1 Department of Immunology and Infectious Diseases, Harvard School of Public
Health, Boston, MA 02115, USA
2 Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess
Medical Center, Boston, MA 02115, USA
3 Institut fuer Biochemie, Freie Universitaet Berlin, Berlin, Germany
* Author for correspondence (e-mail: bburleig{at}hsph.harvard.edu)
Accepted 12 May 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Phosphatidylinositol, Phagocytosis, Invasion, Trypanosoma cruzi, Maturation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The protozoan parasite, Trypanosoma cruzi, invades and replicates
within a wide variety of nucleated cell types, exhibiting tropism for
cardiomyocytes and smooth muscle in vertebrate hosts. More than a decade ago,
it was demonstrated that infective T. cruzi trypomastigotes exploit a
unique actin-independent mechanism to enter non-professional phagocytes, which
involves targeted exocytosis of host cell lysosomes at the site of parasite
attachment (Tardieux et al.,
1992). Prior to invasion, lysosomes are recruited to the plasma
membrane along microtubules in a kinesin-dependent manner
(Rodriguez et al., 1996
),
where they undergo Ca2+-dependent fusion
(Rodriguez et al., 1997
),
delivering membrane required to form the nascent parasitophorous vacuole
(Tardieux et al., 1992
). In
striking contrast to phagocytosis or other actin-driven uptake mechanisms
frequently exploited by intracellular pathogens
(Steele-Mortimer et. al.,
2000
; Coombes and Mahony,
2002
), T. cruzi entry into a variety of non-professional
phagocytic cells is significantly enhanced following disruption of the host
cell actin cytoskeleton (Tardieux et al.,
1992
). While the universality of this concept has recently been
challenged (Procipio et al., 1999;
Rosestolato et al., 2002
)
these observations suggest that depolymerization of the cortical actin
cytoskeleton (Rodriguez et al.,
1995
) in response to parasite-triggered Ca2+ transients
(Tardieux et al., 1994
;
Burleigh et al., 1997
;
Scharfstein et al., 2000
) may
enhance invasion by facilitating docking and fusion of lysosomes with the
plasma membrane (Rodriguez et al.,
1995
; Tardieux et al.,
1992
).
Activation of host cell signaling pathways by infective T. cruzi
trypomastigotes is known to play a critical regulatory role in the cell
invasion process by this pathogen (reviewed by
Burleigh and Woolsey, 2002).
Recent studies show that T. cruzi-induced activation of host cell PI
3-kinase activities correlates with efficient invasion of non-professional
phagocytes (Wilkowsky et al.,
2001
) and macrophages (Todorov
et al., 2000
). This is an intriguing observation considering that
PI 3-kinases direct a broad range of membrane trafficking events and may
therefore participate in the regulation of the lysosome-mediated entry
pathway. In addition, signaling events activated downstream of PI 3-kinase in
response to live trypomastigotes or T. cruzi surface proteins of the
trans-sialidase family (Schenkman
et al., 1991
) are capable of triggering anti-apoptotic cascades in
host cells (Chuenkova et al.,
2001
; Chuenkova and Pereira,
2000
). Thus, PI 3-kinase-dependent T. cruzi invasion,
promoted through the engagement of one or more host cell surface receptors
(Giordano et al., 1994
;
Magdesian et al., 2001
) by
heterogenous parasite surface proteins may have important implications for
intracellular survival of this pathogen in the host
(Chuenkova et al., 2001
).
In this study, we demonstrate that host cell PI 3-kinases activated by infective T. cruzi trypomastigotes early in the cell invasion process regulate lysosome-dependent parasite entry of non-professional phagocytic cells in addition to a major route of T. cruzi entry not previously recognized. We characterize this novel pathogen entry process and demonstrate that acquisition of lysosomal markers by the nascent parasitophorous vacuole is distinct from phagosome maturation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue culture and parasite maintenance
LLcMK2, L6E9 rat myoblasts and mouse embryonic fibroblasts (MEF)
were maintained in DMEM (Gibco) with 10% FBS, 1% penicillin-streptomycin, and
2 mM glutamine (DMEM-10). CHO and FcRIII-CHO cells were maintained in
-MEM (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin.
Mouse embryonic fibroblasts (MEF) were derived from embryos of
p85
-/- p85ß-/- (KO) and
p85
+/- p85ß-/-
(
HET) mice (129 x C57BL/6). The generation of the
p85ß-/- mice and establishment of the embryonic
fibroblasts from crosses with p85
+/- mice will be
described in detail elsewhere (S.M.B. and L.C.C., unpublished). Tissue
culture-derived T. cruzi trypomastigotes (Y strain) were generated by
weekly passage in confluent monolayers of LLcMK2 cells in DMEM
containing 2% FBS as described previously
(Caler et al., 1998
).
Trypomastigotes harvested from culture supernatants were washed three times in
Ringers/BSA prior to use in invasion assays as described
(Tardieux et al., 1992
).
Isolation of primary cardiomyocytes
Primary cultures of ventricular cardiomyocytes from 1-day-old
Sprague-Dawley rats (Charles River Laboratories) were prepared as described
previously (Baliga et al.,
1999). Briefly, neonatal rat pups were humanely sacrificed, their
hearts removed and placed into ice-cold Hanks buffered salt solution (HBSS)
(Worthington Biochemical). Ventricular tissue was digested overnight in a 1%
trypsin/HBSS (Sigma), washed with ice-cold HBSS and transferred into HBSS
containing a 1% collagenase solution (Worthington Biochemical). Isolated cells
were subjected to two rounds of pre-plating onto tissue culture-treated
plastic to enrich for cardiomyocytes. Cell mixtures containing 90-95%
cardiomyocytes were adjusted to the appropriate concentration in DMEM-10
containing 1% sodium pyruvate and plated on laminin-coated tissue culture
plates or coverslips.
Mammalian cell transfection
Cells were seeded onto coverslips in 3.5 cm dishes at
5x104/ml, cultivated overnight, and transfected with 1 µg
DNA by lipofection (FuGENE 6, Roche) for 3-4 hours in complete medium. Cells
were washed and incubated in complete medium for an additional 8-12 hours.
