A Patatin-Like Protein Protects Toxoplasma gondii from Degradation
in a Nitric Oxide-Dependent Manner
Crystal M. Tobin and Laura J. Knoll
Department of Medical Microbiology and Immunology, University of Wisconsin—Madison, Madison, Wisconsin, USA
Toxoplasma gondii is an obligate intracellular parasite that uses immune cells to disseminate throughout its host. T. gondii can
oxide (NO). A T. gondii patatin-like protein called TgPL1 was previously shown to be important for survival in activated macro-
phages. Here we show that a T. gondii mutant with a deletion of the TgPL1 gene (?TgPL1) is degraded in activated macrophages.
purified from Escherichia coli does not have phospholipase activity. This result was not surprising, as TgPL1 contains a G-to-S
parasite survival and replication (3). The PV is comprised of host
cell lipids but excludes host cell proteins, making it distinct from
the endocytic network. Entry by active invasion protects T. gondii
endocytic network, e.g., by phagocytosis, it is killed by standard
host mechanisms (21). Through active invasion, T. gondii can use
host immune cells to disseminate to peripheral tissues, including
During the initial phases of Toxoplasma infection, macro-
tion site. These cells have an array of antimicrobial defenses, such
p47 GTPases, all of which help control parasite growth (20). In
turn, the parasite has developed the ability to alter these immune
cell responses (7). For example, T. gondii has been shown to re-
duce the amount of nitric oxide (NO) produced by macrophages
transcript and protein levels in infected cells (18).
The T. gondii TgPL1 gene has been implicated in the parasite’s
ability to reduce NO levels and survive in macrophages in vitro
(22). TgPL1 encodes a gene with homology to patatin-like phos-
stress response protein in potatoes, are known to have phospho-
conditions, like Arabidopsis homologs, which use the fatty acids
produced as cell signaling intermediates (15). Others are known
virulence factors, like ExoU from Pseudomonas aeruginosa, which
is secreted into host cells and lyses them by using its PLA2activity
(23). Residues conserved among members of this gene family in-
taining the catalytic aspartate (D-X-G/A) (8, 13). All of these
oxoplasma gondii is a protozoan parasite that is able to infect
nucleated cells in warm-blooded hosts, including humans
residues are conserved in TgPL1, except for the catalytic serine
The TgPL1 insertion mutant is degraded in activated macro-
phages despite its ability to invade host cells and establish a non-
fusogenic parasitophorous vacuole. This mutant does not exhibit
defects in acute or chronic infection in the CBA/J mouse model.
Because small amounts of TgPL1 transcript are present in the in-
sertional mutant (21), for this study, we generated a TgPL1 dele-
tion mutant (?TgPL1) to determine the mechanism that TgPL1
uses to protect parasites within activated macrophages.
MATERIALS AND METHODS
Plasmid construction. A construct to replace the open reading frame
firefly luciferase (?HPT::FLUC) was generated. The 5.3 kb upstream of
HPT (5= fragment) was amplified by using Phusion high-fidelity DNA
3=, which adds a 3= XhoI site. The 5.4 kb downstream of HPT (3= frag-
ment) was amplified by using Phusion polymerase and primers 5=-GCG
and 5=-CAACCGAAGTTGGTGTTAGTGACTGA-3=. PCR products
were cloned by using pCR-TOPO (Invitrogen). The 5= fragment was cut
Received 22 June 2011 Returned for modification 21 July 2011
Accepted 10 October 2011
Published ahead of print 17 October 2011
Editor: J. H. Adams
Address correspondence to Laura J. Knoll, email@example.com.
Supplemental material for this article may be found at http://iai.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0019-9567/12/$12.00 Infection and Immunityp. 55–61 iai.asm.org
A TgPL1 knockout construct was generated such that 80 bp upstream
poly(A) addition site was replaced with the chloramphenicol acetyltrans-
ferase (CAT) gene. Four kilobases upstream of TgPL1 (5= fragment) was
amplified by using Phusion polymerase and primers 5=-GATCACTAGT
GCGTCGTATCTGCATGGAGGG-3=, which adds a 5= SpeI site, and 5=-
GATCGTTAACACACCTGAGCGTCTGTTGCC-3=, which adds a 3=
HpaI site. The 3.8 kb downstream of the stop codon (3= fragment) was
amplified by using Phusion polymerase and primers 5=-GATCGGTACC
TAAGCAGTCGCCACTCCAAGAG-3=, which adds a 5= KpnI site, and
5=-GATCCGATCGTGGCTATCATGTCGCGCTGG-3=, which adds a 3=
PvuI site. PCR products were cloned by using pCR-TOPO. The 5=
fragment was subcloned with SpeI and HpaI into pBC-CAT/HPT,
which contains the cat gene from pT/230 (25) and the HPT gene from
pminiHXGPRT-I (9). The 3= fragment was subcloned into the resulting
plasmid with KpnI and PvuI, creating pTgPL1-KO.
