title: The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector.
abstract: The life cycle of theLeishmaniaparasite 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 ofLeishmania majoras they develop in a natural vector,Phlebotomus duboscqiThe 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 thein vivoforms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.IMPORTANCEThe life cycle ofLeishmaniaparasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the gut. Based on RNA-Seq, we provide here the first high-resolution, transcriptomic analysis ofLeishmaniainsect stages during their cyclical developmentin vivo, from tissue amastigotes ingested with the blood meal to infective, metacyclic promastigotes that initiate infection in the mammalian host. The most extensive genetic reprogramming occurred during the early transformation of amastigotes to rapidly dividing procyclic promastigotes in the blood-fed midgut, with major changes in the abundance of mRNAs for surface proteins and metabolism. The post-blood meal-adapted nectomonad stage was characterized by the downregulation of cell cycle-related genes and the upregulation of stress- and starvation-related genes. Finally, the transcriptome of metacyclic promastigotes shifted to a more amastigote-like profile, suggesting their preadaptation to the intracellular host environment.
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Ehud
authors.initials: E
authors.lastname: Inbar
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.|Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA.
authors.forename: V Keith
authors.initials: VK
authors.lastname: Hughitt
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
authors.forename: Laura A L
authors.initials: LA
authors.lastname: Dillon
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kashinath
authors.initials: K
authors.lastname: Ghosh
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.|Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA.
authors.forename: Najib M
authors.identifier: 0000-0001-7970-3312
authors.initials: NM
authors.lastname: El-Sayed
authors.affiliation: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA DSACKS@niaid.nih.gov.
authors.forename: David L
authors.initials: DL
authors.lastname: Sacks
mesh_terms: D000818:Animals; D004199:Disease Vectors; D020869:Gene Expression Profiling; D018320:Leishmania major / Q000235:genetics / Q000254:growth & development*; D010691:Phlebotomus / Q000469:parasitology*; D017422:Sequence Analysis, DNA
keywords: Leishmania; sand fly; transcriptome