Constructs used in transfection include: Akt-PH-GFP and
Akt-PH-GFPR25C (provided by J. Downard, ICRF, UK),
PLC-PH-GFP (provided by T. Meyer, Stanford University),
2xFYVE-GFP (provided by H. Stenmark, Norwegian Radium Hospital, Oslo)
and Rab5-GFP (from M. Desjardins, U. Montreal). Myristolyated (myr)GFP was
constructed as follows: the myristolation sequence and the extreme N-terminal
end (114 nucleotides) of Akt was amplified from the myr-Akt-pcDNA3 (provided
by N. Hay, U. Chicago) by PCR using the following primers: forward:
5'-TTATGGGGAGCAGCAAGAGCAAG-3'; reverse:
5'-TGTAGCCAATAAAGGTGCCATCGTTC-3'. PCR was performed using
pfu polymerase (Strategene) with the accompanying reaction buffer
under the following conditions: denaturation at 94°C, 1 minute; annealing
at 50°C, 1 minute; elongation at 72°C, 2 minute; 40 cycles. The
resulting PCR product was ligated into the CT-GFP Fusion TOPO cloning vector
(Invitrogen) and clones were sequenced to verify orientation and continuity of
the open reading frame.
Immunofluorescence
Coverslips with adherent cells were fixed in 2% paraformaldehyde (PFA)/PBS,
washed with PBS and incubated for 10 minutes in 50 mM NH4Cl/PBS.
Antibody dilutions, incubations and washes were carried out in TBS/BSA (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1% BSA) for staining of external antigens or
permeabilized with TBS/BSA/0.1% saponin to detect intracellular antigens.
Antibody incubations were carried out for 40 minutes with extensive washes for
25 minutes following primary and secondary incubation steps. Parasite and
mammalian DNA was stained with 1.25 µg/ml DAPI (Pierce) for 3 minutes.
Coverslips were washed with PBS and mounted in 10% Mowiol (Calbiochem)
containing 2.5% DABCO (1,4-diazobicyclo-{2,2,2}-octane). Fluorescence images
were obtained with a Nikon TE-300 inverted epifluorescence microscope equipped
with an Orca-100 CCD camera (Hamamatsu) and analyzed using MetaMorph imaging
software (Universal Imaging Corporation).
Quantitative parasite invasion assay
Mammalian cells were plated onto glass coverslips in 3.5 cm2
dishes at a density of 5x104/ml and grown for 48 hours
(80% confluence). Freshly harvested T. cruzi trypomastigotes
were incubated with cells in Ringers/BSA for 15-60 minutes at 37°C.
Following incubation, the remaining extracellular parasites were removed by
washing monolayers 3 times with ice-cold PBS and cells were fixed in 2%
PFA/PBS. Immunostaining was carried out in TBS/BSA using a rabbit anti-T.
cruzi antibody to detect extracellular parasites. Parasite and mammalian
cell nuclei were visualized with 1.25 µg/ml DAPI (Pierce). The number of
intracellular parasites per 200-400 mammalian cells was determined by counting
cells on triplicate coverslips. Drug pretreatments of mammalian cells were as
follows: 20-40 nM wortmannin for 30 minutes; 2 µM cytochalasin D for 10
minutes.
Inert particle and infection pulse-chase assays
Chinese hampster ovary (CHO) cells (for infection and fibronectin-coated
inert particle assays) or FcRIII-CHO cells (for infection and
IgG-coated inert particle assays) were grown on glass coverslips to 80%
confluency (48 hours). Latex beads (3 µm; Sigma) were coated with mouse
fibronectin (0.2 mg/ml; Invitrogen) in PBS at room temperature overnight or
with rabbit IgG (1 mg/ml) in Ringers/BSA for 2 hours at room temperature.
Beads were washed and resuspended in Ringers/BSA at a concentration of
1x108 beads/ml. Latex beads or T. cruzi
trypomastigotes were incubated with cells (under various conditions of drug
pre-treatment) for 10-15 minutes at 37°C, washed extensively with
Ringers/BSA to remove uninternalized beads or parasites and further incubated
at 37°C for the indicated times. Immunofluorescence staining was carried
out on fixed cells to distinguish the remaining extracellular latex beads
(anti-fibronectin or anti-rabbit IgG) from internalized beads detected by DIC
microscopy.
Endocytic tracer internalization
L6E9 myoblasts or CHO cells grown on glass coverslips were incubated with
TR-conjugated 10,000 kDa dextran (TR-dextran) (Molecular Probes) at 0.5 mg/ml
in DMEM with 10% FBS for 3 hours at 37°C, followed by washing and a 3-hour
chase in DMEM-10 prior to infection with T. cruzi trypomastigotes.
TR-dextran-loaded cells were infected with T. cruzi and processed as
described above. For some experiments, cells were washed extensively following
parasite internalization and fresh medium added with or without 40 nM
wortmannin. Coverslips were removed at 10 minute intervals and fixed with 2%
PFA/PBS and immunofluorescence staining carried out as described above.
Supplemental movie
T. cruzi infection of Akt-PH-GFP-expressing L6E9
myoblasts
Cells seeded onto 25 mm2 glass coverslips at a density of
5x104/ml were transfected by lipofection with an Akt-PH-GFP
construct as described above. Coverslips were washed with warm Ringers/BSA and
placed in a temperature-controlled chamber held at 37°C (Nikon).
Transfected cells were located in the field using 100x objective and a
narrow band pass GFP filter (ex: 480 nm; em: 510 nm, Chroma Technologies) and
fluorescent images were collected with a Nikon TE-300 inverted fluorescence
microscope equipped with an Orca-100 CCD camera (Hamamatsu) at time-lapse
intervals of 5 seconds using a computer-controlled shutter system (Sutter)
controlled by MetaMorph imaging software (Universal Imaging). Several images
were taken prior to the addition of 5x107 T. cruzi
trypomastigotes in Ringers/BSA. Time-lapse image files were pseudocolored and
converted to a QuickTime movie (1 frame/3 second). Selected frames were saved
separately, transferred to Adobe Photoshop 6.0 (Adobe Systems) to generate the
composite Fig. 2.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasma membrane invagination defines a novel actin-independent
pathway for T. cruzi entry
Given earlier observations that T. cruzi invasion of
non-professional phagocytic cells involves targeted fusion of host cell
lysosomes with the plasma membrane
(Tardieux et al., 1992), we
predicted that Akt-PH-GFP, which is rapidly recruited to nascent T.
