Uncorrected Version. Published on June 29, 2009 as DOI:10.1189/jlb.0608354
Article
Protein disulfide isomerase (PDI) associates
with NADPH oxidase and is required for
phagocytosis of Leishmania chagasi
promastigotes by macrophages
Célio X. C. Santos,*,1 Beatriz S. Stolf,† Paulo V. A. Takemoto,* Angélica M. Amanso,*
Lucia R. Lopes,‡ Edna B. Souza,§ Hiro Goto,§,2 and Francisco R. M. Laurindo*,2
*Vascular Biology Laboratory, Heart Institute (InCor), and §Tropical Medicine Institute, School of Medicine, and Departments
of †Parasitology and ‡Pharmacology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
RECEIVED JUNE 14, 2008; REVISED MARCH 29, 2009; ACCEPTED APRIL 23, 2009. DOI: 10.1189/JLB.0608354
ABSTRACT
Introduction
PDI, a redox chaperone, is involved in host cell uptake of
bacteria/viruses, phagosome formation, and vascular
NADPH oxidase regulation. PDI involvement in phagocyte
infection by parasites has been poorly explored. Here,
we investigated the role of PDI in in vitro infection of J774
macrophages by amastigote and promastigote forms of
the protozoan Leishmania chagasi and assessed
whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite
redox-dependent way. Binding assays indicate that PDI
preferentially mediates parasite internalization. Bref-A, an
ER-Golgi-disrupting agent, prevented PDI concentration
in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote
phagocytosis was increased further by macrophage
overexpression of wild-type PDI and decreased upon
transfection with an antisense PDI plasmid or PDI siRNA.
At later stages of infection, PDI physically interacted with
L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or
gain-of-function experiments indicated that PMA-driven
NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the
p22phox NADPH oxidase subunit was shown by confocal
colocalization and coimmunoprecipitation. These results
provide evidence that PDI not only associates with
phagocyte NADPH oxidase but also that PDI is crucial for
efficient macrophage infection by L. chagasi. J. Leukoc.
Biol. 86: 000 – 000; 2009.
PDI is a ubiquitous, highly conserved redox chaperone enzyme
from the thioredoxin superfamily, mainly located in the ER,
where it assists redox protein-folding involving oxidation and
multiple intramolecular thiol-disulfide exchanges [1]. Despite
bearing the C-terminal ER retention sequence Lys-Asp-GluLeu, PDI has active, intracellular traffic to different cell compartments [2– 4]. In host cell surface, PDI supports internalization of some bacteria such as Chlamydia [5] and also cholera
and diphtheria toxins [6, 7]. PDI is required for Sindbis virus
infection [8] and also plays a major role in reducing HIV
gp120 protein thiols, an essential step for virus entry into lymphocytes [9, 10]. Proteomic studies in macrophages reported
that ER chaperones such as PDI take part in the formation of
the phagosome during phagocytosis in some parasite models,
including protozoans of the genus Leishmania [11–14]. However, the functional role of PDI in parasite infection remains
poorly explored. Moreover, previous work from our laboratory
revealed PDI functional/physical interaction with NADPH oxidase in vascular cells [15, 16], but so far, the occurrence and
functional implications of a similar association in phagocytes
have not been established.
Leishmania, an obligate intramacrophage parasite, leads to
distinct types of leishmaniases that affect millions of individuals worldwide [17] and more recently, has also emerged as an
important, opportunistic infection among patients bearing
HIV disease [18]. There is a large variety of Leishmania species
leading to different infection manifestations, generally classified as cutaneous (L. major, L. mexicana, and L. braziliensis),
diffuse cutaneous (L. amazonensis), mucocutaneous (L. braziliensis), and visceral (L. donovani, L. chagasi, and/or L. infantum) [19 –21]. The infection cycle in the vertebrate host is ini-
Abbreviations: Bref-A⫽Brefeldin-A, CR1⫽complement receptor 1,
d⫽density, DTNB⫽5,5⬘-dithiobis-(2-nitrobenzoate), ER⫽endoplasmic reticulum, H2O2⫽hydrogen peroxide, LmPDI⫽Leishmania PDI, PAO⫽p-phenylarsine oxide, PDI⫽protein disulfide isomerase, PDI-AS⫽PDI antisense,
PDI-S⫽PDI sense, PI⫽phagocytic index, ROS⫽reactive oxygen species,
siRNA⫽small interfering RNA
0741-5400/09/0086-0001 © Society for Leukocyte Biology
1. Correspondence: Heart Institute (InCor), University of São Paulo School
of Medicine, Vascular Biology Laboratory, Av. Eneas Carvalho Aguiar, 44Annex II, 9th Floor, CEP 05403-000; São Paulo, Brazil. E-mail: celioxcs@
yahoo.com.br
2. These authors contributed equally to this work.
Volume 86, October 2009
Journal of Leukocyte Biology
Copyright 2009 by The Society for Leukocyte Biology.
1
tiated when Leishmania promastigotes are injected into the
skin by the insect vector. In the host, the promastigote is
phagocytized by macrophages and harbored in phagosome/
phagolysosome vesicles, where the parasite is exposed to enzymes, antimicrobial peptides, and ROS generated by NADPH
oxidase activation [22, 23]. Intriguingly, despite confrontation
with such an impressive hostile environment, some parasites
are able to evade such stressful conditions and convert themselves into intracellular amastigotes, resulting in progression to
a disease process. The mechanisms of these processes may be
multiple but likely converge to the mode of regulation of macrophage NADPH oxidase, which thus, may be a key factor governing host-parasite interaction.
