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Reassessment of the Role of Aromatic Amino Acid Hydroxylases and
the Effect of Infection by Toxoplasma gondii on Host Dopamine
Zi T. Wang,
a
Steve Harmon,
b
Karen L. O’Malley,
b
L. David Sibley
a
Department of Molecular Microbiology
a
and Department of Anatomy and Neurobiology,
b
School of Medicine, Washington University in St. Louis, St. Louis, Missouri, USA
Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral
manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid
hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene,
with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the
two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm
previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was overex-
pressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or re-
gional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings sug-
gest alternative roles for the AHH enzymes in T. gondii, since AAH1 is essential for growth in nondopaminergic cells.
The protozoan parasite Toxoplasma gondii is an obligate intra-
cellular parasite that is capable of infecting most warm-
blooded animals. It is a member of the phylum Apicomplexa,
which also contains Plasmodium falciparum, the causative agent of
malaria. Toxoplasma gondii is one of the most widely distributed
parasites in the world, both in geographic location and in the
diversity of its host range (1). Its only definitive hosts are members
of the genus Felis. Exclusively within enterocytes of the gut, it
undergoes a sexual reproductive cycle to form environmentally
resistant and infectious oocysts that are shed in cat feces (2). In all
other hosts, Toxoplasma gondii infection begins as a fast-growing
lytic stage called the tachyzoite. Innate and adaptive immune re-
sponses restrict the growth of tachyzoites, which can respond by
differentiating into bradyzoites, a semidormant stage that exists as
quiescent intracellular cysts in brain and muscle tissue. This
chronic infection can persist for the lifetime of the host (3). Infec-
tions spread among incidental hosts through carnivorism, vertical
transmission, and ingestion of T. gondii oocysts (4).
Rodents that become infected by T. gondii exhibit an unusual
behavioral response: they lose their instinctive aversion to the
odor of cats and instead become mildly attracted to the scent (5–
13). This behavioral manipulation appears specific to the cat (7,
8), and it has been speculated that this facilitates transmission (9).
The exact mechanism of this behavioral manipulation is un-
known, but parasite stimulation of host dopamine pathways in the
brain has been suggested as a cause (14–16). It was observed that
infection of mice by T. gondii caused a 14% increase in whole-
brain dopamine levels upon establishment of chronic infection
(17). Additionally, dopamine receptor antagonist drugs used for
the treatment of schizophrenia block cat attraction in infected
rodents (18,19). T. gondii infection also was described to increase
dopamine content and dopamine release in the dopaminergic cell
line PC12 in vitro (15).
The mechanism by which infection might alter dopamine is
unknown, but it has been suggested that parasite metabolism con-
tributes to elevated dopamine levels (20). The T. gondii genome
contains two genes that encode aromatic amino acid hydroxylases
(AAAH), which carry out the rate-limiting step of dopamine syn-
thesis in metazoans by converting tyrosine into the dopamine pre-
cursor 3,4 dihydroxyphenylalanine (L-Dopa) (20). The two nearly
identical genes AAH1 and AAH2 contain putative signal peptides
targeting them for secretion, and both appear to be functional
tetrahydrobioptern-dependent aromatic amino acid hydroxylases
in vitro (20). AAH1 was reported to be constitutively expressed,
while the expression of AAH2 was reported to increase in the
dormant bradyzoite stage (20). Because of this pattern, AAH2 was
suggested to be the prime candidate effector of the parasite’s ma-
nipulation of host dopamine (20).
We sought to test the hypothesis that the aromatic amino acid
hydroxylases AAH1 and/or AAH2 were responsible for causing
alterations in dopamine metabolism in the host. We successfully
knocked out and complemented the AAH2 gene but, unexpect-
edly, could not observe a parasite effect on host dopamine levels
either in vitro with PC12 cells or in vivo with mouse infection.
Further, we observed that expression of both AAH1 and AAH2
was negligible in tachyzoites, and while they both showed in-
creased expression in bradyzoite stages, the relative expression
level still was very low. Collectively, our findings indicate that
AAH enzymes in T. gondii do not cause global alterations of host
dopamine; rather, they may participate in alternative pathways.
Received 11 August 2014 Returned for modification 19 September 2014
Accepted 22 December 2014
Accepted manuscript posted online 29 December 2014
Citation Wang ZT, Harmon S, O’Malley KL, Sibley LD. 2015. Reassessment of the
role of aromatic amino acid hydroxylases and the effect of infection by
Toxoplasma gondii on host dopamine. Infect Immun 83:1039 –1047.
doi:10.1128/IAI.02465-14.
Editor: J. H. Adams
Address correspondence to L. David Sibley, sibley@wustl.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/IAI.02465-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/IAI.02465-14
March 2015 Volume 83 Number 3 iai.asm.org 1039Infection and Immunity
MATERIALS AND METHODS
Parasite strains. Parasites were propagated by serial passage in human
foreskin fibroblast (HFF) cells grown in Dulbecco’s modified Eagle me-
dium (DMEM; Life Technologies, Carlsbad, CA) containing 10% fetal
bovine serum (FBS) (HyClone, Logan, UT), 10 mM HEPES, pH 7.4, 1
mM glutamine, 10 g/ml gentamicin, under 5% CO
2
at 37°C (D10 me-
dium). The Pru⌬ku80⌬hxg strain (type II) was obtained from John
Boothroyd (21). The ME49 strain (type II) (ATCC 50611; American Type
Culture Collection, Manassas, VA) originally was isolated from sheep
diaphragm (22). The C56 strain (type III) originally was isolated from a
chicken (23). PC12 cells (ATCC CRL-1721) were obtained from the
ATCC and cultured in RPMI 1640 medium (ATCC) supplemented with
10% heat-inactivated horse serum (Life Technologies) and 5% FBS (PC12
medium). SH-SY5Y cells (ATCC CRL-2266), confirmed to be myco-
plasma negative, also were obtained from the ATCC and cultured in a 1:1
mixture of ATCC Eagle’s minimum essential medium and F12 medium
supplemented with 10% FBS (SH-SY5Y medium).
Generation of deletion plasmids. To generate a deletion plasmid tar-
geting the AAH2 locus, regions 1.1 kb upstream and 1.8 kb downstream
from the AAH2 locus were PCR amplified from ME49 genomic DNA
using primers listed in Data Set S1 in the supplemental material and were
cloned into pDONR-p4p1r and pDONR-p2rp3 vectors, respectively (In-
vitrogen). Using the Gateway 3-fragment system, the upstream and
downstream flanks and a central HXGPRT expression cassette cloned into
pDONR-p1p2 were assembled into pDEST-R4R3 to create the plasmid
pHXG-⌬aah2.