amplifying the endogenous promoter and 5= portion of the ORF by using
Phusion polymerase and primers 5=-CAGCAGAAACGCAGATTATG-3=
GAGTTCCTCTTTGCCGTC-3=, which adds an in-frame hemagglutinin
(HA) tag. This fragment was cloned by using pCR-TOPO. A TgPL1
genomic fragment was rescued from the 89B7 insertional mutant as pre-
viously described (22). This fragment was not amplified by PCR because
tently truncated by this method. The rescue plasmid was digested with
SpeI, blunted, and then digested with SacI, and a 3.2-kb fragment corre-
sponding to the 3= end of the TgPL1 locus was gel purified. The vector
containing the 5= end of the locus was digested with KpnI, blunted, and
the rescue plasmid, creating pTgPL1-SacHA.
T. gondii strains, cell culture, and transfections. The type II parasite
strain Prugniaud (Pru) or ME49 was used in all experiments. Parasites
were passaged in confluent human foreskin fibroblasts (HFFs) as previ-
ously described (21). Bone marrow cells (BMCs) were isolated from
C57BL/6 mice (National Cancer Institute, Charles River Laboratories,
Frederick, MD) and developed into bone marrow-derived macrophages
(BMMs) in medium containing 20% L929 cell conditioned medium as
previously described (21). Subsequent assays were performed with
Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine
serum (FBS) supplemented with 2 mM glutamate, 0.5% penicillin-
streptomycin, and 50 mg/ml gentamicin.
To generate a luciferase-expressing wild-type (WT) T. gondii strain,
Pru or ME49 type II parasites were transfected with 50 ?g of p?HPT::
FLUC linearized at the SacII site. Transfected parasites were selected with
340 ?g/ml 6-thioxanthine for 14 days and then cloned by limiting dilu-
tion. Homologous recombination was confirmed by Southern analysis
and luciferase activity, resulting in Pru?HPT::Luc and ME49?HPT::Luc.
To generate a deletion of the TgPL1 gene, Pru?HPT::Luc was trans-
fected with 50 ?g of linearized pTgPL1-KO. Transfections underwent
positive selection in 20 ?M chloramphenicol until no growth was ob-
served for the mock transfection, followed by negative selection with 340
The ?TgPL1 mutant was cloned by limiting dilution and confirmed by
electroporations with 50 ?g of pEndoTgPL1 were selected and cloned as
previously described (22).
T. gondii bradyzoite development was achieved by incubating para-
sites in RPMI 1640 without bicarbonate, 1% FBS, 1% penicillin-
streptomycin, and 42 mM HEPES (pH 8.0).
In vitro growth assay. Host cells were passaged and seeded onto cov-
erslips as previously described (26) and infected with 1 ? 105parasites.
After 3 h, BMMs were left unstimulated, classically activated with 100
ng/ml lipopolysaccharide (LPS) and 100 U/ml gamma interferon (IFN-
nitric oxide synthase inhibitor). Griess reactions were performed as pre-
containing aminoguanidine (22). Additionally, BMMs were treated with
triamine (DETA) nonoate (Cayman Chemicals) plus HEPES or HEPES
alone. After 24 h, coverslips were fixed, permeabilized, and stained with
chronic mouse serum as previously described (22). Parasite growth was
assessed by counting 100 vacuoles per coverslip and categorizing each
vacuole as containing degraded parasites, 1 parasite, 2 parasites, 4 para-
sites, or 8 or more parasites. Samples were blinded to eliminate bias.
Immunofluorescence staining. BMMs were seeded onto coverslips
and infected with T. gondii expressing an HA-tagged version of TgPL1.
