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WOS000297428400012.Pdf Acta Tropica 120 (2011) 231–237 Contents lists available at SciVerse ScienceDirect Acta Tropica journa l homepage: www.elsevier.com/locate/actatropica 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. © 2011 Elsevier B.V. Open access under the Elsevier OA license. 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., 2+ 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 0001-706X © 2011 Elsevier B.V. Open access under the Elsevier OA license. doi:10.1016/j.actatropica.2011.09.001 232 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 32 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 5 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.
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