cruzi vacuoles, would colocalize with host cell lysosomal markers on the
parasite vacuole. Despite our ability to detect lysosome association with
T. cruzi early in the infection process
(Fig. 3A,B), overlap of lamp-1
staining with Akt-PH-GFP on the T. cruzi vacuole was not observed
(Fig. 3C,D). While several
instances of lysosome clustering near Akt-PH-GFP-positive T. cruzi
vacuoles was observed at 15 minutes post-infection,
(Fig. 3C,D), the distribution
of these two markers remained distinct in control (not shown) and infected
cells. Similarly, delivery of endocytosed fluid phase markers from Texas Red
(TR)-dextran-loaded lysosomes to Akt-PH-GFP-positive T. cruzi
vacuoles was not detected (not shown). By 60 minutes post-infection, the
majority of parasite-containing vacuoles were lamp-1-positive
(Fig. 3E,F), and minimal
residual Akt-PHGFP association with T. cruzi was observed. Together
these observations indicate that although rapid lysosome association with
invading or recently internalized trypomastigotes occurs, T. cruzi
internalization and formation of the parasitophorous vacuole is not strictly
dependent upon lysosome fusion with the plasma membrane as previously
suggested (Tardieux et al.,
1992
). Instead, localized production of
PtdInsP3/PtdIns(3,4)P2 at the host
cell membrane-parasite interface prior to and during trypomastigote invasion
suggests that invading parasites associate with host-cell plasma membrane as a
first step in an alternate mode of entry, prior to interaction with the
lysosomal compartment.
|
To examine this possibility, short-term invasion experiments (15 minutes)
were carried out using CHO cells transiently expressing myristoylated GFP
(myr-GFP) or the chimeric PtdIns(4,5)P2-binding protein,
PLC-PH-GFP, both of which associate predominantly with the plasma
membrane (Stauffer et al.,
1998
; McCabe and Berthiaume,
2001
). Similar to the interaction of trypomastigotes with
Akt-PH-GFP-enriched membranes, intimate association of invading
(Fig. 4A,B) and recently
internalized parasites (Fig.
4C) with the plasma membrane markers is observed. Intimate
association of invading trypomastigotes with the host cell plasma membrane is
suggestive of molecular interactions between T. cruzi surface ligands
and host cell receptors and is supported by the exclusion of
membrane-impermeant fluorescent dextran from the nascent T. cruzi
vacuole (data not shown). Although these events resemble actin-dependent
'zippering' processes that occur during receptor-mediated phagocytosis of
inert particles (Swanson and Baer,
1995
) or uptake of some bacterial pathogens
(Mengaud et al., 1996
;
Cossart and Lecuit, 1998
;
Isberg et al., 2000
;
Kwok et al., 2002
), several
observations indicate that host cell actin polymerization is not required for
this novel route of T. cruzi entry. First, examples of F-actin
co-localization with plasma membrane-associated T. cruzi
trypomastigotes were rarely observed (Fig.
4D-F), contrasting markedly with the intense actin staining around
latex beads during uptake into cells (Fig.
4G). In addition, enhancement of parasite internalization
following disruption of the host cell actin cytoskeleton
(Fig. 4H) is a well-documented
feature of the T. cruzi invasion process
(Tardieux et al., 1992
;
Rodriguez et al., 1995
;
Kima et al., 2000
).
Furthermore, we find that cytochalasin D pretreatment failed to inhibit the
early association of invading parasites with plasma membrane markers
(Fig. 4I). Together, these
findings provide the first demonstration that T. cruzi
trypomastigotes enter non-professional phagocytic cells by two distinct
actin-independent routes: the classic lysosome-mediated entry pathway and a
novel pathway that involves intimate association with host cell plasma
membrane markers as an initial step in cell invasion. Moreover, as
50% of
invading or recently internalized parasites were in plasma membrane-derived
vacuoles (Fig. 1D;
Fig. 4I) this novel pathway
constitutes a major route of T. cruzi entry into non-professional
phagocytic cells.
|
Transient interaction of T. cruzi vacuoles with the early
endocytic pathway is minimal
To determine if trypomastigotes that first enter cells in a plasma
membrane-derived vacuole interact with components of the endocytic pathway,
fluorescence microscopy was carried out to examine T. cruzi
co-localization with early endosomal markers
(Fig. 5). Rab5-GFP transiently
expressed in CHO cells was found to associate with T. cruzi
trypomastigotes during the entry process
(Fig. 5A) and immediately
following internalization (Fig.
5B,C). These observations, which support recent findings
(Wilkowsky et al., 2002),
suggest that rapid recruitment of Rab5 to the site of parasite entry may
direct downstream signaling/trafficking events required for T. cruzi
invasion. However, a small fraction of invading parasites were found to
associate with Rab5 during entry (<10%). Association of the PI3P-binding
2xFYVE-domain-GFP chimera (Fig.
5D,E) or endogenous EEA1 (Fig.
5F) with T. cruzi was only detected following complete
internalization of the parasite (Fig.
5E,F). These observations suggest that EEA1/2xFYVE-domain
recruitment to the T. cruzi vacuole occurs at a step subsequent to
Rab5 association and formation of the vacuole as shown previously
(Lawe et al., 2002
).
|
Quantitation of the association of intracellular parasites with EEA1 or
lamp-1 revealed that within the first 10 minutes of infection, 20% of the
T. cruzi-containing vacuoles were EEA1-positive and a similar number
were associated with lamp-1 (Fig.
5G). As intracellular infection progressed, under conditions where
no additional invasion occurred, a decrease in EEA1 association coincided with
increased lamp-1 acquisition by the T. cruzi vacuole
(Fig. 5G). To compare these
events to phagosome maturation in the same cell type, we examined the kinetics
of EEA1 and lamp-1 association with phagosomes formed following
internalization of fibronectin (FN)-coated 3 µm latex beads (LB)
(Fig. 5H). While generally
similar, important differences are noted when comparing these two processes.
Most striking is the relative ability of newly formed vacuoles to associate
with EEA1. Only
20% of T. cruzi vacuoles are EEA1-positive at
early time points of infection (10-20 minutes)
(Fig. 5F), whereas
70% of
LB phagosomes associate with this early endosomal marker shortly after
internalization (Fig. 5H).
Association with EEA1 is transient and lamp-1 is gradually acquired by both
T. cruzi vacuoles and LB phagosomes
(Fig. 5G,H). Few LB phagosomes
are lamp-1-positive at 10 minutes whereas
20% of internalized parasites
associate with lamp-1 at this early time point, probably representing the
fraction of parasites that have gained access to the host cell via the
lysosome recruitment/fusion pathway. These data indicate that a minor fraction
of the total invading parasites exploit the lysosome-mediated entry pathway
for invasion. This finding is consistent for several different cell types
examined. In CHO, L6E9 myoblasts, mouse embryonic fibroblasts, as well as
normal rat kidney fibroblasts (NRK), the cell line in which the lysosome
recruitment/fusion mechanism of T. cruzi entry was originally
described (Tardieux et al.,
1992
), the relative number of lamp-positive T. cruzi
vacuoles ranged from
15-25% at 15 minutes post-infection. Thus,
utilization of a novel alternative route of entry appears to be a common
mechanism of T. cruzi invasion of a variety of non-professional
phagocytic cells.