Parasites were allowed to invade for 3 h. BMMs were classically activated
Samples were fixed after the indicated time for 20 min in 3% formalde-
hyde. Excess formaldehyde was quenched with 0.1 M glycine for 3 min.
Samples were blocked and permeabilized for 1 h at room temperature or
overnight at 4°C. Samples were stained with mouse anti-hemagglutinin
antibody (Covance) and detected with Alexafluor-488-conjugated don-
key anti-mouse secondary antibody and rhodamine-conjugated Dolichos
biflorus agglutinin. Coverslips were mounted in Vectashield mounting me-
age stacks (0.2-?m Z increment) were taken at a ?100 magnification
(PlanApo oil immersion, 1.4 numerical aperture [NA]) using a motorized
wheel, a triple-pass (DAPI-fluorescein isothiocyanate [FITC]-Texas Red)
emission cube, differential interference contrast optics, and a Hamamatsu
Orca-AG charge-coupled-device (CCD) camera operated by OpenLabs 4.0
software (Improvision, Lexington, MA). Fluorescence images were decon-
volved by a constrained iterative algorithm, pseudocolored, and merged by
Induction and purification of TgPL1 from E. coli. The TgPL1 ORF
was amplified by using Accuprime Pfx polymerase (Invitrogen) from
cDNA using primers 5=-GATCAGATGTACACACGCTCCAGTGCAAC
3=, which adds a 5= PciI site, and 5=-GCGGCCGCAGACTCTTCAGACT
TTGCCTCTTCG-3=, which adds a 3= NotI site. This product was cloned
mid was transformed into Rosetta(DE3)pLysS competent E. coli cells
culture grown overnight and grown to an optical density at 600 nm
turing buffer using nickel-nitrilotriacetic acid (NTA) resin (Qiagen). The
protein was then dialyzed into 1? PBS for subsequent assays.
Phospholipase A2activity assay. Phospholipase A2activity assays
were performed as previously described (4). Briefly, 50 ?l of partially
purified TgPL1-6?His from E. coli was added to 50 ?l 2? PLA2buffer
(100 mM Tris-HCl [pH 7.5], 200 mM NaCl, 2 mM EDTA, 4 ?M fluores-
cent phospholipid substrate, 0.2% bovine serum albumin [BSA], 12 mM
CaCl2) and incubated at 37°C for 1 h. Fluorescence was measured at
excitation and emission wavelengths of 345 and 398 nm, respectively.
Mouse infection. For intraperitoneal infections, tachyzoites were
grown to near lysis in HFFs, syringe lysed, and enumerated on a hemocy-
tometer. A total of 2 ? 104parasites were injected into the peritoneal
cavity of 10- to 12-week old female C57BL/6 mice. Infection was allowed
to progress until severe neurological symptoms were evident, at which
time the mice were sacrificed. Mice that survived to the chronic stage of
infection were sacrificed at 25 days postinfection, and their brains were
harvested to assess cyst burden as previously described (22). For oral in-
fections, approximately 106in vitro-derived bradyzoite cysts were fed to fe-
Comparative fitness assay. Growth competition experiments were
Tobin and Knoll
iai.asm.orgInfection and Immunity
parasites of the ?TgPL1 mutant and the ?TgPL1 mutant complemented
were then inoculated into HFF host cells and serially passaged without
drugs every 4 days for 20 days. At every passage, parasites were removed
for plaque assays with or without 1 ?M pyrimethamine, as the comp
parasites contain the mutant DHFR gene, which confers resistance to
pyrimethamine. For in vivo competition studies, C57BL/6 female mice
of the ?TgPL1 mutant and comp. Parasites were harvested by perito-
neal lavage every 4 days for serial passage into another mouse and
plaque assays with and without pyrimethamine selection. Relative fit-
ness was assessed by the slope of the line, represented by the change
recombination. Four kilobases of upstream and 3.8 kb of down-
vector with CAT as a positive selectable marker and HPT as a
negative selectable marker (Fig. 1A). Pru?HPT::Luc parasites
were transfected with plasmid pTgPL1-KO and selected for resis-
tance to both chloramphenicol and 6-thioxanthine. The parental
strain Pru?HPT::Luc was used as the wild type (WT) for all ex-
periments. Clones containing the potential TgPL1 deletion were
analyzed by Southern analysis using a probe of a 500-bp frag-
ment of the TgPL1 5= UTR. For the XbaI-digested samples, the
expected sizes were 4.8 and 3.7 kb for the WT and TgPL1
knockout clones, respectively. For the ApaLI-digested samples,
knockout, respectively. ?TgPL1 exhibited the expected band-
ing pattern (Fig. 1B).
The ?TgPL1 mutant has a growth defect in classically acti-
vated but not naïve macrophages. To determine if the ?TgPL1
mutant exhibits a growth phenotype in macrophages similar to
that of the TgPL1 insertional mutant (21), growth was assessed in
naïve and classically activated macrophages. BMMs were infected
with WT and ?TgPL1 parasites, and growth was assessed by an
immunofluorescence assay (IFA) at 24 h postinfection. Vacuoles
were stained by using serum from chronically infected mice, and
growth was evaluated by tallying the number of parasites per vac-
phages, with over 50% of the vacuoles assessed containing repli-
by nearly 30 to 40% of the vacuoles assessed containing degraded
parasites, whereas wild-type parasites grew to 4 parasites per vac-
for the above-described phenotype, complementation was per-
formed by using the TgPL1 genomic region, which includes the
endogenous promoters and UTRs (22). The introduction of the
TgPL1 genomic region into the ?TgPL1 mutant allows the para-
sites to replicate similarly to WT parasites without the high levels
of degradation seen with ?TgPL1 parasites (Fig. 2B).
absent. To investigate whether NO is necessary for the ?TgPL1
phenotype in classically activated macrophages, growth assays
To reduce the level of NO, the iNOS inhibitor aminoguanidine
was added to macrophages concurrent with LPS and IFN-? acti-
vation and in parallel with the experiments shown in Fig. 2A and
B. No parasite growth defect was seen for the ?TgPL1 mutant
when macrophages were activated in the presence of aminogua-
nidine (Fig. 2C). To fully eliminate the presence of NO, BMMs
the iNOS inhibitor aminoguanidine, ?TgPL1 did not display a
phenotype in iNOS?/?BMMs (Fig. 2D). Whether grown in mac-
rophages with the inhibitor aminoguanidine or in naïve or acti-
vated BMMs from iNOS?/?mice, ?TgPL1 grew similarly to WT
?TgPL1 does not exhibit a growth defect in BMMs with ex-
ogenous nitric oxide. To determine whether NO is sufficient to
cause the ?TgPL1 mutant to have a replication defect, WT and
?TgPL1 parasites were grown in naïve macrophages with the NO
donor DETA NONOate. WT and ?TgPL1 parasites were allowed
NO donor DETA NONOate. WT and ?TgPL1 parasites both
showed a reduction in replication with increasing amounts of the
NO donor (Fig. 3). In samples treated with 200 ?M DETA
NONOate, less than 20% of the vacuoles had more than one par-
asite (Fig. 3D). However, the ?TgPL1 mutant did not exhibit a
ing that exogenously added NO is not sufficient to cause the phe-
notype of the ?TgPL1 mutant.
Peroxynitrite does not inhibit the growth of WT or ?TgPL1
parasites. Reactive oxygen intermediates, such as superoxide, are
also produced in response to stimulation with IFN-?. Superoxide
can react with NO to form peroxynitrite, which is a powerful
oxidizing agent. To determine if peroxynitrite is able to mediate
the degradation of ?TgPL1 parasites in BMMs, Infected BMMs
were treated with 0, 50, 100, or 200 ?M peroxynitrite and incu-
?TgPL1 strategy. White bars indicate 5=- and 3=-flanking regions included in
The black lines indicate non-protein-coding regions of TgPL1 (introns and
UTRs). The light gray boxes indicate the two exons of TgPL1 and the coding
region of CAT. X indicates XbaI sites, and A indicates ApaLI sites. (B) DNAs
from wild-type (WT) and potential knockout parasites were digested with
ApaLI or XbaI and analyzed by Southern analysis. The band sizes were as
expected for the WT and the ?TgPL1 mutant when digested with ApaLI (1.5
and 2.2 kb, respectively) and XbaI (3.7 and 4.8 kb, respectively).
NO Necessary but Not Sufficient To Kill Patatin Mutant
January 2012 Volume 80 Number 1iai.asm.org 57
bated for 24 h, at which point parasite growth was assessed as de-
The ?TgPL1 mutant does not exhibit a virulence defect in
C57BL/6 mice. To examine whether the ?TgPL1 mutant has a
defect in causing disease in mice, 10- to 12-week-old C57BL/6
of 2 ? 104tachyzoites and 100% mortality at a dose of 5 ? 104
tachyzoites in C57BL/6 mice (see Fig. S3B and S3C in the supple-
mental material). To determine if the deletion of TgPL1 affects
levels of cyst formation, cyst burdens from the surviving mice
infected with 2 ? 104tachyzoites were assessed at 25 days postin-
fection. WT- and ?TgPL1-infected mice both had approximately
The virulence of ?TgPL1 parasites was also examined by the oral
inoculation of in vitro-developed bradyzoite cysts, but no signifi-
whether the ?TgPL1 mutant had a growth defect relative to par-
asites that had a functional copy of the TgPL1 gene (comp) in a
comparative fitness assay. Comp parasites were used in favor of
WT parasites because they can be easily differentiated from
?TgPL1 parasites by the presence of the dihydrofolate reductase
(DHFR) marker. The ratio of ?TgPL1 to comp parasites did not
significantly change in tissue culture or in mice over the 20-day
experiment (see Fig. S3A in the supplemental material). Taken
together, these results show that the TgPL1 gene does not play a
Partially purified TgPL1 does not have PLA2activity. TgPL1
belongs to the patatin family of proteins that typically have PLA2
activity; however, TgPL1 has an S-to-G mutation at the predicted
catalytic serine. E. coli-derived TgPL1 with a C-terminal histidine
tag does not exhibit PLA2activity when incubated with a phos-
position (see Fig. S4 in the supplemental material). Surprisingly,
even when the catalytic serine is restored by site-directed mu-
tagenesis, the purified mutant TgPL1 protein does not have PLA2
activity. This lack of PLA2activity could be due to a misfolding of
the enzymatic activity of TgPL1 in T. gondii.
activated macrophages, a hemagglutinin (HA)-tagged version of
TgPL1 (TgPL1-HA) was used to aid the localization of TgPL1.
Macrophages were infected and activated as described above for
the degradation assay. Naïve and activated samples were fixed,
stained, and analyzed by immunofluorescence microscopy. In
parasite dense-granule marker GRA4 at day 1 postinfection (Fig.
macrophages for extended periods of time (5 days postinfection).
These results suggest that TgPL1 may function in the parasito-
phorous vacuole to protect parasites from nitric oxide produced
by these immune cells.
Although the production of NO is important for the host to con-
trol pathogen replication in many systems, little is known about
genes that allow pathogens to evade this host response (5). TgPL1
FIG 2 ?TgPL1 has a NO-dependent growth defect in activated but not naïve bone marrow-derived macrophages. (A to C) Wild-type (WT) parasites, ?TgPL1
parasites, and ?TgPL1 parasites complemented with TgPL1 (comp) were grown in bone marrow-derived murine macrophages with no treatment (naïve) (A),
IFA using serum from chronically infected mice. Vacuoles were first assessed for whether the parasites in each vacuole were intact or degraded. If intact, the
of ?0.01 is indicated by an asterisk.
Tobin and Knoll
iai.asm.org Infection and Immunity
of T. gondii to survive in activated macrophages (21). We have
growth defect. Finally, we have seen that TgPL1 localizes to the
parasitophorous vacuole in naïve and classically activated macro-
the mechanism of how TgPL1 protects T. gondii against degrada-
tion in activated macrophages.
The inability of exogenous NO to degrade ?TgPL1 mutant
parasites may indicate that NO is acting synergistically with other
IFN-?-inducible factors in macrophages to cause this phenotype.
For example, immunity-related GTPases are also induced by
IFN-? and have been associated with the disruption of the T.
gondii vacuole in macrophages and astrocyte cells (2, 17, 19).
These could weaken the vacuole sufficiently to allow the NO to
degrade the parasite. This possibility is unlikely, because
immunity-related GTPases (IRG) strip the parasitophorous vac-
uole from the parasite, leaving free parasites in the host cell cyto-
Alternatively, exogenous NO could have different effects than
The concentration of nitrite in the medium as measured by the
NO to which the parasite is exposed in the macrophage. This idea
is congruent with the presence of both cytosolic and membrane-
of the rest of the cell (28). In this case, dissecting the role of en-
dogenously produced NO versus other IFN-?-inducible genes is
the absence of other IFN-? genes in macrophages.
sary for the control of parasite replication in activated macro-
phages in mice, the role of peroxynitrite in this process had not
been directly assessed (6). We have eliminated the possibility that
degradation occurs because NO combines with reactive oxygen
intermediates to form peroxynitrite. This highly reactive com-
pound contributes to the killing of the fungal pathogen Candida
albicans in NO-producing macrophages (27); however, this com-
pound is less effective than NO at killing parasitic organisms such
as Leishmania major and Giardia lamblia (1, 10). Thus, it is not
surprising that it is ineffective against the T. gondii parasite.
