Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients

Background: Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Methods: Trypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were incubated with metacyclic forms and the number of internalized parasites was counted. Results: All TcI and TcIV T. cruzi strains were poorly infective by the oral route. Parasites were either undetectable or were detected in small numbers in the mouse stomach four days post oral administration. Replicating parasites were found in the stomach and/or in the heart 30 days post-infection. As compared to TcI lineage, the migration capacity of TcIV parasites through the gastric mucin-coated filter was higher but lower than that exhibited by TcVI metacyclic forms previously shown to be highly infective by the oral route. Expression of pepsin-resistant gp90, the surface molecule that downregulates cell invasion, was higher in TcI than in TcIV parasites and, accordingly, the invasion capacity of TcIV metacyclic forms was higher. Gp90 molecules spontaneously released by TcI metacyclic forms inhibited the parasite entry into host cells. TcI parasites exhibited low intracellular replication rate. Conclusions: Our findings indicate that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of metacyclic forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Univ Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilFAPESP: 11/51475-3CNPq: 300578/2010-5Web of Scienc

A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. in these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D H-1 nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INBEQMeDIUniv Fed Uberlandia, Inst Ciencias Biomed, BR-38400 Uberlandia, MG, BrazilUniv São Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Vila Mariana, SP, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Biol Geral, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Vila Mariana, SP, BrazilFAPESP: 2010/51867-6FAPESP: 2012/21153-7FAPEMIG: APQ-00621-11FAPEMIG: APQ-00305-12CAPES: 23038.005295/2011-40Web of Scienc

Moléculas de superfície liberadas pelas formas metacíclicas do trypanosoma cruzi modulam negativamente a invasão celular

Molecules released by pathogenic microorganisms can modulate the interaction with their hosts. We investigated whether metacyclic trypomastigote (MT) forms of diferent strains of Trypanosoma cruzi differencially released extracelular vesicles and soluble proteins, and examined their effect on MT invasion of host cells, focusing on the surface molecules gp82 and gp90. MTs of highly infective CL strain and poorly infective G strain were incubated for 1 h in complete medium (D10) or in PBS++. Conditioned medium (CM), collected after centrifugation of parasites and designated as CM-G and CM-CL, was used for cell invasion assays and Westen blot analysis. Hela cells were incubated for 1 h with MTs of CL strain in D10, or of G strain in PBS++ that induces the scattering/exocytosis of lysosomes and promotes parasite invasion, in the absence or presence of CM. Parasite internalization was significantly reduced in the presence of CM-G but not of CM-CL. Analysis of CM by Western blot, using monoclonal antibodies against gp82 and gp90, which function as promoter and negative regulator of cell invasion, respectively, revealed high levels of gp82 and gp90 in CM-G and low levels in CM-CL. G strain CM generated in PBS++, containing lower amounts of gp90 e gp82 when compared to CM produced in D10, displayed a lower inhibitory effect on invasion by MTs. Preincubation of CM-G (D10) with anti-gp90 or anti-gp82 antibody that does not recognize live MTs neutralized the cell invasion inhibitory activity. Vesicles of diverse size and soluble factors, obtained by fractionation of CM-G and containing gp82 and gp90 molecules, were capable of significantly inhibiting cell invasion by MTs. Our results suggest that factors spontaneously released by MTs, containing high levels of gp82 and gp90, interfer with parasite internalization, gp82 presumably competing for the host cell receptor with the molecule expressed on MT surface, and gp90 further contributing to down modulate invasion.Moléculas liberadas por microrganismos patogênicos podem modular sua interação com os hospedeiros. Nós investigamos se formas tripomastigotas metacíclicas (TMs) de diferentes cepas do Trypanosoma cruzi liberam vesículas extracelulares e proteínas solúveis de maneira diferenciada, e qual o efeito desses fatores na invasão celular, com foco nas moléculas de superfície gp82 e gp90. TMs da cepa CL, altamente infectiva, e da cepa G, pouco infectiva, foram incubados por 1 hora em meio completo (D10) ou em PBS++. O meio condicionado (MC), coletado após centrifugação dos parasitas, e denominado MC-CL e MC-G, foi usado em ensaios de invasão celular e análise por Western blot. Células HeLa foram incubadas por 1 hora com TMs da cepa CL em D10, ou da cepa G em PBS++ que induz espalhamento/exocitose de lisossomos e promove a invasão, na ausência ou na presença de MC. A internalização de parasitas foi significativamente reduzida na presença de MC-G, mas não de MC-CL. Análise de MC por Western blot, usando anticorpos monoclonais contra gp82 e gp90, que funcionam como promotor e regulador negativo da invasão celular, respectivamente, revelou altos níveis de gp82 a gp90 em MC-G e baixos níveis em MC-CL. O MC da cepa G gerado em PBS++, contendo quantidades menores de gp90 e gp82 quando comparado com o MC produzido em D10, mostrou efeito inibitório menor sobre a invasão por TMs. A pré-incubação de MC-G (D10) com anticorpo anti-gp90 ou anti-gp82 que não reconhece TMs vivos neutralizou a atividade inibitória da invasão celular. Vesículas de diversos tamanhos e fatores solúveis, obtidos pelo fracionamento do MC-G e contendo moléculas gp82 e gp90, foram capazes de inibir significativamente a invasão celular de TMs. Nossos resultados sugerem que fatores espontaneamente liberados por TMs, contendo altos níveis de gp82 e gp90, interferem no processo de internalização dos parasitas, gp82 presumivelmente competindo pelo receptor da célula hospedeira com a molécula de superfície dos TMs, e gp90 contribuindo mais ainda para modular negativamente a invasão.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Surface Molecules Released by <i>Trypanosoma cruzi</i> Metacyclic Forms Downregulate Host Cell Invasion

