title: A possible key molecule for the invasion of the Plasmodium berghei ookinetes into the midgut epithelium of Anopheles gambiae mosquitoes.
abstract: BACKGROUND
  In order to identify molecules necessary for the invasion of the mosquito midgut epithelium by plasmodia, interaction assays between both these structures were devised.
  
  
  METHODS
  Enrichment of Plasmodium berghei ookinetes was first carried out using a double 5-17% gradient Nycodenz cushion, which resulted in an enrichment factor of over 800%. Viability of these ookinetes was confirmed by membrane feeding mosquito infection assays, showing no decrease in infection prevalence or intensity, and suggesting that putative surface proteins necessary for the invasion were unaffected.
  
  
  RESULTS
  Protein interaction assays between mosquito midgut and ookinete extracts were optimized yielding Anopheles gambiae mosquito midgut proteins of >220, 200 and 48 kDa that could bind biotinylated P. berghei ookinete extracts, and P. berghei ookinete proteins of 48-45 kDa that could bind biotinylated An. gambiae midgut proteins. Using an An. gambiae midgut biotinylated extract linked to a streptavidin-agarose matrix, P. berghei ookinete proteins of approximately 116, 45 and 21 kDa were obtained. This protein chromotography pull-down assay was reproducibly repeated and spots from 2D eletrophoretic separation were analysed by mass spectrometry. For one spot, a significant match with a putative erythrocyte binding protein from P. falciparum (Pf EBA-165) was obtained. This protein belongs to the erythrocyte binding superfamily of the merozoite stage that is involved in the invasion process of the erythrocytes.
  
  
  CONCLUSIONS
  Our findings suggest that there is a possibility that a homologue of Pf EBA-165 takes part in the ookinete recognition and invasion process of the mosquito midgut by plasmodia ookinetes.
authors.affiliation: Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira, 96, 1349-008 Lisboa, Portugal.
authors.forename: Duarte N
authors.initials: DN
authors.lastname: Toubarro
authors.forename: Diana
authors.initials: D
authors.lastname: Ralha
authors.forename: Sandra
authors.initials: S
authors.lastname: Carvalho
authors.forename: Ana M
authors.initials: AM
authors.lastname: Tomás
authors.forename: A Paulo G
authors.initials: AP
authors.lastname: Almeida
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D000953:Antigens, Protozoan / Q000032:analysis / Q000378:metabolism*; D019921:Biotinylation; D015153:Blotting, Western; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism / Q000469:parasitology; D005260:Female; D006790:Host-Parasite Interactions / Q000502:physiology; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000469:parasitology*; D008024:Ligands; D013058:Mass Spectrometry; D051379:Mice; D008815:Mice, Inbred Strains; D010962:Plasmodium berghei / Q000254:growth & development* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D000953:Antigens, Protozoan; D008024:Ligands