Secondary Mutations Correct Fitness Defects in Toxo With Dinitroaniline Resistance Mutations

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Copyright 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.092494

Secondary Mutations Correct Fitness Defects in Toxoplasma gondii With Dinitroaniline Resistance Mutations

Christopher Ma, Johnson Tran, Catherine Li, Lakshmi Ganesan, David Wood andNaomi Morrissette1

Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697 Manuscript received June 10, 2008

Accepted for publication July 21, 2008

ABS TRACT

Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebratecells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasitescontaining a1-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 andreplication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growthin culture. Weisolated 46 T. gondii lines that havesuppressed microtubule defects associated with the G142S orthe F52Y mutations by acquiring secondary mutations. These compensatory mutations were a1-tubulin

pseudorevertants or extragenic suppressors (the majority alter the b1-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 thea1-tubulin M loop, including one (P364R) thatacts as a compensatory mutation in both F52Y and G142S lines.These lines havereduced dinitroaniline resistancebut mostperformbetter thanparental lines in competitionassays, indicating that there is a trade-off between resistance and replication fitness.

T OXOPLASMA gondii is a human pathogen grouped within the Apicomplexa, a phylum of protozoa that

comprises all obligate intracellular parasites (Levene1988). Infection with this parasite can be life threateningin immunocompromised individuals and cause birthdefects or miscarriage during fetal infection (Black andBoothroyd 2000). Other apicomplexans such as Plasmo-dium spp. and Cryptosporidium spp. have considerablemedical importance or are animal pathogens that affect livestock. Apicomplexans have a specialized apex that con-tains unique organelles that coordinate invasion of host cells (Morrissette and Sibley 2002a). These parasitesaredelimited by a pellicle,a compositestructure formedby the association of the plasma membrane with the innermembrane complex, an assemblage of flattened vesicles.The invasive stages of apicomplexan parasites have twomicrotubule populations: subpellicular microtubules and

spindle microtubules. Subpellicular microtubules arenondynamic; they maintain both apical polarity and thecharacteristic crescent shape of the parasite by interacting

with the cytoplasmic face of the pellicle. Spindle micro-tubules form an intranuclear spindle to coordinatechromosome segregation. Both populations are critically important to parasite survival and replication.

Microtubules are built by the polymerization of a-b-tubulin heterodimers and typically contain 13 pro-tofilaments. Protofilaments are formed by longitudinalhead-to-tail association of heterodimers and laterally associate to assemble the microtubule lattice (Downing

and Nogales 1998). Biochemical and structural studieshave elucidated the roles of tubulin domains, which areimportant to the regulation of microtubule polymeriza-tion and depolymerization. Both a- and b-tubulins haveH1-S2 (N) and M loops, which coordinate contactsbetween adjacent protofilaments (Nogales et al. 1997,1999; Downing and Nogales 1998; Lowe et al. 2001;Li et al. 2002). a-Tubulin contains an eight-amino-acidinsert missing from the analogous region of b-tubulin,

which stabilizes the a-tubulin M loop to promoteprotofilament lateral association. Botha-and b-tubulinsbind to GTP; however, only b-tubulin GTP can be

exchanged or hydrolyzed while a-tubulin GTP plays astructural role in this subunit (David-Pfeuty et al. 1977;Sage et al. 1995a,b; Nogales et al. 1998; A nders andBotstein 2001). The b-tubulin subunits are stimulatedto hydrolyze GTP in a polymerization-dependent fash-ion by association of a GTPase-activating domain fromthe a-tubulin subunit of the adjacent dimer within theprotofilament. Structural evidence indicates that GTP-bound dimers have a conformation that facilitatesprotofilament contacts by both a- and b-subunits. Afterhydrolysis, GDP-tubulin dimers have a kinked confor-

1Corresponding author: Department of Molecular Biology and Biochem-istry, University of California, Irvine, CA 92697-3900.E-mail: [email protected]

Genetics 180: 845–856 (October 2008)



mation that is believed to weaken lateral contacts topromote microtubule disassembly (Nogales et al. 2003;

W ang et al. 2005; W ang and Nogales 2005; Nogales

and W ang 2006a,b).Protozoan parasites are sensitive to dinitroaniline

compounds, which disrupt microtubules to inhibit replication (Stokkermans et al. 1996; Bogitsh et al.1999; Makioka et al. 2000; Traub-Cseko et al. 2001;