PI 3-kinase inhibition blocks lysosome association with invading
T. cruzi trypomastigotes
Inhibition of host cell PI 3-kinases significantly impairs T.
cruzi invasion (Fig. 6A)
as previously described (Wilkowsky et al.,
2001). Since our data clearly demonstrate that this pathogen
exploits two distinct routes to enter non-professional phagocytic cells, we
examined the effect of wortmannin pretreatment on the relative ability of
T. cruzi to immediately associate with lysosomal (lamp-1) or plasma
membrane markers. Strikingly, early lamp-1 association with invading or
intracellular parasites was completely abolished by this treatment, strongly
suggesting that host cell PI 3-kinase activity is required for the
lysosome-mediated entry pathway. In contrast, the relative ability of
trypomastigotes to associate with the plasma membrane marker PLC
-PH-GFP
was unchanged (
50% of total cell-associated parasites) in wortmannin
pretreated cells (Fig. 6B). A
fraction (
22%) of intracellular parasites is not associated with either
marker in untreated cells, which may reflect the population associated with
early endosomes (Fig. 5G). Since this fraction increases upon inhibition of PI 3-kinase activity, it
appears as though the dynamics of the T. cruzi interaction with this
compartment are significantly altered by wortmannin pretreatment, possibly
impacting parasite progression into, or out of, this compartment.
|
To specifically address the role of class I PI 3-kinases in the
lysosome-mediated entry pathway, we examined the relative ability of T.
cruzi to associate with lamp-2 in mouse embryonic fibroblasts (MEF)
lacking and ß isoforms of p85
(p85
-/-ß-/-; 'KO') as compared to
MEF heterozygous for p85
(p85
+/-ß-/-; '
HET') (S.M.B.
and L.C.C., unpublished). Although a dramatic reduction (70%) in infectivity
of p85-deficient fibroblasts was observed
(Fig. 6C), the immediate
association of T. cruzi with host cell lysosomes was not abrogated in
these fibroblasts (Fig. 6D). In
addition, the kinetics of subsequent lamp-2 acquisition by the T.
cruzi vacuole was not altered in cells lacking p85 expression
(Fig. 6D). Overall, these data
demonstrate that disruption of PI 3-kinase signaling in host cells, either
using membrane-permeant drugs or abrogation of signaling through class I PI
3-kinases, leads to a dramatic reduction (>60%) in the ability of T.
cruzi to infect host cells and indicates that both routes of T.
cruzi entry are affected by PI 3-kinase inhibition. While the
lysosome-mediated entry pathway is exquisitely sensitive to
wortmannin-pretreatment, signaling through class I PI 3-kinase is not required
for this route of entry, and suggests that other wortmannin-sensitive PI
3-kinases are involved in this process. However, the potential contribution of
an alternatively spliced form of p85 (p55
) which is still expressed in
p85-deficient fibroblasts (S.M.B. and L.C.C., unpublished) cannot be
excluded.
T. cruzi vacuole maturation is distinct from phagosome
maturation
T. cruzi-containing vacuoles formed by plasma membrane
invagination transiently associate with early endosomal markers and gradually
acquire lysosomal markers in a process that is reminiscent of phagosome
maturation (Desjardins et al.,
1994). A critical step in the phagosome maturation process is the
generation of PtdInsP3 at the phagosome membrane following
recruitment of the class III PI 3-kinase, VPS34
(Vieira et al., 2001
), such
that acquisition of lysosomal markers by the phagosome can be efficiently
blocked by the addition of PI 3-kinase inhibitors immediately following
particle uptake (Fratti et al.,
2001
). To examine whether T. cruzi vacuole
maturation is sensitive to PI 3-kinase inhibition, wortmannin was added to
cells at 10 minutes post-infection. To our surprise, wortmannin failed to
alter the kinetics of either lamp-1 (Fig.
7A) or endocytosed TR-dextran
(Fig. 7B) acquisition by the
T. cruzi vacuole, despite the ability of this treatment to block EEA1
association with the T. cruzi vacuole (not shown). Control
experiments clearly demonstrate that wortmannin effectively arrests maturation
of phagosomes formed following uptake of IgG-coated latex beads by
FcR
IIICHO cells (Fig.
7C). Similar results were obtained with LY492002 (data not shown).
These findings highlight fundamental differences in the maturation of T.
cruzi vacuoles compared to latex bead phagosomes and suggest that
acquisition of lysosomal markers by the T. cruzi vacuole may occur by
an unprecedented PI 3-kinase-independent pathway.
|
To explore this possibility, we exploited the ability of wortmannin to
block the lysosome-mediated entry pathway
(Fig. 6B), which provided a
useful approach to specifically examine the maturation of T. cruzi
vacuoles formed following internalization at the plasma membrane. In cells
pretreated with wortmannin, the kinetics of lamp-1 acquisition mimics that
observed for untreated control cells, except that the 'set point' is lower
because of the absence of lysosome-mediated entry
(Fig. 7D; pre). This
result can be interpreted in two ways: either lamp-1 acquisition by the T.
cruzi vacuole is completely refractory to PI 3-kinase inhibition or cells
are recovering from drug pretreatment. To distinguish between these
possibilities experiments were carried out in which wortmannin was added back
to pre-treated cells following parasite internalization for 10 minutes to
prevent recovery (Fig. 7D;
pre + post). Under conditions of continued PI 3-kinase inhibition,
lamp-1 acquisition by the T. cruzi vacuole was completely abolished,
indicating that an early PI 3-kinase-dependent step is clearly required for
T. cruzi vacuole maturation. However, this step becomes refractory to
inhibition within 10 minutes of parasite entry in marked contrast to phagosome
maturation as shown here and previously
(Fratti et al., 2001).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A critical question raised by our findings is how such a prominent pathway
for T. cruzi invasion escaped earlier detection. First, novel use of
plasma membrane-GFP markers coupled with time-lapse fluorescence imaging in
the present study afforded a definitive view of the early interactions of
T. cruzi trypomastigotes with the host cell plasma membrane. This
methodology presented a clear advantage over previous attempts to localize
surface-targeted membrane proteins in the nascent T. cruzi vacuole by
immunofluorescence staining (Procipio et al., 1999;
Kima et al., 2000). Secondly,
the original paper describing the unusual lysosome-mediated entry pathway
failed to report the frequency of this event
(Tardieux et al., 1992
). On
the contrary, quantitation is emphasized in our study where we consistently
find that the fraction of intracellular trypomastigotes that immediately
associates with host cell lysosomes is minimal (
20%). While we cannot
absolutely rule out the possibility that the fluorescence visualization
techniques used to detect lamp-1 and TR-dextran association with the T.
cruzi vacuole are inefficient and result in an underestimation of the
number of parasites, we are able to account for
90% of the intracellular
parasites: (50% plasma membrane; 20% lysosome; 20% EEA1) shortly after T.
cruzi internalization. Thus, our data do not support gross
under-representation of lysosome-associated parasites. Moreover, the existence
of a significant, lysosome-independent route for T. cruzi entry may
offer an explanation for previous observations showing that interference with
the function of synaptotagmin VII, an important component of
Ca2+-regulated lysosome exocytosis machinery
(Reddy et al., 2001
), resulted
in an
90% inhibition of Ca2+-dependent lysosome exocytosis
(Martinez et al., 2000
), while
the effect on T. cruzi invasion was considerably less profound
(Caler et al., 2000
).
