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Cellular Microbiology (2015)

doi:10.1111/cmi.12472

Trypanosoma cruzi extracellular amastigotes trigger


the protein kinase D1-cortactin-actin pathway during
cell invasion
Alexis Bonfim-Melo,1 Bianca Ferrarini Zanetti,2
den Ramalho Ferreira,1 Sandy Vandoninck,3
Sang Won Han,2 Johan Van Lint,3
Renato Arruda Mortara1 and Diana Bahia1,4*
1
Departamento de Microbiologia, Imunologia e
Parasitologia, Escola Paulista de Medicina,
Universidade Federal de So Paulo (EPM-UNIFESP),
So Paulo, Brazil.
2
Interdisciplinary Center for Gene Therapy
(CINTERGEN), Universidade Federal de So Paulo
(UNIFESP), So Paulo, Brazil.
3
Department of Cellular and Molecular Medicine,
University of Leuven, Leuven, Belgium.
4
Departamento de Biologia Geral, Instituto de Cincias
Biolgicas, Universidade Federal de Minas Gerais
(ICB-UFMG), Belo Horizonte, Brazil.
Summary
Trypanosoma cruzi extracellular amastigotes
(EAs) display unique mechanisms for cell invasion
that are highly dependent on host actin filaments.
Protein kinase D1 (PKD1) phosphorylates and
modulates the activity of cortactin, a key regulator
of actin dynamics. We evaluated the role of host
cortactin and PKD1 in actin filament dynamics
during HeLa cell invasion by EAs. Host cortactin,
PKD1 and actin are recruited by EAs based on
experiments in fixed and live cells by time lapse
confocal microscopy. EAs trigger PKD1 and
extracellular signal-regulated kinase 1/2 activation,
but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and noninfective epimastigotes both triggered distinct
host responses and did not recruit the molecules
studied herein. EA invasion was influenced by
depletion or overexpression of host cortactin and
PKD1, respectively, suggesting the involvement of
both proteins in this event. Collectively, these

Received 26 September, 2014; accepted 31 May, 2015. *For correspondence. E-mail dianabahia@hotmail.com; Tel. +55 31 3409-2568;
Fax +55 31 3409-2567.

results show new host cell mechanisms subverted


during EA internalization into non-phagocytic
cells.

Introduction
Trypanosoma cruzi is the flagellated parasite that causes
Chagas disease, which is endemic in Latin America
(Clayton, 2010). Extracellular amastigotes (EAs) are alternative infective forms of T. cruzi that, together with classic
infective bloodstream trypomastigotes, sustain a parasite
cycle in mammalian hosts colonizing professional and
non-professional phagocytic cells (Andrews et al., 1987;
Ley et al., 1988). Although bloodstream or metacyclic
trypomastigote cell invasion engages membrane wound
repair and lysosome exocytosis and/or phosphatidylinositol 3 kinase-dependent mechanisms (Burleigh, 2005;
Fernandes et al., 2011), EA internalization occurs through
parasite-induced actin-rich membrane expansion on the
host cell surface (Mortara, 1991; Procpio et al., 1998).
EA adhesion to Vero or HeLa host cells induces
recruitment and colocalization of actin with several host
proteins, such as extracellular matrix components,
integrins and actin-binding proteins (Procpio et al., 1999).
Cytochalasin D strongly suppresses EA internalization
(Mortara, 1991; Procpio et al., 1998). Recruitment of host
cell proteins and regulation of actin filaments are likely
mediated by the host small GTPase Rac1, but not RhoA or
Cdc42 (Fernandes and Mortara, 2004). We recently
showed that EA internalization by non-phagocytic
cells involves specific, sequential recruitment of
phosphatidylinositol phosphate markers, suggesting that
the parasites trigger phagocytosis-like machinery in nonphagocytic cells (Fernandes et al., 2013). To date, involvement of host cell kinases in T. cruzi cell invasion has been
evaluated mostly by analyzing inhibitor effects (Procpio
et al., 1998; Fernandes et al., 2006; Magdesian et al.,
2007), and the details regarding actin-dependent EA host
cell invasion signalling pathways are still poorly understood (Ferreira et al., 2012).
Cortactin is involved in actin-related cellular processes
ranging from lamellipodium protrusion and extracellular
matrix degradation to uptake of intracellular pathogens,
such as bacteria and parasites (Chen et al., 2003;

