title: In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus.
abstract: Leishmania infantum is the protozoan parasite responsible for zoonotic visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been reported in this area. The life cycle of the parasite is digenetic. The promastigote stage develops within the gut of phlebotomine sand flies, whereas amastigotes survive and multiply within phagolysosomes of mammalian host phagocytes. The major vector of L. infantum in Spain is Phlebotomus perniciosus. The axenic culture model of promastigotes is generally used because it is able to mimic the conditions of the natural environment (i.e. the sand fly vector gut). However, infectivity decreases with culture passages and infection of laboratory animals is frequently required. Enrichment of the stationary phase population in highly infective metacyclic promastigotes is achieved by negative selection with peanut agglutinin (PNA), which is possible only in certain Leishmania species such as L. major and L. infantum. In this study, in vitro infectivity and differential gene expression of cultured PNA-negative promastigotes (Pro-PNA(-)) and metacyclic promastigotes isolated from the sand fly anterior thoracic midgut (Pro-Pper) have been compared.
  
  
  
  In vitro infectivity is about 30 % higher in terms of rate of infected cells and number of amastigotes per infected cell in Pro-Pper than in Pro-PNA(-). This finding is in agreement with up-regulation of a leishmanolysin gene (gp63) and genes involved in biosynthesis of glycosylinositolphospholipids (GIPL), lipophosphoglycan (LPG) and proteophosphoglycan (PPG) in Pro-Pper. In addition, differences between Pro-Pper and Pro-PNA(-) in genes involved in important cellular processes (e.g. signaling and regulation of gene expression) have been found.
  
  
  
  Pro-Pper are significantly more infective than peanut lectin non-agglutinating ones. Therefore, negative selection with PNA is an appropriate method for isolating metacyclic promastigotes in stationary phase of axenic culture but it does not allow reaching the in vitro infectivity levels of Pro-Pper. Indeed, GIPL, LPG and PPG biosynthetic genes together with a gp63 gene are up-regulated in Pro-Pper and interestingly, the correlation coefficient between both transcriptomes in terms of transcript abundance is R (2) = 0.68. This means that the correlation is sufficiently high to consider that both samples are physiologically comparable (i.e. the experiment was correctly designed and performed) and sufficiently low to conclude that important differences in transcript abundance have been found. Therefore, the implications of axenic culture should be evaluated case-by-case in each experimental design even when the stationary phase population in culture is enriched in metacyclic promastigotes by negative selection with PNA.
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas (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.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), calle Ramiro de Maeztu, 9, 28040, Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Alonso
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), calle Ramiro de Maeztu, 9, 28040, Madrid, Spain.
authors.forename: María A
authors.initials: MA
authors.lastname: Degayón
authors.affiliation: Laboratorio de Ecología Molecular, Centro de Astrobiología, (Instituto Nacional de Técnica Aeroespacial "Esteban Terradas"-Consejo Superior de Investigaciones Científicas), ctra. de Ajalvir Km 4, 28850, Torrejón de Ardoz, Madrid, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Moreno-Paz
authors.affiliation: Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Virología e Inmunología Sanitarias (Instituto de Salud Carlos III), ctra. Majadahonda-Pozuelo s/n, 28220, Majadahonda, Madrid, Spain.
authors.forename: Maribel
authors.initials: M
authors.lastname: Jiménez
authors.affiliation: Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Virología e Inmunología Sanitarias (Instituto de Salud Carlos III), ctra. Majadahonda-Pozuelo s/n, 28220, Majadahonda, Madrid, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Molina
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), calle Ramiro de Maeztu, 9, 28040, Madrid, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Larraga
mesh_terms: D000818:Animals; D005075:Biological Evolution; D001692:Biological Transport; D002460:Cell Line; D004734:Energy Metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D006801:Humans; D018314:Leishmania infantum / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics; D008954:Models, Biological; D019887:Peanut Agglutinin / Q000494:pharmacology; D010691:Phlebotomus / Q000469:parasitology; D025941:Protein Interaction Mapping; D059748:Proteolysis; D015398:Signal Transduction
chemical_list: D019887:Peanut Agglutinin; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania
keywords: Differential gene expression; Infectivity; Leishmania infantum; Metacyclic promastigotes; Peanut lectin agglutination; Phlebotomus perniciosus