A consistent feature of T. cruzi invasion of non-professional
phagocytic cells is facilitation of the process by disruption of the host cell
actin cytoskeleton (Tardieux et al.,
1992; Rodriguez et al.,
1995
; Kima et al.,
2000
). While the existence of an actin-associated pathway for
T. cruzi invasion of non-professional phagocytic cells has also been
suggested (Procopio et al.,
1999
; Rosestolato et al.,
2002
), we find that F-actin co-localization with invading
trypomastigotes is minimal and that envelopment of infective T. cruzi
trypomastigotes in a confined plasma membrane-derived vacuole does not require
host cell actin polymerization. On the contrary, the overall capacity for cell
invasion by T. cruzi is enhanced by cytochalasin D pretreatment, in
support of previous reports (Tardieux et
al., 1992
; Rodriguez et al.,
1995
; Kima et al.,
2000
). Time-lapse fluorescence imaging showed motile
trypomastigotes penetrating the cell at an activated region of the plasma
membrane where the lipid products of class I PI 3-kinases accumulate. As
signaling through class I PI 3-kinases has been implicated in the
reorganization of actin microfilaments
(Greenwood et al., 1998
;
Hooshmand-Rad et al., 2000
) it
is possible that this early parasite-induced signaling pathway may participate
in actin remodeling events during the cell invasion process. It has already
been established that intracellular Ca2+ transients triggered in
host cells by infective T. cruzi trypomastigotes results in a rapid,
transient disruption of the cortical actin cytoskeleton
(Rodriguez et al., 1995
) while
the role of PI 3-kinases in this process remain to be determined. Given the
dramatic reduction of T. cruzi invasion of fibroblasts and myoblasts
following inhibition of host cell Ca2+ signaling
(Tardieux et al., 1994
;
Rodriguez et al., 1995
) and
class I PI 3-kinase-dependent signaling, it is likely that both of these early
signaling pathways function as global regulators of the T. cruzi
invasion process. However, as class I PI 3-kinases are not strictly required
for lysosome-mediated entry, despite the exquisite sensitivity of this process
to wortmannin pretreatment, our findings suggest that preferential activation
of class I PI 3-kinase signaling may favor entry by the plasma membrane
invagination route and activation of a distinct wortmannin-sensitive pathway
may direct entry through the targeted lysosome exocytosis.
The molecular events regulating the T. cruzi-dependent activation
of class I PI 3-kinases have yet to be established. However, the intimate
association of invading trypomastigotes with
PtdInsP3/PtdIns (3,4)P2-enriched host
cell plasma membrane is suggestive of specific ligand-receptor interactions
which directly or indirectly trigger PI 3-kinase activation. Obvious
candidates for such signaling ligands are members of the T. cruzi
trans-sialidase/gp85 family of surface glycocproteins
(Pereira-Chioccola and Schenkman,
1999). These abundant parasite proteins have been implicated in
mediating attachment of T. cruzi to mammalian cells
(Magdesian et al., 2001
;
Ming et al., 1993
;
Pereira et al., 1996
) and
stimulation of PI 3-kinase/Akt-dependent pro-survival pathways in neuronal
cells was shown to be mediated by trans-sialidase
(Chuenkova et al., 2001
;
Chuenkova and Pereira, 2000
).
Although the complexities of the early interactions of T. cruzi
trypomastigotes with mammalian host cell interactions are still emerging, the
extent to which specific early signaling pathways are engaged by this parasite
during the infective process are likely to have important downstream
consequences with respect to regulation of host cell gene expression
(Vaena de Avalos et al., 2002
)
and intracellular survival (Chuenkova et
al., 2001
).
Transient residence within an acidic, lysosomal compartment is a
prerequisite for T. cruzi progression into the host cell cytosol
where replication takes place (Andrews et
al., 1990; Ley et al.,
1990
). This requirement is fulfilled directly when parasites enter
host cells using the lysosome recruitment pathway
(Tardieux et al., 1992
) and
indirectly following the alternate route of entry described here. Parasites
that first enter cells in a plasma membrane-derived vacuole gradually
accumulate the lysosomal markers, lamp-1 and endocytosed TR-dextran, with
kinetics similar to those observed for the maturation of latex bead
phagosomes. These results predict that acquisition of lysosomal markers by the
T. cruzi vacuole would follow the ordered process reported for the
maturation of latex bead phagosomes
(Desjardins et al., 1994
).
Nascent phagosomes undergo a series of transient fusion and fission events
with early and late endosomes, progressively assuming the properties of a
degradative lysosome (Desjardins et al.,
1994
; Duclos et al.,
2003
). Recent studies have identified the generation of
PtdInsP3 on the phagosome membrane, through the activity
of the class III PI 3-kinase, VPS34
(Vieira et al., 2001
) and the
subsequent recruitment of the Rab5 effector protein, EEA1, via its
PI3P-binding FYVE domain (Stenmark et al.,
1996
; Lawe et al.,
2000
), as critical steps in the maturation process
(Fratti et al., 2001
;
Vieira et al., 2001
).
Phagosome maturation can be effectively blocked by microinjection of
antibodies to EEA1 or VPS34 or by the addition of PI 3-kinase inhibitors to
cells shortly after phagocytic uptake
(Fratti et al., 2001
). The
importance of EEA1 in phagosome maturation is underscored in studies of the
mycobacterial vacuole. This intracellular pathogen survives in cells by
avoiding fusion with host lysosomes
(Sturgill-Koszycki et al.,
1994
; Clemens and Horwitz,
1995
), exploiting a strategy whereby EEA1 is excluded from the
mycobacterial vacuole (Fratti et al.,
2001
).