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cellular microbiology

A. Bonfim-Melo et al.

Lambotin et al., 2005; Eiseler et al., 2010; Rajadurai


et al., 2012). A key regulator of the actin cytoskeleton
(Daly, 2004), cortactin, displays a unique structure that
interacts with the Arp2/3 complex, which includes newly
formed actin filaments and a variety of actin binding/
regulating proteins (Daly, 2004). Cortactin activity is also
regulated by phosphorylation. Extracellular signalregulated kinase (ERK) phosphorylation of cortactin
activates neuronal WiskottAldrich syndrome protein
(N-WASP) dependent actin polymerization, whereas
Src phosphorylation causes an opposite effect
(Martinez-Quiles et al., 2004). During host cell invasion,
the bacterium Campylobacter jejuni induces ERK activation and phosphorylation of cortactin that in turn promotes
its association with N-WASP and culminates with uptake
of bacteria (Samuelson and Konkel, 2013). Src
phosphorylation of cortactin is important for actin polymerization during host cell invasion by other bacteria (Shigella flexneri and Staphylococcus aureus) or parasites
(Cryptosporidium parvum) (Chen et al., 2003; Bougnres
et al., 2004; Agerer et al., 2005).
Protein kinase D1 (PKD1), one of the cortactinregulating kinases, has been recently described (De
Kimpe et al., 2009). PKD1 belongs to a three-member
family of serinethreonine kinases involved in apoptosis,
immune responses, regulation of Golgi function, cell
migration and proliferation, as reviewed in Van Lint et al.
(2002) and Fu and Rubin (2011). PKD1 colocalizes with
actin and the Arp2/3 complex at the leading edge of
lamellipodia, playing a role in cellular migration through
phosphorylation of cortactin (De Kimpe et al., 2009). Cells
that express a constitutively active PKD1 mutant or a
phospho mimetic cortactin at the PKD1 phosphorylation
site display reduced lamellipodium protrusion and cell
migration (Eiseler et al., 2010). The authors suggested
that cortactin phosphorylation by PKD1 at serine 298
increases its affinity to the Arp2/3 complex and WAVE2
(WASP family verprolin-homologous protein-2), slowing
down actin dynamics and eventually inhibiting cell migration (Eiseler et al., 2010).
PKD1 has also gained increasing interest as a downstream mediator of plasma membrane receptors, such as
tyrosine kinase receptors and G protein-coupled receptors (Van Lint et al., 2002; Fu and Rubin, 2011). Herein,
we show that T. cruzi EAs activate host signalling
and induce cortactin phosphorylation, leading to actindependent host cell invasion.

of these two components has never been assessed


during actin-dependent host cell invasion by intracellular
pathogens (Bowden et al., 1999; De Kimpe et al., 2009).
To investigate whether both proteins are involved in EA
host cell invasion, we first investigated their recruitment
to actin-rich cup-like structures induced by EAs in HeLa
cells (Procpio et al., 1999; Fernandes et al., 2013).
We established HeLa cell lineages that constitutively
overexpressed green fluorescent protein (GFP)-tagged
cortactin or PKD1 by lentiviral transduction and evaluated
corecruitment with actin to EA invasion sites. Using time
lapse confocal microscopy, we observed corecruitment
and colocalization of LifeAct-RFP and cortactin-GFP to
EA adhesion sites (Fig. 1A, arrowheads). Both actin and
cortactin were recruited and colocalized from initial
contact of an EA until the end of the observation period
(Fig. 1B; Video S1, 48 min). PKD1-GFP HeLa cells were
used to investigate recruitment of kinase and actin. We
observed corecruitment and colocalization of PKD1-GFP
with LifeAct-RFP to EA adhesion sites (Fig. 2A, arrowheads). Both molecules colocalized from initial contact
throughout the internalization period that culminated with
parasite entry at around 48 min (Fig. 2B, arrowhead;
Video S2).
Time lapse experiments demonstrated that EAs were
internalized within 510 min (data not shown) and up to
40 min after initial actin recruitment (Fig. 2B, Video S2,
arrowhead). We also observed that several EAs attached
and recruited actin, but were not internalized during the
experimental period (usually ranging from 120 to 180 min;
Video S1 and S2, asterisks), a behaviour previously unnoticed (Fernandes et al., 2013).
Fixed PKD1-GFP HeLa cells previously incubated
with EAs for 1 h were used to evaluate recruitment of all
three molecules, and actin and cortactin were visualized
after immunofluorescence staining. We observed
corecruitment and spatial colocalization of all three molecules (Fig. 3) to EA membrane expansion cups at the
invasion sites. Additionally, a three-dimensional reconstruction model clearly shows accumulation of cortactin
and PKD1-GFP in the actin-rich cup-like structures (Video
S3). These results show that EAs are able to induce
recruitment of host cortactin and PKD1 along with actin to
actin-rich cup-like structures during EA internalization.

Results

Internalization of intracellular pathogens initiates with


the attachment phase that triggers host cell endocytic
machinery (Carabeo, 2011). To address this point, we
evaluated the importance of EA viability. Although heatkilled EAs (hkEA) promptly attached to HeLa cells, recruitment by these parasite forms was not detected,

EAs recruit actin, cortactin and PKD1 to actin-rich


cup-like structures
PKD1 and cortactin participation in invadopodia and
lamellipodia regulation has been reported, but association

Host cell molecules are not recruited by heat-killed


amastigotes or non-infective epimastigotes

2015 John Wiley & Sons Ltd, Cellular Microbiology

PKD1 and cortactin in T. cruzi cell invasion

Fig. 1. Host actin and cortactin colocalize at EA invasion sites. HeLa cells expressing cortactin-GFP were transfected with LifeAct-RFP.
Interactions with EAs (MOI 50:1) were evaluated by time lapse confocal microscopy at a frame interval of 8 min. Both cell types were
prestained with Hoechst (white); images show a single focal plane.
A. Corecruitment and colocalization of actin (red) and cortactin (green) to the EA adhesion site at selected time points (arrowheads).
B. Complete time lapse acquisition from (A) showing the interaction of an EA (arrowhead) with a HeLa cell. These observations are
representative of at least 40 interactions from two independent experiments. Bars = 10 m.
2015 John Wiley & Sons Ltd, Cellular Microbiology

