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Vol. 18, Issue 12, 4711-4720, December 2007

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Mutations in -Tubulin Confer Dinitroaniline Resistance at a Cost to Microtubule Function

Christopher Ma^*,, Catherine Li^*,, Lakshmi Ganesan^*, Jean Oak^*, Susan Tsai^*, David Sept, and Naomi S. Morrissette^*

*Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697; and Department of Biomedical Engineering and the Center for Computational Biology, Washington University School of Medicine, St. Louis, MO 63110

Submitted April 27, 2007; Revised September 6, 2007; Accepted September 7, 2007
Monitoring Editor: Kerry Bloom

Protozoan microtubules are sensitive to disruption by dinitroanilines, compounds that kill intracellular Toxoplasma gondii parasites without affecting microtubules in vertebrate host cells. We previously isolated a number of resistant Toxoplasma lines that harbor mutations to the 1-tubulin gene. Some of the mutations are localized in or near the M and N loops, domains that coordinate lateral interactions between protofilaments. Other resistance mutations map to a computationally identified binding site beneath the N loop. Allelic replacement of wild-type 1-tubulin with the individual mutations is sufficient to confer dinitroaniline resistance. Some mutations seem to increase microtubule length, suggesting that they increase subunit affinity. All mutations are associated with replication defects that decrease parasite viability. When parasites bearing the N loop mutation Phe52Tyr are grown without dinitroaniline selection, they spontaneously acquired secondary mutations in the M loop (Ala273Val) or in an -tubulin–specific insert that stabilizes the M loop (Asp367Val). Parasites with the double mutations have both reduced resistance and diminished incidence of replication defects, suggesting that the secondary mutations decrease protofilament affinity to increase parasite fitness.

These authors contributed equally to this work.

Address correspondence to: Naomi Morrissette (nmorriss{at}uci.edu).