Generation of AAH2 cleanup and complementation plasmids. To
generate a plasmid to remove HXGPRT from the AAH2 locus, the 1.1-kb
upstream and 1.8-kb downstream regions were PCR amplified from
ME49 genomic DNA with primers that added a 20-bp overlap. The pieces
then were fused by PCR and cloned into pDONR-p4p1r to create plasmid
p⌬aah2. To complement AAH2, the cDNA of AAH2 was amplified from
a Pru⌬ku80⌬hxg cDNA sample, and the 1.1-kb upstream region with an
⬃20-bp overlap into the 5=end of the AAH2 coding sequence and the
1.8-kb downstream region with an ⬃20-bp overlap into the 3=end of the
AAH2 coding sequence were amplified from Pru⌬ku80⌬hxg genomic
DNA (gDNA). The three pieces were fused by PCR and cloned into
pDONR-p4p1r to create the plasmid p::AAH2.
Generation of AAH1/AAH2 tagging and AAH2 BAG1 overexpres-
sion plasmids. To generate plasmids to tag AAH1/AAH2, primers were
designed to amplify 1.5 kb of the 3=end of the gene with the addition of a
Ty epitope (24) before the stop codon and to amplify 600 bp of the 3=UTR
of the gene with the same addition. These two pieces were fused by PCR
and cloned into pDONR-p4p1r. A 1.5-kb region 1.5 kb downstream from
the stop codon was cloned into pDONR-p2rp3, and the pieces were com-
bined with HXGPRT-pDONR-p1p2 to create the tagging plasmids
pAAH1::Ty and pAAH2Ty. To generate a plasmid to drive AAH2 with the
BAG1 promoter, primers were designed to amplify 900 bp of the 5=UTR
of BAG1 with an ⬃20-bp overlap into the AAH2 coding sequence, the
cDNA of AAH2 with a Ty epitope insertion, and 1.5 kb of the AAH2 3=
UTR with an ⬃20-bp overlap into the Ty tag and AAH2 coding sequence.
These three fragments were fused with end-overlap PCR and cloned into
p2rp3. This fusion was combined with a 900-bp region upstream of AAH2
in pDONR-p4p1r and the HXGPRT cassette in pDONR-p1p2 into
pDEST-R4R3 to create the plasmid pBAG1::AAH2Ty.
Generation of parasite transgenic lines. To generate transgenic lines,
Pru⌬ku80⌬hxg parasites were transfected with DNA PCR amplified from
the critical regions of recombinant plasmids. Following the transfection of
amplified DNA (5 to 15 g), parasites were allowed to recover on HFF
monolayers for 24 h before positive selection for the HXGPRT cassette
with 25 g/ml mycophenolic acid (Sigma-Aldrich) supplemented with 50
g/ml xanthine (Sigma-Aldrich) (25) or negative selection against HXG-
PRT with 340 g/ml 6-thioxanthine (26). Resistant parasites were cloned
by limiting dilution into 96-well plates containing HFF monolayers and
screened by PCR.
In vitro differentiation. Parasites were differentiated using 48 h of
treatment with sodium bicarbonate-free RPMI 1640 medium containing
1% FBS and 50 mM HEPES, pH 8.1, at 37°C without CO
2
(27). Parasites
in PC12 cells were differentiated using 48 h of treatment with PC12 me-
dium supplemented with 50 mM HEPES, pH 8.1. Parasites in SH-SY5Y
cells were differentiated using 48 h of treatment with SH-SY5Y medium
supplemented with 50 mM HEPES, pH 8.1.
Infection of mice. Parasites were harvested by syringe lysis of infected
HFF cultures at 24 h postinfection using a 22-gauge needle. They then
were purified by passage through a 3.0-m polycarbonate filter (GE Wa-
ter and Process Technologies, Beaumont, TX), counted by a hemocytom-
eter, and diluted in fresh D10 medium. Eight-week-old female CD1 mice
(Charles River Laboratories, Wilmington, MA) were injected intraperito-
neally (i.p.) in a volume of 200 l containing 10
3
,2⫻10
2
,or10
4
parasites
and monitored daily. One month postinfection, mice were euthanized by
CO
2
asphyxiation followed by cervical dislocation serially across treat-
ment groups to maximize consistency, and brains were removed for anal-
ysis. Animal studies were approved by the Institutional Animal Studies
Committee (School of Medicine, Washington University in St. Louis).
Plaque assay. Parasites were syringe lysed from infected HFF mono-
layers, purified with a 3.0 m polycarbonate filter, and counted by hemo-
cytometer. Parasites were serially diluted, and 10
3
parasites (100 l) were
seeded onto confluent HFF monolayers in 6-well plates and grown for 11
days at 37°C, 5% CO
2
. HFFs were fixed with 70% ethanol for 10 min and
then incubated with 0.5% crystal violet in distilled H
2
O for 10 min,
washed, and scanned with an EPSON Perfection V500 photo scanner.
Immunofluorescence microscopy. Infected monolayers cultured on
glass coverslips were fixed and permeabilized with 4% formaldehyde con-
taining 0.5% Triton X-100 for 15 min. Slips were stained with primary
antibody that reacts to the Ty tag (24) and fluorescein isothiocyanate
(FITC)-conjugated Dolichos biflorus lectin (DBL) (Sigma, St. Louis, MO)
diluted at 1:1,000 in phosphate-buffered saline (PBS)–5% FBS containing
5% NGS (normal goat serum; Sigma). Primary antibodies were visualized
using goat anti-mouse or goat anti-rabbit IgG conjugated to Alexa-594
(Invitrogen Molecular Probes, Carlsbad, CA). Slides were analyzed with a
Zeiss AxioSkop wide-field epifluorescence microscope equipped with an
AxioCam charge-coupled-device (CCD) camera, and images were cap-
tured using AxioVision v3.1 (Carl Zeiss Inc., Thornwood, NY). Using
Photoshop CS3 (Adobe, San Jose, CA), images were cropped, color levels
were adjusted, and then images were assembled.
qPCR. Tachyzoite and bradyzoite parasites were harvested into calci-
um-free PBS by syringe lysis of infected HFF cultures and filtration with a
3.0 m polycarbonate filter. RNA was harvested using a Qiagen RNeasy
kit (Qiagen, Valencia, CA). cDNAs were prepared from total RNA (1.0
g) using 50 M oligo(dT) (20) and Superscript III reverse transcriptase
(RT; Invitrogen) according to the manufacturer’s protocols. PCR primers
for reference and stage-specific genes were described previously (27), and
primers for AAH1 and AAH2 were designed using Primer Express soft-
ware, version 1.0 (Applied Biosystems, Forster City, CA, USA). Real-time
quantitative PCR (qPCR) was performed using a SmartCycler (Cepheid,
Sunnyvale, CA) in a reaction volume of 25 l containing SYBR Advantage
qPCR premix (Invitrogen), 400 nM each primer, and 1 l of cDNA. The
reaction conditions were 50°C for 2 min, 95°C for 10 min, and then 45
cycles of 95°C for 15 s and 62°C for 30 s. Data analysis was conducted using
SmartCycler (Cepheid) software. Relative gene expression levels were cal-
culated as fold change using the threshold cycle (C
T
) formula 2
⫺⌬⌬CT
,
where ⌬C
T
⫽C
T
(actin) ⫺C
T
(target gene) and ⌬⌬C
T
⫽⌬C
T
(tachyzoite-stage RNAs) ⫺⌬C
T
(bradyzoite-stage RNAs) (28). The
housekeeping gene encoding actin (ACT1) was used as a reference
control.
