Phosphatidylserine exposure by Toxoplasma gondii is fundamental to balance the immune response granting survival of the parasite and of the host.

Abstract

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS⁺ and PS⁻ subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS⁺ and lower for the PS⁻ subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS⁺ subpopulation and the total populations of tachyzoites. However, the PS⁻ subpopulation was not able to inhibit NO production. PS⁺ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS⁻ subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS⁺ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS⁻ subpopulation. Moreover, mice infected with the PS⁺ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS⁺ and PS⁻ subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth.

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Phosphatidylserine Exposure by Toxoplasma gondii Is
Fundamental to Balance the Immune Response Granting
Survival of the Parasite and of the Host
Thiago Alves Teixeira dos Santos1,2, Juliana de Arau ´jo Portes1,3, Joa ˜o Claudio Damasceno-Sa ´2, Lucio
Ayres Caldas3, Wanderley de Souza3, Renato Augusto DaMatta2, Sergio Henrique Seabra1*
1Laborato ´rio de Tecnologia em Bioquı ´mica e Microscopia (LTBM), Colegiado de Cie ˆncias Biolo ´gicas e da Sau ´de, Centro Universita ´rio Estadual da Zona Oeste (UEZO), Rio
de Janeiro, Brazil, 2Laborato ´rio de Biologia Celular e Tecidual, Centro de Biocie ˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF),
Campos dos Goytacazes, Brazil, 3Laborato ´rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı ´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
(UFRJ), Rio de Janeiro, Brazil
Abstract
Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms
of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing
secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase,
NO production inhibition and consequently persisting in these cells. Here PS+and PS2subpopulation of tachyzoites were
separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and
pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when
compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population,
intermediate for the PS+and lower for the PS2subpopulation. Production of NO by activated macrophages was inhibited
after infection with the PS+subpopulation and the total populations of tachyzoites. However, the PS2subpopulation was
not able to inhibit NO production. PS+subpopulation invaded macrophages by active penetration as indicated by tight-
fitting vacuoles, but the PS2subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles
containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional
phagocytic cell line where only the PS+tachyzoites were found inside these cells in tight-fitting vacuoles. Both
subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were
seen in the peritoneal cavity of mice infected with the PS2subpopulation. Moreover, mice infected with the PS+
subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS+and PS2
subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are
required to maintain the balance between inflammation and parasite growth.
Citation: dos Santos TAT, Portes JdA, Damasceno-Sa ´ JC, Caldas LA, de Souza W, et al. (2011) Phosphatidylserine Exposure by Toxoplasma gondii Is Fundamental
to Balance the Immune Response Granting Survival of the Parasite and of the Host. PLoS ONE 6(11): e27867. doi:10.1371/journal.pone.0027867
Editor: Emmanuel Dias-Neto, AC Camargo Cancer Hospital, Brazil
Received July 7, 2011; Accepted October 26, 2011; Published November 29, 2011
Copyright: ? 2011 dos Santos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by FAPERJ (Research Support Foundation of Rio de Janeiro) and CNPQ (National Research Council of Brazil). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: seabra@pq.cnpq.br
Introduction
Toxoplasmosis is caused by Toxoplasma gondii, an obligate
intracellular protozoan [1]. This parasite has a worldwide
distribution, in a broad host range, and is considered one of the
most successful on earth [1,2]. Although up to one third of the
world human population is infected with T. gondii [3], most
infections are asymptomatic, but severe clinical manifestations can
arise in immunocompromised individuals [1].
Although NO production by activated macrophages controls T.
gondii proliferation [4–7], the parasite regulates NO production
and can persist in activated macrophages [4,5,7–9]. Our group has
showed that approximately 50% of the total population of T. gondii
exposes phosphatidylserine (PS) on their outer leaflet of the plasma
membrane, this mechanism is involved in the inhibition of NO
production of infected activated macrophages [4]. Inhibition of
NO allows T. gondii to persist in infected macrophages [4,5,7–9]. A
reduced expression of inducible NO synthase [4,5,7,8] and
disappearance of nuclear factor kappa B (NF-kB) from the nucleus
of activated T. gondii infected macrophages occurs in a Trans-
forming Growth Factor-beta1 (TGF-b1) dependent way [4].
However, the molecular mechanism that controls these evasion
processes is not well known.
PS is a phospholipid present at the plasma membrane, which is
a major ligand involved in the uptake of apoptotic cells [10].
Phagocytosis of apoptotic cells by macrophages induces a
noninflammatory response based on the exposure of PS [11] that
leads to TGF-b1 secretion [11,12]. Due to this property, PS has
been related to the evasion mechanism of Leishmania amazonensis
from macrophages, a concept known as ‘‘apoptotic mimicry’’
[13,14]. It was demonstrated that this protozoan exposes PS and
this is involved in the internalization process, causing alternative
activation of macrophage through the induction of TGF-b1
secretion, interleukin (IL)-10 synthesis, and inhibition of NO
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production [15,16]. Similarly, trypomastigotes of Trypanosoma cruzi
exposes PS reducing iNOS expression after infection of activated
macrophages [17]. Apoptotic mimicry has also been implicated in
the entrance of the vaccinia virus into host cells [18].
The mechanism of T. gondii invasion involves the formation of a
moving junction between the parasite and the host cell plasma
membrane. The plasma membrane invaginates concomitantly
with the formation of the parasitophorous vacuole. This process is
known as active invasion [19]. However, the entrance of
tachyzoites can also occur by an internalization pathway that
involves the host cell [19,20], as recently indicated in a study using
dynasore [21], an inhibitor of the endocytic pathway [22].
Here we showed the immunopathological mechanisms behind
the interaction of isolated PS+and PS2subpopulation of T. gondii
with host cells. Morphological analyses of their interactions with
dynasore showed that PS+parasites invaded macrophages by
active penetration, while PS2parasites entered by phagocytosis.
Only the PS+subpopulation of T. gondii was able to inhibit NO of
activated macrophages. A non-professional phagocytic cell line
was actively invaded by the PS+subpopulation, but no tachyzoites
were internalized in this cell line when the PS2subpopulation was
used. Furthermore, infected mice with the separated subpopula-
tions were killed faster than the ones infected with the total
population. High levels of inflammation were found in mice
infected with the PS2subpopulation, and increased parasite
burden was present in mice infected with the PS+subpopulation,
explaining the low survival of mice infected with both subpopu-
lations. Collectively, these results indicate that the escape
mechanism of T. gondii is dependent on the exposure of PS and
suggests that both subpopulations of T. gondii are necessary for a
successful infection and survival.
Results
1. The isolation method to obtain PS+and PS2
subpopulations was efficient
The isolation method of the PS+
subpopulation was assayed by flow cytometer. This analysis
showed a clear shift to the right of the histogram indicating that
the isolated PS+subpopulation was exposing high levels of PS
(Figure 1A). A 96% purity was obtained for the PS+subpopula-
tion. In addition, the isolated PS2subpopulation did not stain with
annexin V (Figure 1B); a 98% purity was obtained.
and PS2
tachyzoite
2. The PS2subpopulation of tachyzoite entered
macrophages through phagocytosis and only the PS+
subpopulation inhibited NO of activated macrophages
Both isolated PS subpopulations were less capable of infecting
resident macrophages after 1 h when compared to the total T.
gondii population (Figure 2A and 2B). However, the PS+
subpopulation was better able to grow in resident macrophages
after 24 and 48 h when compared to the PS2subpopulation
(Figure 2B). To further understand the entrance mechanism of
both PS subpopulations in resident macrophages, dynasore was
used before and during the interaction. The entrance capacity of
the PS+subpopulation did not vary with increasing concentrations
of dynasore (Figure 2C); however, the entrance capacity of the
PS2subpopulation lowered with higher dynasore concentrations
(Figure 2D). Moreover, only the PS+subpopulation was able to
inhibit NO production after 24 and 48 h of infection of activated
macrophages (Figure 2E). As demonstrated before, the total
population inhibited NO production like the PS+subpopulation
(not shown). Furthermore, the amount of nitrite measured in the
supernatant of activated macrophages that interacted with the
PS2subpopulation was statistically equal to the amount produced
by noninfected activated macrophages (Figure 2E).
3. PS2tachyzoites were in loose-fitting vacuoles while
the PS+were in tight-fitting vacuoles
Loose-fitting vacuoles observed after the interaction of macro-
phages with the PS2subpopulation (Figures 3A, 3C, 3E and 4A)
are regarded as an indicator of phagocytosis as the entrance
mechanism of tachyzoites [19]. Interaction of macrophages with
the PS+subpopulation resulted in tight-fitting vacuoles (Figures 3B,
3D, 3F and 4B), indicating that active penetration [19] was the
main entrance mechanism in macrophages for this subpopulation.
Tight and loose-fitting vacuoles were counted by light microscopy
and interactions with the PS2subpopulations resulted in about
76% of loose-fitting vacuoles and the PS+subpopulation in 78% of
tight-fitting vacuoles.
Analysis by transmission electron microscopy of the interaction
between the non-professional phagocytic LLC-MK2 and isolated
subpopulations of T. gondii showed that the PS2parasites could not
infect the host cells (Figure 4C). However, the PS+parasites were
able to invade this cell lineage (Figure 4D) by active penetration as
judged by the tight-fitting vacuole.
4. Both PS subpopulations were able to kill mice faster
than the total population
Mice infected with PS+or PS2subpopulations of T. gondii had a
mean survival time of 5 days, while mice infected with the total
population of parasites had a mean survival time of 7 days
(Figure 5A). Immediately following the death of the mice, spleen
and liver were collected for pathological analysis. Leukocytes of
the inflammatory infiltrate and parasites were counted and cell
numbers in tissues of mice infected with the PS subpopulations
were compared to the numbers found in tissue of mice infected
with the total population. Macrophages and lymphocytes were the
only leukocyte types seen and the former the only one with
significant differences between the infections. Spleens infected with
the PS+ subpopulation presented a significant reduction (2.75 fold)
in the number of macrophages and a significant increase (2.20
fold) of tachyzoites (5B2 and 5B3). The infection with the PS2
subpopulation resulted in a significant increase of macrophages
(1.91 fold) in relation to the infection with the total population
(5B1 and 5B3). In livers, the infection with the PS+ subpopulation
decreased significantly the number of macrophages (3.00 fold) and
increased significantly the number of tachyzoites (2.67 fold) (5B5
and 5B6). The infection with the PS2 subpopulation increased
significantly the number of macrophage (1.91 fold) and decreased
significantly the number of tachyzoites (2.00 fold) (5 B4 and 5B6).
Discussion
Toxoplasma gondii is an obligate intracellular parasitic protozoan
that causes toxoplasmosis. The success of the infection by this
parasite depends on the evasion mechanisms against the host’s
immune response. However, the parasite population must be
controlled to avoid host death, ensuring parasite survival and
subsequent passage to the next generation of the host. The balance
between parasite evasion mechanism and host control of the
parasite is not understood. In studies with L. amazonensis it has been
reported that exposure of PS on the surface of the parasites is one
of the main mechanisms of inhibition of the immune response
[15,16]; leading to the concept of ‘‘apoptotic mimicry’’ [13,14].
When a cell initiates the apoptotic process, PS on the outer
extracellular leaflet of the plasma membrane signals the inhibition
of the inflammatory response of macrophages [11,23]. In the quest
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to better understand the behavior of the infection caused by the
PS+and PS2subpopulations of T. gondii, interactions were
performed in vitro and in vivo after the isolation of these
subpopulations by magnetic separation. Flow cytometry assured
the efficacy of PS subpopulations isolation.
A reduced infection index in resident macrophages was
observed when both PS subpopulations were used to infect these
cells compared to the total T. gondii population. To better
understand this result, analyses of the size of the vacuoles
containing tachyzoites were performed by light, scanning and
transmission electron microscopy, through which we could
observe that PS2parasites were inside loose-fitting vacuoles and
PS+parasites inside tight-fitting vacuoles. In addition, interaction
between isolated subpopulations and the non-professional phago-
cytic cell line, LLC-MK2, showed that only PS+parasites were
internalized. It has been demonstrated that tachyzoites that infect
the host cells by active penetration end up in tight-fitting vacuole,
whereas the ones that enter through phagocytosis are found in
loose-fitting vacuole [19]. Thus, our results indicate that PS2
subpopulation enter macrophages through a phagocytic mecha-
nism. On the other hand, the PS+subpopulation was found in
macrophages and in the LLC-MK2 cell line in tight-fitting
vacuoles. Because this type of vacuoles is an indicator of active
penetration [19], it is reasonable to conclude that only the PS+
Figure 1. Phosphatidylserine (PS)+and PS2subpopulations of Toxoplasma gondii were magnetically separated and analyzed by flow
cytometry after annexin-V staining. (A) PS+subpopulation of T. gondii. (B) PS2subpopulation of T. gondii. The black lines represent control
parasites without Annexin – V and the gray lines refer to the isolated subpopulations. Results from one representative experiment out of five.
doi:10.1371/journal.pone.0027867.g001
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tachyzoite were able to use this entrance mechanism to invade
host cells. Furthermore, this difference in entrance mechanism
between both PS subpopulations and the fact that the LLC-MK2
cell line is not a professional phagocyte, explains why PS2
tachyzoites were not found inside this cell line.
Dynasore has been described as an inhibitor of the endocytic
pathway because it inhibits dynamin [22]. Dynamin has recently
been implicated in actin dynamics [24], thus, its use can alter all
cell processes that involve this cytoskeleton filament, including
phagocytosis. Moreover, phagocytosis also depends on the fusion
of endomembrane compartments to the plasma membrane to
form the phagosome, a process known as ‘‘focal exocytosis’’ [25].
Because dynamin is necessary for the fusion and scission of vesicles
[26], it is sound to infer that dynasore is a potential inhibitor of
phagocytosis. The use of dynasore reduced the entrance of the
PS2subpopulation, but no alteration was found when the PS+
subpopulation was treated. This corroborates with phagocytosis as
the internalization mechanism in macrophages by the PS2
subpopulation. Furthermore, this compound was not able to
inhibit the active penetration mechanism of PS+subpopulation
indicating that dynasore only worked at host cell actin filaments.
Another interesting datum was that the infection index in
resident macrophages was higher for the total population when
compared to both PS subpopulations. This result can be explained
Figure 2. Interaction of Toxoplasma gondii total population, PS+or PS2subpopulations of tachyzoites with resident (A, B, C, D) or
activated (E) macrophages. (A) Infection index of resident macrophage after interaction with total population. (B) Infection index of resident
macrophage after interaction with PS+or PS2T. gondii subpopulations. *Significant difference from total population (p#0.05) by variance analysis
(ANOVA). #Significant difference from the PS+subpopulation (p#0.05) by variance analysis (ANOVA). (C–D) Infection index of resident macrophage
that interacted with PS+(C) or PS2(D) T. gondii subpopulation in the presence of dynasore. *Significant difference from the untreated samples
(p#0.05) by variance analysis (ANOVA). (E) Nitric oxide production of noninfected activated macrophages or after interaction with PS+or PS2
subpopulations of T. gondii. *Significant difference from the noninfected values (p#0.05) by the Student t test. Results are from at least three
independent experiments.
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by the fact that PS+and PS2subpopulations entered macrophages
by active penetration or phagocytosis, respectively. Because the
total population was a combination of both subpopulations that
enter through two distinct mechanisms, the infection index for the
total population was higher compared to each PS subpopulations,
which enter by one or the other mechanism. The infection index
in resident macrophages was higher for the PS+subpopulation
than for PS2parasites. This result indicates that only PS+
tachyzoites are capable of developing in resident macrophages.
Because the PS2subpopulation enters macrophages through
phagocytosis, this may lead to more effective killing mechanisms,
suppression of growth and lower infection index.
It has been reported that 50% of the tachyzoite population are
found in macrophages in loose-fitting vacuoles [20]. On the other
hand, it has also been reported that 80% of the tachyzoite
population that enter macrophages are found in tight-fitting
vacuoles and 20% in loose-fitting vacuoles [19]. This might be
explained by the percentage of the PS+tachyzoite that composes
the population. In our model, the PS+subpopulation varies from
50 to 80% of the total population (unpublished results). All
together the datum indicates that PS exposure may be important
to trigger the active penetration process. Nevertheless, further
studies are necessary to better understand the consequences of PS
exposure on parasite-host cell interactions.
Quantification of NO production during interaction with
isolated tachyzoite subpopulations, as well as with the total
population of T. gondii, showed that only the total and the PS+
subpopulation were able to inhibit the production of this
microbicidal agent, whereas PS2subpopulation was not able to
inhibit NO production in activated macrophages. These results
confirm the hypothesis that only parasites that expose PS can
inhibit NO production by macrophages [4]. Thus, only these
tachyzoites are able to escape this microbicidal system ensuring the
persistence of T. gondii in activated macrophages. This is a clear
confirmation that apoptotic mimicry is used by T. gondii [4,27].
Infection of mice with both subpopulations and the total
population of T. gondii showed that both isolated subpopulations
caused an early death compared to mice infected with the total
population. Light and transmission electron microscopy of liver
and spleen of infected mice revealed that mice infected by the PS+
subpopulation died through hyper parasitemia, indicating that the
immune system of these mice was not able to contain and control
the proliferation of this subpopulation. On the other hand, mice
infected with the PS2subpopulation died of a high inflammatory
Figure 3. Light and scanning electron microscopy of macrophage vacuoles with phosphatidylserine (PS)+and PS2tachyzoites of
Toxoplasma gondii. (A), (C) and (E) Macrophage vacuoles after interaction with PS2subpopulation of T. gondii. Note the loose-fitting vacuole around
the parasite caused by phagocytosis entrance. (B), (D) and (F) Macrophage vacuoles after interaction with PS+subpopulation of T. gondii. Note the
tight-fitting vacuoles around the parasite, indicating active penetration. Results are from three independent experiments.
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process probably generated by their immune systems as a response
against this subpopulation of tachyzoites. This is likely to happen
because PS2tachyzoites were not capable of inhibiting the
antiparasitic activity caused by NO, inducing a higher inflamma-
tory response not balanced by the PS+subpopulation. The
examined liver and spleen of mice infected with the total
population of T. gondii revealed the presence of mild parasitosis
and inflammatory infiltrate, suggesting that the higher survival rate
of these mice compared with the ones infected with separated PS
subpopulations was due to an equilibrium between the host
immune response and the proliferation of the T. gondii.
In conclusion, T. gondii of the RH strain can be divided into two
subpopulations with different PS exposure capacities. These
subpopulations gain access to macrophages in two distinct
manners resulting in completely different final outcomes: survival
or death of the parasite. Both subpopulations may be necessary to
maintain a balance in the host between parasite death and growth.
This balance may be achieved by the different PS subpopulations
assuring parasite and host survival resulting in an evident
advantage to the RH strain of T. gondii.
Materials and Methods
1. Peritoneal macrophages and LLC-MK2
Peritoneal macrophages were obtained by peritoneal washing of
Swiss mice with Hank’s solution [5]. Macrophages were plated
over glass coverslips in 24-well plates. After 1 h of adherence at
37uC, cells were washed with Hank’s solution and cultured with
DMEM containing 5% FBS at 37uC in a 5% CO2atmosphere.
LLC-MK2 (ATCC number CCL-7TM) was maintained in 25 cm2
cell culture flasks bottles with DMEM containing 10% FBS. Cells
were infected with the separated PS subpopulations of tachyzoites
(see item 4) and fixed for transmission electron microscopy (item
11).
2. Ethics Statement
This study was carried out in strict accordance with the
Brazilian Law #11794/08. The animal studies protocol was
reviewed and approved by the Committee on the Ethics of Animal
Experiments of the Universidade Estadual do Norte Fluminense
(Permit Number: 98).
3. Tachyzoites of Toxoplasma gondii
Tachyzoites of T. gondii (RH strain) were maintained by
inoculation into the peritoneal cavity of mice every 2 or 3 days
[5]. After this period, a wash of the peritoneal cavity was
performed with Hank’s solution followed by centrifugation at
100 g for 5 min. The collected supernatant was centrifuged at
1000 g for 10 min. Parasites were resuspended in DMEM and
counted in a Neubauer chamber.
4. Isolation of PS+and PS2subpopulations
Parasites were collected from infected mice as described above
and 26108tachyzoites were incubated with annexin V conjugated
to magnetic microspheres (Miltenyi Biotec). After 40 min, the cell
suspension was added to a magnetic column, retaining only the
PS+parasites; PS2parasites were eluted, collected and saved. PS+
parasites were removed from the column using standard
manufacturer’s instructions.
Figure 4. Transmission electron microscopy of vacuoles containing Toxoplasma gondii after interactions with macrophages or the
non-phagocytic cell line LLC-MK2. (A) Interaction of macrophages with PS2subpopulation of T. gondii. (B) Interaction of macrophages with PS+
subpopulation of T. gondii. (C) Interaction of LLC-MK2 with PS2subpopulation of T. gondii. (D) Interaction of LLC-MK2 with PS+subpopulation of T.
gondii. Note tight-fitting vacuoles after interaction of host cells with PS+tachyzoites; PS2tachyzoites were not able to enter LLC-MK2 cells and were
found in loose-fitting vacuoles after interaction with macrophages. Results are from two independent experiments.
doi:10.1371/journal.pone.0027867.g004
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5. Flow cytometry analysis of the subpopulations
separation method
After magnetic separation, PS+, PS2subpopulations of T. gondii
and the unseparated total population were incubated with
annexin-V - alexa 488 and propidium iodide for 1 h [4]. After
incubation, parasites were analyzed in a flow cytometer BD
Xcalibur. The analyses on histogram were performed in the
WinMDI 2.9 program for PC.
6. Activation of peritoneal macrophages and interaction
with parasites
Interactions were performed with non-activated (resident) and
macrophages activated for 24 h right after adherence. Activation
was performed with 50 U/ml of murine recombinant interferon-c
(IFN-c, Sigma) and 100 ng/ml of Escherichia coli lipopolysaccharide
(0111:B4 LPS, Sigma). The cells were washed and either PS2, PS+
or the total population of tachyzoites was added with a ratio of 10
parasites to 1 macrophage for 1 and 24 h. In some experiments
macrophages were incubated with crescent mM concentrations of
dynasore for 1 h before and during the interaction [21].
7. Sample preparation for morphological analysis and
determination of the index of infection
After methanol fixation, cells were washed, stained with Giemsa
(diluted 106in distilled water), dehydrated in a series of acetone-
xylene solution, mounted on Entellan and observed under an
Figure 5. Analysis of Toxoplasma gondii infection in vivo. C57/BL6 mice were infected with PS+, PS2subpopulations or the total population of T.
gondii. (A)Survival curveof mice afterthe infection with T. gondii. Kaplan Meier analysis p=0.0273.(B)Lightmicroscopy of spleenandlivertissue of C57/
BL6 mice afterinteraction with PS+or PS2subpopulationof T. gondii. Bars=100 mm. (B1–3) Spleenimages after interactionwith the PS2(B1), PS+(B2) or
the total population of T. gondii (B3). Note the presence of inflammatory cells in B1 (arrows) and the presence of parasites in B2 and B3 (arrows). (B4–6)
Liver images after interaction with PS2(B4), PS+(B5) or the total population of T. gondii (B6). Note similar results obtained for the spleen tissue. Inset –
Transmission electron microscopy: B2, Bar=2 mm; B4, Bar=16 mm. Results are from two independent experiments with 6 animals per group.
doi:10.1371/journal.pone.0027867.g005
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optical microscope Axioplan – ZEISS. Images were captured with
an MRc5 AxioCam digital camera and processed with the
Axiovision program. The percentage of infected cells and the
number of intracellular parasites per macrophage were counted
and the infection index was obtained by multiplying both numbers
[4,5]. The number of tight-fitting or loose-fitting vacuoles
containing T. gondii was also counted. Analysis of variance
(ANOVA) was used to compare mean values; a p,0.05 was
considered significant.
8. Scanning electron microscopy
Coverslips containing adhered infected macrophages were fixed
for 1 h in 4% recently prepared formaldehyde, 2.5% glutaralde-
hyde in sodium cacodylate buffer 0.1 M, pH 7.4. Cells were
dehydrated in acetone. Cells were critical point dried and had
their upper part scraped off with adhesive tape, revealing the
internal organization of vacuoles [21,28]. After that, cells were
metalized with gold by sputtering (25 nm thick) and observed in a
JSM 6490 LQ Jeol scanning electron microscope.
9. Analysis of nitric oxide production
The production of NO was indirectly assessed by reading nitrite
in the culture supernatants by the Griess reagent. The superna-
tants were mixed at a ratio of 1:1 with the Griess reagent (1
volume of 1% Sulfanilamide in 5% phosphoric acid in deionized
water with an equal volume of 0.1% N-[1-naphthyl] ethylenedi-
amine in deionized water). After 10 minutes, the mixture was read
in an ELISA reader (540 nm) and quantification of NO
production was based on a standard curve sodium nitrite in
DMEM [29]. The Student t test was used to compare mean
values, a p,0.05 was considered significant.
10. In vivo infections
C57BL/6 mice were intraperitoneally inoculated with 16104of
the PS2, PS+subpopulation or the total population of tachyzoites.
Deaths of mice were recorded daily and a Kaplan Meier survival
curve and statistical analysis performed. Spleens and livers of
recently dead mice were collected and processed for transmission
electron microscopy.
11. Light microscopy, transmission electron microscopy
and quantification of the cell types in spleen and liver of
infected mice
LLC-MK2 and macrophages interacted with the PS2or PS+
subpopulations of T. gondii for 30 min were washed and fixed with
the same solution used for scanning electron microscopy (item 8).
Small fragments of recently obtained spleens and livers were fixed
with the same solution. Samples were washed, post-fixed with 1%
osmium tetroxide, dehydrated with acetone and embebbed in
epoxy resin. Semi-thin sections were stained with 1% toluidine
blue solution (light microscopy) and ultrathin sections (80 nm)
were stained with uranyl acetate and lead citrate (transmission
electron microscopy). Ultrathin sections were observed in a Tecnai
Spirit transmission electron microscope at 120 KV.
For quantification of the cell types found in spleen and liver of
infected mice (with the total tachyzoite population, the PS2 or the
PS+ subpopulation), alternative semi-thin sections of both tissues
were obtained from 2 infected mice that had recently died in the
groups. A total of six sections per animal from each group were
stained with 1% toluidine blue solution and cells were counted in 4
randomly chosen microscopic fields. Mean numbers of cells were
compared between tissues from mice infected with the total
population and the PS subpopulations. The Student t test was used
to compare significant differences (p,0.05) of the mean values.
Acknowledgments
The authors thank Francisco Medros Junior and Eliandro Lima for
technical assistance and Andre `a C. Ce ´sar for reviewing the manuscript.
Author Contributions
Conceived and designed the experiments: TATS LAC WS RAD SHS.
Performed the experiments: TATS JAP JCDS LAC WS RAD SHS.
Analyzed the data: TATS JAP JCDS LAC WS RAD SHS. Contributed
reagents/materials/analysis tools: WS RAD SHS. Wrote the paper: TATS
LAC WS RAD SHS.
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