A. Bonfim-Melo et al.

Fig. 2. Host actin and PKD1 colocalize at EA invasion sites. HeLa cells expressing PKD1-GFP were transfected with LifeAct-RFP.
Interactions with EAs (MOI 50:1) were evaluated by time lapse confocal microscopy at a frame interval of 8 min. Both cell types were
prestained with Hoechst (white); images show a single focal plane.
A. Corecruitment and colocalization of actin (red) and PKD1 (green) to the EA adhesion site at selected time points (arrowheads).
B. Complete time lapse acquisition from (A) showing the interaction of an EA (arrowhead) with a HeLa cell. These observations are
representative of at least 40 interactions from two independent experiments. Bars = 10 m.
2015 John Wiley & Sons Ltd, Cellular Microbiology

PKD1 and cortactin in T. cruzi cell invasion

Fig. 3. Host actin, cortactin and PKD1 colocalize at EA invasion sites. EAs (MOI 10:1) were added to HeLa cells expressing PKD1-GFP,
immunostained for actin and cortactin and analysed by confocal microscopy. Both cell types were stained with DAPI (white); images show a
single focal plane. Corecruitment and colocalization of actin (red), cortactin (blue) and PKD1 (green) to the EA adhesion site (arrowhead).
These observations are representative of at least 60 interactions from two independent experiments. Bar = 10 m.

suggesting that parasite viability is required for EA internalization (Fig. 4A and B), which is in agreement with
previous observations (Fernandes et al., 2013). No
recruitment of actin, cortactin or PKD1 was observed in
cells incubated with inert particles such as latex beads
or zymosan, reinforcing the notion that parasite viability
is necessary for host molecule recruitment (data not
shown). We examined whether viable, non-infective
epimastigote forms could induce recruitment. In contrast
to EAs, epimastigotes attached poorly to mammalian
cells and did not induce recruitment of host molecules
(Fig. 4C and D), even in cells co-stimulated by live EAs
(Fig. 4C and D). These results show that actin, cortactin
and PKD1 recruitment is driven by viable EAs.

EAs trigger PKD1 and ERK activation, but not Src


family kinase (SFKs), and selective site-specific
phosphorylation of cortactin
We examined the PKD1/cortactin signalling pathway
induced by EAs. HeLa cells were incubated with EAs for
various time periods and the phosphorylation status of
pathway components was probed by Western blotting
with specific anti-phospho proteins.
Incubation of HeLa cells with EAs resulted in a timedependent increase in PKD1 phosphorylation at serine
910, an autophosphorylation site, indicating its activation
(Fig. 5A). We also investigated ERK and Src activity, as
2015 John Wiley & Sons Ltd, Cellular Microbiology

these are key cortactin kinases (Martinez-Quiles et al.,


2004). EAs induced ERK activation (Fig. 5B) that led to
cortactin phosphorylation at the corresponding substrate
residue (serine 405, Fig. 5C). In contrast, parasites did
not induce SFK activation (tyrosine 416, Fig. 5D) or consequent cortactin phosphorylation (tyrosine 421 and 466,
Fig. 5E and F). These results indicate that specific host
signalling pathways can modulate recruitment and regulation of actin filaments during EA internalization.

Heat-killed EAs and non-infective forms induced distinct


kinase activation and cortactin phosphorylation patterns
Different from live EAs, hkEAs or epimastigotes did not
induce sustained PKD1 activation (Fig. 6A and F).
However, dead parasites or epimastigotes activated ERK
with a distinct profile from that observed in live EAs
(Fig. 6B and G). This ERK activation, however, did not
result in consistent alterations in cortactin phosphorylation
levels (Fig. 6C and H). Regarding SFKs, we observed
unexpected activation at earlier (hkEA) or later
(epimastigotes) time points (Fig. 6D and I). In both
instances, cortactin phosphorylation was only increased
at later time points (Fig. 6E and J). These results confirm
that parasite viability and infectivity are required to induce
a specific set of host signalling that leads to efficient
recruitment of cortactin, PKD1 and actin to EA invasion
sites necessary for parasite internalization.

A. Bonfim-Melo et al.

Fig. 4. Host actin, cortactin and PKD1 are not recruited by heat-killed parasites or epimastigote forms. HeLa cells with and without PKD1-GFP
were incubated with heat-killed EAs (A and B) or epimastigotes (C and D) (MOI 10:1), stained for cortactin and/or actin and analysed by
epifluorescence microscopy.
A,B. Arrowheads show attached hkEAs not recruiting host actin, cortactin or PKD1.
C,D. White arrowheads show no recruitment by epimastigotes and black arrowheads indicate recruitment by live EAs. These
observations are representative of at least 100 interactions from three independent experiments. DAPI (blue) stains kinetoplasts and nuclei.
Bars = 10 m.

