The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

Abstract

The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.

Proteomics and Leishmaniasis: Potential Clinical Applications

“…Nectomonads displayed changes related to cell cycle arrest and starvation and stress, including autophagic pathways. Further development to metacyclic stage was accompanied by amastigote‐like profiles of genes related to surface proteins and metabolism, suggesting a preadaptation to the intracellular stage in the vertebrate host . Proteomic comparison of the different insect stages would add important information to the findings reported in this study.…”

“…A recent study compared the transcriptomes of four distinct stages of L. major development in its natural vector . The differentiation from tissue amastigotes to procyclic promastigotes was accompanied by the largest gene expression changes, including reduction of transcripts of amastins and upregulation of transcripts of several cell surface proteins, sugar and amino acid transporters, and genes associated with glucose metabolism and cell cycle progression.…”

The mRNA-bound proteome ofLeishmania mexicana: novel genetic insight into an ancient parasite

“…Our data ( Fig 5A) supports this finding albeit on a broader timescale than an hourly timecourse and in a different Leishmania species, suggesting this lack of correlation between mRNA and protein expression may be common to all Leishmania. Certainly transcript expression potentiates Leishmania protein expression and thereby provides useful insight into gene expression that would be prohibitively expensive and experimentally challenging to obtain on the proteomic level currently [19].…”

“…The cell cycle analysis of PCF cell cultures mid-log at 5x10 6 cells/ml showed high replication efficiency (S: 19%, G2/M: 35%), sensitivity to human serum incubation and heightened Histone h4 (h4) transcript expression ( Fig 1B, C and D). We introduce Histone H4 (LmxM.36.0020) here as a novel transcript marker of PCF stage L.mexicana parasites, investigated due to its specific expression in sandfly-derived L.major PCF cells [19]. Stationary META form parasites were harvested at ~4x10 7 cells/ml 7 days post-incubation in Grace's media as described [20] and validated by reduced replication and protein synthesis [21](S: 3%; G2/M: 19%), resistance to human complement lysis [22] and heightened expression of sherp transcript [23]( Fig 1B, C and D).…”

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

“…Comparing Leishmania major culture promastigotes and murine macrophage amastigotes, or culture promastigotes and human macrophage amastigotes, at least 30% of the genes were differentially expressed (q-value <0.05) [13][14][15]. Apart from a recent work investigating L. major stages inside the Phlebotomus papatasi sand fly [16], little is known of molecular markers and RNA differential expression between the Leishmania promastigote stages developing in the midgut of the sand fly vector, particularly for L. infantum inside its natural sand fly vector, Lutzomyia longipalpis. In order to fill this knowledge gap, we performed a comprehensive RNAseq investigation to assess L. infantum gene expression in the midgut of Lutzomyia longipalpis at six time points corresponding to each developmental stage, from procyclic to infective metacyclic promastigotes.…”