Bhattacharya et al. 2002). Given that these com-pounds are inactive against vertebrate or fungal tubu-lins, the mechanism of dinitroaniline action is of particular interest for development of new antimicro-bial therapies. Our assays, as well as data from others,indicate that wild-type T. gondii parasites have an IC50

value of 0.25 mm for the dinitroaniline oryzalin and dis-play aberrant phenotypes in culture at 0.5 mm oryzalin(Stokkermans et al. 1996; Morrissette and Sibley2002b). Although extracellular parasites are refractory tothe effects of microtubule-disrupting drugs, during in-tracellular growth, parasite microtubules are dynamicand sensitive to disruption by submicromolar concen-

trations of oryzalin or other dinitroanilines.Dinitroaniline selectivity is associated with restricted

binding to sensitive (plants and protozoa) but not toresistant (vertebrates and fungi) tubulin (Hess andBayer 1977; Morejohn et al. 1987; Chan and Fong1990; Hugdahl and Morejohn 1993). Studies usingmolecular dynamics analysis and compound dockingindicate that dinitroanilines interact with a consistent site on multiple conformations of a-tubulin derivedfrom independent molecular dynamics models of T.gondii , Leishmania spp., and Plasmodium spp. tubulins(Morrissette et al. 2004; Mitra and Sept 2006).Parallel analysis of vertebrate (bovine) a-tubulin revealsthat dinitroanilines have nonspecific, low-affinity inter-actions and no consensus binding site, consistent within vivo and in vitro observations that these compoundsdo not bind to vertebrate tubulin or disrupt vertebratemicrotubules. This work predicts a binding site onparasite a-tubulin that is located beneath the H1-S2(N) loop. Since this loop participates in protofilament interactions, the binding site location suggests that dinitroanilines disrupt lateral contacts in the microtu-bule lattice. Consistent with this notion, a comparison of a-tubulin molecular dynamics simulations in the pres-ence and absence of a bound drug suggests that

dinitroaniline binding profoundly limits flexibility of the a-tubulin H1-S2 loop, which is drawn in toward thecore of the tubulin dimer (Mitra and Sept 2006).

T. gondii is a haploid organism for most of its life cycle,although it is capable of sexual recombination during atransient diploid zygote stage. There are three a- andthree b-tubulin genes in the genome: the a2 geneappearsto be gamete stage specific and the divergent a3gene may be used to construct the conoid, an unusualstructure built of tubulin sheets rather than micro-tubules (Hu et al. 2002). The a1- and b1-tubulin genes

are the dominantly expressed forms in the proliferative(tachyzoite) stage of the parasite studied here. We havepreviously identified a number of a1-tubulin point mutations associated with oryzalin resistance in T. gondii (Morrissette et al. 2004; Ma et al. 2007). The mutationsare distributed throughout the linear sequence of a1-tubulin. However, when mapped onto a model of T.gondii a1-tubulin based on the structure of the verte-

brate tubulin dimer, many of the mutations cluster inspecific domains of the protein. For example, many mutations are located in or near domains that areassociated with microtubule stability or in the core of a-tubulin, adjacent to or within the dinitroaniline-binding site identified by computational methods.Our working model suggests that most tubulin muta-tions confer dinitroaniline resistance by one of two pos-sible mechanisms: (1) by increasing subunit affinity

within the microtubule to compensate for the action of a bound drug and (2) by reducing the affinity of thetubulin for dinitroanilines by changes to the compoundbinding site.

Our previous observations that diverse mutations toa1-tubulin are sufficient for conferring resistance todinitroanilines such as oryzalin in the protozoan parasiteT. gondii might suggest that dinitroaniline-binding siteligands are not appropriate for development of antipar-asitic agents. However, in many cases, drug resistancemutations isolated under laboratory conditions are not observed in clinical settings since parasites are incapableof robust growth in natural reservoirs. For example, asubset of laboratory-selected dihydrofolate reductase(DHFR) mutations thatconfer pyrimethamine resistancein Plasmodium falciparum are either rare or not observedin the field (Hankins et al. 2001; Hastings et al. 2002;Bates et al. 2004). Moreover, studies that have exploitedgrowth competition assays to assess the fitness of T. gondii strains bearing wild-type or pyrimethamine-resistant DHFR genes have concluded that even mutant strainsthat behave similarly to wild-type parasites in vitro candisplay growth defects in vivo (Fohl and R oos 2003). Weshow here that dinitroaniline-resistant parasites havereduced fitness reflected by increased rates of replicationdefects and poor performance in growth competitionassays. These lines rapidly and spontaneously acquirecompensatory mutations that correct the fitness defectsand reduce resistance to dinitroanilines. We conclude

that drugs that selectively target parasite microtubulesremain a realistic option for the development of new antiparasitic therapies.