Western blotting. Western blots were done as previously described
(29), with the following antibodies: rabbit ␣GRA2 and mouse monoclo-
nal antibody (MAb) BB2. Primary antibodies were detected with fluores-
cently conjugated IRDye secondary antibodies (LI-COR, Lincoln, NE)
and visualized on an Odyssey infrared imaging system (LI-COR).
Wang et al.
1040 iai.asm.org March 2015 Volume 83 Number 3Infection and Immunity
ELISA. High-binding enzyme-linked immunosorbent assay (ELISA)
plates (Corning, Corning, NY) seeded with parasite antigen (sonicated
RH strain lysate in PBS at 10
5
parasites per well) were incubated with
serum from chronically infected mice (collected 1 month postinfection)
diluted 1:1,000 in PBS-Tween containing 0.1% bovine serum albumin
(BSA) for1hatroom temperature. Antibodies were detected with a
1:5,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-
mouse IgG (Jackson Laboratories, Sacramento, CA) for1hatroom tem-
perature. HRP activity was captured using BD OptEIA substrate reagent A
and substrate reagent B (BD Biosciences, Franklin Lakes, NJ). Absorbance
at 450 nm was read using an EL-800 universal microplate reader (Bio-Tek
Instruments Inc., Winooski, VT).
Cyst count. Chronically infected animals were euthanized and the
brains removed. Brains were homogenized and stained with DBL as pre-
viously described (27). Dilutions of stained homogenates were examined
using a Zeiss wide-field epifluorescence microscope. Three separate ali-
quots were counted per brain sample, and total brain cyst load was deter-
mined based on the total volume of the brain homogenate (0.6 to 1.0 ml)
and the average count per volume (15 to 20 l).
In vitro sample preparation. Six-well plates were seeded with 300 l
of a 0.01% solution of type IV collagen (Sigma) in 0.1 M glacial acetic acid.
After three 1-ml PBS washes to remove residual acetic acid, PC12 cells
were seeded at 10
6
cells each into each well and allowed to adhere for 24 h.
Filtered parasites were seeded onto PC12s at a multiplicity of infection
(MOI) of 1 (one parasite per PC12 cell) and allowed to invade for 4 h. Cells
were washed three times with PBS to remove noninvaded parasites and
then returned to standard or differentiation culture conditions for 48 h.
SH-SY5Y cells were seeded onto 6-well plates at 1 ⫻10
6
cells per well.
PC12 or SH-SY5Y cells were dislodged from the plate using mechanical
pressure from a pipette, counted by hemocytometer, pelleted at 5,000
RCF (relative centrifugal force), and resuspended into 500 l ice-cold 0.1
N perchloric acid and 0.4 M sodium metabisulfite in distilled H
2
O (PCA
buffer). Cells were homogenized on ice with a Branson sonifier cell dis-
ruptor 185 (three 5-s pulses, power of 3/10) (Danbury, CT) and centri-
fuged at 14,000 RCF for 15 min at 4°C. The supernatant was filtered
through a 0.22-m filter and diluted into MD-TM buffer (75 mM
NaH
2
PO
4
-H
2
O, 1.7 mM octane sulfonic acid, 100 l/liter triethylamine,
25 M EDTA, 10% acetonitrile, pH 3.0; ESA, Bedford, MA) for analysis
with high-performance liquid chromatography (HPLC).
In vivo sample preparation. Chronically infected mice were eutha-
nized. The brain was removed and cut sagitally along the midline. Half of
each brain was weighed, transferred to 500 l ice-cold PCA buffer, and
homogenized by a hand homogenizer, followed by sonication on ice with
a Branson sonifier cell disruptor 185 (three 10-s pulses, power of 3/10).
Homogenates were spun at 14,000 RCF for 15 min, and the supernatant
was collected and filtered through a 0.22-m filter. Supernatants were
diluted into MD-TM buffer for HPLC analysis.
HPLC. HPLC analysis was performed, as described in reference 30,
using a Coulochem electrochemical detector (ESA, Bedford, MA) with an
ESA MD 150- by 3.2-mm column. The mobile phase consisted of ESA
MD-TM buffer. HPLC detection of samples was calibrated using standard
samples of dopamine (DA), dopachrome (Dopac), and homovanillic acid
(HVA) at 0.1, 0.5, or 1.0 ng/100 l. The retention times of each catechol-
amine was determined. The total area under the HPLC trace for five rep-
licate runs of each catecholamine was measured to create the reference
curve for subsequent quantitative analysis of catecholamine amounts (see
Fig. S1 in the supplemental material).
Statistical testing. qPCR data were analyzed by two-way analysis of
variance (ANOVA) with Tukey’s multiple-comparison test. In vitro
HPLC data were analyzed by one-way ANOVA. Cyst count data and in
vivo mouse dopamine data were analyzed by the Kruskal-Wallis nonpara-
metric test and Dunn’s multiple-comparison test. Cyst count versus do-
pamine data were analyzed by linear regression. Statistical analysis was
done in Prism 6 for Mac OSX (GraphPad Software, La Jolla, CA).
RESULTS
Deletion of AAH2 in the type II Pru⌬ku80⌬hxg background.
Previous work has described two highly similar AAH genes in T.
gondii (20). During the course of this work, assembly 9 of the T.
gondii ME49 genome was released (ToxoDB.org). In this version,
only one of the AAH genes is found in the chromosome (i.e.,
AAH2), while the other one is on an unassembled contig (i.e.,
AAH1 on contig TgME49_asmbl.1705). Because this differs from
the genomic organization of the genes in assembly 8 (where they
were found in tandem on chromosome V), we PCR mapped and
sequenced the regions flanking AAH1 and AAH2 from the Pru
strain. Our findings were consistent with the arrangement of the
previous ME49 genome assembly 8 (see Fig. S2 in the supplemen-
tal material).
To examine the role of AAH2, a double homologous recombi-
nation strategy was employed to target the gene for deletion (Fig.
1A). We transfected knockout constructs into the background
strain Pru⌬ku80⌬hxg (21) and selected for positive transfectants
using HXG selection (25). The gene was successfully knocked out,
creating the knockout strain Pru⌬ku80⌬hxg⌬aah2, as shown by
the absence of an expected 4.8-kb band encompassing the AAH2
gene (Fig. 1B). The knockout line then was complemented with a
cDNA copy of the AAH2 gene driven by its endogenous 5=and 3=
UTRs, creating the complement strain Pru⌬ku80⌬hxg⌬aah2::
AAH2. The complement was confirmed by PCR, showing the
presence of a shorter 1.6-kb band consistent with the cDNA of
AAH2 (Fig. 1B). Despite repeated attempts (more than 3) to delete
AAH1 using a similar strategy, we were unable to obtain knock-
outs of this gene (data not shown), suggesting it is essential.