Macrophage NADPH oxidase comprises catalytic subunit Nox2
(or gp91phox), which heterodimerizes with p22phox at the
plasma membrane. The regulatory subunits p47phox, p67phox,
p40phox, and Rac1 stay in the cytosol in the dormant complex
configuration [24, 25]. Upon pathogen exposure and during
phagocytosis, oxidase subunits assemble at phagosome/phagolysosome vesicles, and the enzyme complex becomes activated [26,
27]. A number of mechanisms regulate all such steps [28, 29],
and particularly, the thiol redox state is well known to affect
phagocytic [30] and nonphagocytic [15, 31] NADPH oxidase. In
the present study, we explored the role of PDI in mediating parasite infection. We present evidence that PDI not only associates
with NADPH oxidase controlling its activity, but PDI is also required for efficient infection/phagocytosis of the promastigote of
L. chagasi by the macrophage.
MATERIALS AND METHODS
Chemicals
Bacitracin, DTNB, DTT, PAO, and H2O2 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Bref-A was from Calbiochem (San Diego,
CA, USA). Mouse anti-PDI mAb were from Stressgen Biotechnologies Corp.
(San Diego, CA, USA; SPA-891 and SPA-890) and the neutralizing RL90 antibody from ABR Affinity BioReagents (Golden, CO, USA; MA3-019). Goat polyclonal antibody against p22phox was from Santa Cruz Biotechnology (Santa
Cruz, CA, USA; sc-11712), and mouse polyclonal anti-Leishmania antibody was
produced in the mouse at the Tropical Medicine Institute of São Paulo, University of São Paulo (São Paulo, Brazil) [33].
Macrophage culture and Leishmania strain
Male hamsters and BALB/c mice, supplied by the Animal Breeding Facility of
University of São Paulo Medical School, were maintained in the Animal Facility of the Tropical Medicine Institute of São Paulo, University of São Paulo.
Peritoneal resident cells were obtained from the peritoneal cavity, suspended
in RPMI-1640 medium (Gibco, Grand Island, NY, USA), supplemented with
100 IU/mL penicillin, 100 ␮g/mL streptomycin, and 2% heat-inactivated
BALB/c mouse serum, and plated in 100 mm culture plates (density of 2⫻106
cells/plate). After 2 h incubation at 37°C in 5% CO2, macrophages were
washed three times with culture medium to remove nonadherent cells, grown
for 24 h, and used for experiments. Murine macrophage-like J774 cells were
grown in DMEM supplemented with 10% FBS, penicillin G (100 IU/ml), and
streptomycin (100 ␮g/ml). L. chagasi (MHOM/BR/72/strain 46) was maintained in hamsters. For the experiments, the spleens of L. chagasi-infected
hamsters were removed aseptically and the amastigotes purified as described
[33]. Amastigotes were grown initially in Novy-Nicolle-McNeal culture medium
overlayered by RPMI-1640 medium (Gibco) with 10% heat-inactivated FCS,
and the derived promastigotes were expanded, maintained in 199 medium
with 10% heat-inactivated FCS at 25°C, and used for experiments when in sta-
2 Journal of Leukocyte Biology
Volume 86, October 2009
tionary phase [33]. Of note, although amastigotes isolated from in vivo lesions
can bear some inhibitory host proteins attached to their surface, lesion-derived
amastigotes still mimic the most suitable conditions found in the course of
host infection and were therefore used in our study.
Macrophage infection
Macrophages (d⫽2⫻105 cell/well; 24-well plate) were incubated or not
with thiol inhibitors: DTNB, bacitracin, PAO, and neutralizing PDI RL-90
mAb for 1 h (37°C, 5% CO2) and subsequently washed 3⫻ with PBS. Nonopsonized parasites, fixed or not in 4% paraformaldehyde for 2 h, were
washed, diluted in complete culture medium, and added to the macrophage medium (multiplicity macrophages/parasites of 1:5 to 1:20; final incubation volume, 500 ␮l). In some cases, parasites were serum-opsonized
30 min prior to infection using mouse serum of infected animals; also,
some promastigotes were treated previously with 1 mM DTT (reducing
agent) or 1 mM H2O2 (oxidant agent) for 1 h and then washed 3⫻ with
PBS. Incubations with DTT or H2O2 induced respective lethality of 12%
and 20%, and only a living parasite number was computed for infection
experiments. Phagocytosis was assessed for 4 h, a period in which the process reaches its maximum for both stages of the parasite (data not shown).
After 4 h of infection with parasites, cells were washed with PBS, fixed with
methanol for 10 min, stained with Giemsa for 15 min, and processed for
assessment of parasitism under light microscopy (Zeiss Axiovert 200M). PI
was estimated as number of parasites/infected cell multiplied by the percentage of infected cells [34]. A total of ca. 400 macrophages/glass slide
was analyzed under light microscopy.
Binding assay
To verify if PDI inhibitors could influence promastigote binding or internalization by J774, binding assay was performed similarly, as described previously [35, 36]. Briefly, macrophages (d⫽3⫻105 cell/well; 24-well plate)
were pretreated with thiol inhibitor as above, and promastigotes were
added (10:1) for 1 h at 4°C, when excess of parasites was washed way.
Next, reaming cells were kept for an additional 4 h at 37°C, and PI was
determined as described above.
Transient transfection with cDNA plasmids or siRNA
Constructs were made in mammalian cell pcDNA3.1 expression vector-encoding sequences for catalase or for wild-type human PDI (BC010859) in a
PDI-S or PDI-AS direction (the later two, kindly provided by Dr. Mariano
Janiszewski, University of São Paulo). PDI siRNA was 5⬘-GACCUCCCCUUCAAAGUUGTT-3⬘, and control-scrambled siRNA was 5⬘-CGUACUCCUAACAGCGCUCTT-3⬘. J774 macrophages (d⫽1⫻107cell/100 mm dish) were
maintained in DMEM medium with 10% FBS in the presence of antibiotics. Macrophages grown for 24 h in six-well dishes were transiently transfected (in the presence of FBS and antibiotics) with 5 ␮g with PDI-S or
PDI-AS plasmids or with 100 pmol siRNA duplexes using LipofectamineTM
2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad,
CA, USA). Experiments were performed 24 h after transfection with cDNA.
Cells transfected with PDI siRNA were used after 48 h. Transfection efficiency was confirmed by Western blot analysis, except in the case of catalase, which was confirmed by its increased activity in total macrophage homogenates.
Assessment of ROS production
H2O2 production by J774 cells (105 cells/9-well plate), activated or not with
PMA (160 ng/ml), was measured by the Amplex Red Kit assay according to
the manufacturing company (Invitrogen). This method is based on the oxidation of 10-acetyl-3,7-dihydro-phenoxazine by peroxidase, which in the
presence of H2O2, produces Resorufin, which was monitored at 575 nm in
a microplate spectrophotometer (SpectraMaxTM 340, Molecular Devices,
Sunnyvale, CA, USA). H2O2 production was estimated using H2O2 standard
curve (␧Resorufin⫽5.4⫻104 M–1 cm–1).
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Santos et al. PDI is required for Leishmania spp. phagocytosis
Membrane fraction preparation
Cell membrane fractions were obtained by sequential centrifugation, as described previously [15]. Briefly, J774 cells or parasites grown in 100 mm
dishes were washed with cold PBS, harvested, homogenized in lysis buffer
composed of 50 mM Tris, pH 7.4, containing 0.1 mM EDTA, 0.1 mM
EGTA, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 mM PMSF, sonicated, and centrifuged (18,000 g for 15 min) to separate mitochondria and
nuclei. Supernatants were centrifuged further at 100,000 g for 1 h to obtain an enriched membrane fraction. Samples were maintained at – 80°C
until use. The J774 membrane fraction was used for Western blotting experiments. The parasite membrane fraction was used for the determination
of total thiol content, as detailed in Table 1.
Confocal microscopy
For immunofluorescence staining, cells were grown on glass slides. J774
cells were transfected as described above with PDI-S and PDI-AS plasmid.
After 24 h, cells were fixed in 4% paraformaldehyde, washed in PBS,
blocked for 1 h with 5% BSA/PBS, and incubated overnight with the following antibodies: anti-PDI (1:2000, SPA-891) and anti-p22phox (1:50,
Santa Cruz Biotechnology). Immunodetection was performed with anti-mouse
IgG Alexa Fluor-546 (1:150, Invitrogen), Alexa Fluor-488 (1:1500, Invitrogen),
or anti-goat conjugated to Rhodamine (1:100). For experiments addressing
colocalization between PDI and Leishmania staining, J774 cells were grown
on glass slides and incubated with L. chagasi (parasite:cell⫽5:1) for 30 min
or 4 h, fixed as above, and blocked with 2% BSA/PBS. Glass slides were
then incubated overnight with rabbit anti-PDI 1:200 (Stressgen Biotechnologies Corp., SPA-890) and mouse anti-Leishmania polyclonal serum 1:500 or
PBS 2% BSA (negative control). Immunodetection was performed with anti-mouse Alexa-660 (1:400, Invitrogen) and anti-rabbit Alexa 488 (1:200,
Invitrogen). Fluorescence image studies were performed using a Zeiss laserscanning confocal microscope LSM 510 META. Setting conditions were
normalized using a negative control glass slide.
Immunoprecipitation and Western analysis
Cells were lysed in Nonidet lysis buffer containing Tris/HCl (pH 7.4, 20
mM), NaCl (150 mM), Na4P2O7 (10 mM), okadaic acid (10 nM), Na3VO4
(2 mM), leupeptin (2 ␮g/ml), pepstatin (2 ␮g/ml), trypsin inhibitor (10
␮g/ml), PMSF (44 ␮g/ml), and Nonidet P-40 (1% v/v), left on ice for 10
min, and then centrifuged at 10,000 g/10 min. Immunoprecipitation experiments were performed using 500 ␮g total homogenate protein. Following preclearing with protein A/G Sepharose (Amersham Biosciences, Piscataway, NJ, USA), in the absence or presence of nonspecific IgG, proteins
were precipitated using an anti-PDI antibody (ABR Affinity BioReagents).
Immunoprecipitates, as well as total cell and parasite homogenates, were
heated (95°C, 10 min) and run on SDS-polyacrylamide gels, transfered to
nitrocellulose, and incubated with the following antibodies: anti-PDI (1:
1000, SPA-891), anti-p22phox (1:1000), and anti-Leishmania (1:500). Proteins were revealed by chemiluminescence; densitometric analysis of bands
TABLE 1. Dithiol Levels in Membrane Fraction of Different
Forms of L. chagasi
DTNB⫹
PAO
PAO/Promastigote
PAO/Amastigote
a
% Inhibitiona
100.0 ⫾ 0.0
82.0 ⫾ 4.2
94.0 ⫾ 2.1
Percent inhibition was obtained by competing assay of DTNB with
different concentrations of PAO (1, 25, 50, 100 ␮M) in the absence
or presence of parasite membrane fraction for 30 min. Slopes were
determined as ⌬432 nm/⌬(PAO, ␮M)/min. Basal thiol levels were
4.8 ⫾ 1.2 ␮mol thiol/mg parasite membrane fraction protein for
promastigotes.
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was performed with the Scion Image program (a commercial version of the
National Institutes of Health Image program).
Statistics
Values are expressed as mean ⫾ se. Statistical comparisons were performed
with Student’s t-test for unpaired data or one-way ANOVA, followed by Student-Newman-Keuls test at 0.05 significance level.
RESULTS
Pharmacological antagonism of surface/PDI thiols
modulates parasite phagocytosis by macrophages
The role of thiol/PDI in Leishmania phagocytosis of two different stages of L. chagasi—amastigote and promastigote—was
investigated using the PI (Figs. 1– 4). Macrophages were preincubated prior to infection (see Materials and Methods) with
pharmacological thiol reagents that act by different mechanisms: DTNB is a membrane-impermeable thiol oxidant that
forms mixed disulfides with thiol groups [31]; PAO forms coordination bonds via As(3⫹) with dithiols such as those from
CXXC thioredoxin motifs, including PDI [9]; and bacitracin
acts as a general PDI competitive inhibitor via redox thiols
[15, 37]. Results in Figure 1 show that phagocytosis of promastigotes but not amastigotes was inhibited by all such thiol reagents, thus suggesting different surface thiol redox profiles in
distinct parasite stages. Accordingly, average total dithiol protein content in promastigote and amastigote membrane fractions was estimated as 18% versus 6%, respectively (Table 1).
A more specific role of PDI in promastigote uptake was indicated further by significant infection index inhibition by a
known neutralizing anti-PDI mAb (Fig. 2A), which is directed
against an epitope containing active site thiols [15, 37]. PDI/
thiol antagonists also inhibited L. chagasi promastigote phagocytosis in mouse peritoneal macrophages (Fig. 2B). The identity and function of a LmPDI have been described [38, 39];
however, thiol inhibitors and anti-PDI antibody had similar
effects on infection when paraformaldehyde-fixed parasites
were used (data not shown), thus indicating that macrophage
rather than parasite PDI is likely to contribute to modulation
of infection. To check if macrophage PDI-mediated promastigote infection is preferentially related to parasite binding or
internalization, we performed a promastigote-binding assay
(Table 2). Although pretreatment with thiol reagents still has
a pronounced effect in the PI in incubations first kept at 4°C
for 1 h and then at 37°C for 4 h, infection for only at 4°C for
1 h showed a marginal effect of such a thiol inhibitor, and we
did not observe any substantial differences upon basal levels
on such an index (Table 2). Thus, at these conditions, results
indicate that binding is unaffected by PDI inhibitors, as opposed to phagocytosis.
PDI traffic to membranes during phagocytosis
During parasite infection, total macrophage PDI expression
was essentially unchanged over the time course of our experiments (Fig. 3A). This is consistent with the fact that the levels
of other ER chaperones, such as Grp78 and Grp94, were also
not changed significantly following promastigote infection in
J774 cells (unpublished data from our laboratory). This obserVolume 86, October 2009
Journal of Leukocyte Biology
3
nia antibody with no detectable cross-reactivity with J774 cells
at confocal and immunoprecipitation assays (Fig. 5). As expected, confocal analysis revealed that after 30 min of incubation, parasites were typically attached at the macrophage surface (Fig. 5B), and after 4 h of incubation, most parasites were
inside the phagocyte (Fig. 5C). Under the later condition, we
detected only a low-degree colocalization between Leishmania
and macrophage PDI (central cells, yellow staining, Figs. 5, C
and D), and PDI appeared to be present in vesicle-like structures surrounding the parasite.
To verify further possible specific molecular interaction
between parasite protein and PDI, we performed immunoprecipitation assays. Thus, PDI from promastigote-infected
J774 macrophages was immunoprecipitated and subse-
Figure 1. Effect of thiol inhibitors on phagocytic index of L. chagasi
amastigote and promastigote. J774 cells (105) were pretreated with
DTNB (750 ␮M), bacitracin (500 ␮M), or PAO (5 ␮M) for 1 h. After
washing, cells were infected with L. chagasi amastigote or promastigote
for 4 h (parasite:cell⫽10:1). PI was determined as described in Materials and Methods. Data are mean ⫾ se of three independent experiments. *, P ⬍ 0.05 (ANOVA).
vation suggests that modulatory roles of PDI on cell-surface
thiols, phagocytosis, and NADPH oxidase activity (shown below; see Figs. 7–10) could involve protein traffic from ER to
other compartments, rather than changes in PDI degradation/
synthesis. In fact, macrophage preincubation with Bref-A, an
ER-Golgi disruption agent, promoted a decrease in membrane
PDI concentration (Fig. 3B) and was accompanied by a significant decrease in L. chagasi promastigote phagocytosis
(Fig. 3B).
Interventions affecting macrophage PDI expression
levels induce parallel changes in promastigote
L. chagasi phagocytosis
To investigate more specifically the role of PDI in the phagocytic process, we transfected J774 macrophages with PDI-S or
PDI-AS plasmids, as well as with PDI siRNA (see Materials and
Methods). Over the time course of our experiments, PDI expression levels increased by 150% with PDI-S and decreased by
30% or 65% with PDI-AS or PDI siRNA, respectively (Fig. 4,
inset). In parallel, PDI-S-transfected cells showed an increase
in PI, and PDI-AS transfection, which acts as a dominant-negative, promoted a significant, ⬃40% decrease in such an index.
A similar decrease in phagocytosis was obtained after PDI silencing with siRNA (Fig. 4).
Interaction between macrophage PDI and L. chagasi
promastigote
Having collected data supporting an important functional role
of macrophage PDI on phagocytosis, we looked for more direct evidence for the interaction between PDI and L. chagasi
(Figs. 5 and 6). We selected a specific polyclonal anti-Leishma4 Journal of Leukocyte Biology
Volume 86, October 2009
Figure 2. Effect of thiol inhibitors on PI of promastigotes. (A) J774
cells (105) or mouse peritoneal macrophages (105 cells) were incubated or not with DTNB (750 ␮M), bacitracin (500 ␮M), or neutralizing anti-PDI mAb (ABR Affinity BioReagents, 1:100) and were infected
with promastigote (parasite:cell⫽10:1), similarly as described in Figure
1. PI was determined as described in Materials and Methods. (Inset)
Results when parasites were serum-opsonized (opz) prior to infection.
Of note, thiol inhibitor concentrations correspond to IC50 values obtained with J774 cells. As a control, macrophages exposed to antihistone antibody had no significant effect on parasite uptake, thus providing anti-PDI specificity (data not shown). Data are mean ⫾ se of
three independent experiments. *, P ⬍ 0.05 (ANOVA).
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Santos et al. PDI is required for Leishmania spp. phagocytosis
Figure 3. PDI expression and effect of Golgi-ER disruption on phagocytosis of L. chagasi promastigote by J774 cells. (A, upper panel) Representative Western blotting showing total PDI expression after 4 h of macrophage (105 cells/well) infection with promastigote (parasite:cell⫽5:1); lower
panel is the graphic showing the corresponded densitometry. (B and C) J774 cells (107 cells/plate) were exposed to Bref-A for 15 min and infected with parasites for 4 h (parasite:cell⫽5:1). Specifically, B, upper panel, represents Western blotting, showing that during infection, Bref-A
prevented PDI enrichment at a cell membrane fraction; lower panel is the graphic showing the respective densitometry. (C) Graphic showing that
PI was determined as described in Materials and Methods. Densitometries were performed as described in Materials and Methods, and data are
mean ⫾ se of three independent experiments. *, P ⬍ 0.05, versus control (ANOVA).
quently blotted with anti-Leishmania antibody (see Materials
and Methods). In macrophages infected with Leishmania for
4 h but not in noninfected macrophages or in macrophages
infected for only 1 h, the immunoprecipitate revealed a specific protein band of ⬃95 kDa protein (Fig. 6B, Lane 5),
clearly detected in Leishmania homogenates (Fig. 6A). Moreover, incubation between purified bovine PDI (Sigma Chemical Co., P3818) and parasites did not yield any detectable
association (Fig. 6B, Lane 3). Overall, these data indicate
that a more stable physical association between PDI and
parasite is associated with its sustained interaction with the
macrophage milieu. Additional control experiments using
yeast particles or amastigote parasites did not result in any
detectable association with PDI (data not shown). In addition, although macrophage PDI can potentially interact with
many other soluble Leishmania proteins, including its own
parasite PDI (LmPDI) [38, 39], our PDI antibody demonstrated no cross-reactivity versus Leishmania (stripping analysis of Fig. 6A; data not shown).
significantly by an average of 30% in macrophages preincubated with catalase (250 U/ml; data not shown). To gain
some insight into how redox status of the parasite affects
phagocytosis, we reduced (DTT) or oxidized (H2O2) the
promastigote previously and offered them to J774 cells, preincubated or not with thiol inhibitors (see Materials and
Methods; Fig. 7). Of note, reduced and oxidized promastigotes had a similar and significant decrease in PI versus the
native parasite. However, when macrophages were exposed
previously to thiol/PDI inhibitors, and then parasites were
offered in a reduced or oxidized state, the reduced parasites were taken up more efficiently than oxidized ones (Fig.
7; see PI ratio, R, depicted in detached line). Besides, parasites offered in the reduced state were able to overcome the
inhibition promoted by DTNB or bacitracin significantly
(Fig. 7). These results suggest that surface macrophage thiols, including PDI, may act in reducing the parasite and enable the parasite to undergo phagocytosis efficiently.
Redox status of parasite affects phagocytosis
PDI regulates NADPH oxidase activity in J774
macrophages
Promastigote phagocytosis is accompanied by NADPH oxidase-derived ROS production [22, 23, 25, 26]. We confirmed that promastigote but not amastigote phagocytosis
increases spontaneous ROS production ⬃2.7-fold versus
basal in J774 cells after infection, reaching a maximum after
2 h (data not shown). Of note, ROS production appears to
help sustain promastigote phagocytosis, as PI was decreased
Although a number of mechanisms may underlie PDI effects
on parasite phagocytosis, the thiol redox state not only affects
promastigote uptake as shown above but also, is long known to
affect phagocytic [30] and nonphagocytic [15, 31] NADPH
oxidase. To confirm more specifically whether macrophage
NADPH oxidase activity is functionally dependent on PDI, loss- or
gain-of-function strategies for PDI were used, and ROS produc-
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Volume 86, October 2009
Journal of Leukocyte Biology
5
TABLE 2. Effect of PDI Inhibitors on Binding and
Internalization of L. chagasi Promastigote by J774 Cells
Incubationa
4°C/1 h
None
DTNB (150 ␮M)
Bacitracin (100 ␮M)
4°C over 37°C/4 h
None
DTNB (150 ␮M)
Bacitracin (100 ␮M)
Phagocytic index
47.9 ⫾ 5
45.8 ⫾ 8
48.0 ⫾ 7
48.3 ⫾ 6
35.2 ⫾ 2b
30.0 ⫾ 3b
a
J774 cells were first exposed to thiol inhibitors similar to those described in Figures 1 and 2 and then incubated with promastigote
(multiplicity macrophage/parasite, 10/1) at 4°C/1 h. Parasites were
washed away and kept or not for an additional 4 h at 37°C, when PI
was estimated as described in Materials and Methods. The values correspond to the mean ⫾ se of three independent experiments. b P ⬍
0.05 versus incubation.
revealed p22phox protein, indicating mutual association
(Fig. 10, left panel). Analogous results were obtained with
reverse immunoprecipitation of p22phox and Western analysis of PDI (Fig. 10, right panel).
DISCUSSION
Figure 4. Effect of PDI expression changes on phagocytosis. J774 cells
(107) were transfected with PDI-S or PDI-AS plasmids and PDI siRNA
and its scrambled control and were infected with promastigote (parasite:cell⫽20:1) for 4 h. PI was determined as described in Materials
and Methods. Data are mean ⫾ sd of four independent experiments.
*, P ⬍ 0.05, versus control (ANOVA). (Inset) Representative Western
blotting of PDI expression in J774 cells and the respective densitometry values.
PDI has been described to play a role in host cell uptake of some
bacteria/viruses. Herein, we reported that PDI is also required
tion was measured after PMA exposure (Fig. 8). Cells overexpressing PDI with PDI-S showed a marked (60 –70% vs. control)
increase in H2O2 generation after 15 min of PMA addition.
Contrarily, transfection with PDI-AS plasmid, as well as PDI
siRNA duplex, led to significant inhibition of oxidase activity
at levels similar to cells overexpressing catalase (Fig. 8). Transfections were confirmed by Western blotting of PDI expression
(see inset of Fig. 4).
PDI associates with macrophage NADPH oxidase
We next investigated a possible physical association between
the PDI and NADPH oxidase complex, in this case, focused on
the p22phox subunit. Confocal colocalization experiments
were performed in cells transfected with PDI-S or PDI-AS plasmids and stimulated with PMA (Fig. 9). Importantly, transfection with either plasmid did not affect p22phox expression
levels (Fig. 9, C and D). The colocalization between PDI and
p22phox was evident in PDI-S-transfected cells (Fig. 9E) and
less evident after PDI silencing (PDI-AS, Fig. 9F). In unstimulated PDI-S cells, PDI also exhibited evident colocalization
with p22phox (data not shown). Further investigation was
performed with immunoprecipitation experiments. After
PMA exposure, Western analysis of PDI immunoprecipitates
6 Journal of Leukocyte Biology
Volume 86, October 2009
Figure 5. Confocal microscopy images of J774 cells only (A) or infected (B–D) with promastigotes (parasite:cellⴝ5:1) for 30 min (B) or
4 h (C and D). PDI is stained in green (A–D) and Leishmania in red
(B–D). Punctuated staining for Leishmania after 4 h of infection (central cells, C) suggests its conversion to amastigote form. Nucleic acids
were labeled with 4⬘,6-diamidino-2-phenylindole (blue).
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Santos et al. PDI is required for Leishmania spp. phagocytosis
Figure 6. To verify possible physical association between PDI and
Leishmania protein in the course of infection, we performed an immunoprecipitation assay. (A) Representative Western blotting of total
J774 macrophage (M␾) homogenates and total promastigote parasite
homogenate (duplicate of distinct batches), revealed with antibody
against Leishmania. (B) Representative immunoprecipitation (Lanes
1–5) of the indicated systems with antibody against PDI and revealed
with anti-Leishmania antibody. Lanes are: 1, Macrophage homogenate
showing no antibody cross-reactivity; 2, immunoprecipitation-negative
control; 3, promastigote parasites incubated for 1 h in the presence of
exogenous (100 ␮M) PDI in PBS; 4, macrophages infected with promastigote parasites for 1 h; 5, same as Lane 4 but infection time of
4 h. Note the corresponding Leishmania protein band ⬃95 kDa after
4 h of infection (Lane 5, *) but not after 1 h of infection (Lane 4) or
after exogenous PDI incubation with parasites (Lane 3). Of note, reverse immunoprecipitation using anti-Leishmania antibody was not successful because of poor efficiency of this antibody in the assay (data
not shown).
for efficient parasite infection, specifically with promastigote of L.
chagasi. PDI effects involve its direct interaction with parasite protein(s) and are paralleled by its concomitant modulation of redox processes. Particularly, we show that PDI associates with macrophage NADPH oxidase and contributes to sustain its activity.
These findings have considerable implications for understanding
the role of PDI and more generally, ER chaperones in phagocytosis, as well as the pathophysiology and redox regulation of macrophage NADPH oxidase and may help design novel Leishmania
therapeutic targets.
Although most eukaryotic PDI stays at the ER lumen, a considerable pool recycles to the cell surface [1– 4]. Recently, proteomic studies described PDI as part of phagolysosome formation in several parasite models including Leishmania [11–14],
but clear evidence of PDI participation in parasite infection
had not been provided. To the best of our knowledge, this is
the first description of a role of macrophage PDI as a mediator of parasite infection/phagocytosis. Importantly, although
our studies did not address the role of ER in mediating phagocytosis [40, 41], our data provide compelling evidence for at
least a supportive role of an ER chaperone in a host-parasite
interaction. This is consistent with previous data in which ER
chaperone knockdown in Dictyostelium down-regulated phagocytosis [42]. Another connection between PDI and Leishmania
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could be MHC class-I, considering that PDI was reported to exert
redox processing/selection of antigen-derived peptides [43].
The mechanisms by which host PDI was associated more
specifically to promastigote uptake and not amastigote of
L. chagasi are yet unknown but may likely involve parasite thiol
content and also, the different ability of the two parasite forms
to trigger NADPH oxidase activation in the course of the
in vitro infection (see discussion above). Additionally, both
forms of Leishmania are known to use distinct macrophage
pathways during phagocytosis. Macrophage-mediated parasite
internalization involves different cell receptors including Fc␥,
CR1/3/4, mannose, and phosphatidylserine [44]. Not much is
known about the molecular mechanism involving Leishmania
promastigote uptake by macrophages, but although the serumopsonized promastigote undergoes phagocytosis via CR, requiring RhoA GTPase [36], nonopsonized promastigotes of
L. donovani are internalized by Fc␥ and CR3 receptors associated to Rac1 and Cdc2 [35, 36]. PDI involvement in such
pathways is not known. Interestingly, though, Rac1 is a key
regulatory subunit of NADPH oxidase [24 –27], and we have
described recently that several NADPH oxidase subunits display significant interaction with PDI in a redox-dependent
manner during neutrophil oxidase activation [16]. Of note,
Leishmania spp. are presumably opsonized rapidly with serum
components, including complement soon after inoculation
into a host. In our studies, a serum-opsonized promastigote of
L. chagasi increased PI versus nonopsonized parasites significantly, similar to findings reported for L. donovani [36]. In
Figure 7. Effect of redox state of promastigote on phagocytosis under
macrophage PDI inhibition. J774 cells were preincubated or not with
thiol inhibitors as in Figure 2. In parallel, promastigote parasites were
pretreated with 1 mM DTT or 1 mM H2O2 for 1 h, as described in
Materials and Methods. Cells were incubated with parasites (parasite:
cell⫽5:1) for 4 h. PI was determined as described in Materials and
Methods. R represents ratio between average PI obtained with reduced/oxidized parasites. Data are mean ⫾ se of three independent
experiments (*, P⬍0.05, vs. control; ANOVA).
Volume 86, October 2009
Journal of Leukocyte Biology
7
Figure 8. Effect of PDI expression on J774 macrophage respiratory
burst (measured as H2O2 production). J774 cells (107) were transfected as described in Materials and Methods with PDI-S or PDI-AS
plasmids, PDI siRNA, or its scrambled control or catalase cDNA (see
Fig. 4, inset, for corresponding changes in PDI protein expression).
After 24 h of incubation, cells were counted, and 105 cells/well were
transfered to a 96-well microplate in the presence of Amplex-Red reagent. H2O2 accumulation was assessed 15 min after adding or not
160 ng/ml PMA. Data are mean ⫾ se of three independent experiments. *, P ⬍ 0.05, versus control (ANOVA).
such condition, thiol inhibitors had a similar inhibitory effect
in nonopsonized or serum-opsonized promastigote uptake by
J774 cells (Fig. 2A, inset). Additionally, as the physical association between PDI and parasite proteins may not be extremely
strong, as suggested by lack of their substantial colocalization,
and as PDI is present in vesicles surrounding parasites after
4 h of infection (Fig. 5, C and D), it is possible that PDI effects on parasite occur more indirectly, via macrophage vesicle
signaling pathways.
Our data also indicated a clear, functional, as well as spatial/physical interaction between PDI and at least the p22phox
NADPH oxidase subunit, revealed through colocalization and
coimmunoprecipitation experiments, in line with our previous
results in vascular smooth muscle cells [15]. The precise nature of this interaction in the macrophage, as well as in the
smooth muscle cell, remains under investigation. The spatial
resolution of the methods used in our study indicates that
such interaction does not necessarily imply direct PDI binding
to a specific NADPH oxidase subunit but could mean solely
the sharing of a common cellular microdomain. In any case, it
is known that PDI binding to other proteins or peptides may
not occur via thiol groups but rather via hydrophobic interactions, although thiol groups contribute to stabilize such
interaction [45]. The role of the Nox subunit thiols in the
Nox-PDI interaction is not obvious, considering that none
of Nox isoforms and its known associated regulatory subunits exhibits thioredoxin dithiol motifs. Moreover, PDI/
oxidase interaction likely occurs in the context of complex
8 Journal of Leukocyte Biology
Volume 86, October 2009
cellular mechanisms involving exit from the ER and further
traffic to post-Golgi compartments and membranes [1–5].
Regardless of the pathways involved, however, the involvement with PDI adds a novel, thiol redox-dependent component, providing potential crosstalk between NADPH oxidase
and phagocytosis.
In fact, we observed a clear role of macrophage thiols and
more specifically, of PDI in sustaining in vitro Leishmania infection/phagocytosis. The parallel inhibition by PDI antagonists
of phagocyte NADPH oxidase-mediated ROS production and
of L. chagasi promastigote phagocytosis supports a role for PDI
in redox-dependent mechanisms coupling ROS production
with thiol pathways underlying Leishmania infection. It is unclear at present whether large protein complex(es) bearing
PDI and Nox would be involved directly in the control of
phagocytosis or whether both proteins converge into this process as separate entities. In this context, the chaperone activity
of PDI might contribute to stabilize assembly of the NADPH
oxidase complex at the phagosome. Although it is not yet established whether PDI support of phagocytosis is a particular
feature of Leishmania infection, preliminary evidence from our
Figure 9. Confocal microscopy images of PDI and p22phox in J774
cells. Macrophages (107 cells) were transfected with PDI-S or PDI-AS
plasmids, similarly to experiments in Figure 4, and stimulated with
PMA. PDI is represented by green staining (Alexa Fluor 488-conjugated secondary antibody) in A and B; p22phox is depicted as red (Alexa Fluor 546-conjugated secondary antibody) staining in C and D;
and their colocalization is shown as yellow staining in E and F; blue
represents nuclear staining. The insets show PDI-p22phox colocalization superimposed to Nomarski.
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Santos et al. PDI is required for Leishmania spp. phagocytosis
Figure 10. Coimmunoprecipitation of PDI and p22phox subunits in
J774 cells. Protein extracts from J774 cells after PMA exposure (160
ng/ml) were subjected to immunoprecipitation (IP), followed by Western blotting (WB) analysis. Left, Immunoprecipitation with anti-PDI
antibody and Western analysis for p22phox in 15% SDS-PAGE. Lanes
are: 1, immunoprecipitation-negative control, and 2, total cell extract.
Right, Immunoprecipitation with anti-p22phox antibody and Western
analysis for PDI in 8% SDS-PAGE. Lanes are: 1, PDI standard, and 2,
total cell extract. The 50-kDa band is an IgG heavy chain.
laboratory indicates that PDI antagonism also inhibits yeast
(Saccharomyces cerevisae) phagocytosis by J744 cells or neutrophils (data not shown). This suggests that the redox-dependent mediation of phagocytosis by PDI may be a more general
phenomenon. An important implication of our study is that
redox modulation of the parasite may bear correlation with its
susceptibility to phagocytosis, which translates, in the case of
Leishmania, into its infectiveness. Here, particularly important
was the investigation of the comparative role of thiols in mediating promastigote rather than amastigote infection. Although
promastigote and amastigote are susceptible to ROS in vitro
[46], only the promastigote succeeds in establishing infection,
eliciting ROS derived from NADPH oxidase activity much
more efficiently than amastigote, overall more than tenfold
(data not shown; ref. [47]). As pointed out before, these differences may be related to the fact that the Leishmania amastigote has approximately threefold lower levels of dithiol protein content than the promastigote (Table 1; ref. [49]). In
addition, amastigote inhibits p22phox translocation to the
phagosome and promotes gp91phox heme degradation during
phagocytosis [32, 47]. Additionally, amastigote has detoxifying
ROS enzymes such as catalase [50].
Our results are in line with previous observations that promastigote phagocytosis seems to require a mild oxidizing environment created by NADPH oxidase-derived ROS [51]. This
idea is supported by the observed, significant decrease in promastigote phagocytic indexes in the presence of exogenous
catalase. In fact, exposure to sublethal concentrations of oxidants causes a stress response in the promastigote parasite
[52], which potentially reinforces the idea that mild oxidative
stress precedes proliferation and transformation between life
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stages of protozoans, such as Leishmania and other trypanosomatidae [49, 52, 53]. Such oxidant production might favor a
local ambient, in which signaling events related to phagocytosis, e.g., kinase activation and phosphatase inhibition, might be
favored. In parallel, our results suggest that PDI-associated
thiol reactions play a key role in the parasite-phagosome interaction. Upon thiol and/or PDI antagonism, which generally
inhibited phagocytosis, phagocytosis increased when promastigotes were reduced previously (Fig. 7). In other words,
forced parasite thiol reduction could substitute partially for
the effects of PDI. Together, these data indicate that macrophage PDI and possibly other surface thiols have a direct effect in reducing the promastigote. This redox association is
similar to that reported for diphtheria and HIV incorporation
by lymphocytes [9, 10]. Mechanisms underlying the apparent
contradictory occurrence of parasite reduction in an oxidizing
environment during phagocytosis are not entirely clear but may
involve temporal or spatial dissociation of the two processes and
are in line with the versatile catalytic proprieties of PDI. Interestingly, the effect of PAO on thiols/PDI (Fig. 2) is of particular
interest, as this arsenic compound may act similarly to antimonials, widely used in Leishmaniases chemotherapy [54, 55].
Overall, our studies support a redox nature of the phagocytosis/infection process in macrophages, in which PDI participates at least in two ways. At initial steps, by means of
thiol exchange reactions, macrophage PDI appears to promote parasite reduction, which may be important for promastigote internalization, and PDI-NADPH oxidase interaction increases ROS production, providing an intraphagosomal-oxidizing milieu, favoring promastigote infection,
although the mechanisms of such effects deserve further
study. At later stages after infection, macrophage PDI associates more stably with parasite protein(s). Those results have
considerable implications regarding a better understanding
of the pathophysiology of Leishmania infection and more
generally of the role of ER chaperones in host-parasite interaction. Moreover, these results suggest that PDI and
more generally, macrophage thiol redox-centered interventions, might be relevant, therapeutic targets against Leishmania infection.
ACKNOWLEDGMENTS
This work was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP), Conselho Nacional de Pesquisa
(CNPq)-Instituto do Milênio Redoxoma, Fundação EJ Zerbini (InCor), and Financiadora de Estudos é Projetos (FINEP). We thank
Dr. Alcione Vendramine for helpful discussion.
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KEY WORDS:
thiols 䡠 chaperone 䡠 PDI 䡠 Nox 䡠 parasite 䡠 infection
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