MATERIALS AND METHODS

Culture of Toxoplasma lines: T. gondii tachyzoites (thestandard RH strain and mutants derived from the RH strain)

were propagated in human foreskin fibroblast (HFF) cells inDMEM with 10% FBS as previously described (R oos et al.1994). Oryzalin (Riedel-deHaen, Germany) stock solutions

846 C. Ma et al.



analyzed the sequences of T. gondii a1- and b1-tubulingenes for mutations associated with suppression of defects. We identified both a1-tubulin pseudorevertantsand extragenic mutations in b1-tubulin and other(unidentified) genes. We isolated 27 independent linesfrom the F52Y line and identified mutations that localize to a1-tubulin or b1-tubulin in 22 of these lines(Figure 2 and Table 1). Two of the a1-tubulin mutationsare located in the M or H1-S2 (N) loops and sevenmutations are located in the eight-amino-acida-tubulin-specific insert. Six of the lines have mutations to theb1-tubulin gene; two of these are in the M or H1-S2 (N)loops. The G142S resistance mutation alters theGGGTGSG tubulin motif, which is associated withbinding the phosphate portion of GTP. Of the 24G142S-derived lines characterized here, 13 acquired asecondary mutation in the a1-tubulin gene (Figure 3and Table 2). One of these mutations is associated withGTP binding (S171A), two localized in the eight-amino-acid a-tubulin-specific insert, and one is in the M loop.

We also isolated 13 lines with extragenic suppressors, with 11 of these occurring in the b1-tubulin gene and 2being in other, unidentified loci. During the course of these studies we did not identify any true revertants of F52Y or G142S parasites.

Growth competition assays indicate that the derivedlines have increased fitness relative to the parentalstrains: We have exploited an established GFP-express-ing T. gondii line to assess the relative fitness of the F52Y and G142S parasite lines and the derived progeny (K imet al. 2001). Both the GFP-expressing line and the

tubulin mutant lines are derived from RH strain T.gondii . We co-infected HFF cells with an equivalent number of GFP-expressing parasites and parasites of each tubulin mutant line and exploited green fluores-cence to follow the relative rates of replication andgrowth over serial passage (Figure 4). Parasites fromlysed flasks were used to inoculate new flasks such that they would lyse the host cell monolayer in 2 days. Theremainder of each sample was analyzed by flow cytom-etry. In initial experiments, the parasites were labeled

with the DG52 (anti-SAG1) antibody to ensure that theentire parasite population was visualized for analysis(Figure 4 inset). Once we were confident that our gatinghad captured the parasite population, we omitted theDG52 labeling, with consistent results. At each timepoint, the total number of parasites and the number of GFP-labeled parasites were determined. Nonfluorescingparasites represent the fraction of the population ex-pressing the specific tubulin mutation(s). These growthcompetition assays indicate that the F52Y and G142S

lines compete poorly with wild-type GFP parasites(Figure 4). After 6 days, the G142S line is not detectableand at 8 days the F52Y line cannot be detected. In con-trast, the b1-P61A and a1-P364R lines that are derivedfrom the G142S and F52Y parasites are still present at thistime, although their declining percentage in the pop-ulation indicates that they are less fit than the GFP-RHline. As a control, we also carried out growth competitionfor wild-type RH parasites vs. the GFP-expressing para-sites. Over time, we reproducibly observed that the GFP-expressing parasites were at a disadvantage relative to the

Figure 1.—(A) The F52Y and G142S a-tubulin point mutations confer 13 and 1 mm oryzalin resistance when integrated into wild-type (sensitive) T. gondii parasites. The F52Y mutation (red) is located in the H1-S2 (N) loop (red) of a1-tubulin. The com-putationally determined dinitroaniline-binding site is below this loop (oryzalin in orange). Interactions between the H1-S2 (N)loop and the M loop of adjacent dimers coordinate lateral associations between protofilaments in the microtubule lattice. TheG142S mutation is located in the core of a1-tubulin and the G142 residue represents the initial glycine in the GGGTGSG motif (teal), which is characteristic of tubulins. This motif is part of the GTP-binding site, which is essential for correct folding of thetubulin dimer and interacts with the phosphate portion of GTP (purple). (B) (Top) Immunofluorescence images of extracellularparasites of the G142S line labeled with DG52 (red labels the parasite surface) and DAPI (blue labels the parasite DNA). (Bottom)DNA distribution only. (1) A normal-appearing parasite has a crescent shape and DNA staining of the nucleus (arrow) and api-

coplast DNA (arrowhead). (2–6) Parasite ‘‘monsters’’ result from replication defects due to improper coordination of spindlemicrotubule (nuclear division) and subpellicular microtubule (cytokinesis) functions. Although the extracellular parasites in3 and 4 are similar to intracellular replicating stages (see diagram in Ma et al. 2007), parasites cannot complete division outsideof host cells and their abnormal shape prevents invasion of new host cells. In addition, 2, 5, and 6 illustrate parasites with diffuse(2), improperly segregated (three nuclei) (5), and nonsegregated (6) nuclear staining (compare to 1).