The parasite AAH2 is not essential for growth, differentia-
tion, host infection, or oral transmission. To test for overall
growth, we compared the ability of knockout and wild-type par-
asites to form plaques on HFF monolayers. A plaque assay showed
no significant defect in growth rate in the knockout (Fig. 1C; also
see Fig. S3 in the supplemental material). We next tested the ability
of the parasite to convert from the lytic tachyzoite stage to the
dormant bradyzoite stage in vitro under stress induced by high pH
(8.1), a well-established model for in vitro differentiation (31).
Bradyzoite differentiation under high pH was normal, as mea-
sured by FITC-conjugated Dolichos biflorus lectin specific to N-
acetyl galactosamine that is found in the bradyzoite cyst wall (32)
(Fig. 2; also see Fig. S4 in the supplemental material).
We next used quantitative real-time PCR to test for abnormal-
ities in bradyzoite induction at the transcriptional level. We exam-
ined the change in transcription of the tachyzoite-specific genes
SAG1 and SAG2A, bradyzoite-specific genes BAG1 and LDH2
(27), as well as our genes of interest, AAH1 and AAH2.Asex-
pected, no significant differences in expression levels were seen
with either the tachyzoite or the bradyzoite samples across geno-
types (Fig. 3A). Of note, contrary to previous reports that AAH1 is
constitutive but AAH2 is bradyzoite specific (20), the expression
of both AAH1 and AAH2 appeared to go up in bradyzoites (Fig.
3A). As expected, the transcript for AAH2 was absent from the
knockout but restored in the complement (Fig. 3A). However,
absolute expression of these genes remained very low; thus, rela-
tive fold change was not indicative of strong expression in the
bradyzoite stage. This finding is consistent with data found in
ToxoxDB. Based on microarray data for the type II ME49 strain
(33), the expression levels of AAH1 and AAH2 are in the 15th and
Infection by Toxoplasma gondii Does Not Alter Dopamine
March 2015 Volume 83 Number 3 iai.asm.org 1041Infection and Immunity
30th percentiles among all T. gondii genes in tachyzoites and go up
to the 35th and 40th percentiles, respectively, in bradyzoites. As a
comparison, BAG1, a bradyzoite-specific protein that is induced
in bradyzoites (34), has an expression level in the 60th percentile
in tachyzoites, going up to the 100th percentile in bradyzoites.
Expression of AAH1 and AAH2 is very low in tachyzoites or
bradyzoites. We sought to identify the localization of the parasite
hydroxylases using both immunofluorescence and Western blot
analysis. Using a double homologous recombination strategy with
HXG selection as previously described, AAH1 and AAH2 were
separately tagged with a Ty epitope (24), and parasites were
screened for expression. Western blot analysis failed to identify
detectable levels of either protein in bradyzoites (Fig. 3B). In
tachyzoites (not shown) and in bradyzoites, no signal was visible
for either Ty-tagged AAH protein by immunofluorescence (Fig.
3C). To differentiate between an absent signal due to minimal/low
expression levels from an artifact of protein folding, another par-
asite line was created in which the BAG1 promoter was used to
overexpress Ty-tagged AAH2 (see Fig. S5 in the supplemental ma-
terial). The resulting parasite line showed a clear Ty epitope signal
by Western blotting (Fig. 3B) and immunofluorescence (Fig. 3C)
when grown under bradyzoite-inducing conditions. However, we
were unable to demonstrate cross-reactivity with the mammalian
tyrosine hydroxylase antibody previously reported in the litera-
ture (15), even in the AAH2-overexpressing line (see Fig. S6). We
also were unable to detect dopamine in PC12 cells using a previ-
ously reported technique (15 and data not shown). Taken to-
gether, these results suggest that our inability to detect proteins
derived from AAH1 and AAH2 under native conditions is due to
low levels of expression in both tachyzoites and bradyzoites.
Infection with T. gondii does not affect host dopamine levels
in vitro.We investigated the effect of T. gondii infection on in vitro
dopaminergic PC12 cell cultures in vitro and found that infection
with tachyzoites did not significantly alter dopamine content (Fig.
4A and Table 1). To test for a dopaminergic effect in the brady-
zoite stage, we stressed infected cells by raising the pH to 8.1, a
treatment known to induce differentiation (31). Immunofluores-
cence analysis confirmed the presence of bradyzoite vacuoles
within PC12 cells (see Fig. S7 in the supplemental material). Al-
though we observed a 10-fold decrease in dopamine content per
cell under the high-pH stress conditions, no difference in dopa-
mine content was observed between infected and uninfected cells
(Fig. 4B). In tachyzoite and bradyzoite cultures, neither deletion
of AAH2 nor bradyzoite-specific overexpression of AAH2 resulted
in significant differences in dopamine content. To rule out the
possibility that increases in dopamine content were masked by the
high endogenous dopamine stores of PC12 cells, which can reach
up to 20 ng/10
5
cells, we repeated the experiment with SH-SY5Y
cells that produce but do not store dopamine and again observed
no increase in dopamine levels (data not shown).
Infection with T. gondii does not affect global or regional
host dopamine levels in vivo.We initially tested for changes in
the ability to form cysts in host brains in vivo. One thousand
tachyzoites of each strain were injected i.p. into 6- to 8-week-old
CD1 female mice, and brains were assayed 1 month postinfection
for cyst formation. Both mutant and complement strains showed
normal parasite cyst burden relative to that of wild-type parasites
(Fig. 5A). Additionally, there was no difference in average cyst size
between wild-type and ⌬aah2 knockout parasites (see Fig. S8 in
the supplemental material). We also tested the ability of these cysts
to survive digestion to induce oral transmission of infection. Five
cysts were fed by oral gavage to naive 6- to 8-week-old CD1 mice,
and serum was collected 1 month postinfection to assay infec-
tivity by ELISA. All strains caused seroconversion in mice upon
FIG 1 Generation of an AAH2-deficient strain and genetic complementation. (A) Schematic of AAH2 knockout strategy. (Left) HXGPRT construct flanked by
5=and 3=regions from the AAH2 genomic locus was used to knock out the AAH2 gene in the Pru⌬ku80⌬hxg background by double homologous crossover. MPA,
mycophenolic acid; Xa, xanthine, which was used for selection. (Center) The deletion strain (⌬aah2::HXG) was transfected with cleanup construct to remove the
HXGPRT drug marker. (Right) The deletion strain (⌬aah2::HXG) was complemented by replacing HXGPRT with a cDNA copy of AAH2. (B) Diagnostic PCR
of the wild-type (Pru⌬ku80⌬hxg), deletion mutant (Pru⌬ku80⌬hxg⌬aah2), and complement (Pru⌬ku80⌬hxg⌬aah2::AAH2) lines. Arrows show the respective
primers used to confirm the genetic architecture (see Table S1 in the supplemental material). Expected product sizes were the following: AAH1, 4.820 kb; AAH2,
4.820 kb; AAH2 cDNA, 1.698 kb. (C) Plaque assay measuring in vitro growth of strains on HFF monolayers stained with crystal violet.
