolism. g-h. Heatmaps and cluster analyses depicting differences in the number of DE genes up-regulated (g) and down-regulated (h) belonging to different sorts of secreted protein molecular function. The heatmaps are color-coded according to the legends on the right. Legends: S: other; Metal: metalloprotease; Lipoc: lipocalin; Met-li: lipase; Met-nu: nuclease; Mucin; Protea: protease; Protin: protease inhibitor; Uk: unknown protein. DE was considered significant for transcripts displaying LFC either lower than − 1 or higher than 1 and FDR q-value lower than 0.05