title: Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.
abstract: BACKGROUND
  Leishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been recently reported in central Spain. Leishmania spp. parasites are transmitted to the mammalian host by the bite of sand flies. The primary vector of L. infantum in Spain is Phlebotomus perniciosus. For decades, research on these parasites has involved the axenic culture model of the promastigote stage including gene expression profiling studies performed in the post-genome era. Unlike the controversial axenic culturing of amastigotes, promastigote cultures are generally accepted and used, although with the precaution of avoiding excessive culture passage.The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. Shotgun DNA microarray hybridization analyses allowed for transcriptome comparison of these promastigotes with amastigotes obtained by infection of the U937 cell line. The results have been compared with those described in published expression analyses using axenic promastigotes.
  
  
  RESULTS
  A total of 277 up-regulated genes were found through this hybridization experiment. The comparison of these particular results with published gene expression profile analyses performed using the same experimental procedure to study cultured promastigotes in stationary phase versus amastigotes revealed considerable differences (approximately 95% of the up-regulated genes were different). We found that the up-regulation rate is lower in amastigotes than in sand fly-derived promastigotes, which is in agreement with the over-expression of genes involved in gene expression regulation and signaling in those promastigote populations.
  
  
  CONCLUSIONS
  The up-regulation rate is lower in intracellular amastigotes than in promastigotes obtained from the sand fly gut. This was also reported by us using the promastigote culture model and is an evidence for the hypothesis of promastigote preadaptation towards life in the intracellular environment. Regarding transcript abundance, the set of differentially regulated genes is notably different when using promastigotes from the sand fly foregut instead of axenic cultures.
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Científicas, Calle Ramiro de Maeztu, 9, 28040 Madrid, Spain. pjalcolea@cib.csic.es.
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Alcolea
authors.forename: Ana
authors.initials: A
authors.lastname: Alonso
authors.forename: Manuel J
authors.initials: MJ
authors.lastname: Gómez
authors.forename: Marina
authors.initials: M
authors.lastname: Postigo
authors.forename: Ricardo
authors.initials: R
authors.lastname: Molina
authors.forename: Maribel
authors.initials: M
authors.lastname: Jiménez
authors.forename: Vicente
authors.initials: V
authors.lastname: Larraga
mesh_terms: D000818:Animals; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D018314:Leishmania infantum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008018:Life Cycle Stages; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D010586:Phagocytes / Q000166:cytology / Q000469:parasitology; D010691:Phlebotomus / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020298:U937 Cells; D015854:Up-Regulation
chemical_list: D012333:RNA, Messenger