Wang et al.
1042 iai.asm.org March 2015 Volume 83 Number 3Infection and Immunity
feeding, indicating they are capable of normal oral transmis-
sion (see Fig. S9).
We observed no significant difference in whole-brain dopa-
mine levels between mice infected with wild-type, ⌬aah2,or
⌬aah2::AAH2 parasites compared to PBS controls (Fig. 5B). How-
ever, because the overall cyst burden was relatively low, ⬃100 cysts
per brain, we repeated whole-brain dopamine assays with the
more cyst-competent ME49 strain. At 1 month postinfection, cyst
burden in ME49-infected mice was significantly higher (Fig. 5A),
but brain dopamine levels were 20% lower than those of unin-
fected mice (Fig. 5B). This likely was due to persistent acute infec-
tion, as concurrent illness also was observed. Consequently, infec-
tion with ME49 parasites was repeated at a lower dose (200
tachyzoites), and brain dopamine was assayed 2 months postin-
fection, after acute illness was no longer visible. Despite slightly
increased cyst burdens relative to those of Pru infection, brain
dopamine levels still were not significantly different from those for
uninfected mice (Fig. 5B). Overall there was no correlation rela-
tionship between cyst density and whole-brain dopamine for
ME49-infected mice (Fig. 5C). We further investigated the possi-
bility of changes in dopamine at the regional level, examining the
striatum of infected and uninfected mice, and again we found no
difference in dopamine levels between control and infected mice
(see Fig. S10 in the supplemental material).
Finally, because all of the previous experiments were done with
type II strains, we repeated the experiment using the type III C56
strain, reported previously (17). Five mice were infected i.p. with
our standard dose (10
3
tachyzoites) and five mice with the dose
used by Stibbs (10
4
tachyzoites) (17). Using either method, the
brain cyst density was significantly lower at 1 month postinfection
than that with the type II Pru or ME49 strains (e.g., 2/5 mice
infected with 10
3
parasites and 3/5 mice infected with 10
4
parasites
showed brain cyst densities below the detection limit of 20 cysts
per brain [Fig. 5A]). Brain dopamine levels were not significantly
different from those of uninfected mice at either dose (10
3
tachyzoites, P⫽0.13; 10
4
tachyzoites, P⫽0.20; Mann-Whitney
test) (Fig. 5B).
DISCUSSION
Although T. gondii’s ability to manipulate rodent behavioral re-
sponses to the cat is well documented (5–13) and several studies
suggest that the parasite manipulates dopamine signaling in the
host brain (15,17,19,20,35), no parasite effectors have been
found for these host alterations. We sought to test the hypothesis
that the parasite genes AAH1 and/or AAH2, which encode the
catecholamine biosynthetic enzyme tyrosine hydroxylase, were
responsible for the alterations of host dopamine levels observed in
vitro (15) and in vivo (17). However, we were unable to demon-
strate T. gondii infection having any effect on dopamine levels in
the catecholaminergic cell line PC12 or in infected mouse brains.
Furthermore, contrary to previous reports that described tyrosine
hydroxylase within parasite bradyzoite cysts (15), we observed
that levels of both AAH1 and AAH2 were extremely low and un-
detectable at native expression levels in both tachyzoites and bra-
dyzoites, consistent with low expression levels observed in mi-
croarray data (previously reported in ToxoDB). Collectively, these
studies indicate that AAH genes do not lead to global changes in
dopamine production in the host, although they may contribute
to local differences. Additionally, our findings suggest that AAH
genes are involved in a different function, since AAH1 is essential
for growth in nondopaminergic cells.
We did not observe changes in global brain dopamine levels
with either the Pru strain or the C56 strain, unlike what was pre-
viously reported (17). However, closer examination of this origi-
nal study indicates that the significant difference reported can be
attributed to very low variance in the sample rather than dramatic
differences in the average values (17). Brain dopamine values in
our sample values approximate those reported previously yet
show higher variance, consistent with the expectation for animal
studies. Our results are consistent with other reports that investi-
gated dopamine and neurotransmitter levels in infected rodents.
For example, Goodwin et al. (36) described minimal changes ob-
served in dopamine, norepinephrine, and serotonin concentra-
tions in the frontal cortex and striatum of chronically infected
FIG 2 Differentiation into bradyzoites in vitro. (A) Formation of cysts by
wild-type, ⌬aah2, and ⌬aah2::AAH2 parasites in tachyzoite and bradyzoite
conditions in vitro. Partial and fully formed cysts were enumerated based on
staining with DBL. There was no significant difference in cyst formation in
bradyzoite-induced parasites (P⫽1.00) and no significant difference in cyst
formation (P⫽0.66) or partial cyst formation (P⫽0.88) in tachyzoite con-
ditions (both determined by one-way ANOVA; n⫽3 experiments). (B) Rep-
resentative pictures of intact, partial, and absent cyst formation in parasite
vacuoles. Blue, 4=,6-diamidino-2-phenylindole (DAPI); red, GRA7; green,
DBL. Scale bar, 10 m.
Infection by Toxoplasma gondii Does Not Alter Dopamine
March 2015 Volume 83 Number 3 iai.asm.org 1043Infection and Immunity
mice. Similarly, Gatkowska et al. (37) noted profound changes in
the ratios of dopamine, serotonin, and norepinephrine to their
metabolites during acute infection, but which returned to baseline
upon the establishment of chronic infection. Additionally, we did
not observe differences in dopamine or related metabolite levels in
the striatum, a region of the brain rich in dopaminergic terminals.
It was recently suggested that expression of AAH enzymes in bra-
dyzoites (detected with a commercial anti-tyrosine hydroxylase
[TH] antibody) leads to elevated dopamine surrounding tissue
cysts in vivo (15). We were unable to replicate these findings here,
as the commercial antibody to TH did not react to T. gondii in our
hands, even in the strain that overexpressed AAH2. Additionally,
attempts to localize dopamine in PC12 cells were not successful,
presumably because this small metabolite rapidly diffuses in fixed
cells and tissues. Despite not observing global or regional changes,
our experiments cannot rule out an effect of AAH enzymes on
localized or transient dopamine levels in the host, which could in
turn affect behavior. Further experiments using microdialysis
monitoring may reveal highly localized changes to dopamine in
the CNS and may be useful in studying the role of parasite infec-
tion and AAH enzymes on host signaling pathways.
Previous studies have indicated that infection with T. gondii
FIG 3 Differentiation and expression levels in tachyzoites and bradyzoites. (A) Quantitative real-time PCR comparing gene expression of bradyzoites relative to
that of tachyzoites of wild-type, knockout (⌬aah2), and complemented (⌬aah2::AAH2) parasites. Stage-specific markers SAG1,BAG1,SAG2A, and LDH2, along
with AAH1 and AAH2, were monitored with gene-specific primers (see Table S1 in the supplemental material). Results are means ⫾standard deviations (SD)
(n⫽3 experiments). Excluding the expected differences in AAH2 expression, expression differences between the genes probed were not significant (P⫽0.19 by
two-way ANOVA). (B) Western blot of bradyzoites expressing a Ty epitope-tagged AAH1 or AAH2 or a tagged copy of AAH2 driven by the BAG1 promoter. Red,
GRA2; green, Ty. Expected protein sizes: AAH1 and AAH2, 55 kDa; GRA2 (dense granule protein 2), 28 kDa. (C) Immunofluorescent assay of AAH1-Ty-,
AAH2-Ty-, and BAG1::H2Ty-tagged parasites differentiated into bradyzoites. Blue, DAPI; green, DBL; red, Ty. Scale bar, 10 m.
