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Acta Tropica 120 (2011) 231–237

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Acta Tropica

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Characterization of the infective properties of a new genetic group of

Trypanosoma cruzi associated with bats

∗

Fernando Yukio Maeda, Renan Melatto Alves, Cristian Cortez, Fabio Mitsuo Lima, Nobuko Yoshida

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently described.

Received 19 July 2011

Here we characterized a T. cruzi strain from this new genetic group, which could be a potential source

Received in revised form 23 August 2011

of infection to humans. Metacyclic trypomastigotes (MT) of this strain, herein designated BAT, were

Accepted 1 September 2011

compared to MT of well characterized CL and G strains, as regards the surface proﬁle and infectivity

Available online 7 September 2011

toward human epithelial HeLa cells. BAT strain MT expressed gp82, the surface molecule recognized by

monoclonal antibody 3F6 and known to promote CL strain invasion by inducing lysosomal exocytosis,

Keywords:

as well as mucin-like molecules, but lacked gp90, which functions as a negative regulator of invasion in

Trypanosoma cruzi

G strain. A set of experiments indicated that BAT strain internalization is gp82-mediated, and requires

New genotype

the activation of host cell phosphatidylinositol 3-kinase, protein kinase C and the mammalian target of

Metacyclic forms

Cell invasion, Bats rapamycin. MT of BAT strain were able to migrate through a gastric mucin layer, a property associated

with p82 and relevant for oral infection. Gp82 was found to be a highly conserved molecule. Analysis

of the BAT strain gp82 domain, containing the cell binding- and gastric mucin-binding sites, showed 91

and 93% sequence identity with G and CL strains, respectively. Hela cell invasion by BAT strain MT was

inhibited by puriﬁed mucin-like molecules, which were shown to affect lysosome exocytosis required

for MT internalization. Although MT of BAT strain infected host cells in vitro, they were less effective than

G or CL strains in infecting mice either orally or intraperitoneally.

1. Introduction Metacyclic trypomastigotes (MT) of T. cruzi from bats were

found to invade cultured cells, followed by intracellular develop-

Trypanosoma cruzi, the protozoan parasite that causes Chagas’ ment of parasites (Marcili et al., 2009). How do they enter host

disease, is constituted of genotypically heterogeneous populations cells, which MT molecules are involved in the process, what sig-

that may differ considerably in their phenotypic characteristics. In naling pathways are triggered during MT-target cell interaction, to

2009, by reviewing the available knowledge, an expert committee what extent does this new genetic group differ from other T. cruzi

reached a new consensus for T. cruzi intraspeciﬁc nomenclature: lineages? These are questions that remain to be elucidated. Differ-

the known T. cruzi isolates and strains should be referred to by ent T. cruzi strains may vary greatly in their infectivity in vitro and

six discrete typing units, TcI-TcVI (Zingales et al., 2009). Recently, in vivo, and these differences are associated with the differential

phylogenetic analyses using SSU rDNA, cytochrome b and histone engagement of surface molecules and triggering of distinct signal-

H2B genes, and genotyping method targeting ITS1 rDNA, revealed ing pathways in both cells (Neira et al., 2002; Ferreira et al., 2006;

a new genotype of T. cruzi, which is associated with bats and is Cortez et al., 2006a; Covarrubias et al., 2007). For instance, strains

not clustered within any of the previously deﬁned lineages (Marcili G (TcI) and CL (TcVI), belonging to highly divergent genetic groups

et al., 2009). This new group is formed exclusively by highly homo- and associated with marsupial and human infection, respectively

geneous bat isolates from anthropic areas that were endemic for (Briones et al., 1999), are characterized by their differential infec-

Chagas’ disease, indicating that bats may be important reservoirs tivity toward cultured mammalian cells as well as in mice (Yoshida,

and potential source of T. cruzi infection to humans (Marcili et al., 2006). MT of poorly infective G strain apparently use the mucin-like

2009). It is therefore relevant to investigate the infective properties glycoproteins to enter human epithelial cells whereas MT of highly

of the parasite isolated from bat. invasive CL strain rely on the surface molecule gp82 (Ruiz et al.,

1998), which induces a Ca -dependent disruption of the host cell

actin cytoskeleton (Cortez et al., 2006b) that facilitates lysosomal

exocytosis and parasite internalization (Martins et al., 2011). There

∗ are also evidences that successful establishment of CL strain in mice

Corresponding author.