EA invasion is modulated by expression of host


cortactin or PKD1
Given that EAs induce recruitment of cortactin and PKD1
to actin-rich cups, we investigated whether expression of
these proteins could affect EA internalization. We established HeLa cells with constitutive knockdown of cortactin
following shRNA lentiviral transduction and control cells
expressing a non-target shRNA and compared them with
non-transduced HeLa cells (Fig. S1). All cells were incubated with EAs, washed, fixed with Bouin and stained with
Giemsa. Intracellular parasites were counted under bright
field microscopy (Fernandes et al., 2013). We observed a

reduction in the rate of EA cell invasion in the shRNA


cortactin groups compared with both control groups
(Fig. 7A), suggesting that cortactin promotes EA host cell
invasion.
Taking into account that cortactin activity is tightly associated with actin cytoskeleton regulation (Daly, 2004), we
examined whether reduced cortactin expression, in addition to inhibiting EA internalization, would also decrease
actin recruitment to EA invasion sites. We incubated EAs
with HeLa cells, stained for actin and quantified recruitment under epifluorescence microscopy. Despite reduced
EA internalization by HeLa cells following cortactin knockdown, we observed no differences in EA-induced actin
2015 John Wiley & Sons Ltd, Cellular Microbiology

PKD1 and cortactin in T. cruzi cell invasion

Fig. 5. PKD1, ERK and cortactin, but not SFKs, are


phosphorylated during EA interaction. Prestarved HeLa cells
(medium without FCS) were incubated with EA (MOI 10:1) for the
indicated time points. Lysates were resolved by SDS-PAGE and
analysed by Western blotting with the following antibodies: (A)
pPKD1 S910: activated PKD1; (B) pERK T202/Y204: activated
ERK1/2; (C) pCort S405: cortactin phosphorylated by ERK1/2; (D)
pSFK Y416: activated SFKs; (E,F) pCort Y421 or Y466: cortactin
phosphorylated by SFKs. Anti-actin antibody was used as a loading
control. These observations are representative of at least five
independent experiments.

recruitment (Fig. 7B), indicating that cortactin is not


directly involved in actin polymerization during EA internalization. Interestingly, we were still able to detect
residual cortactin recruitment to EA invasion sites in
cortactin-depleted HeLa cells (Fig. S2).
More EAs were internalized when we used HeLa cells
overexpressing PKD1 tagged with GFP compared with
cytosolic GFP or non-transduced control groups (Fig. 8),
suggesting that PKD1 also promotes EA internalization.

Discussion
T. cruzi presents to mammalian cells with three different
infective forms: metacyclic trypomastigotes, bloodstream
trypomastigotes and EAs. Many studies have shown that
metacyclic and bloodstream trypomastigotes as well as
EAs possess distinct mechanisms for host cell invasion
(Mortara et al., 2005; 2008; Ferreira et al., 2012). EAs
display actin-dependent invasion in non-phagocytic cells
2015 John Wiley & Sons Ltd, Cellular Microbiology

similar to phagocytosis-driven professional phagocytes


(Fernandes et al., 2013). Despite these observations,
detailed molecular mechanisms, including involvement of
regulatory proteins, are still unknown (Ferreira et al.,
2012). We evaluated the role of the host actin regulating
protein cortactin and one of its kinases, PKD1. Both proteins play important roles in actin-related events, but have
not been studied during T. cruzi host cell interactions. We
observed that both proteins are recruited to the actin-rich
projections formed during EA internalization in fixed and
live cells, suggesting their participation in this process.
Cortactin and PKD1 are also phosphorylated during
EAhost cell interaction. PKD1 autophosphorylation at
serine 910 indicates that this protein is activated, although
we did not examine the precise mechanism of activation.
Because PKD1 translocates to the plasma membrane, it
could possibly be activated after phosphorylation by a
protein kinase C, a very ubiquitous mechanism of PKD
activation (Zugaza et al., 1996; Fu and Rubin, 2011). Both
kinases are translocated to the inner leaflet of the plasma
membrane after phospholipase C (PLC) activation,
which in turn culminates in simultaneous production of
diacylglycerol and calcium release from the endoplasmic
reticulum (ER) (Wei et al., 2009). Such a signalling
pathway would be in accordance with previous observations that showed reduced EA internalization (from G and
CL strains) in HeLa cells treated with PLC or ER-Ca2+
releasing inhibitors (Fernandes et al., 2006). Kunkel et al.
(2007) also showed robust PKD1 activation after an
increase in intracellular Ca2+ concentration in a PLCdependent, nPKC-independent manner. Therefore, a host
PLC/PKC signalling pathway could be a possible mechanism underlying PKD1 activation during EA interaction.
Cortactin phosphorylation modulates binding to actinrelated partners (Daly, 2004). In this study, we observed
the phosphorylation of host cortactin on ERK, but not on
SFKs residues, during EA interactions with cells. ERK
phosphorylation of cortactin positively regulates its capacity to stimulate N-WASP in Arp2/3-dependent actin polymerization (Martinez-Quiles et al., 2004). In contrast, SFK
phosphorylation of cortactin inhibits N-WASP activity
(Martinez-Quiles et al., 2004). Our results suggest
that EAs induce cortactin phosphorylation at sites that
control and stimulate N-WASP/Arp2/3-dependent actin
polymerization. The role of ERK and SFK phosphorylation
of cortactin regarding host cell invasion by intracellular
pathogens varies in the literature. Although SFK
phosphorylation of cortactin is important in host invasion by
Shigella spp., Staphylococcus aureus and Neisseria
meningitidis (Hoffmann et al., 2001; Bougnres et al.,
2004; Agerer et al., 2005), several recent reports have
depicted involvement of serine phosphorylation of
cortactin in pathogen internalization. ERK phosphorylation
and SFK dephosphorylation of cortactin are important in

A. Bonfim-Melo et al.

Fig. 6. Heat-killed parasites and non-infective forms induce distinct PKD1, ERK, SFKs and cortactin phosphorylation. Prestarved HeLa cells
(medium without FCS) were incubated with hkEA or epimastigote forms (MOI 10:1) for the indicated time points. Lysates were resolved by
SDS-PAGE and analysed by Western blotting with the following antibodies: (A,F) pPKD1 S910: activated PKD1; (B,G) pERK T202/Y204:
activated ERK1/2; (C,H) pCort S405: cortactin phosphorylated by ERK1/2; (D,I) pSFK Y416: activated SFKs; (E,J) pCort Y421: cortactin
phosphorylated by SFKs. These observations are representative of at least four independent experiments.