The initial mouse studies done with CBA/J mice infected with
the TgPL1 insertional mutant showed that there was no virulence
a complete deletion in C57BL/6 mice that have high Th-1 polar-
ization, which is important for the production of IFN-? and,
hence, NO. The results of these studies showed that TgPL1 is not
essential to the parasite’s ability to cause disease during the acute
stage of infection. It is also expendable for cyst formation in an
oral infection model. This result may indicate that survival in ac-
tivated macrophages is not important for these processes or that
the ?TgPL1 mutant parasite compensates for its defect by using
alternative pathways to grow and reach distal sites of the host.
naïve macrophages, respectively, where the ?TgPL1 mutant has
no growth defect, or in dendritic cells, where growth of ?TgPL1
has not been assessed.
Unlike other patatin-like phospholipases associated with
FIG 3 Addition of an NO donor is not sufficient for the degradation phenotype. Bone marrow-derived macrophages were infected with wild-type (WT) and
?TgPL1 parasites and treated with 0 ?M (A), 50 ?M (B), 100 ?M (C), or 200 ?M (D) the NO donor DETA NONOate. Parasite growth was assessed at 24 h
postinfection as described in the legend of Fig. 1.
NO Necessary but Not Sufficient To Kill Patatin Mutant
January 2012 Volume 80 Number 1 iai.asm.org 59
pathogens, TgPL1 seems to be acting more like the plant stress
response proteins rather than cytolytic PLA2enzymes, such as
mental stresses, plant patatins change their location from within
low-pH vesicles to the cytoplasm (16). The localization of TgPL1
directly at the interface of the host-parasite interaction, even be-
fore the onslaught of NO caused by classical activation. Future
studies will investigate the levels and timing of TgPL1 that are
TgPL1 fused with dense granules to localize TgPL1 into parasito-
phorous vacuoles even without stress will allow the necessary lev-
the functional domains of TgPL1 will shed light on how T. gondii
evades the antimicrobial effects of NO.
We sincerely thank Dana Mordue for helpful conversations, Mary Pat
Craver for the construction of plasmid pBC-CAT/HPT, David Sibley for
the ?-tubulin antibody, and Jay Bangs for the use of his microscope.
This research was supported by National Institutes of Health (NIH)
National Research Service award GM072125 (C.M.T.) and by NIAID
1. Assreuy J, et al. 1994. Production of nitric oxide and superoxide by
activated macrophages and killing of Leishmania major. Eur. J. Immunol.
2. Butcher BA, et al. 2005. p47 GTPases regulate Toxoplasma gondii survival
in activated macrophages. Infect. Immun. 73:3278–3286.
3. Carruthers V, Boothroyd JC. 2007. Pulling together: an integrated model
of Toxoplasma cell invasion. Curr. Opin. Microbiol. 10:83–89.
4. Cassaing S, et al. 2000. Toxoplasma gondii secretes a calcium-
independent phospholipase A(2). Int. J. Parasitol. 30:1137–1142.
5. Chakravortty D, Hensel M. 2003. Inducible nitric oxide synthase and
control of intracellular bacterial pathogens. Microbes Infect. 5:621–627.
6. Chang HR, Pechère JC. 1989. Macrophage oxidative metabolism and
intracellular Toxoplasma gondii. Microb. Pathog. 7:37–44.
7. Denkers EY, Kim L, Butcher BA. 2003. In the belly of the beast: subver-
sion of macrophage proinflammatory signalling cascades during Toxo-
plasma gondii infection. Cell. Microbiol. 5:75–83.
A2reveals a novel topology and catalytic mechanism. Cell 97:349–360.
9. Donald RG, Carter D, Ullman B, Roos DS. 1996. Insertional tagging,
cloning, and expression of the Toxoplasma gondii hypoxanthine-
xanthine-guanine phosphoribosyltransferase gene. Use as a selectable
marker for stable transformation. J. Biol. Chem. 271:14010–14019.
10. Fernandes PD, Assreuy J. 1997. Role of nitric oxide and superoxide in
Giardia lamblia killing. Braz. J. Med. Biol. Res. 30:93–99.
11. Fohl LM, Roos DS. 2003. Fitness effects of DHFR-TS mutations associ-
ated with pyrimethamine resistance in apicomplexan parasites. Mol. Mi-
12. Hill D, Chirukandoth S, Dubey J. 2007. Biology and epidemiology of
13. Hirschberg HJ, Simons JW, Dekker N, Egmond MR. 2001. Cloning,
lipase A. Eur. J. Biochem. 268:5037–5044.
14. Kim S-K, Karasov A, Boothroyd JC. 2007. Bradyzoite-specific surface
15. La Camera S, et al. 2005. A pathogen-inducible patatin-like lipid acyl
hydrolase facilitates fungal and bacterial host colonization in Arabidopsis.
Plant J. 44:810–825.
16. Laxalt AM, Munnik T. 2002. Phospholipid signalling in plant defence.
Curr. Opin. Plant Biol. 5:332–338.
17. Ling YM, et al. 2006. Vacuolar and plasma membrane stripping and
autophagic elimination of Toxoplasma gondii in primed effector macro-
phages. J. Exp. Med. 203:2063–2071.
18. Lüder CGK, Algner M, Lang C, Bleicher N, Gross U. 2003. Reduced
plasma gondii facilitates parasite replication in activated murine macro-
phages. Int. J. Parasitol. 33:833–844.
FIG 4 TgPL1 localization change in activated macrophages. An HA-tagged version of TgPL1 was used to assess its localization in naïve macrophages at 1 day
postinfection (Day 1) and activated macrophages at either 1 day (Day 1-Act) or 5 days (Day 5-Act) postinfection. TgPL1 partially colocalizes with the
dense-granule protein GRA4, indicating that TgPL1 is outside the parasite in the parasitophorous vacuole space. The white scale bar is 5 ?m. The scales for all
panels are identical. DIC, differential interference contrast.
Tobin and Knoll
iai.asm.org Infection and Immunity
19. Melzer T, Duffy A, Weiss LM, Halonen SK. 2008. The gamma interferon Download full-text
plasma vacuolar disruption and induces parasite egression in IFN-
gamma-stimulated astrocytes. Infect. Immun. 76:4883–4894.
20. Miller CM, Boulter NR, Ikin RJ, Smith NC. 2009. The immunobiology
of the innate response to Toxoplasma gondii. Int. J. Parasitol. 39:23–39.
21. Mordue D, Sibley L. 1997. Intracellular fate of vacuoles containing Toxo-
plasma gondii is determined at the time of formation and depends on the
mechanism of entry. J. Immunol. 159:4452–4459.
22. Mordue DG, Scott-Weathers CF, Tobin CM, Knoll LJ. 2007. A patatin-
like protein protects Toxoplasma gondii from degradation in activated
macrophages. Mol. Microbiol. 63:482–496.
23. Sato H, Frank DW. 2004. ExoU is a potent intracellular phospholipase.
Mol. Microbiol. 53:1279–1290.
24. Senda K, Yoshioka H, Doke N, Kawakita K. 1996. A cytosolic phospho-
lipase A2from potato tissues appears to be patatin. Plant Cell Physiol.
25. Soldati D, Boothroyd JC. 1995. A selector of transcription initiation in
the protozoan parasite Toxoplasma gondii. Mol. Cell. Biol. 15:87–93.
26. Tobin C, Pollard A, Knoll L. 2010. Toxoplasma gondii cyst wall formation
in activated bone marrow-derived macrophages and bradyzoite condi-
tions. J. Vis. Exp. 2010:pii?2091.
27. Vazquez-Torres A, Jones-Carson J, Balish E. 1996. Peroxynitrite con-
tributes to the candidacidal activity of nitric oxide-producing macro-
phages. Infect. Immun. 64:3127–3133.
28. Vodovotz Y, Russell D, Xie QW, Bogdan C, Nathan C. 1995. Vesicle
membrane association of nitric oxide synthase in primary mouse macro-
phages. J. Immunol. 154:2914–2925.
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