While T. cruzi-containing vacuoles associate with the early
endosomal markers, Rab5 and EEA1, early in the infective process, our data
strongly suggest that EEA1 recruitment to the parasite vacuole is not a strict
requirement for progression to a lysosomal compartment. Relatively few T.
cruzi vacuoles become EEA1 positive (20%) following infection of
myoblasts as compared to latex bead phagosomes (
70%) in the same cell
type. Furthermore, the kinetics of lamp-1 and lysosomal TR-dextran acquisition
by the T. cruzi vacuole was found to be insensitive to PI 3-kinase
inhibition by concentrations of wortmannin that disrupt EEA1 localization with
the parasite vacuole. In contrast, maturation of latex bead phagosomes in CHO
cells was significantly attenuated by the addition of wortmannin, similar to
that shown previously for macrophages
(Fratti et al., 2001
).
Additional investigation of the T. cruzi vacuole maturation process
exposed the requirement for a very early wortmannin-sensitive step that was
only effectively blocked with a combination of wortmannin pretreatment of
cells and post-treatment following parasite internalization. Further studies
are required to delineate the early molecular events that regulate T.
cruzi vacuole maturation; our data strongly suggest that this process is
clearly distinct from classical phagosome maturation with respect to
interactions with the early endosomal compartment as well as susceptibility to
PI 3-kinase inhibition. Since immediate association of invading
trypomastigotes with host cell lysosomes (i.e. the lysosome-mediated entry
pathway) is also blocked by wortmannin pretreatment, PI 3-kinases are
implicated in this entry pathway as well, although neither process appears to
rely on class I PI 3-kinases. These findings suggest that lysosome-dependent
entry and T. cruzi vacuole maturation may be mechanistically related,
temporally distinct processes.
In this study we have defined a novel pathway for T. cruzi
trypomastigotes entry and a unique vacuole maturation process that appears to
bypass the requirement for association with EEA1. While some pathogens
skillfully avoid fusion with lysosomes, entry into and escape from this acidic
compartment is a prerequisite for T. cruzi intracellular survival
(Ley et al., 1990). Thus, it
is tempting to speculate that T. cruzi-induced signaling pathways may
accelerate or even bypass the early maturation steps to ensure its successful
delivery to lysosomes. With an increased understanding of the complexities of
pathogen-specific strategies for intracellular survival
(Fratti et al., 2001
;
Coppens et al., 2000
;
Ghigo et al., 2002
), it is
becoming difficult to retain the classical concept of phagosome maturation
(Desjardins et al., 1994
).
Instead, the mechanisms by which pathogen-containing vacuoles interact with
intracellular compartments (or markers for these compartments) suggest that
intracellular pathogens navigate the maturation process as a network. The
challenge and excitement will be to elucidate the molecular mechanisms
underlying this subversive activity that holds great potential to reveal novel
regulators of the endocytic trafficking in mammalian cells.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrews, N. W., Hong, K. S., Robbins, E. S. and Nussenzweig, V. (1987). Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp. Parasitol. 64,474 -484.[Medline]
Andrews, N. W., Abrams, C. K., Slatin, S. L. and Griffiths, G. (1990). A T. cruzi-secreted protein immunologically related to the complement component C9: evidence for membrane pore-forming activity at low pH. Cell 61,1277 -1287.[CrossRef][Medline]
Araki, N., Johnson, M. T. and Swanson, J. A. (1996). A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell. Biol. 135,1249 -1260.[Abstract]
Baliga, R. R., Pimental, D. R., Zhao, Y. Y., Simmons, W. W., Marchionni, M. A., Sawyer, D. B. and Kelly, R. A. (1999). NRG-1-induced cardiomyocyte hypertrophy. Role of PI-3-kinase, p70(S6K), and MEKMAPK-RSK. Am. J. Physiol. 277,H2026 -2037.[Medline]
Burleigh, B. A. and Woolsey, A. M. (2002). Cell signalling and Trypanosoma cruzi invasion. Cell. Microbiol. 4,701 -711.[CrossRef][Medline]
Burleigh, B. A., Caler, E. V. Webster, P. and Andrews, N. W.
(1997). A cytosolic serine endopeptidase from Trypanosoma
cruzi is required for the generation of Ca2+ signaling in mammalian
cells. J. Cell Biol.
136,609
-620.
Caler, E. V., Morty, R. E., Burleigh, B. A. and Andrews, N.
W. (2000). Dual role of signaling pathways leading to
Ca2+ and cyclic AMP elevation in host cell invasion by
Trypanosoma cruzi. Infect. Immun.
68,6602
-6610.
Caler, E. V., Vaena de Avalos, S., Haynes, P. A. Andrews, N. W.
and Burleigh, B. A. (1998). Oligopeptidase B-dependent
signaling mediates host cell invasion by Trypanosoma cruzi.
EMBO J. 17,4975
-4986.
Celli, J., Olivier, M. and Finlay, B. B.
(2001). Enteropathogenic Escherichia coli mediates
antiphagocytosis through the inhibition of PI 3-kinase-dependent pathways.
EMBO J. 20,1245
-1258.
Chuenkova, M. V., Furnari, F. B., Cavenee, W. K. and Pereira, M.
A. (2001). Trypanosoma cruzi trans-sialidase: a
potent and specific survival factor for human Schwann cells by means of
phosphatidylinositol 3-kinase/Akt signaling. Proc. Natl. Acad. Sci.
USA. 98,9936
-9941.
Chuenkova, M. V. and Pereira, M. A. (2000). A
trypanosomal protein synergizes with the cytokines ciliary neurotrophic factor
and leukemia inhibitory factor to prevent apoptosis of neuronal cells.
Mol. Biol. Cell 11,1487
-1498.
Clemens, D. L. and Horwitz, M. A. (1995). Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181,257 -270.[Abstract]
Coombes, B. K. and Mahony, J. B. (2002). Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells. Cell. Microbiol. 4,447 -460.[CrossRef][Medline]
Coppens, I., Sinai, A. P. and Joiner, K. A.
(2000). Toxoplasma gondii exploits host low-density
lipoprotein receptor-mediated endocytosis for cholesterol acquisition.
J. Cell Biol. 149,167
-180.
Cossart, P. and Lecuit, M. (1998). Interactions
of Listeria monocytogenes with mammalian cells during entry and
actin-based movement: bacterial factors, cellular ligands and signaling.
EMBO J. 17,3797
-3806.
Cox, D., Tseng, C. C., Bjekic, G. and Greenberg, S.
(1999). A requirement for phosphatidylinositol 3-kinase in
pseudopod extension. J. Biol. Chem.
274,1240
-1247.
Desjardins, M., Huber, L. A., Parton, R. G. and Griffiths, G. (1994). Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J. Cell Biol. 124,677 -688.[Abstract]
Duclos, S. and Desjardins, M. (2000). Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell. Microbiol. 2, 365-377.[CrossRef][Medline]
Duclos, S., Corsini, R. and Desjardins, M.