HeLa cell invasion by Coxiella burnetii and for cortactin


capacity to bind N-WASP (Rosales et al., 2012). Similarly,
Campylobacter jejuni induces and requires cortactin
phosphorylation by ERK, but not by SFK, and host cell
invasion is inhibited in cells depleted of cortactin and
N-WASP (Samuelson and Konkel, 2013). Interestingly,
Campylobacter jejuni also controls Rac1 and Cdc42
GTPases (Krause-Gruszczynska et al., 2007), similar to
T. cruzi EAs (Fernandes and Mortara, 2004). Therefore,
despite a large phylogenetic gap, both pathogens appear
to share similar host cell invasion mechanisms.
PKD1 phosphorylation of cortactin regulates its affinity
to the WAVE2 complex and reduces actin dynamics
(Eiseler et al., 2010). Although we were not able to
observe PKD1 phosphorylation of cortactin at the Ser 298
site directly (a PKD1 phosphorylation site in cortactin
identified by De Kimpe et al., 2009), our results suggest
that PKD1 plays a positive role in EA host cell invasion.
Invasion was augmented in cells overexpressing a PKD1GFP construct compared with control cells. Considering
lamellipodium protrusion, cortactin phosphorylated by
PKD1 at serine 298 increases its binding capacity to filamentous actin, reduces actin turn over and inhibits cell
migration (Eiseler et al., 2010). PKD1 reduces actin
dynamics by phosphorylating other actin-related proteins,

such as Ras and Rab interactor 1 protein and slingshot 1


L protein phosphatase (Eiseler et al., 2009; Ziegler et al.,
2011). On the other hand, earlier reports showed that
PKD1 activity is related to ERK activation implying that
PKD1 can also modulate cortactin activity indirectly
(Hausser et al., 2001; Brndlin et al., 2002). Rezaee et al.
(2013) have recently described that PKD activity is
required for cortactin phosphorylation by ERK and actin
remodelling in respiratory syncytial virus infection; therefore, a similar signalling pathway regulating actin dynamics in EA host cell invasion may occur. Nevertheless,
PKD1 involvement in an actin-independent mechanism
and the participation of other PKD1 substrates cannot be
ruled out as this kinase also regulates other cellular processes (Wei et al., 2009).
In addition to increased internalization by PKD1-GFP
overexpressing HeLa cells, we observed reduced EA
internalization in HeLa cells depleted of cortactin, suggesting that both proteins promote EA invasion. Cortactin
knockdown in HeLa cells could possibly interfere with
the activity of endogenous dynamin 2, a GTPase responsible for vesicle scission during diverse endocytic processes (Chappie and Dyda, 2013). Cells depleted of
cortactin displayed reduced dynamin 2 recruitment to
clathrin-coated pits and decreased receptor-mediated
2015 John Wiley & Sons Ltd, Cellular Microbiology

PKD1 and cortactin in T. cruzi cell invasion

Fig. 7. Host cortactin depletion reduces EA host cell invasion but not EA-driven actin recruitment.
A. HeLa cells transduced with shRNA for cortactin (Cort), non-targeting (scramble, Scr) or non-transduced () were incubated with EA (MOI
10:1) for 2 h, and intracellular parasites (white arrowheads) were counted under bright field microscopy. Data are representative of four
independent experiments (mean SD, *** mean P < 0.001, n = 12).
B. HeLa cells transduced with shRNA for cortactin (Cort), non-targeting (scramble, Scr) or non-transduced () were incubated with EAs (MOI
5:1) for 30 min, immunostained for actin and analysed using epifluorescence microscopy. Data are representative of three independent
experiments (mean SD, P > 0.05, n = 9). Bars = 10 m.

endocytosis, such as transferrin uptake (Zhu et al., 2005).


Although convincing evidence of receptor-mediated
T. cruzi internalization is still lacking, it has already been
shown that trypomastigotes are less infective in HeLa
cells overexpressing a dominant-negative dynamin 2 construct (Wilkowsky et al., 2002). Additionally, in cortactindepleted cells we were still able to detect residual
cortactin recruitment to EA invasion sites. However, with
the available techniques it was not possible to assess the
contribution of this recruitment to EA uptake and it was
thus not possible to rule out a cortactin-independent
pathway.
Actin recruitment to EA adhesion sites was not reduced
in cortactin-depleted cells (Fig. 8). This may have
occurred because cortactin recruitment is dependent on
actin polymerization and not the opposite (Zhu et al.,
2005). Although N-WASP and WAVE2 proteins are
referred to as type I nucleation promoting factors (NPF I)
as they nucleate actin monomers at pre-existing actin
filaments, cortactin is a type II NPF linking protein
because it modulates and mediates the interactions of
several NPF I proteins (Daly, 2004). Therefore, cortactin
acts on actin-regulating proteins rather than directly promoting actin polymerization. We observed actin-recruiting
EAs during in vivo experiments that were internalized
2015 John Wiley & Sons Ltd, Cellular Microbiology

Fig. 8. Host PKD1 overexpression increases EA cell invasion.


