Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers

Abstract

Background: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking.

Methodology/principal findings: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum.

Conclusions: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.

Fig 1. Parasite growth and overall analysis of Leishmania sequencing.

A. Phase contrast images of the Leishmania parasites at different stages obtained from midguts at different time points. B. Principal component analysis (PCA) describing the position of each Leishmania time point in the expression space. Expression space was generated based on the log₂ TPMs (transcripts per million) of the significantly differentially expressed transcripts across six time points. The Eigenvalues and % variance for PC1 and PC2 were 806.1 and 70.58% and 174.6 and 15.3%, respectively. C. Total number of differentially expressed transcripts between Leishmania time points. D. Enrichment of DE transcripts for each Leishmania stage in pairwise comparisons, as color coded in the legend. E. Venn diagrams depicting the number of DE transcripts unique and shared amongst pairwise comparisons of Leishmania stages. DE was considered significant for transcripts displaying FDR (false discovery rate) q-value lower than 0.05 and LFC (log₂ fold change) either lower than -0.5 or higher than 0.5. PRO2d: procyclics at day 2. NEC4d: nectomonds at day 4. LEP6d: leptomonads 6 at days. LEP8d: leptomonads at 8 days. MET12d: metacyclics at day 12. MET14d: metacyclics at day 14.

Fig 2. Analysis of differentially expressed (DE) transcript enrichment in different Leishmania stages.

A. Principal Component Analysis (PCA) analysis of all the DE transcripts in all time points based on the log₂ fold change (LFC) of every pairwise combination of Leishmania time points. Each quadrant in the expression space was label from 1^st to 4^th and the transcripts mapped to the respective quadrants were color coded in Spring Green (1^st), Dodge Blue (2^nd), Blue Violet (3^rd), and Red (4^th). The Eigenvalues and % variance for PC1 and PC2% were 20.69 and 95.35% and 0.68 and 3.15%, respectively. B. Expression analysis per quadrant. The average TPM across time points for every DE transcript mapped in each quadrant was plotted. Horizontal bars indicate median values and differences were statistically significant (* Mann Whitney test, p < 0.0001). Color coding as in A. C. Expression analysis per quadrant per time point. The average TPM for each time point for every DE transcript mapped in each quadrant was plotted. Mean TPM as shapes and SEM (standard error of mean) bars are depicted. Based on the differences observed in B and C, the quadrants in A were labeled to describe the up-regulated transcripts expressed in high and low abundance (as defined by PC1) and expressed early and late time points (as defined by PC2). D-F. Leishmania DE transcripts up-regulated in each stage mapped onto the expression space. D. Bubble plot mapping the procyclic up-regulated transcripts (Royal blue) and the nectomonad up-regulated ones (Sea green) on the transcriptional space. Scale in gray represents the log₂ fold change corresponding to the diamenter of the bubbles. E. Bubble plot mapping the nectomonad up-regulated ones (Sea green) and the leptomonad up-regulated ones (Saddle brown) on the transcriptional space. F. Bubble plot mapping the leptomonad up-regulated ones (Saddle brown) and the metacyclic up-regulated ones (Fuchsia) on the transcriptional space. Differences were statistically significant at p < 0.001 (Chi-square test). DE was considered significant for transcripts displaying FDR q-value lower than 0.05 and LFC either lower than -0.5 or higher than 0.5. PRO2d: procyclics at day 2. NEC4d: nectomonds at day 4. LEP6d: leptomonads at 6 days. MET14d: metacyclics at day 14.

Fig 3. Heatmap depicting temporal expression of selected DE genes.

A. Genes encoding histone proteins. H1: histone H1. H2A: histone H2A; H2B: histone H2B; H3: histone H3. H4: histone H4. B. Metacyclogenesis-related genes. HASPa: hydrophilic acylated surface protein a; HASPb: hydrophilic acylated surface protein b; SHERP: small hydrophilic endoplasmic reticulum-associated protein; META1: META domain-containing protein. C. Genes involved in phosphoconjugate sythesis. Arabinosyl: phosphoglycan beta 1,2 arabinosyltransferase; Glycosyl: glycosyltransferase family-like protein; Galactosyl: phosphoglycan beta 1,3 galactosyltransferase; Mannosyl: mannosyltransferase-like protein; PPG4: proteophosphoglycan; LPG3: glucose regulated protein 94. GenBank gene Ids and color intensity scale are also depicted on the left. PRO2d: procyclics at day 2; NEC4d: nectomonads at day 4; LEP6d: leptomonads at day 6; LEP8d: leptomonads at day 8; MET12d: metacyclics at day 12; MET14d: metacyclics at day 14.

Fig 4. Chromosome displaying at least three-fold enrichment of DE genes across time Leishmania stages.

A. Chromosomes displaying enrichment of DE genes from the procyclic to the nectomonad stage. B. Chromosomes exhibiting decrease in the proportion of DE genes from the procyclic to the nectomonad stage. C. Chromosomes displaying enrichment of DE genes from leptomonad to metacyclic stage. D. Chromosomes exhibiting decrease in the proportion of DE genes from the leptomonad to the metacyclic stage. PRO2d: procyclics at day 2. NEC4d: nectomonds at day 4. LEP6d: leptomonads at 6 days. MET14d: metacyclics at day 14.

Fig 5. Candidate Leishmania stage-specific markers.

Venn diagrams highlighting (in white) the numbers of DE genes between (A) procyclics, (B) nectomonad, (C) leptomonad, and (D) metacyclic. E. Overall expression profile patterns of the candidate Leishmania stage-specific markers. Number of candidate genes per stage are shown in the inset. PRO2d: procyclics at day 2. NEC4d: nectomonds at day 4. LEP6d: leptomonads 6 at days. LEP8d: leptomonads at 8 days. MET12d: metacyclics at day 12. MET14d: metacyclics at day 14.