(2003). Remodeling of endosomes during lysosome biogenesis
involves 'kiss and run' fusion events regulated by rab5. J. Cell
Sci. 116,907
-918.
Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S. and
Deretic, V. (2001). Role of phosphatidylinositol 3-kinase and
Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation
arrest. J. Cell Biol.
154,631
-644.
Ghigo, E., Capo, C., Tung, C. H., Raoult, D., Gorvel, J. P. and
Mege, J. L. (2002). Coxiella burnetii survival in
THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma
mediates its restoration and bacterial killing. J.
Immunol. 169,4488
-4495.
Giordano, R., Chammas, R., Veiga, S. S., Colli, W. and Alves, M. J. (1994). Trypanosoma cruzi binds to laminin in a carbohydrate-independent way. Braz. J. Med. Biol. Res. 27,2315 -2318.[Medline]
Greenwood, J. A., Pallero, M. A., Theibert, A. B. and
Murphy-Ullrich, J. E. (1998). Thrombospondin signaling of
focal adhesion disassembly requires activation of phosphoinositide 3-kinase.
J. Biol. Chem. 273,1755
-1763.
Hackstadt, T. (2000). Redirection of host vesicle trafficking pathways by intracellular parasites. Traffic. 1,93 -99.[CrossRef][Medline]
Hooshmand-Rad, R., Hajkova, L., Klint, P., Karlsson, R.,
Vanhaesebroeck, B., Claesson-Welsh, L. and Heldin, C. H.
(2000). The PI 3-kinase isoforms p110 and p110ß have
differential roles in PDGF- and insulin-mediated signaling. J. Cell
Sci. 113,207
-214.
Insall, R. H. and Weiner, O. D. (2001). PIP3, PIP2, and cell movement - similar messages, different meanings? Dev. Cell 6, 743-747.
Ireton, K., Payrastre, B., Chap, H., Ogawa, W., Sakaue, H.,
Kasuga, M. and Cossart, P. (1996). A role for
phosphoinositide 3-kinase in bacterial invasion.
Science 274,780
-782.
Isberg, R. R., Hamburger, Z. and Dersch, P. (2000). Signaling and invasin-promoted uptake via integrin receptors. Microbes Infect. 2, 793-801.[CrossRef][Medline]
Kima, P. E., Burleigh, B. A. and Andrews, N. W. (2000). Surface-targeted lysosomal membrane glycoprotein-1 (Lamp-1) enhances lysosome exocytosis and cell invasion by Trypanosoma cruzi. Cell Microbiol. 2, 477-486.[CrossRef][Medline]
Kjeken, R., Mousavi, S. A., Brech, A., Griffiths, G. and Berg, T. (2001). Wortmannin-sensitive trafficking steps in the endocytic pathway in rat liver endothelial cells. Biochem. J. 357,497 -503.[CrossRef][Medline]
Kwok, T., Backert, S., Schwarz, H., Berger, J. and Meyer, T.
F. (2002). Specific entry of Helicobacter pylori
into cultured gastric epithelial cells via a zipper-like mechanism.
Infect. Immun. 70,2108
-2120.
Lawe, D. C., Chawla, A., Merithew, E., Dumas, J., Carrington,
W., Fogarty, K., Lifshitz, L., Tuft, R., Lambright, D. and Corvera, S.
(2002). Sequential roles for phosphatidylinositol 3-phosphate and
Rab5 in tethering and fusion of early endosomes via their interaction with
EEA1. J. Biol. Chem.
277,8611
-8617.
Lawe, D. C., Patki, V., Heller-Harrison, R., Lambright, D. and
Corvera, S. (2000). The FYVE domain of early endosome antigen
1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding.
Critical role of this dual interaction for endosomal localization.
J. Biol. Chem. 275,3699
-3705.
Ley, V., Robbins, E. S., Nussenzweig, V. and Andrews, N. W. (1990). The exit of Trypanosoma cruzi from the phagosome is inhibited by raising the pH of acidic compartments. J. Exp. Med. 171,401 -413.[Abstract]
Li, G., D'Souza-Schorey, C., Barbieri, M. A., Roberts, R. l., Klippel, A., Williams, L. T. and Stahl, P. D. (1995). Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc. Natl. Acad. Sci. USA 92,10207 -10211.[Abstract]
Magdesian, M. H., Giordano, R., Ulrich, H., Juliano, M. A.,
Juliano, L., Schumacher, R. I., Colli, W. and Alves, M. J.
(2001). Infection by Trypanosoma cruzi. Identification
of a parasite ligand and its host cell receptor. J. Biol.
Chem. 276,19382
-19389.
Marshall, J. G., Booth, J. W., Stambolic, V., Mak, T., Balla,
T., Schreiber, A. D., Meyer, T. and Grinstein, S. (2001).
Restricted accumulation of phosphatidylinositol 3-kinase products in a
plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis.
J. Cell Biol. 153,1369
-1380.
Martinez, I., Chakrabarti, S., Hellevik, T., Morehead, J.,
Fowler, K. and Andrews, N. W. (2000). Synaptotagmin VII
regulates Ca2+-dependent exocytosis of lysosomes in fibroblasts.
J. Cell Biol. 148,1141
-1149.
McCabe, J. B. and Berthiaume, L. G. (2001).
N-terminal protein acylation confers localization to cholesterol,
sphingolipid-enriched membranes but not to lipid rafts/caveolae.
Mol. Biol. Cell 12,3601
-3617.
Mengaud, J., Ohayon, H., Gounon, P., Mege, R. M. and Cossart, P. (1996). E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell 84,923 -932.[Medline]
Ming, M., Chuenkova, M., Ortega-Barria, E. and Pereira, M. E. (1993). Mediation of Trypanosoma cruzi invasion by sialic acid on the host cell and trans-sialidase on the trypanosome. Mol. Biochem. Parasitol. 59,243 -252.[CrossRef][Medline]
Patki, V., Virbasius, J., Lane, W. S., Toh, B. H., Shpetner, H.
S. and Corvera, S. (1997). Identification of an early
endosomal protein regulated by phosphatidylinositol 3-kinase. Proc.
Natl. Acad. Sci. USA 94,7326
-7330.