HeLa cells overexpressing PKD1-GFP, cytosolic GFP or
non-transduced () were incubated with EAs (MOI 10:1), and
intracellular parasites (white arrowheads) were counted under
bright field microscopy. Data are representative of three
independent experiments (mean SD, *** mean P < 0.001, n = 9).
Bars = 10 m.

10 A. Bonfim-Melo et al.
within 5 min along with others that displayed delayed
(> 120 min) or blocked internalization (Videos S1 and S2).
This unusual invasive behaviour can explain similar
recruitment by EAs with distinct internalization in
cortactin-depleted cells.
To evaluate parasite factors that regulate host cell
signalling, we used dead EAs and non-infective
epimastigotes. Previous studies have shown that these
forms had reduced or absent adhesion and internalization
in non-professional phagocytic cells, although signalling
events surrounding these interactions have not been
studied (Nogueira and Cohn, 1976; Fernandes et al.,
2013). We showed that these forms did not induce recruitment of host molecules involved in EA internalization.
However, epimastigotes and dead EAs both triggered signalling patterns that were very distinct from live EAs.
These observations confirm that EAs induce specific signalling in host cells that promotes actin polymerization
and culminates in parasite internalization. Host signalling
modulation could be stimulated by parasite surface molecules given that epimastigotes lack important surface
molecules required for host protein activation during
T. cruzi internalization and mammalian cell invasion
(Wilkowsky et al., 2001; Yoshida and Cortez, 2008).
Although hkEAs express the molecules necessary for
attachment, their ability to trigger internalization is likely
compromised (Fernandes et al., 2013). These results
show that live EAs trigger specific signalling in host cells,
which is crucial for promoting their internalization.
In conclusion, we showed that EAs control host
cortactin and PKD1 signalling pathways in an actindependent mechanism of invasion. EA interaction induces
recruitment and distinct phosphorylation of both proteins.
This host signalling is specifically induced by live and
infective parasites. Modulation of host cortactin and PKD1
expression confirmed the positive role of these molecules
in EA invasion. These results corroborate previous studies
suggesting phagocytic induction by EAs during internalization by non-phagocytic cells (Fernandes et al., 2013).
Experimental procedures
Antibodies, reagents and plasmids
Rabbit anti-pS910 PKD1 (SAB4300075), rabbit anti-cortactin (C
7112) and mouse anti-actin (A2228) were obtained from SigmaAldrich. Rabbit anti-pT202/Y204 ERK1/2 (#9101S) and rabbit
anti-pY416 SFKs (#2101S) were obtained from Cell Signaling.
Rabbit anti-pY421 cortactin (AB3852) was obtained from
Millipore and rabbit anti-pY466 cortactin (PA114115) was
obtained from Thermo Scientific. Rabbit anti-PKD1 (1986-1) was
obtained from Epitomics. Rabbit anti-pS405 cortactin (AB-100-1)
was obtained from Protea and mouse anti-cortactin (sc-55578)
was from Santa Cruz. The secondary antibodies goat anti-mouse
AlexaFluor 568 (A11004) and goat anti-rabbit AlexaFluor 488
(A11008) were from Invitrogen. Goat anti-rabbit IgG peroxidase

(A6154) and goat anti-mouse IgG peroxidase (A4416) were


obtained from Sigma-Aldrich.
4,6-Diamidino-2-phenylindole (DAPI) and Hoechst 33342
were obtained from Invitrogen. Puromycin and polybrene were
obtained from Sigma-Aldrich. FuGene HD was obtained from
Roche. Protein assay dye reagent concentrate was obtained
from Bio-Rad.
LifeAct-RFP was purchased from ibidi. pEGFP-N1 PKD1 was
kindly provided by Dr. Angelika Hausser from Universitt Stuttgart
(Hausser et al., 2002). pEGFP-N1 cortactin has been previously
described (Eiseler et al., 2010). Lentiviral plasmids pMDL g/p
RRE, pRSV REV, pCi-VSVG and pLL-CNG were previously
described (Rubinson et al., 2003; Zanetti et al., 2013). pLKO.1puro TCR1.5 plasmids encoding shRNA sequences were purchased from Sigma. Cortactin sense strand sequence: CAC GAA
TAT CAG TCG AAA CTT. Scramble sense strand sequence: CAA
CAA GAT GAA GAG CAC CAA. For PKD1 and cortactin lentiviral
construction, respective genes were inserted into a pLL-CNG
plasmid. The fragment of PKD1 fused to GFP was removed from
pEGFP-N1 PKD1 first by digestion with restriction enzyme XhoI,
with sequentially blunting of DNA ends (Quick Blunting Kit, NEB),
followed by digestion with BsrGI endonuclease. The fragment
was inserted into lentiviral vector pLL-CNG that was digested
with NheI, with subsequent blunting of DNA ends using the same
kit, followed by digestion with BsrGI to use one blunt and one
stick end (BsrGI) for cloning. Cortactin fused to a GFP fragment
was removed from pEGFP-N1 cortactin and inserted into pLLCNG using EcoRI and XbaI sites on both plasmids. All restriction
enzymes were purchased from NEB.

Parasites and mammalian cells


T. cruzi G strain has been previously characterized (Yoshida,
1983). EAs were obtained after 1416 h of incubation at 37C
from trypomastigotes from tissue culture in liver infusion tryptose
(LIT) medium pH 5.8 with 10% foetal calf serum (FCS) as previously described (Ley et al., 1988; Mortara, 1991; Fernandes
et al., 2013). Parasites were pre-incubated in complete RPMI
medium with 10% FCS for 1 h prior to each assay to recover from
the acidic pH of the LIT medium.
HeLa (human cervical adenocarcinoma cells) and Vero (green
monkey epithelial kidney cells) cells were obtained from the
Instituto Adolfo Lutz. Both cell lineages were maintained in RPMI
medium with 10% FCS supplemented with antibiotics (10 g ml1
streptomycin and 100 U ml1 penicillin, Sigma-Aldrich) in a
humidified atmosphere at 37C and 5% CO2. HEK293T (human
kidney embryonic cells, kindly provided by Dr. Silvia Boscardin
from ICB-USP) were cultivated in Dulbeccos modified essential
medium (DMEM; Sigma-Aldrich) with 10% FCS and antibiotics
(10 g ml1 streptomycin and 100 U ml1 penicillin, SigmaAldrich) in a humidified atmosphere at 37C and 5% CO2.

Lentiviral transduction and HeLa lineage establishment


Viral vectors were produced following the method described in
Barde et al. (2010). Briefly, 2 106 HEK293T cells were plated on
10 cm2 plates containing DMEM with 10% FCS, 0.2 mM
glutamine, 10 U ml1 penicillin and 10 g ml1 streptomycin. Four
10 cm2 plates were used to produce each batch of viral vector.
One day after plating, the medium was replaced with fresh
2015 John Wiley & Sons Ltd, Cellular Microbiology

PKD1 and cortactin in T. cruzi cell invasion


medium, and 20 g of transfer vector (pLL-PKD1 or pLLcortactin), 5 g pRSV-REV, 10 g pMDLg/pRRE and 6 g pCiVSVG were used to transfect each plate of HEK293T cells by the
calcium phosphate coprecipitation method. The cell culture
supernatant was collected after 24 and 48 h, filtered using a
0.45 m syringe and concentrated by centrifugation at 14 000
r.p.m. at 4C for 2 h in a sucrose gradient (Sorvall RC 5C Plus,
SA600 rotor, Thermo Scientific). The concentrated viruses were
stored at 80C until use.
Viruses were titrated based on Barde et al. (2010). In a 24-well
plate, 2 104 HeLa cells were plated per well. After 24 h, various
quantities of virus were added to each well in the presence of
8 g ml1 polybrene. After 48 h, transduced cells were counted
using a fluorescence microscope, and virus titre was expressed
as transduction units (TU) ml1: TU ml1 = (% GFP-positive
cells total number of cells)/virus volume (ml). GFP-positive cells
were enriched using a cell sorter (FACSAriaII). HeLa cells
expressing cortactin or PKD1-GFP at a rate of more than 98%
were used for invasion assays.

11

After brief centrifugation (20 200 g, 10 min), lysates were quantified by the Bradford reaction (Bradford, 1976), resolved
by SDS-PAGE, transferred to nitrocellulose membranes and
phosphorylation state was evaluated by Western blotting. Blocking was performed in 5% non-fat milk in PBS-T (PBS Tween-20
0.1% + phosphatase inhibitors, Na3VO4 and NaF) for 1 h at RT
primary antibodies were incubated in PBS-T + 5% bovine
serum albumin overnight at 4C and secondary antibodies
were incubated with 5% non-fat milk in PBS-T for 1 h at RT
Chemiluminescent reaction was performed using ECL Prime
(Amersham, GE) and the chemiluminescent signal was acquired
using Alliance 2.7 equipment (UVitec).

Invasion assays
Suspensions of 1.5 105 HeLa cells were plated in 24-well
plates. The next day, EAs (MOI 10:1) were added and incubated
for 2 h. After a gentle wash with PBS, slides were fixed with
Bouin, stained with Giemsa and intracellular parasites counted
under an optical microscope (Fernandes et al., 2013).

Immunofluorescence
HeLa cells were fixed with 3.5% paraformaldehyde for 15 min
at room temperature (RT). Primary and secondary antibodies
were diluted in PGNS solution [PBS pH 7.2 with 0.1% gelatin
(Sigma-Aldrich), 0.1% sodium azide (Sigma-Aldrich) and 0.2%
saponin (Sigma-Aldrich)] and sequentially incubated at RT in a
humidified chamber for 1 h. Slides were mounted in pH 9.0 buffered glycerol solution with 9 mM p-phenylenediamine (Platt and
Michael, 1983) and evaluated under epifluorescence microscopy
(BX51, Nikon). Images were acquired with ImagePro Plus
(MediaCybernetics) and processed with ImageJ (Schneider
et al., 2012) and/or Photoshop CS5 (Adobe).

Time lapse analysis and confocal microscopy


For time lapse analysis of live cells, HeLa cells were plated in a
Hi-Q4 dish (ibidi) and transfected with LifeActin-RFP (ibidi) and
FuGene HD (Roche) after 24 h according to the manufacturers
instructions. Supernatant was changed 24 h later and experiments conducted within 4896 h post transfection. On the day of
the experiment, HeLa cells and EAs were stained with Hoechst
(10 M) for 15 min at RT time lapse acquisition was performed
under physiological conditions (humidified atmosphere at 37C
and 5% CO2) in a TCS SP5 II Tandem Scanner (Leica) confocal
microscope with a 63 1.40 NA objective. Fixed samples were
also imaged with this instrument. Image processing, analysis and
multidimensional reconstructions were performed with Imaris
v.7.4.2 software (Bitplane).

Statistical analysis
All results are presented as the mean standard deviation. Differences were considered statistically significant when P < 0.001,
as determined by one-way analysis of variance and Tukeys
multiple comparison post-test. Analyses and graphs were
created using Prism 5 (GraphPad) software.

Acknowledgements
The authors wish to acknowledge the support of a FAPESP grant
(2007/50551-2) and CAPES. R.A.M. and D.B. are recipients of
CNPq fellowships. A.B.-M. is recipient of a FAPESP fellowship
(2010/14190-8). We thank Dr. Ulisses Gazos Lopes (Instituto de
Biofsica, UFRJ) for providing us lentiviral plasmids. We would
like to thank Mario Costa Cruz and Dr. Fernando Real (EPM,
UNIFESP) for initial technical support and valuable advice
regarding confocal microscopy experiments and video/image
analyses on Imaris software. We also thank Dr. Alexandre
Salgado Basso and Daniela Teixeira (EPM, UNIFESP) for
technical support with cytometry experiments. BioMed
Proofreading LLC (http://www.biomedproofreading.com) is also
acknowledged. D.B. wishes to thank Erika Alves for stimulating
discussion and initial experiments with cortactin and PKD.

Conflict of interest
All authors declare no competing interests.

Western blotting
For signalling assays, 3 106 HeLa cells were plated in a 10 cm
plate and starved for 48 h (three incubations in medium without
FCS). On the following day, the medium was replaced with fresh
medium (without FCS) containing AEs (MOI 10:1) and incubated
under culture conditions for various time intervals ranging from 0
to 120 min. Cells were washed, harvested and extracted (50 mM
tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM
PMSF, 1 mM iodoacetamide, 2 mM Na3VO4 and 1 mM NaF).
2015 John Wiley & Sons Ltd, Cellular Microbiology

Authors contributions
Conception of the study: D.B. Designed the experiments:
A.B.-M., S.W.H., J.V.L., R.A.M. and D.B. Performed the
experiments: A.B.-M., B.F.Z., E.R.F. and S.V. Interpretation of the results and data analysis: A.B.-M., J.V.L.,
R.A.M. and D.B. Wrote the manuscript: A.B.-M., R.A.M.
and D.B.

12 A. Bonfim-Melo et al.
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publishers web-site:
Fig. S1. Cortactin knockdown in HeLa cells. HeLa cells were
transduced or not () with lentiviral particles encoding a shRNA
targeting cortactin mRNA (Cort) or a scramble shRNA sequence
(no target mRNA, Scr), selected with puromycin and expanded.
Cortactin expression was evaluated by (A) immunofluorescence
with a mouse anti-cortactin or by (B) Western blotting with a
rabbit anti-cortactin as previously described. DAPI (blue) was
used to stain cell nuclei and anti-actin was used as a protein
loading control.
Fig. S2. Residual cortactin recruitment by EAs in cortactin
knockdown HeLa cells. Cortactin depleted (Cort) or nontransduced () HeLa cells were incubated with EAs (MOI 10:1)
stained for cortactin and actin and analysed by epifluorescence
microscopy. White arrowheads show cortactin recruitment
(residual in Cort group) to the EA invasion sites. DAPI (blue)
stains kinetoplasts and nuclei. Bars = 20 m.
Video S1. EAs induce corecruitment and colocalization of host
actin and cortactin in live HeLa cells. HeLa cells expressing
cortactin-GFP were transfected with LifeAct-RFP and interactions with EAs (MOI 50:1) were evaluated by time lapse confocal
microscopy at a frame interval of 8 min. Both cell types were

14 A. Bonfim-Melo et al.
prestained with Hoechst (white). The video shows a single focal
plane. EA (arrowhead) inducing corecruitment and colocalization
of actin (red) and cortactin (green, Cort) in a HeLa cell. Asterisks
show EAs that recruited actin, but did not invade during the
experimental period. Bar = 10 m.
Video S2. EAs induce corecruitment and colocalization of host
actin and PKD1 in live HeLa cells. HeLa cells expressing PKD1GFP were transfected with LifeAct-RFP, and interactions with
EAs (MOI 50:1) were evaluated by time lapse confocal microscopy at a frame interval of 8 min. Both cell types were prestained
with Hoechst (white). The video shows a single focal plane. EA
(arrowhead) inducing corecruitment and colocalization of actin

(red) and cortactin (green, Cort) in a HeLa cell. Asterisks show


EAs that recruited actin, but did not invade during the experimental period. Bar = 10 m.
Video S3. EAs induce corecruitment and colocalization of
cortactin and PKD1 with actin in HeLa cells. HeLa cells expressing PKD1-GFP (green) were incubated with EAs (MOI 50:1),
immunolabelled for actin (red) and cortactin (blue) and analysed
by confocal microscopy. The video shows three-dimensional
reconstruction of two cup-like actin-rich structures formed
beneath EA adhesion sites where cortactin and PKD1
colocalized. Both cell types were prestained with DAPI
(white).

2015 John Wiley & Sons Ltd, Cellular Microbiology