E-mail address: [email protected] (N. Yoshida). by the oral route, which is the main mode of T. cruzi transmission in

232 F.Y. Maeda et al. / Acta Tropica 120 (2011) 231–237

some regions (Coura, 2006), is critically dependent on gp82 (Neira 2.3. Southern blot analysis and pulsed ﬁeld gel electrophoresis

et al., 2003; Cortez et al., 2003). In this study we aimed at charac-

terizing a T. cruzi strain from bat, herein designated BAT, focusing For Southern blot analysis, T. cruzi DNA was digested with dif-

on the expression of surface molecules implicated in parasite–host ferent restriction enzymes, separated by electrophoresis on 0.8%

cell interaction and on the mechanisms of cell invasion. agarose gel and blotted onto nylon membranes. Hybridization with

the probe, which consisted of a DNA fragment corresponding to ORF

of gp82 gene (whole insert of gp82 cDNA clone) labeled with [ P],

and washings were performed as detailed (Araya et al., 1994). For

2. Materials and methods

pulsed ﬁeld gel electrophoresis, agarose blocks containing genomic

◦

DNA were prepared, incubated at 50 C for 16 h in lysis solution

2.1. Parasites, mammalian cell culture and invasion assays

containing 10 mM Tris–HCl, pH 8.0, 500 mM EDTA, 1% sarkosyl,

1 mg/ml proteinase K, equilibrated in TE, washed and stored in

A strain of a new lineage of T. cruzi (Marcili et al., 2009), isolated ◦ 7

0.5 M EDTA at 4 C. Small portions (equivalent to 10 parasites)

from Myotis levis in São Paulo, was kindly provided by Dr. Marta

were electrophoresed (1.2% agarose gel in 0.5× TBE) at 80 V for

M.G. Teixeira from Universidade de São Paulo, Brazil. In addition

132 h in Gene Navigatore System (Pharmacia), from pulse times

to this strain, designated BAT, we used T. cruzi strains G, isolated

varying from 90 to 800 s. DNA from Hansenula wingei was used as

from an opossum in the Brazilian Amazon (Yoshida, 1983), and

reference. After transfer to nylon membranes, chromosomal DNA

CL, isolated from the domiciliary insect vector Triatoma infestans 32

bands were hybridized with the [ P]-labeled insert of gp82 cDNA

in the southern state of Rio Grande do Sul, Brazil, in a dwelling

clone and revealed by exposure to X-ray ﬁlm (Hyperﬁlm-MP, Amer-

where people were infected (Brener and Chiari, 1963). The parasites

sham).

were maintained cyclically in mice and in liver infusion tryptose

medium containing 5% fetal bovine serum. For differentiation of

2.4. Production and puriﬁcation of J18 and GST

epimastigotes into MT, Grace’s medium (Invitrogen) and TC100

medium (Vitrocell, Brazil) were also used. MT from cultures at the

The recombinant protein J18, containing the full-length T. cruzi

stationary growth phase were puriﬁed by passage through DEAE- TM

gp82 sequence (GenBank data base, accession number L14824)

cellulose column, as described (Teixeira and Yoshida, 1986). HeLa

in frame with glutathione S-transferase (GST), was produced in

cells, the human carcinoma-derived epithelial cells, were grown

◦ E. coli DH5-␣ by transforming the bacteria with a pGEX-3 construct

at 37 C in Dulbecco’s Minimum Essential Medium (DMEM) sup-

comprising the gp82 gene. Details of the construction and the puriﬁ-

plemented with 10% fetal calf serum, streptomycin (100 ␮g/ml)

cation of J18, as well as of GST, are described elsewhere (Cortez et

and penicillin (100 U/ml) in a humidiﬁed 5% CO2 atmosphere. Cell

al., 2006b).

invasion assays were carried out as detailed elsewhere (Yoshida

et al., 1989), by seeding the parasites onto each well of 24-well

2.5. Puriﬁcation of mucin-like molecules from T. cruzi

plates containing 13 mm diameter round glass coverslips coated

with 1.5 × 10 HeLa cells. The multiplicity of infection (MOI) was

We followed the procedure described by Acosta-Serrano et al.

10:1 for CL strain and 20:1 for G and BAT strains. After 1 h incuba- 10

(2001). Cultures (total of 5 × 10 parasites for each preparation)

tion with MT, the duplicate coverslips were washed in PBS, ﬁxed in

were centrifuged, and the pellet was freeze-dried and placed

Bouin solution, stained with Giemsa, and sequentially dehydrated

in a sonicating water bath for 10 min with 10 ml of chloro-

in acetone, a graded series of acetone:xylol and xylol. The number

form/methanol/water (1:2:0.8, by volume). After centrifugation at

of intracellular parasites was counted in 250 stained cells.

2000 × g for 5 min, and two more extraction of the pellet, the insol-

uble material served as a source of delipidated parasites whereas

the pooled fractions (30 ml) were placed in a round-bottom ﬂask

2.2. Isolation of a cDNA clone containing the C-terminal domain

and dried by rotatory evaporation. The residue was extracted

of BAT strain gp82

with 20 ml of butan-1-ol/water (2:1, by volume) The butan-1-ol

phase contained the lipid fraction (F1) and the aqueous phase (F2)

Complementary DNA (cDNA) from BAT strain MT (1 × 10 ) was

contained epimastigote mucins. F2 was washed twice with water-

obtained using the AccessQuick RT-PCR System (Promega) on

saturated butan-1-ol and concentrated. The delipidated parasites

total RNA extracted by TRIzol (Invitrogen). Following cDNA synthe-

were extracted (three times) by sonication with 10 ml of 9% butanol

sis, the strategy for the ampliﬁcation of C-terminal domain of BAT

in water, and the pooled soluble material containing mucins (F3)

strain gp82 was based on its presumed similarity with CL/G strain

was concentrated. The mucins were resuspended in 2 ml of buffer

gp82. The forward primer 5 -GGATCCATGTTCGTCAGCAGCCTGCTG-

A (0.1 M ammonium acetate in 5% ppropan-1-ol (v/v)) and fraction-

3 corresponded to a sequence that precedes the epitope for

ated on an octyl-Sepharose column (10 × 0.5 cm), pre-equilibrated

mAb 3F6 and contained ATG plus an artiﬁcial Bam HI site; the

in buffer A. After washing the column with buffer A, and elution

reverse primer 5 - GAATTCGTTCAGTGGGCGGTTGTACAAGAAGA-3

with a linear gradient over 100 ml at a ﬂow rate of 12 ml/h, start-

corresponded to a sequence that follows the highly con-

ing with 15 ml of buffer A and ending with 60% (v/v) propan-1-ol in

served VTVKNVFLYNR motif characteristic of all members of the

water, fractions (2 ml) were analyzed by silver staining of SDS-PAGE

gp85/trans-sialidade superfamily and contained a stop codon plus

gels, as well as by immunoblotting using the available monoclonal

an artiﬁcial Eco RI site. A total of 40 cycles of denaturing, anneal- antibodies.

◦ ◦ ◦

ing and elongation at 94 C for 20 s, 55 C for 30 s and 72 C for

1 min, respectively, were performed. After puriﬁcation, using Pure-

2.6. Parasite migration through the gastric mucin layer

Link kit (Iinvitrogen) the PCR product was cloned in the plasmid

vector pGEM-T Easy (Promega). Following ligation to the vector,

Polycarbonate transwell ﬁlters (3 ␮m pores, 6.5 mm diameter,

the product was transformed in Escherichia coli strain DH5␣, and

␮

Costar, Cambridge MA) were coated with 50 l of a preparation

the colonies grown in LB broth. Clones containing the expected

containing 10 mg/ml gastric mucin from porcine stomach (porcine

771 bp fragment after restriction analysis with Eco RI and Bam HI

stomach mucin type III, Sigma) in water. Parasites, in 600 ␮l PBS,

were sequenced using ABI 3130XL Genetic Analyzer and BigDye × 7

were added to the bottom of 24-well plates (2 10 parasites/well)

◦

Terminator v3.1 (Applied Biosystems).

and incubated for 1 h at 37 C. Thereafter, the mucin-coated

F.Y. Maeda et al. / Acta Tropica 120 (2011) 231–237 233

transwell ﬁlters were placed onto parasite-containing wells, and on the parasite surface and are implicated in interactions with

100 ␮l PBS were added to the ﬁlter chamber. At different time host cells. Gp90, which acts as a negative modulator of cell

◦

points of incubation at 37 C, 10 ␮l were collected from the ﬁlter invasion (Málaga and Yoshida, 2001), was undetectable in BAT

chamber for determination of parasite number and the volume in strain whereas the invasion-promoting and mAb 3F6-reactive gp82

this chamber was corrected by adding 10 ␮l PBS. (Ramirez et al., 1993) was expressed at levels comparable to G

and CL strains (Fig. 1A). Mucin-like glycoproteins were revealed

2.7. Exocytosis assay in BAT strain by mAb 2B10 but not by mAb 10D8 (Fig. 1A), indi-

cating that they lack galactofuranose residues that are part of the

Conﬂuent monolayers of HeLa cells, grown in 24-well plates in epitope for mAb 10D8 (Yoshida, 2006), an antibody that inhibits

␮

DMEM were washed twice in PBS and incubated in 300 l DMEM G strain infectivity (Yoshida et al., 1989). Overall, the surface pro-

without phenol red. After 1 h, the supernatants were collected and ﬁle of BAT strain displayed higher similarity to CL strain than to G

the cells were lysed in DMEM or PBS containing 1% NP-40, and strain. To determine the infectivity of BAT strain, as compared to

␮

30 l of 1 M sodium acetate pH 4.0 was added to decrease pH. Sam- G and CL strains, MT were incubated with HeLa cells for 1 h and

ples were centrifuged for 5 min at 13,000 g and the supernatants the number of intracellular parasites was counted after ﬁxation,

␮ ␮

were collected, 20 l aliquots were diluted with 60 l citrate buffer staining with Giemsa and serial dehydration. The rate of inter-

␮ ␤ d

and 160 l of 100 mM 4-nitrophenyl N-acetyl- - -glucosaminide nalization of BAT strain (MOI = 20) was signiﬁcantly higher than

◦

(Sigma) were added. After 1 h incubation at 37 C, the reaction that of G strain (MOI-20) and comparable to that of CL strain at

␮

was stopped by adding 720 l of 200 mM sodium borate pH 9.8 MOI = 10 (Fig. 1B). Next, we examined whether gp82 was impli-

and absorbance was measured at 405 nm in a Labsystems Mul- cated. In one set of experiments, MT were pre-incubated for 15 min

tiskan MS plate reader. Exocytosis was expressed as % of total with mAb 3F6 and then added to Hela cells. After 1 h incubation, the

␤

-hexosaminidase activity (supernatant + cell extract). cells were processed for parasite counting. MAb 3F6 signiﬁcantly

inhibited parasite internalization (Fig. 1C). To further assess the

2.8. Indirect immunoﬂuorescence assays involvement of gp82, Hela cells were pre-incubated for 15 min in

absence or in the presence of J18, the recombinant protein contain-

To visualize parasites co-localized with lyososome marker, MT ing the full length gp82 sequence fused to GST, or GST as control,

◦

were incubated with adherent HeLa cells for 1 h at 37 C. After ﬁx- at 40 ␮g/ml, and MT of BAT strain were added. After 1 h incubation,

ation with 4% p-formaldehyde in PBS for 30 min, the cells were in the presence of J18 or GST, the cells were processed as above.

incubated with 50 mM NH4Cl in PBS for 30 min, washed 3 times Parasite invasion was inhibited by J18, but not by GST (Fig. 1D).

and permeabilized by 30 min treatment with 1% saponin in PGN These results indicate that, similar to CL strain (Ramirez et al., 1993),

(PBS containing 0.15% gelatin and 0.1% sodium azide). Follow- BAT strain relies on gp82 molecule to enter host cells. In another

ing 1 h incubation at room temperature with mouse anti-human set of experiments, we tested the effect of drugs that affect cell

Lamp-2 (H4B4 monoclonal antibody), diluted 1:5 in PGN, and 3 signaling and were previously shown to inhibit CL strain MT inva-

washes in PBS, the cells were incubated for 1 h in PGN with Alexa sion (Martins et al., 2011). HeLa cells were treated for 30 min with

Fluor 568-conjugated anti-mouse IgG (Invitrogen), diluted 1:200, 100 nM of wortmannin, an inhibitor of lipid kinase phosphoinositol

␮

and 10 g/ml DAPI (4 ,6 1-diamino-2-phenylindole dihydrochlo- 3 kinase (PI3K), phorbol myristate acetate (PMA), a drug that can

ride) for visualization of nucleus. Images were acquired in Olympus downregulate protein kinase C (PKC), or rapamycin, which inhibits

BX51, equipped with an Olympus DP71 CCD camera, using Image mammalian target of rapamycin (mTOR). After removal of the drug,

Pro Plus 6.2 software (Media Cybernetic Inc.). the parasites were added. Following 1 h incubation, along with the

untreated controls, the cells were processed for counting of inter-

2.9. Oral infection of mice with BAT strain MT nalized parasites. All three drugs diminished invasion of BAT strain

(Fig. 1E). As the gp82-mediated invasion of CL strain MT is inhibited

To examine the infectivity of BAT strain MT in vivo, four to ﬁve by the referred drugs, and is associated with lysosomal exocytosis

week-old female Balb/c mice, bred in the animal facility at Uni- that contributes for parasitophorous vacuole formation (Martins

versidade Federal de São Paulo, were used. All procedures and et al., 2011), we checked whether BAT strain MT co-localized with

experiments conformed with the regulation of the institutional lysosome marker during invasion. HeLa cells were incubated with

Ethical Committee for animal experimentation, and the study was MT for 1 h and were then processed for immunoﬂuorescence using

approved by the Committee (#CEP 0117/11). Mice were infected anti-Lamp-2 antibody. Parasites co-localized with Lamp-2 could be

with metacyclic forms by the oral route (10 parasites per mouse), visualized (Fig. 1F).

using a plastic tube adapted to a 1 ml syringe. Starting on day

10 post-inoculation, parasitemia was monitored twice a week by 3.2. High identity of gp82 sequences deduced from cDNA clones

␮

examining 5 l blood samples collected from the tail, at the phase of BAT, G and CL strains

contrast microscope.

We have obtained a cDNA clone (GenBank accession number

2.10. Statistics JN116557), as described in Section 2.2, which putatively codes

for the carboxy-terminal domain of BAT strain gp82. The amino

To determine signiﬁcance of data Student’s t test, the program acid sequence deduced from this cDNA clone, designated F11, dis-

GraphPad InStat was used. played 91% and 93% identity, respectively, with the corresponding

sequences deduced from clones J18 (GenBank L14824) and R31

3. Results (GenBank AF128843), derived from highly divergent strains G and

CL (Fig. 2A), conﬁrming the high conservation of gp82 sequence

3.1. Surface proﬁle and infectivity of metacyclic trypomastigotes among genetically distant T. cruzi populations. As regards the func-

(MT) of BAT strain tionally relevant sites of gp82, such as the host cell binding site,

which appears to be formed by a juxtaposition of two separate

To compare the MT surface proﬁle of BAT strain with that of sequences represented by peptides p4 and p8 (Manque et al., 2000),

G and CL strains, we used monoclonal antibodies (mAbs) directed the gastric mucin binding site represented by peptide p7 (Staquicini

either to gp90, gp82 or mucin-like molecules, which are expressed et al., 2010), and the epitope for mAb 3F6 represented by peptide

234 F.Y. Maeda et al. / Acta Tropica 120 (2011) 231–237

Fig. 1. Surface proﬁle and infectivity of metacyclic trypomastigotes (MT) of T. cruzi BAT strain. (A) MT of BAT strain and reference strains G and CL were processed for

Western blot analysis, using the indicated monoclonal antibodies directed to surface molecules gp90, gp82 and mucin-like gp35/50. (B) Cell invasion assays were performed

by incubating HeLa cells with BAT strain (MO = 20), G strain (MOI = 20) or CL strain (MOI = 10) for 1 h. After ﬁxation and Giemsa staining, the number of intracellular parasites

was counted in a total of 250 cells. The values are the means SD of four independent experiments performed in duplicate. (C) BAT strain MT, untreated or pretreated with

mAb 3F6, were incubated for 1 h with HeLa cells, which were then processed as in (B) for parasite counting. (D) HeLa cells, untreated or pretreated with the recombinant

␮

protein J18 or GST, at 40 g/ml, were incubated with BAT strain MT and processed for parasite counting. (E) HeLa cells were treated with the indicated drug, at 50 nM. After

washing out the drugs, the cells were incubated for 1 h with the parasites, ﬁxed and stained with Giemsa. Values in (B–E) are the means SD of three independent assays

performed in duplicate. In all cases, MT invasion was signiﬁcantly inhibited ( p < 0.05) by the indicated treatment. (F) HeLa cells were incubated with BAT strain MT for 1 h

and then processed for immunoﬂuorescence using anti-Lamp-2 antibody and DAPI. Parasite associated with Lamp-2 are indicated by white arrow. Scale bar = 10 ␮m.

p3, a few amino acid changes was detected in BAT as compared to performed by hybridizing the same probe with chromosomal size

G and CL strains (Fig. 2B). The observed substitutions presumably fragments separated by pulsed ﬁeld gel electrophoresis, revealed

do not result in substantial changes in the properties of BAT strain marked differences of BAT strain as compared to G and CL strains

gp82, as judged by the ability of mAb 3F6 and the recombinant (Fig. 3B).

protein J18 in inhibiting BAT strain entry into host cells (Fig. 1C

and D). It should be noted that the pair of contiguous glutamic acid 3.3. Migration of BAT strain MT through gastric mucin and

residues and of aspartic acids in p4 and p8 sequences, previously invasion of host cells

shown to be required for cell binding of gp82 (Manque et al., 2000),

was conserved in the 3 strains (Fig. 2B). The genomic organiza- The ability of MT to traverse the gastric mucin layer, which is

tion of BAT strain gp82 gene family was also examined. Southern critically dependent on gp82, is an important step for reaching the

blot of genomic DNA digested with restriction enzyme Bam HI, Eco target epithelial cells in oral infection, as previously shown for CL

RI, Hind III or Xho I, was hybridized with the insert of cDNA clone strain (Staquicini et al., 2010). We examined whether MT of BAT iso-

J18. The proﬁle of BAT strain differed considerably from that of late exhibited such a property. Transwell ﬁlters coated with gastric

G and CL strains (Fig. 3A). Chromosomal mapping of gp82 genes, mucin were placed onto parasite-containing wells, samples from

F.Y. Maeda et al. / Acta Tropica 120 (2011) 231–237 235

Fig. 2. Sequences of gp82 carboxy-terminal domain of different T. cruzi strains. (A) Shown are the aminoacid sequences deduced from cDNA clones F11 (BAT strain), J18 (G

strain) and R31 (CL strain). Overall, BAT strain sequence was 91% and 93% identical to G and CL sequences. (B) The sequences represented by peptides p4 and p8, identiﬁed

as the host cell binding site of gp82, as well as the sequences p3 and p7, identiﬁed as the epitope for mAb 3F6 and the gastric mucin-binding site, are shown, with asterisks

indicating the aminoacid residues of BAT strain gp82 that differ from G and CL strains.

the ﬁlter chamber were collected at different time points and the 3.4. Inhibition of MT invasion of host cells by T. cruzi mucin

number of parasites counted. Along the time, increasing number of molecules that exhibit lysosomal exocytosis-inhibiting properties

MT was recovered from the transwell chamber (Fig. 4A). Next, cell

invasion assays in the presence of gastric mucin were performed. Mucin-like molecules expressed on the surface of metacyclic

It has been shown that gp82-expressing CL strain metacyclic forms forms have been implicated in target cell invasion of G strain

efﬁciently invade HeLa cells regardless whether gastric mucin is (Yoshida et al., 1989). As mucins are expressed at high levels in

present or not, in contrast to gp82-deﬁcient T. cruzi strains whose BAT strain (Fig. 1A), we investigated whether they played a role

internalization is impaired by gastric mucin (Cortez et al. (2003). in invasion. HeLa cells were incubated with MT in absence or

Also shown was that CL strain metacyclic forms, as well as gp82, in the presence of mucins puriﬁed from BAT, as well as from G

are devoid of submaxillary mucin-binding property, and parasite and CL strains. Internalization of BAT strain MT was inhibited by

invasion is reduced in the presence of this mucin (Staquicini et al., mucins from all three strains, the homologous mucins exhibiting

2010). In assays in which BAT strain MT were incubated with HeLa the highest effect (Fig. 5A). Next, the lysosomal exocytosis-inducing

cells in the presence of 2 mg/ml of gastric or submaxillary mucin, a activity of mucin molecules was examined. HeLa cells were incu-

␮

marked decrease in parasite invasion was observed in the presence bated with mucin, at 20 g/ml, and 1 h later the lysosomal enzyme

␤

of submaxillary but not of gastric mucin (Fig. 4B). -hexosaminidase was measured in the supernatant as well as in

the cell extract. Exocytosis was signiﬁcantly reduced by BAT strain

mucin (Fig. 5B). Although G and CL strain mucins also diminished

Fig. 4. Migration of BAT strain MT through gastric mucin and host cell invasion. (A)

Transwell ﬁlters coated with gastric mucin were placed onto parasite-containing

Fig. 3. Genomic organization of gp82 genes in T. cruzi strains. (A) Southern blot wells. At different time points, samples from the ﬁlter chamber were collected and

of genomic DNA digested with the indicated restriction enzymes was hybridized the number of parasites counted. Values represent the means SD of three exper-

with the whole insert of gp82 cDNA clone (J18) labeled with [ P]. (B) Chromosomal iments performed in triplicate. (B) Gastric or submaxillary mucin was added to

bands of parasites were separated by pulsed ﬁeld gel electrophoresis, transferred to HeLa cells 15 min before addition of parasites. After 1 h, the cells were ﬁxed and

nylon membrane and hybridized with the [ P]-labeled probe as above. Numbers Giemsa-stained. The number of internalized parasites was counted in a total of 250

correspond to molecular sizes. Note the differences between BAT strain and the cells. Values are means SD of three independent assays performed in duplicate.

other two strains. Submaxillary mucin signiﬁcantly inhibited ( p = 0.0001).

236 F.Y. Maeda et al. / Acta Tropica 120 (2011) 231–237

gp82 plays a central role in selectively binding to gastric mucin, a

property critical for the parasite migration through the mucus layer

toward the underlying target cells (Staquicini et al., 2010). Also of

note is that gp82 is resistant to degradation by pepsin (Cortez et al.,

2006a).