Pereira, M. E., Zhang, K., Gong, Y., Herrera, E. M. and Ming, M. (1996). Invasive phenotype of Trypanosoma cruzi restricted to a population expressing trans-sialidase. Infect. Immun. 64,3884 -3892.[Abstract]
Pereira-Chioccola, V. L. and Schenkman, S. (1999). Biological role of Trypanosoma cruzi trans-sialidase. Biochem. Soc. Trans. 27,516 -518.[Medline]
Procopio, D. O., Barros, H. C. and Mortara, R. A. (1999). Actin-rich structures formed during the invasion of cultured cells by infective forms of Trypanosoma cruzi. Eur. J. Cell Biol. 78,911 -924.[Medline]
Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes. Cell. 106,157 -169.[Medline]
Rodriguez, A., Martinez, I., Chung, A., Berlot, C. H. and
Andrews, N. W. (1999). cAMP regulates
Ca2+-dependent exocytosis of lysosomes and lysosome-mediated cell
invasion by trypanosomes. J. Biol. Chem.
274,16754
-16759.
Rodriguez, A., Rioult, M. G., Ora, A. and Andrews, N. W. (1995). A trypanosome-soluble factor induces IP3 formation, intracellular Ca2+ mobilization and microfilament rearrangement in host cells. J. Cell Biol. 129,1263 -1273.[Abstract]
Rodriguez, A., Samoff, E., Rioult, M. G., Chung, A. and Andrews, N. W. (1996). Host cell invasion by trypanosomes requires lysosomes and microtubule/kinesin-mediated transport. J. Cell Biol. 134,349 -362.[Abstract]
Rodriguez, A., Webster, P., Ortego, J. and Andrews, N. W.
(1997). Lysosomes behave as Ca2+-regulated exocytic
vesicles in fibroblasts and epithelial cells. J. Cell
Biol. 137,93
-104.
Rosestolato, C. T., Dutra, M., de Souza, W. and de Carvalho, T. M. (2002). Participation of host cell actin filaments during interaction of trypomastigote forms of Trypanosoma cruzi with host cells. Cell Struct. Funct. 2, 91-98.[CrossRef]
Scharfstein, J., Schmitz, V., Morandi, V., Capella, M. M., Lima,
A. P., Morrot, A., Juliano, L. and Muller-Esterl, W. (2000).
Host cell invasion by Trypanosoma cruzi is potentiated by activation
of bradykinin B(2) receptors. J. Exp. Med.
192,1289
-1300.
Schenkman, S., Jiang, M. S., Hart, G. W. and Nussenzweig, V. (1991). A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Cell 65,1117 -1125.[Medline]
Schulte, R., Zumbihl, R., Kampik, D., Fauconnier, A. and Autenrieth, I. B. (1998). Wortmannin blocks Yersinia invasin-triggered internalization, but not interleukin-8 production by epithelial cells. Med. Microbiol. Immunol. (Berl). 187, 53-60.[CrossRef][Medline]
Scianimanico, S., Desrosiers, M., Dermine, J. F., Meresse, S., Descoteaux, A. and Desjardins, M. (1999). Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes. Cell. Microbiol. 1,19 -32.[CrossRef][Medline]
Sinai, A. P. and Joiner, K. A. (1997). Safe haven: the cell biology of nonfusogenic pathogen vacuoles. Annu. Rev. Microbiol. 51,415 -462.[CrossRef][Medline]
Stauffer, T. P., Ahn, S. and Meyer, T. (1998) Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr. Biol. 8, 343-346.[Medline]
Steele-Mortimer, O., Knodler, L. A. and Finlay, B. B. (2000). Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton. Traffic 1, 107-118.[CrossRef][Medline]
Steele-Mortimer, O., Meresse, S., Gorvel, J. P., Toh, B. H. and Finlay, B. B. (1999). Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway. Cell Microbiol. 1, 33-49.[CrossRef][Medline]
Stenmark, H., Aasland, R., Toh, B. H. and D'Arrigo, A.
(1996). Endosomal localization of the autoantigen EEA1 is
mediated by a zinc-binding FYVE finger. J. Biol. Chem.
271,24048
-24054.
Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H. L., Fok, A. K., Allen, R. D., Gluck, S. L., Heuser, J. and Russell, D. G. (1994). Lack of acidification in mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263,678 -681.[Medline]
Swanson, J. A. and Baer, S. C. (1995). Phagocytosis by zippers and triggers. Trends Cell Biol. 5,89 -93.[CrossRef]
Tardieux, I., Nathanson, M. H. and Andrews, N. W. (1994). Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients. J. Exp. Med. 179,1017 -1022.[Abstract]
Tardieux, I., Webster, P., Ravesloot, J., Boron, W., Lunn, J. A., Heuser, J. E. and Andrews, N. W. (1992). Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells. Cell 71,1117 -1130.[Medline]
Todorov, A. G., Einicker-Lamas, M., de Castro, S. L., Oliveira,
M. M. and Guilherme, A. (2000). Activation of host cell
phosphatidylinositol 3-kinases by Trypanosoma cruzi infection.
J. Biol. Chem. 275,32182
-32186.
Toker, A. and Cantley, L. C. (1997). Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387,673 -676.[CrossRef][Medline]
Vaena de Avalos, S. G., Blader, I. J., Fisher, M., Boothroyd, J.
C. and Burleigh, B. A. (2002). Immediate/early response to
Trypanosoma cruzi infection involves minimal modulation of host cell
transcription. J. Biol. Chem.
277,639
-644.
Vieira, O. V., Botelho, R. J., Rameh, L., Brachmann, S. M.,
Matsuo, T., Davidson, H. W., Schreiber, A., Backer, J. M., Cantley, L. C. and
Grinstein, S. (2001). Distinct roles of class I and class III
phosphatidylinositol 3-kinases in phagosome formation and maturation.
J. Cell Biol. 155,19
-25.
Watson, R. T. and Pessin, J. E. (2001). Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4. Exp. Cell Res. 271, 75-83.[CrossRef][Medline]
Watton, S. J. and Downward, J. (1999). Akt/PKB localisation and 3'-phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction. Curr. Biol. 9, 433-436.[CrossRef][Medline]
Wilkowsky, S. E., Barbieri, M. A., Stahl, P. and Isola, E. L. (2001). Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion. Exp. Cell Res. 264,211 -218.[CrossRef][Medline]
Wilkowsky, S. E., Barbieri, M. A., Stahl, P. and Isola, E. L. (2002). Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7. Biochem. Biophys. Res. Commun. 291,516 -521.[CrossRef][Medline]
Xu, S., Cooper, A., Sturgill-Koszycki, S., van Heyningen, T.,
Chatterjee, D., Orme, I., Allen, P. and Russell, D. G.
(1994). Intracellular trafficking in Mycobacterium
tuberculosis and Mycobacterium avium-infected macrophages.
J. Immunol. 153,2568
-2578.
Related articles in JCS: