Transcriptome analysis of Leishmania infantum PNA+ and PNA- promastigotes by complete shotgun DNA microarrays

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in Mediterranean areas and also acts as an opportunistic parasite in HIV patients. Metacyclic promastigotes are transmitted during bloodmeals of the sand-fly host after development. Metacyclogenesis can be micmiked in axenic cultures and peanut lectin (PNA) agglutination followed by two-step centrifugation allows the separation of procyclic and metacyclic promastigotes in L. major. The purpose of this study is to isolate both fractions simultaneously from the same population of L. infantum in stationary phase of axenic culture and compare their expression profiles through DNA microarrays, specially focusing on metacyclic promastigotes. Whole-genome shotgun DNA microarrays were constructed and used to analyse the stationary-phase procyclic and metacyclic expression profiles. Four biological replicates of the experiment were performed and analysed, so that 322 clones with meaningful values of stage-specific regulation were selected. We found several genes dealing with primary metabolism, differentiation in procyclic promastigotes and with development of infectivity in metacyclic promastigotes. The differences we have found between the procyclic (PNA+) and metacyclic (PNA-) transcriptomes demonstrate that negative selection of metacyclic promastigotes through PNA agglutination is suitable in L. infantum and both fractions can be isolated. In addition, up-regulation of genes implied in lipophosphoglycan (LPG), proteophosphoglycan (PPG) and glycoprotein biosynthesis indicate that metacyclic promastigotes are related with infectivity. Keywords: comparative hybridization between cDNAs from procyclic PNA+ and metacyclic PNA- promastigotes of L.infantum -CONSTRUCTION OF SHOTGUN DNA GENOME LIBRARY IN PUC18 VECTOR -PCR AMPLIFICATION WITH EXPAND LONG TEMPLATE. 29952 AMPLIFICATIONS -CONTROL ITEMS: positive control genes from Leishmania sp., genomic DNA from L.infantum, herring sperm DNA; negative control genes (from the nif regulon) from Leptospirillum ferrooxidans (BACTERIA), buffer 1xSSC -SPOTTING OF 30576 PCR PRODUCTS/CONTROL ITEMS: ARRAY CONSTRUCTION -LEISHMANIA GROWTH CURVE AND PNA+/- SEPARATION BY CENTRIFUGATION AFTER AGGLUTINATION -TOTAL RNA EXTRACTION, PURIFICATION, AMPLIFICATION AND CDNA SYNTHESIS/INDIRECT LABELLING -HYBRIDIZATION AND SCANNING (DUAL CHANNEL) -ANALYSIS: RAW DATA (GENEPIX 6.0) -NORMALIZATION: LOWESS PER PIN (ALMAZEN SOFTWARE) -COMPARATIVE EXPERIMENT OF THREE BIOLOGICAL REPLICATES BY T-TEST -FILTERING OF INTERESTING SPOTS (THAT MAY CONTAIN DIFFERENTIALLY REGULATED GENES) -CULTURES OF THE SELECTED CLONES, MINIPREPS AND SEQUENCING REACTIONS. -ASSEMBLY OF SEQUENCES, BLAST, PROCESSING WITH A CUSTOM PERL SCRIPT AND MAPPING AGAINST THE GENOME OF L.INFANTUM SEQUENCED BY THE SANGER CENTER WITH A GBROWSE APPLICATION. -CLUSTERING: GENE ONTOLOGY MOLECULAR FUNCTIONS