FIG 4 Production of dopamine in infected PC12 cells. (A) Comparison of
infection with wild-type, knockout (⌬aah2), complement (⌬aah2::AAH2),
or BAG1-2Ty overexpressor tachyzoites in PC12 cells (P⫽0.40 by one-way
ANOVA). Results are means ⫾SD (n⫽2 to 5 replicates). The yaxis shows
the ratio of dopamine content per PC12 cell relative to uninfected PC12s.
(B) Comparison of dopamine content in PC12 cells grown under brady-
zoite conditions (P⫽0.4239 by one-way ANOVA). Results are means ⫾
SD (n⫽2to4).
TABLE 1 Dopamine and metabolite content determined by HPLC
c
Stage and strain Dopac
a
Dopamine HVA
b
Tachyzoite
Pru⌬ku80⌬hxg 0.84 ⫾0.71 20.43 ⫾1.95 2.71 ⫾0.35
⌬aah2 0.60 ⫾0.51 16.84 ⫾2.25 2.42 ⫾0.20
⌬aah2::AAH2 0.65 ⫾0.63 18.38 ⫾3.90 2.28 ⫾0.86
Uninfected 0.25 ⫾0.37 17.48 ⫾5.68 1.99 ⫾0.56
Bradyzoite
Pru⌬ku80⌬hxg 0.02 ⫾0.03 0.96 ⫾0.21 1.38 ⫾0.10
⌬aah2 0.07 ⫾0.01 1.37 ⫾0.78 2.55 ⫾0.31
⌬aah2::AAH2 0.25 ⫾0.19 1.54 ⫾0.12 1.88 ⫾0.17
Uninfected 0.01 ⫾0.01 0.71 ⫾0.00 1.09 ⫾0.82
a
3,4-Dihydroxyphenylacetic acid.
b
Homovanillic acid.
c
Results are reported as ng catecholamine/10
5
cells. Means ⫾standard deviations are
shown from 3 technical replicates each (tachyzoites) and 2 each (bradyzoites) for one
representative experiment.
Wang et al.
1044 iai.asm.org March 2015 Volume 83 Number 3Infection and Immunity
increases dopamine production in PC12 cells (15) and implied
that the AAH1 and AAH2 genes contribute to this by producing
the precursor L-Dopa (20). However, we failed to reproduce the
finding that infection elevates dopamine production in PC12 and
also did not observe any change in SH-SY5Y cells when infected
with either tachyzoites or bradyzoites. Given this finding, it was
not surprising that no further changes were observed in dopamine
production in these cell lines using mutants that lack AAH2. Based
on immunofluorescence staining, qPCR, and microarray data, it
also does not appear that AAH genes are highly expressed in bra-
dyzoites. Finally, we did not observe increased levels of host do-
pamine production using a strain of T. gondii that overexpresses
AAH2. This overexpressing strain was useful for demonstrating
that AAH2 appears to be secreted from bradyzoites and is detect-
able in the cyst matrix, as previously described (15). However,
under native conditions it appears to be expressed at very low
levels.
Collectively, our findings fail to support the original hypothe-
sis that T. gondii AAH enzymes lead to globally elevated dopamine
levels in the host. Instead, it is plausible that AAH genes are in-
volved in other metabolic functions in the parasite. This possibil-
ity is supported by the fact that AHH1 appears to be essential in
nondopaminergic cells. Not only did repeated attempts to knock
out this gene by double homologous cross over fail but we also
have been unable to disrupt this gene using the much more effi-
cient CRISPR system, which was recently described for T. gondii
(38). The function of AAH1 might be revealed using recently de-
scribed strategies for inducible knockdown (39). Possible alterna-
tive activities of the hydroxylases include the production of dity-
rosine, which has previously been described in the oocyst walls of
Eimeria (40,41). Microarray data describe the upregulation of
AAH genes in the oocyst stages of T. gondii (42), suggesting they
participate in this process. However, such a role is unlikely to
explain the essentiality of AAH1 in tachyzoites. Alternatively, be-
cause tetrahydrobiopterin likely is consumed as a cofactor by these
hydroxylases, the AAH genes may serve as a sink for the output of
the pterin pathway, which has been partially characterized in pre-
vious studies (43). Finally, although T. gondii is not known to
produce melanin, which plays an important role in fungal viru-
lence (44), the AAH enzymes could provide precursors to other
catechol metabolites that are important in growth and/or viru-
lence of T. gondii.
Although we did not find evidence for global changes in dopa-
mine levels, infection by the parasite causes a number of global
changes in the rodent brain. For example, infection causes low-
level inflammation throughout the brain (45), affects the activity
level of infected neurons (46), alters microRNA expression in cells
(35), and causes changes in host gene expression in the frontal
cortex of mice (12). Since chronic infection leads to activated mi-
croglia around many, but not all, tissue cysts (47), a potential
cause of signaling and behavioral abnormalities would be inflam-
mation. Interestingly, it was discovered that mice infected by at-
tenuated parasites incapable of persisting as cysts in the brain still
exhibited attraction to cat odor months after acute infection had
been completely cleared and parasites were no longer detectable in
the brain by PCR (13). These findings suggest that alteration or
rewiring of the mouse brain during acute infection leads to alter-
ations in rodent behavior. Such interactions are likely to be com-
plex and will require extensive further study to uncover the basis
for altered behavior in chronically infected animals.
FIG 5 Dopamine content in the brain of control and infected mice. (A) Par-
asite cyst burden in whole brains of CD1 mice examined at 1 (1 mo) or 2
months (2 mo) postinfection. Infection with ME49 at 1 mo showed signifi-
cantly higher cyst burden (P⫽0.0003 by Kruskal-Wallis test with Dunn’s
multiple comparisons for Pru strains and ME49). Infection with the type III
C56 strain showed significantly lower cyst burden (P⫽0.0003 by Kruskal-
Wallis test with Dunn’s multiple comparisons for Pru strains and C56), with 5
of 10 mice showing cyst burdens below the detectable limit of 20 cysts/brain
(not plotted). (B) Dopamine levels in total brain homogenates in uninfected
and infected mice at 1 (1mo) or 2 (2mo) months postinfection. Dopamine
levels were not significantly different between infected or uninfected animals
or between infection strains (P⫽0.075 by Kruskal-Wallis test with Dunn’s
multiple comparisons test). (C) Linear regression analysis between cyst density
and brain dopamine concentration in mice infected with ME49 for 1 month
(1mo) or 2 months (2mo). R
2
⫽0.1333 (red). If the highest point in the linear
regression was removed, R
2
⫽0.1175 (blue).