In addition to gp82, BAT strain MT express mucin-like molecules

at high levels. These molecules may also play a role in parasite inter-

nalization. Host cell invasion was reduced in the presence of mucins

puriﬁed from BAT isolate. The ﬁnding that BAT strain mucins

reduced the levels of lyosomal exocytosis, i.e., they had an oppo-

site effect of gp82, further reinforces the role played by exocytosis

in MT invasion. We presume that gp82-mediated parasite–host

cell interaction prevails over that mediated by mucins during BAT

strain invasion. If the situation were the other way around, the

Fig. 5. Effect of T. cruzi mucins on host cell exocytosis and invasion by BAT strain MT. impairment of lysosomal exocytosis, which contributes to para-

(A) The indicated T. cruzi mucins were added to HeLa cells 15 min before parasites. sitophorous vacuole formation (Tardieux et al., 1994; Rodríguez

After 1 h incubation, the cells were ﬁxed and Giemsa-stained for parasite counting.

et al., 1999; Fernandes et al., 2011), would result in low infection

Values are means SD of four independent assays performed in duplicate. Invasion

* ** rate. G strain MT, which rely predominantly on mucin molecules,

was signiﬁcantly inhibited ( p < 0.01, p < 0.05) by mucins of different strains. (B)

are poorly invasive (Yoshida, 2006). By contrast, highly invasive CL

Hela cells were incubated for 1 h with the indicated T. cruzi mucins and the released

lysosomal enzyme ␤-hexosaminidase was measured. Values are means ± SD of three strain MT depend mostly on gp82 and minimally, if at all, on mucin

independent assays performed in duplicate. The difference between exocytosis of

molecules (Ramirez et al., 1993). BAT and CL strain mucins lack

Hela cells treated with BAT strain mucin and the control was signiﬁcant ( p < 0.05).

recognition by mAb 10D8, which reacts with an epitope containing

galactofuranose residues in G strain mucins (Yoshida, 2006) and

lysosomal exocytosis, the difference from the control was not sta- reduces parasite infectivity (Yoshida et al., 1989). It is possible that

tistically signiﬁcant. this structural difference determines the differential interaction of

mucins from BAT, CL and G strains with host cells.

3.5. In vivo infection by BAT strain MT We have analyzed here only one strain from each of the three

genetic groups, therefore we can not assert that these strains are

To determine the infectivity of BAT strain MT by the oral route, a representatives of the respective lineages. As regards TcI, the meta-

group of mice (n = 5) was infected orally (10 parasites per mouse) cyclic forms of 7 strains that we have analyzed so far, including

and the course of infection was monitored. Blood samples were those isolated from marsupial or from wild triatomine in differ-

examined for the presence of parasites up to 30 day post infection. ent geographical regions, displayed similar surface proﬁle and their

Parasitemia was not detectable, what is in contrast to infection ability to infect human epithelial cells was associated with the

by CL strain MT that consistently resulted in patent parasitemias expression of gp90 on the surface (Ruiz et al., 1998; Yoshida, 2006).

(Cortez et al., 2003, 2006a; Covarrubias et al., 2007). Intraperitoneal Thus, G strain that originated from a marsupial in the Brazilian

injection was also quite inefﬁcient. On average, from 10 infected Amazon may be a bona ﬁde representative of TcI associated with

mice we could recover parasites in hemoculture from two to three the wild transmission cycle. However, as TcI that predominates

mice, whereas positive hemoculture is invariably obtained from all in northern South America is also associated with human disease,

mice infected with G or CL strain. chagasic cardiomyopathy being commonplace in countries such as

Venezuela (Miles et al., 2009), it would be of interest to investi-

4. Discussion gate the infective properties of TcI strains isolated from Chagasic

patients. We have found in a previous study that metacyclic forms

Our results have indicated that BAT strain MT invade host cells of T. cruzi strains (presumably TcII), isolated from acute cases of

in a manner similar to CL strain MT, i.e., they engage the surface Chagas’ disease in Brazil and expressing gp90 at high levels, exhib-

molecule gp82 and trigger the activation of mTOR, PI3K and PKC ited reduced capacity to enter host cells in vitro but efﬁciently

to promote their internalization through lysosome exocytosis. The infected mice by the oral route, provided that they expressed

involvement of gp82 in BAT strain MT entry into host cells was pepsin-susceptible gp90 isoform, which was digested upon contact

implied from the ﬁndings that parasite internalization was inhib- with gastric juice (Cortez et al., 2006a; Covarrubias et al., 2007).

ited by monoclonal antibody directed to gp82, as well as by the The inter-lineage hybrids TcV and TcVI are apparently the main

recombinant protein based on gp82. As regards the lysosome exo- causes of severe acute and chronic Chagas disease in the greater

cytosis, its role in invasion was deduced from experiments showing Gran Chaco region and neighbouring countries where T. infestans is

that down regulation of mTOR, PI3K and PKC, previously shown to the principal domestic vector (Miles et al., 2009). Metacyclic forms

affect mobilization of lysosomes from the perinuclear region to the of CL strain (TcVI), isolated from T. infestans, was characterized by

cell periphery (Martins et al., 2011), diminished MT internalization. expressing gp90 at low levels and by high infectivity in vitro as

Of interest was the ﬁnding that BAT strain gp82 shares high well as in vivo (Yoshida, 2006). If this is a common feature of TcV

sequence identity with gp82 of genetically divergent G and CL and TcVI, it remains to be investigated. BAT strain distinguished

strains, isolated from different sources in widely distant geograph- from all T. cruzi strains examined to date by lack of reaction with

ical regions. G strain derived from a marsupial captured in the monoclonal antibodies directed to gp90. More strains have to be

Brazilian Amazon while CL strain, associated with human infec- analyzed to assess whether this is a general characteristic within

tion, was isolated in the far south of the country. The conservation this new genetic group.

of p82 molecule may be associated with its crucial role in the Metacyclic forms of BAT strain used in this study were poorly

establishment of infection by the oral route, which is possibly a infective in mice. This may not necessarily indicate that this genetic

mode of transmission prevalent among insectivorous mammalian group associated with bats have a low potential to infect humans.

hosts since ancient times. Insect stage metacyclic forms are well One should bear in mind that, as stressed above, only one isolate of

equipped to efﬁciently infect by the oral route, selectively invading this group was examined. TcI, for instance, was initially thought to

the gastric mucosal epithelium (Hoft, 1996; Hoft et al., 1996), and be innocuous to humans, but it is now clear that parasites of this

F.Y. Maeda et al. / Acta Tropica 120 (2011) 231–237 237

lineage may cause severe clinical manifestations. In central Brazil, invasion of gastric mucosal epithelium in orally infected mice. Microbes Infect.

8, 36–44.

TcI was identiﬁed in 12 acute cases of Chagas’ disease (Luquetti

Cortez, M., Atayde, V., Yoshida, N., 2006b. Host cell invasion mediated by

et al., 1986), and Anez˜ et al. (2004) have found the predominance

Trypanosoma cruzi surface molecule gp82 is associated with F-actin disas-

of TcI human isolates in Venezuela allied to the higher prevalence sembly and is inhibited by enteroinvasive Escherichia coli. Microbes Infect. 8,

1502–1512.

of severe symptoms of the disease. Another report that contradicts

Coura, J.R., 2006. Transmission of chagasic infection by oral route in the natural

the assumption of innocuousness of TcI refers to a Bolivian patient

history of Chagas’ disease. Rev. Soc. Bras. Med. Trop. 39 (Suppl. 3), 113–117.

with Chagas disease with accompanying AIDS, and severe central Covarrubias, C., Cortez, M., Ferreira, D., Yoshida, N., 2007. Interaction with host fac-

tors exacerbate Trypanosoma cruzi cell invasion capacity upon oral infection. Int.

nervous system involvement, whose cerebrospinal ﬂuid showed

J. Parasitol. 37, 1609–1616.

TcI parasite population (Burgos et al., 2008). An interesting question

Fernandes, M.C., Cortez, M., Flannery, A.R., Tam, C., Mortara, R.A., Andrews, N.W.,

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Ferreira, D., Cortez, M., Atayde, V.D., Yoshida, N., 2006. Actin cytoskeleton-dependent

The ﬁrst fossil evidence of triatomine-trypanosomatid association

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of debate is whether T. cruzi infection spread from bats to other

Hoft, D.F., Farrar, P.L., Kratz-Owens, K., Shaffer, D., 1996. Gastric invasion by Try-

mammals, including humans.

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Rodrigues, I., 1986. Trypanosoma cruzi: zymodemes associated with acute and

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Metacyclic forms of BAT strain, belonging to a new genotype of

Málaga, S., Yoshida, N., 2001. Targeted reduction in expression of Trypanosoma

T. cruzi associated with bats from anthropic areas, have the abil-

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Characterization of the cell adhesion site of Trypanosoma cruzi metacyclic stage

the highly infective T. cruzi CL strain, which also relies on gp82 for

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of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses

C and the mammalian target of rapamycin. This leads to lysoso-

using SSU rDNA, cytochrome b and histone H2B genes and genotyping bases on

mal exocytosis, an event required for parasite internalization. BAT

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bats has still to be evaluated.

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do Estado de São Paulo (FAPESP #2006/61450-0) and Conselho

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Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin

and invasion of epithelial cells. Infect. Immun. 71, 557–561.

#301409/2007-2 and #470726/2007-5), Brazil.

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