title: Secondary mutations correct fitness defects in Toxoplasma gondii with dinitroaniline resistance mutations.
abstract: Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing alpha1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild-type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were alpha1-tubulin pseudorevertants or extragenic suppressors (the majority alter the beta1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified seven novel mutations that localize to an eight-amino-acid insert that stabilizes the alpha1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but most perform better than parental lines in competition assays, indicating that there is a trade-off between resistance and replication fitness.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Ma
authors.forename: Johnson
authors.initials: J
authors.lastname: Tran
authors.forename: Catherine
authors.initials: C
authors.lastname: Li
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Ganesan
authors.forename: David
authors.initials: D
authors.lastname: Wood
authors.forename: Naomi
authors.initials: N
authors.lastname: Morrissette
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D004136:Dinitrobenzenes / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D013424:Sulfanilamides / Q000494:pharmacology; D014122:Toxoplasma / Q000235:genetics* / Q000378:metabolism; D014274:Trifluralin / Q000031:analogs & derivatives / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: D000814:Aniline Compounds; D004136:Dinitrobenzenes; D013424:Sulfanilamides; D014404:Tubulin; D050257:Tubulin Modulators; C027392:ethafluralin; C012465:oryzalin; D014274:Trifluralin
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