848 C. Ma et al.



wild-type RH T. gondii . Therefore, the growth competi-tion using GFP-RH parasites underestimates the fitnessdefects associated with the dinitroaniline resistance mu-tations and the associated suppressed lines. We analyzedall of the F52Y- and G142S-derived lines after 2 weeks inserial passage with GFP-RH parasites to assess relativefitness relationships in competition with wild-type para-sites (Figures 5 and 6). In most cases, lines with com-pensatory mutations do not fully recover robust growth.The most effective F52Y-derived lines with respect tofitness are point mutations at T51A (thea-tubulin H1-S2loop), P364A (the a-tubulin insert), and G34S (theb-tubulin H1-S2 loop), which were still all ,50% of the

culture after 2 weeks (Figure 5 and Table 3). The most effective G142S-derived lines are S171A (an a-tubulinGTP-binding site residue), L230V (located in helix 7 of a-tubulin), and V363A (in the a-tubulin insert). TheL230V line is present in50% of the culture after 2 weeks(Figure 6 and Table 3). However, since the GFP-RHparasites have a fitness defect relative to unlabeled RHstrain wild-type parasites, it is still likely that the L230V line would compete unfavorably with wild-type T. gondii .

The derived lines have diminished oryzalin resistancerelative to the parental strains: The F52Y and G142S

parasite lines display 13 and 1.0 mm oryzalin resistance,respectively. Lines derived from these parental strains dis-play reduced oryzalin resistance (Figures 5 and 6 andTable 3). With respect to the F52Y-derived lines, the P364R line retains the highest level of oryzalin resistance (7 mm)and competes relatively well with the GFP-RH parasites.Since the G142S line parasites have 1 mm oryzalin resis-tance, the range of resistance observed in the derivedlines is substantially lower. A number of the lines (theQ256H, S287P, and V363A mutations in a-tubulin andthe P358R and A393V mutations in b-tubulin) do not have greater resistance to oryzalin than that observedfor wild-type parasites (,0.5 mm by morphological

criteria). Lines with secondary mutations at S171A andL230V retain the highest levels of oryzalin resistance inthe context of the greatest degree of overall fitness inthe competition assay. All 46 lines bearing compensa-tory mutations are associated with diminished oryzalinresistance.

DISCUSSION

Previous genetic studies have identified secondary mutations that correct tubulin-associated defects. In

Figure 2.—Location of compensatory mutationsobtained from the F52Y line mapped on a modelof the Toxoplasma tubulindimer. The a1-subunit is

white and the b1-subunit is blue. (A) Most mutationsfall between the M and H1-S2 loops (red) facing the

inner lumen of the micro-tubule. The F52Y mutationis located in the H1-S2 loopof a1-tubulin (red) and sec-ondary mutations in a1-tu-bulin (black text) occur at T51A, V181I, A273V,K280N, N293D, A295G,S300T, M313T, P360A,T361P, P364A, P364R,G366R, D367V, L368F, andR373C (orange/yellow).Mutations in yellow localizeto the a-tubulin-specific in-sert. Extragenic suppres-

sors in the b1-tubulinsubunit (white text) occurat G34S, G82D, P261S,G269V, L273V, andH396Q. (B) The b1-tubu-lin mutation P261S is theonly mutation that facesthe outer surface of the di-mer within the microtu-

bule lattice, while the b-subunit mutation H396Q is located at the dimer–dimer interface within the protofilament. (C) Themutations T361P, P364A, P364R, G366R, D367V, and L368F occur in the a-tubulin-specific insert (yellow) and the mutationsP360A and R373C are adjacent to this insert (orange).

Secondary Mutations in Tubulin 849



studies of colchicine resistance, intragenic mutationscorrect defects associated with the D45Y mutation to b-tubulin, which confers colchicine resistance in CHOcells (W ang et al. 2004). This substitution is located inthe H1-S2 loop and, since it increases microtubulestability, cells that express it are hypersensitive to taxol.Lines that suppress the taxol hypersensitivity and asso-ciated temperature-sensitive defects have point muta-tions at V60A (the H1-S2 loop) or Q292H (near the Mloop) of b-tubulin that alter microtubule stability. When

wild-type CHO cells express a tagged b-tubulin con-struct bearing the D45Y mutation, the microtubulearray is strikingly denser relative to the array observed

with expression of wild-type tubulin alone. Inclusion of secondary mutations in theb-tubulin constructs reduces

the microtubule array, making it similar to that observed with expression of wild-type tubulin. When b-tubulinconstructs with theV60A and Q292H secondary mutationsalone are expressed, the microtubule arrays are dramat-ically reduced relative to those observed with wild-typetubulin expression. These observations are strikingly similar to our results described here. The F52Y mutationis predicted to increase microtubule stability to conferdinitroaniline resistance, but also causes increased rates of replication defects. Secondary mutations are located inregions such as the H1-S2 loop, the M loop, and the a-

tubulin insert domain, which are predicted to decreasemicrotubule stability. We attemptedto express GFP-tagged

vertebrate tubulin with analogous mutations in COS-7cells to visualize differential assembly of these tubulins.Unfortunately, expression appeared to be somewhat toxicand the patterns were too variable to be used to assess therelative stability of tubulins with these mutations ( J. Tranand N. Morrissette, data not shown).

