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 wholegenome 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.T oxoplasma gondii is an intracellular parasite that infects a broad range of mammals and birds from around the world (1). The ubiquitous prevalence of the parasite is due to several aspects of the parasite's life cycle, including the ability to infect all mammalian nucleated cells, the ability to reside in intermediate hosts for long periods by forming dormant bradyzoite cysts, and the ability to produce millions of environmentally resistant oocysts shed from felids, the definitive host for T. gondii (1). Due to its high prevalence, humans are commonly exposed to the parasite through consumption of undercooked meat or ingestion of oocysts that contaminate food and water (2, 3). Infection by the parasite very rarely leads to complications in healthy individuals, but in individuals with compromised immune systems, the parasite can cause disease and even death if untreated (4). Due to concerted efforts to study this opportunistic pathogen, many new molecular biology techniques have been developed to interrogate the biology of the parasite and its interaction with the host (5, 6).One powerful approach has been the use of forward genetics in Toxoplasma gondii. Within its intermediate hosts, such as the mouse, the parasite is haploid and divides by mitosis, whereas following development of enteric stages in felids, the parasite is shed as a spore-like stage that undergoes meiosis in the environment. Sexual recombination can generate recombinant genotypes when multiple T. gondii genotypes coinfect a felid. The ability to complete the life cycle in the laboratory has been utilized to create several genetic crosses that have yielded important insights into virulence mechanisms employed by th...

Identification of Small Molecule Inhibitors That Block the Toxoplasma gondii Rhoptry Kinase ROP18

“…T. gondii RH strain parasites were maintained by passage in HFFs, cultured in DMEM 3% FBS, as previously described 26 . The genotypes of parasites used in recruitment assays were RHΔ ku80 Δ hxgprt Δ uprt::CBR (herein described as wild type) and RHΔ ku80 Δ rop18::HXGPRT Δ uprt::CBR (herein described as Δ rop18 mutant ), as described previously 58 . All cultures were determined to be mycoplasma negative using the e-Myco plus kit (Intron Biotechnology).…”

Genetic Mapping of Pathogenesis Determinants inToxoplasma gondii

“…This break can then be repaired by nonhomologous end joining to create frame-shift or stop mutants, or by homologous recombination to create allelic replacements or knockouts when targeting constructs are employed. CRISPR has been successfully applied to all these genetic manipulation strategies in T. gondii, and in strains that were previously refractory to genetic manipulation (7,8,71,107,112). The power of this very efficient approach promises to open a myriad of possibilities for the study of this parasite.…”

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

“…After these methodological reports were published, the CRISPR/Cas9 system was used in T. gondii for gene knockout and functional studies of several proteins (Behnke et al. ,b; Long et al. ; Rugarabamu et al.…”

“…Behnke et al. () combined forward and reverse genetics approaches for mapping the sinefungin resistance gene ( SNR1 ), and then they disrupted it by CRISPR/Cas9 to confirm it renders sinefungin (SFN) resistant parasites. In another work, the same group used a modified version of their T. gondii CRISPR/Cas9 plasmid (Shen et al.…”

“…After these methodological reports were published, the CRISPR/Cas9 system was used in T. gondii for gene knockout and functional studies of several proteins (Behnke et al 2015a,b;Long et al 2016b;Rugarabamu et al 2015;Wang et al 2015Wang et al , 2016. Behnke et al (2015b) combined forward and reverse genetics approaches for mapping the sinefungin resistance gene (SNR1), and then they disrupted it by CRISPR/Cas9 to confirm it renders sinefungin (SFN) resistant parasites. In another work, the same group used a modified version of their T. gondii CRISPR/Cas9 plasmid (Shen et al 2014), to express two different sgRNAs that targeted regions upstream and downstream the tandem array of ROP5 genes in T. gondii genome, and completely replaced the tandem with the DHFR marker delivered in a DNA donor cassette (Behnke et al 2015a).…”