Infection by Toxoplasma gondii Does Not Alter Dopamine
March 2015 Volume 83 Number 3 iai.asm.org 1045Infection and Immunity
ACKNOWLEDGMENTS
We are grateful to Jennifer Barks and Qiuling Wang for technical assis-
tance and Stephen Beverley, Tim Holy, Beau Ances, Robert Yolken, and
Mikhail Pletnikov for helpful advice.
This work was supported in part by a grant from the NIH (AI059176).
REFERENCES
1. Dubey JP. 2007. The history and life cycle of Toxoplasma gondii, p 1–17. In
Weiss LM, Kim K (ed), Toxoplasma gondii the model Apicomplexan: per-
spectives and methods. Academic Press, Elsevier, New York, NY.
2. Frenkel JK, Dubey JP, Miller NL. 1970. Toxoplasma gondii in cats: fecal
stages identified as coccidian oocysts. Science 167:893– 896. http://dx.doi
.org/10.1126/science.167.3919.893.
3. Dubey JP, Thulliez P. 1993. Persistence of tissue cysts in edible tissues of
cattle fed Toxoplasma gondii oocysts. Am J Vet Res 54:270 –273.
4. Dubey JP, Dubey JP. 2010. Toxoplasmosis of animals and humans, 2nd
ed. CRC Press, Boca Raton, FL.
5. Kaushik M, Knowles SC, Webster JP. 2014. What makes a feline fatal in
Toxoplasma gondii’s fatal feline attraction? Infected rats choose wild cats.
Integr Comp Biol 54:118 –128. http://dx.doi.org/10.1093/icb/icu060.
6. Kaushik M, Lamberton PH, Webster JP. 2012. The role of parasites and
pathogens in influencing generalised anxiety and predation-related fear in
the mammalian central nervous system. Hormones Behav 62:191–201.
http://dx.doi.org/10.1016/j.yhbeh.2012.04.002.
7. Lamberton PH, Donnelly CA, Webster JP. 2008. Specificity of the Tox-
oplasma gondii-altered behaviour to definitive versus non-definitive host
predation risk. Parasitology 135:1143–1150. http://dx.doi.org/10.1017
/S0031182008004666.
8. Vyas A, Kim KS, Giacomini N, Boothroyd JC, Sapolsky RM. 2007.
Behavioral changes induced by Toxoplasma infection of rodents are
highly specific to aversion of cat odors. Proc Natl Acad SciUSA104:6442–
6447. http://dx.doi.org/10.1073/pnas.0608310104.
9. Berdoy M, Webster JP, Macdonald DW. 2000. Fatal attraction in rats
infected with Toxoplasma gondii. Proc R Soc Lond B Biol Sci 267:1591–
1594. http://dx.doi.org/10.1098/rspb.2000.1182.
10. Kannan G, Moldovan K, Xiao JC, Yolken RH, Jones-Brando L,
Pletnikov MV. 2010. Toxoplasma gondii strain-dependent effects on
mouse behaviour. Folia Parasitol 57:151–155. http://dx.doi.org/10
.14411/fp.2010.019.
11. Golcu D, Gebre RZ, Sapolsky RM. 2014. Toxoplasma gondii influences
aversive behaviors of female rats in an estrus cycle dependent manner.
Physiol Behav 135:98 –103. http://dx.doi.org/10.1016/j.physbeh.2014.05
.036.
12. Xiao J, Kannan G, Jones-Brando L, Brannock C, Krasnova IN, Cadet
JL, Pletnikov M, Yolken RH. 2012. Sex-specific changes in gene ex-
pression and behavior induced by chronic Toxoplasma infection
in mice. Neuroscience 206:39 – 48. http://dx.doi.org/10.1016/j.neuroscience
.2011.12.051.
13. Ingram WM, Goodrich LM, Robey EA, Eisen MB. 2013. Mice infected
with low-virulence strains of Toxoplasma gondii lose their innate aversion
to cat urine, even after extensive parasite clearance. PLoS One 8:e75246.
http://dx.doi.org/10.1371/journal.pone.0075246.
14. Webster JP, McConkey GA. 2010. Toxoplasma gondii-altered host be-
haviour: clues as to mechanism of action. Folia Parasitol 57:95–104. http:
//dx.doi.org/10.14411/fp.2010.012.
15. Prandovszky E, Gaskell E, Martin H, Dubey JP, Webster JP, McConkey
GA. 2011. The neurotropic parasite Toxoplasma gondii increases dopa-
mine metabolism. PLoS One 6:e23866. http://dx.doi.org/10.1371/journal
.pone.0023866.
16. Skallova A, Kodym P, Frynta D, Flegr J. 2006. The role of dopamine in
Toxoplasma-induced behavioural alterations in mice: an ethological and
ethopharmacological study. Parasitology 133:525–535. http://dx.doi.org
/10.1017/S0031182006000886.
17. Stibbs HH. 1985. Changes in brain concentrations of catecholamines and
indoleamines in Toxoplasma gondii infected mice. Ann Trop Med Para-
sitol 79:153–157.
18. Jones-Brando L, Torrey EF, Yolken R. 2003. Drugs used in the treatment
of schizophrenia and bipolar disorder inhibit the replication of Toxo-
plasma gondii. Schizophr Res 62:237–244. http://dx.doi.org/10.1016
/S0920-9964(02)00357-2.
19. Webster JP, Lamberton PH, Donnelly CA, Torrey EF. 2006. Parasites as
causative agents of human affective disorders? The impact of anti-
psychotic, mood-stabilizer and anti-parasite medication on Toxoplasma
gondii’s ability to alter host behaviour. Proc Biol Sci 273:1023–1030. http:
//dx.doi.org/10.1098/rspb.2005.3413.
20. Gaskell EA, Smith JE, Pinney JW, Westhead DR, McConkey GA. 2009.
A unique dual activity amino acid hydroxylase in Toxoplasma gondii.
PLoS One 4:e4801. http://dx.doi.org/10.1371/journal.pone.0004801.
21. Fox BA, Falla A, Rommereim LM, Tomita T, Gigley JP, Mercier C,
Cesbron-Delauw MF, Weiss LM, Bzik DJ. 2011. Type II Toxoplasma
gondii KU80 knockout strains enable functional analysis of genes required
for cyst development and latent infection. Eukaryot Cell 10:1193–1206.
http://dx.doi.org/10.1128/EC.00297-10.
22. Lunde MN, Jacobs L. 1964. Properties of Toxoplasma Lysates Toxic to
Rabbits on Intravenous Injection. J Parasitol 50:49 –51.
23. Sibley LD, Boothroyd JC. 1992. Virulent strains of Toxoplasma gondii
comprise a single clonal lineage. Nature 359:82– 85.
24. Bastin P, Bagherzadeh Z, Matthews KR, Gull K. 1996. A novel epitope
tag system to study protein targeting and organelle biogenesis in Trypano-
soma brucei. Mol Biochem Parasitol 77:235–239.
