title: Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker.
abstract: Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDR(r) and type 10 VAND-SNF(r). The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA sibley@wusm.wustl.edu mike_behnke@yahoo.com.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Behnke
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Asis
authors.initials: A
authors.lastname: Khan
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA sibley@wusm.wustl.edu mike_behnke@yahoo.com.
authors.forename: L David
authors.initials: LD
authors.lastname: Sibley
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000633:toxicity; D026905:Amino Acid Transport Systems / Q000235:genetics*; D000981:Antiprotozoal Agents / Q000633:toxicity*; D004351:Drug Resistance / Q000235:genetics*; D008040:Genetic Linkage; D005819:Genetic Markers; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics*; D040641:Quantitative Trait Loci; D012641:Selection, Genetic; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics*
chemical_list: D026905:Amino Acid Transport Systems; D000981:Antiprotozoal Agents; D005819:Genetic Markers; D015800:Protozoan Proteins; D000241:Adenosine; C006235:sinefungin