Many of the secondary mutations are identical orquite similar to previously identified mutations that decrease microtubule stability in Caenorhabditis elegans and Arabidopsis thaliana. C. elegans sensory neuron func-tion requires unusual 15-protofilament microtubules(Chalfie and Thomson 1979, 1982; Chalfie 1982;Chalfie et al. 1986; Savage et al. 1989, 1994). These mi-

crotubules are built from dimers encoded by distinct a- (mec-12 1) and b- (mec-7 1) tubulin genes (Savage et al.1989, 1994; Gu et al. 1996; Fukushige et al. 1999). A number of mec-7 and mec-12 alleles have been identifiedin screens for sensory neuron defects. In addition,mutations in Arabidopsis that convert linear growth of plant parts (such as roots and stems) to a twisted phe-notype have altered cortical microtubule arrays. Thistwisting morphology can be phenocopied by treat-ment of wild-type plants with the microtubule inhibitorpropyzamide. Many of the twisting mutants have muta-

TABLE 1

F52Y secondary mutations

Mutant Codon change Location Other nearby mutations

T51A ACC to GCC AT H1-S2 loop F52I, F52L, F52Y: Tg dinitroanilineR (Morrissette et al. 2004) V181I GTT to ATT AT b-5 to a-5 loop S180P, A180T: Attwisting (monomerinterface)(Ishida et al. 2007a) A273V GCG to GTG AT M loop I275T: Tg dinitroanilineR (Morrissette et al. 2004)K280N AAG to AAC AT M loop K279A-K281A: Sc benomylss (R ichards et al. 2000), A281T:

At twisting (Ishida et al. 2007a)N293D AAC to GAC AT a-helix 9 A295V: Tg dinitroanilineR (Morrissette et al. 2004) A295G GCT to GGT AT a-helix 9 A295V: Tg dinitroanilineR (Morrissette et al. 2004)S300T AGC to ACC AT a-9 to b-8 loop S300T: Ant. fish coldR (Detrich et al. 2000), M301T:

Tg dinitroanilineR (Morrissette et al. 2004)M313T ATG to ACG AT b-sheet 8 D310A-K312A: Sc benomylss (R ichards et al. 2000)P360A CCC to GCC AT near insert G354E: Ce mec-12 (Fukushige et al. 1999)T361P ACT to CCT AT insert P364A CCT to GCT AT insert P364R CCT to CGT AT insert Also suppresses G142S G366R GGT to CGT AT insert D367V GAC to GTC AT insert L368F TTG to TTC AT insert R373C CGC to TGC AT b-sheet 10 D373A-R374A: Sc benomylss (R ichards et al. 2000)G34S GGT to AGT BT H1-S2 loop G34S: Ce mec-7 (Savage et al. 1989)G82D GGC to GAC BT a-2 to b -3 loop F85L: Tg suppressor of G142SP261S CCT to TCT BT a-8 to b -7 loop F261V: Hs paclitaxelR (Monzo et al. 1999)G269V GGG to GTG BT b-sheet 7 G269D: Ce mec-7 (Savage et al. 1989)L273V CTC to GTC BT M loop T274I: Hs epothilone/taxaneR (Giannakakou et al. 2000)H396Q CAC to CAG BT a-helix 11 A393T: Ce mec-7 (Savage et al. 1989); A394T:

At twisting (Ishida et al. 2007a)

At, Arabidopsis thaliana ; Ant. fish, Antarctic fish; Ce, Caenorhabditis elegans ; Hs, Homo sapiens ; Sc, Saccharomyces cerevisiae ; Tg, Toxo- plasma gondii ; R , resistant; ss, supersensitive.

850 C. Ma et al.



tions to a- and b-tubulin that decrease microtubulestability and increase plant sensitivity to microtubule-disrupting drugs (Thitamadee et al. 2002; A be et al.2004; Nakajima et al. 2004, 2006; Shoji et al. 2004; A beand Hashimoto 2005; Ishida and Hashimoto 2007;Ishida et al. 2007a,b). The b-tubulin mutation G34S (aF52Y suppressor) was previously identified as a mild,

recessive mec-7 allele (Savage et al. 1989). Other mec-7 alleles (P61L/S, G269D, R318G, and A393T) alterequivalent residues to different substitutions in T. gondii b-tubulin (the F52Y suppressor G269V and the G142Ssuppressors P61A, R318C, and A393V). The G142S sec-ondary mutation I355E in a-tubulin is adjacent to thelocation of the mec-12 allele G354E. Moreover, the A393

