title: Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.
abstract: Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.
authors.affiliation: Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada G1V 4G2; Département de Microbiologie, Infectiologie, et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 0A6.
authors.forename: Élodie
authors.initials: É
authors.lastname: Gazanion
authors.affiliation: Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada G1V 4G2; Département de Microbiologie, Infectiologie, et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 0A6.
authors.forename: Christopher
authors.identifier: 0000-0003-4834-4956
authors.initials: C
authors.lastname: Fernández-Prada
authors.affiliation: Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada G1V 4G2; Département de Microbiologie, Infectiologie, et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 0A6.
authors.forename: Barbara
authors.initials: B
authors.lastname: Papadopoulou
authors.affiliation: Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada G1V 4G2; Département de Microbiologie, Infectiologie, et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 0A6.
authors.forename: Philippe
authors.initials: P
authors.lastname: Leprohon
authors.affiliation: Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada G1V 4G2; Département de Microbiologie, Infectiologie, et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 0A6 Marc.Ouellette@crchul.ulaval.ca.
authors.forename: Marc
authors.initials: M
authors.lastname: Ouellette
mesh_terms: D000981:Antiprotozoal Agents / Q000494:pharmacology; D003360:Cosmids / Q000235:genetics; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics*; D017125:Genes, Protozoan; D059014:High-Throughput Nucleotide Sequencing; D018314:Leishmania infantum / Q000235:genetics*; D010743:Phospholipids / Q000235:genetics
chemical_list: D000981:Antiprotozoal Agents; D010743:Phospholipids
keywords: Cos-Seq; Leishmania; functional cloning; next-generation sequencing; resistance