25. Donald RGK, Roos DS. 1998. Gene knock-outs and allelic replacements
in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run
mutagenesis. Mol Biochem Parasitol 91:295–305.
26. Pfefferkorn ER, Bzik DJ, Honsinger CP. 2001. Toxoplasma gondii:
mechanism of the parasitostatic action of 6-thioxanthine. Exp Parasitol
99:235–243. http://dx.doi.org/10.1006/expr.2001.4673.
27. Fux B, Nawas J, Khan A, Gill DB, Su C, Sibley LD. 2007. Toxoplasma
gondii strains defective in oral transmission are also defective in develop-
mental stage differentiation. Infect Immun 75:2580 –2590. http://dx.doi
.org/10.1128/IAI.00085-07.
28. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data
using real-time quantitative PCR and the 2(-delta-delta C(T)) method.
Methods 25:402– 408. http://dx.doi.org/10.1006/meth.2001.1262.
29. Shen B, Sibley LD. 2014. Toxoplasma aldolase is required for metabolism
but dispensable for host-cell invasion. Proc Natl Acad Sci U S A 111:3567–
3572. http://dx.doi.org/10.1073/pnas.1315156111.
30. Lotharius J, O’Malley KL. 2000. The parkinsonism-inducing drug
1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation.
A novel mechanism of toxicity. J Biol Chem 275:38581–38588. http://dx
.doi.org/10.1074/jbc.M005385200.
31. Soête M, Camus D, Dubremetz JF. 1994. Experimental induction of
bradyzoite-specific antigen expression and cyst formation by the RH
strain of Toxoplasma gondii in vitro. Exp Parasitol 78:361–370.
32. Boothroyd JC, Black M, Bonnifoy S, Hehl A, Knoll L, Manger ID,
Ortega-Barria E, Tomavo S. 1997. Genetic and biochemical analysis of
development in Toxoplasma gondii. Philos Trans R Soc Lond 352:1347–
1354.
33. Behnke M, Wooten JC, Lehmann M, Radke J, Lucas O, Nawas J, Sibley
LD, White M. 2010. Coordinated progression through two subtranscrip-
tions underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One
5:e12354. http://dx.doi.org/10.1371/journal.pone.0012354.
34. Bohne W, Gross U, Ferguson DJP, Hessemann J. 1995. Cloning and
characterization of a bradyzoite-specifically expressed gene (hsp30/bag1)
of Toxoplasma gondii, related to genes encoding small heat-shock proteins
of plants. Mol Microbiol 16:1221–1230.
35. Xiao J, Li Y, Prandovszky E, Karuppagounder SS, Talbot CC, Jr,
Dawson VL, Dawson TM, Yolken RH. 2014. MicroRNA-132 dysregula-
tion in Toxoplasma gondii infection has implications for dopamine sig-
naling pathway. Neuroscience 268:128 –138. http://dx.doi.org/10.1016/j
.neuroscience.2014.03.015.
36. Goodwin D, Hrubec TC, Klein BG, Strobl JS, Werre SR, Han Q, Zajac
AM, Lindsay DS. 2012. Congenital infection of mice with Toxoplasma
gondii induces minimal change in behavior and no change in neurotrans-
mitter concentrations. J Parasitol 98:706 –712. http://dx.doi.org/10.1645
/GE-3068.1.
37. Gatkowska J, Wieczorek M, Dziadek B, Dzitko K, Dlugonska H. 2013.
Sex-dependent neurotransmitter level changes in brains of Toxoplasma
gondii infected mice. Exp Parasitol 133:1–7. http://dx.doi.org/10.1016/j
.exppara.2012.10.005.
38. Shen B, Brown KM, Lee TD, Sibley LD. 2014. Efficient gene disruption
in diverse strains of Toxoplasma gondii using CRISPR/CAS9. mBio
5:e01114 –14. http://dx.doi.org/10.1128/mBio.01114-14.
39. Andenmatten N, Egarter S, Jackson AJ, Jullien N, Herman JP, Meissner
M. 2012. Conditional genome engineering in Toxoplasma gondii uncovers
Wang et al.
1046 iai.asm.org March 2015 Volume 83 Number 3Infection and Immunity
alternative invasion mechanisms. Nat Methods 10:125–127. http://dx.doi
.org/10.1038/nmeth.2301.
40. Belli SI, Wallach MG, Luxford C, Davies MJ, Smith NC. 2003. Roles of
tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-
dihydroxyphenylalanine-mediated protein cross-linking in development
of the oocyst wall in the coccidian parasite Eimeria maxima. Eukaryot Cell
2:456 – 464. http://dx.doi.org/10.1128/EC.2.3.456-464.2003.
41. Belli SI, Wallach MG, Smith NC. 2003. Cloning and characterization of
the 82 kDa tyrosine-rich sexual stage glycoprotein, GAM82, and its role in
oocyst wall formation in the apicomplexan parasite, Eimeria maxima.
Gene 307:201–212. http://dx.doi.org/10.1016/S0378-1119(03)00451-7.
42. Fritz HM, Buchholz KR, Chen X, Durbin-Johnson B, Rocke DM,
Conrad PA, Boothroyd JC. 2012. Transcriptomic analysis of toxoplasma
development reveals many novel functions and structures specific to
sporozoites and oocysts. PLoS One 7:e29998. http://dx.doi.org/10.1371
/journal.pone.0029998.
43. Wang Q, Hauser V, Read M, Wang P, Hanson AD, Sims PF, Hyde JE.
2006. Functional identification of orthologous genes encoding pterin re-
cycling activity in Plasmodium falciparum and Toxoplasma gondii. Mol
Biochem Parasitol 146:109 –112. http://dx.doi.org/10.1016/j.molbiopara
.2005.11.002.
44. Revankar SG, Sutton DA. 2010. Melanized fungi in human disease. Clin
Microbiol Rev 23:884 –928. http://dx.doi.org/10.1128/CMR.00019-10.
45. Munoz M, Liesenfeld O, Heimesaat MM. 2011. Immunology of Toxo-
plasma gondii. Immunol Rev 240:269 –285. http://dx.doi.org/10.1111/j
.1600-065X.2010.00992.x.
46. Haroon F, Handel U, Angenstein F, Goldschmidt J, Kreutzmann P,
Lison H, Fischer KD, Scheich H, Wetzel W, Schluter D, Budinger E.
2012. Toxoplasma gondii actively inhibits neuronal function in chroni-
cally infected mice. PLoS One 7:e35516. http://dx.doi.org/10.1371/journal
.pone.0035516.
47. Evans AK, Strassmann PS, Lee IP, Sapolsky RM. 2014. Patterns of
Toxoplasma gondii cyst distribution in the forebrain associate with indi-
vidual variation in predator odor avoidance and anxiety-related behavior
in male Long-Evans rats. Brain Behav Immun 37:122–133. http://dx.doi
.org/10.1016/j.bbi.2013.11.012.
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