Figure 3.—Location of compensatory mutations obtained from the G142S line mapped on a model of the Toxoplasma tubulindimer (the a1-subunit is white and the b1-subunit is blue). The G142S mutation (yellow) is located in the GTP-binding domain of a-tubulin. (A) Most mutations (orange/yellow) are on the surface of the dimer that faces the inner lumen of the microtubule. Sec-ondary mutations in a1-tubulin (black text) occur at I93V, G131R, S171A, I209V, L230V, Q256H, S287P, P348L, I355E, V363A, P364R,

V371G, and F418I. Suppressors in the b1-tubulin subunit (white text) occur at residues A18P, P61A, F85L, K122E, H264Q, Q291E,R318C, N337T, P358R, M363V, and A393V. G142S a1-tubulin mutations at V363A and P364R occur in the a-tubulin-specific insert (yellow). (B) Thea1-tubulin mutation at Q256 and theb1-tubulin mutation at H264 are the only mutations that face the outer surfaceof the dimer within the microtubule lattice. The b-subunit mutation A393V is located at the dimer–dimer interface within the protofila-ment. (C) The primary resistance mutation at G142 is located within the tubulin motif (teal) of the a-tubulin GTP-binding site.The GTP moiety is purple and residues contributing to GTP binding outside of the GGGTGS tubulin motif are red. The primary mutation G142S is located in the binding site and interacts with the a-phosphate. The S171A mutation also locates to the bindingsite; S171 interacts with the ribose portion of GTP (Gigant et al. 2000; Lowe et al. 2001).




residue is also identified as a threonine substitutioncausing twisting in Arabidopsis b-tubulin (A394T). TheF52Y compensatory mutations V181I and K280N in a-tubulin and H396Q in b-tubulin and the G142S muta-tion P348L (a-tubulin) are near the twisting mutationsS180P, A180T, A281T, and T349I (a-tubulin) and A394T(b-tubulin). Finally, the G142S suppressor Q291E is ad-

jacent to a second glutamine at 292 that is a mec-7 allele

(Q292P). This residue alsoinfluences microtubule stability in the context of epothilone resistance (Q292G) and sup-presses hyperstabilized microtubules (Q292H) in D45Y colchicine-resistant cells (He et al. 2001; W ang et al. 2004).

One of the novel findings presented in this work is theidentification of a set of seven mutations that localize tothe a-tubulin-specific insert (TVVPGGDL). The com-pensatory mutations in the F52Y line that localize to theinsert are T361P, P364A, P364R, G366R, D367V, andL368F and in the G142S line mutations are V363A andP364R. To our knowledge, the insert substitutions rep-

resent the first description of mutations located in thishighly conserved domain. We hypothesize that thesesubstitutions impair the ability of the insert to promoteM-loop lateral contacts with the adjacent protofilament.Indeed, the D367V mutation eliminates a salt bridge that occurs with R229 (Ma et al. 2007). Strikingly, the P364R mutation functions as a compensatory mutation in bothlines, albeit more successfully in the F52Y line (compare

competition results in Figures 5 and 6 and Table 3). Although the G142S/P364R line does not competeeffectively enough with wild-type parasites to remain inthe culture at 14 days, it was isolated as a line withimproved growth over the G142S line and at some subtlelevel is likely to improve fitness of theparental line. This isalso likely to be the explanation for the F52Y suppressor

A295G in competition at 14 days (Table 3).The studies presented here show a correlation be-

tween acquisition of increased fitness and the appear-ance of mutations in the a1- or b1-tubulin genes. While

TABLE 2

G142S secondary mutations

Mutant Codon change Location Other nearby mutations

I93V ATC to GTC AT b-sheet 3 H89A-E91A: Sc benomylss (R ichards et al. 2000)G131R GGT to CGT AT a-3 to b -4 loop D128A-D131A: Sc colds, benomylss (R ichards et al. 2000)S171A TCG to GCG AT b-5 to a -5 loop K167A-E169A: Sc benomylss (R ichards et al. 2000)I209V ATC to GTC AT a-helix 6 A208F: At lefty pseudorevertant (Thitamadee et al. 2002)

L230V CTG to GTG AT a-helix 7 I231T: Tg dinitroanilineR

(Morrissette et al. 2004)Q256H CAG to CAC AT a-helix 8 E255A: Sc dominant lethal (R ichards et al. 2000)S287P TCT to CCT AT M-loop S287P: Antarctic fish coldR (Detrich et al. 2000)P348L CCC to CTC AT a-10 to b-9 loop T349I: At twisting (Ishida et al. 2007a)I355E GAA to ATC AT b-sheet 9 G354E: Ce mec-12 (Fukushige et al. 1999); C356Y:

Sp lethalts (R adcliffe et al. 1998) V363A GTT to GCT AT insert P364R CCT to CGT AT insert Also suppresses F52Y

V371G GTC to GGC AT b-9 to b -10 loop D373A-R374A: Sc benomylss (R ichards et al. 2000)F418I TTC to ATC AT a-helix 12 E415K: Ce mec-12 (Fukushige et al. 1999); E416A-E418A:

Sc slow growth, benomylss (R ichards et al. 2000) A18P GCC to CCC BT a-helix 1 A19K Sc required for taxol binding (Gupta et al. 2003)P61A CCG to GCG BT H1-S2 loop V60A: CHO D45Y supp. (W ang et al. 2004); P61L, P61S:

Ce mec-7 (Savage et al. 1989)F85L TTC to TTG BT a-2 to b-2 loop G82D : Tg compensatory for F52Y K122E AAG to GAG BT a-helix 3 R121A-R122A: Sc benomylss (R eijo et al. 1994)H264Q CAC to CAG BT a-8 to b-7 loop L263Q: Dm E194K suppr. (Fackenthal et al. 1995); G269D:

Ce mec-7 (Savage et al. 1989)Q291E CAG to GAG BT a-helix 9 Q292G: Hs epothiloneR (He et al. 2001); Q292P: Ce mec-7

(Savage et al. 1989);Q292H: D45Y supp. (W ang et al. 2004)

R318C CGT to TGT BT b-sheet 8 F317I, R318G: Ce mec-7 (Savage et al. 1989); R318A-K320A:Sc benomylR (R eijo et al. 1994)

N337T AAC to ACC BT a-10 to b -9 loop K336A-D339A: Sc recessive lethal (R eijo et al. 1994)P358R CCG to CGG BT b-9 to b-10 loop C354S, C354A: Sc benomylR , cold stable (R eijo et al. 1994)M363V ATG to GTG BT b-9 to b-10 loop A364T: Hs paclitaxelR (Giannakakou et al. 2000)

A393V GCT to GTT BT a-helix 11 A393T: Ce mec-7 (Savage et al. 1989); A394T: At twisting (Ishida et al. 2007a)

At, A. thaliana ; Ce, C. elegans ; CHO, Chinese hamster ovary cells; Dm, Drosophila melanogaster ; Hs, H. sapiens ; Sc, S. cerevisiae ; Sp,Schizzosacharomyces pombe ; R , resistant; s, sensitive; ss, supersensitive; ts, temperature sensitive.

852 C. Ma et al.



the improved fitness observed in parasite lines derivedfrom the F52Y and G142S parental strains is likely due tosecondary tubulin mutations, other (nontubulin) mu-tations may also contribute to the observed phenotypesof decreased resistance and enhanced growth. Indeed,five of the lines derived from the F52Y suppressor screenand two lines derived from the G142S suppressor screenhave improved growth but do not have altered a1-, a3-,

b1-, or b3-tubulin genes, suggesting that nontubulincompensatory mutations do arise. Moreover, although

we have created allelic replacements for two a-tubulinpseudorevertants in wild-type parasites (Ma et al. 2007),

we cannot create allelic replacements for the b-tubulinsuppressors since we cannot select for these in the

absence of dinitroaniline selection. When we analyzedthe allelic replacements for the a-tubulin pseudorever-tants (F52Y/A273V and F52Y/D367V) in the competitionassay, they performed differently from the equivalent lines with these spontaneous mutations. Surprisingly, inboth cases the transgene versions of the lines hadgreater fitness levels than the spontaneous mutant lines(data not shown). The suppressed lines described here

were isolated after 4–6 weeks of growth in media without oryzalin. This represents the shortest time in which wecould observe parasites with new mutations. In contrast,it takes at least 6 weeks to isolate transgene-bearinglines. We predict that further growth of lines withimpaired fitness in the absence of oryzalin selection

Figure 4.—To assess rela-tive fitness of the F52Y andG142S lines as well as thestrains derived from theselines, equivalent numbersof parasites from a specificmutant line and wild-typeRH strain parasites express-ing GFP were inoculated in-to culture and the relative

percentage of each popula-tion was quantified overtime during serial passageusing flow cytometry. Therelative parasite concentra-tions were quantified at the time of complete host cell lysis when the lysate

was passed into a new flaskof host cells containing par-

asites. The red line indicates the behavior of G142S parasites in competition and the orange line is the G142S suppressor lineb-P61A (located in the b-tubulin H1-S2 loop). The green line follows a competition assay containing F52Y and the teal line rep-resents behavior of the F52Y secondary mutation a-P364R (located in the a-tubulin-specific insert). The G142S parasites are out-competed by GFP-RH parasites by day 6 whereas theb-P61A suppressor line persists until day 24. The F52Y parasites are eliminatedat day 12 but, in the presence of the a-P364R secondary mutation, they were still present, albeit at reduced levels, when the com-

petition was terminated at 30 days. The purple line traces the control competition between wild-type (untransfected RH strainparasites) and the GFP-transfected ‘‘wild-type’’ RH strain line. Unlabeled parasites have a growth advantage over GFP-transfectedparasites. (Inset) A representative FACS dot plot of DG52-labeled (red) parasites showing relative numbers of GFP-expressing wild-type Toxoplasma and a mutant line after a single passage.

Figure 5.—Replicationfitness and oryzalin resis-tance in F52Y parasitesand the derived lines. Solid

bars: the percentage of mu-tant parasites relative toGFP-RH parasites afterseven passages (14 days).The assays were carriedout in triplicate and errorbars indicate standard errorof the mean. Shaded bars:oryzalin resistance levelsobserved for F52Y parasitesand the derived lines.




would select for additional mutations that collectively contribute to increased fitness.

Previous studies have established that there is atradeoff between carrying genes that confer drug re-

sistance and the fitness cost of these altered alleles,particularly in the absence of drug selection. Recent field studies from Malawi indicate that P. falciparum parasites in this region lost resistance to chloroquine in,10 years after sulfadoxine–pyrimethamine treatment

replaced chloroquine treatment for individuals suffer-ing from malaria (Laufer et al. 2006; V ogel 2006).These field observations, as well as the controlledlaboratory studies of DHFR mutations in P. falciparum

and T. gondii described in the Introduction, indicatethat the existence of mutant lines that express resistant forms of target proteins does not rule out the de-

velopment and use of agents that inhibit these proteins. With respect to the studies presented here, we believe

Figure 6.—Replicationfitness and oryzalin resis-tance in G142S parasitesand the 24 derived lines.Solid bars: the percentageof mutant parasites relativeto GFP-RH parasites afterseven passages (14 days).The assays were carriedout in triplicate and errorbars indicate standard errorof the mean. Shaded bars:oryzalin resistance levelsobserved in G142S parasitesand the derived lines.

TABLE 3

Resistance and fitness levels

Mutation% decreaseresistance

% decreasepopulation

(day 14) Mutation% decreaseresistance

% decreasepopulation

(day 14)

F52Y-parental 0 100 G142S-parental 0 100F52Y-aT51A 31 43 G142S-aI93V 75 89F52Y-a V181I 54 60 G142S-aG131R 75 97F52Y-a A273V 73 86 G142S-aS171A 50 24F52Y-aK280N 88 87 G142S-aI209V 75 89F52Y-aN293D 62 84 G142S-aL230V 50 0F52Y-a A295G 46 100 G142S-aQ256H 100 87F52Y-aS300T 46 80 G142S-aS287P 100 96F52Y-aM313T 96 86 G142S-aP348L 75 84F52Y-aP360A 46 92 G142S-aI355E 75 85F52Y-aT361P 62 80 G142S-a V363A 100 25F52Y-aP364A 46 30 G142S-aP364R 50 100F52Y-aP364R 23 69 G142S-a V371G 75 93

F52Y-aG366R 77 78 G142S-aF418I 50 84F52Y-aD367V 73 92 G142S-b A18P 75 90F52Y-aL368F 62 92 G142S-bP61A 50 74F52Y-aR373C 85 94 G142S-bF85L 50 76F52Y-bG34S 73 14 G142S-bK122E 75 94F52Y-bG82D 62 75 G142S-bH264Q 75 88F52Y-bP261S 46 91 G142S-bQ291E 75 95F52Y-bG269V 85 88 G142S-bR318C 75 96F52Y-bL273V 81 95 G142S-bN337T 75 96F52Y-bH296Q 62 91 G142S-bP358R 100 81

G142S-bM363V 50 86G142S-b A393V 100 85

854 C. Ma et al.



that although dinitroanilines or other microtubule-disrupting compounds do not yet exist in an appropri-ate form for treatment of parasite infections, selectively targeting protozoan tubulin remains a viable strategy foreliminating parasite infections.

We thank Dan Sackett for commenting on this manuscript, DavidSibley and Michael Grigg for helpful conversations on competitionassays, and Sheryl Tsai for bringing clarity to PyMOL commands. We

thank Candice Kwark and Brian Luk, high school student participants

in the American Cancer Society summer research program at theUniversity of California at Irvine(UCI),who helpedselectand analyze

the lines described here. Research presented in this article wassupported by National Institutes of Health grants AI055790 and

AI067981 to N.S.M. L.G. and D.W. are undergraduate students inthe campus-wide honors program at UCI and L.G. was supported by a

UCI summer undergraduate research program fellowship.

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Secondary Mutations Correct Fitness Defects in Toxo With Dinitroaniline Resistance Mutations

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