<div><p>Background</p><p>The question whether metacylic trypomastigote (MT) forms of different <i>T</i>. <i>cruzi</i> strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using <i>T</i>. <i>cruzi</i> strains that differ widely in the ability to invade cells.</p><p>Methodology/Principal Findings</p><p>Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca²⁺ and Mg²⁺ (PBS⁺⁺). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS⁺⁺, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS⁺⁺ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion.</p><p>Conclusion/Significance</p><p>Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.</p></div

Change in the expression of surface gp90 and gp82 proteins in <i>T</i>. <i>cruzi</i> G strain metacyclic forms after incubation in different media.

<p>Parasites were incubated or not in D10 or PBS⁺⁺ for 1h at 37°C. After centrifugation, the supernatant was discarded and the parasites were incubated for 1 h with monoclonal antibody directed to gp90 or gp82. Following fixation and reaction with Alexa Fluor 488-conjugated anti-IgG, the parasites were analyzed by flow cytometry. Controls consisted of parasites incubated with the second antibody only. Shown in the lower panel are images of parasites visualized by epifluorescence microscope, with 100X objective.</p

Reversal of the inhibitory effect of G-CM on PBS⁺⁺-induced lysosome spreading by specific antibodies.

<p>HeLa cells were incubated for 30 min in PBS⁺⁺ in absence (-) or in the presence of CL-CM, G-CM, G-CM preincubated with mAb 5E7, mAb 2C2, anti-J18 or anti-C03 antibody and processed for confocal immunofluorescence analysis using anti-LAMP2 antibody, Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, with 63X objective. Scale bar: 15 μm. HeLa cells incubated in PBS⁺⁺ in the presence of r-gp90 served as control for inhibition of lysosome spreading.</p

Effect of gp82 contained in G-CM on MT invasion.

<p>A-B) HeLa cells were incubated for 1 h in PBS⁺⁺ with G strain MT in absence or in the presence of G-CM generated in D10 alone, or preincubated with anti-gp82 mAb 3F6 (A) or with polyclonal anti-gp82 antibodies (B). After fixation and Giemsa-staining, the number of intracellular parasites was counted in 250 cells. Values are the means ± SD of three independent assays performed in duplicate. The inhibitory effect of G-CM was significantly reverted by anti-gp82 polyclonal antibody anti-C03 (*P<0.005). C) HeLa cells were incubated for 30 min in absence or in the presence of 20 μg/ml recombinant gp82 (r-gp82) and processed for confocal immunofluorescence analysis as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004883#pntd.0004883.g005" target="_blank">Fig 5B</a>.</p

Release of vesicles by <i>T</i>. <i>cruzi</i> metacyclic tripomastigotes.

<p>A) Parasites were washed in PBS and processed for analysis by scanning electron microscopy. Scale-bars: 1–5 μm). B) After 1 h incubation in particle-free D10 medium or in PBS⁺⁺, the parasites were centrifuged and the supernatant collected for fractionation. Large particles (V2) and smaller particles (V16), obtained after 2 h and 16 h ultracentrifugation respectively, were quantified by nanoparticle tracking analysis. Shown is the particle size distribution in conditioned medium from G and CL strain in D10 and PBS⁺⁺. Data are representative of 3 independent experiments. The number of particles released by G and CL strains was significantly different (*P<0.05). C) The fractions V2, V16 and vesicle-free (VF) fractions obtained from G-CM were analyzed by Western blot using monoclonal antibodies directed to gp90 and gp82. D) HeLa cells were incubated for 1 h with G strain MT in PBS⁺⁺ or in PBS⁺⁺ plus V2, V16 or VF fractions of G-CM generated in D10 or PBS⁺⁺. Data are representative of 3 independent experiments (*P<0.05, **P<0.01, ***P<0.005).</p

Inhibitory effect of gp90 molecule on PBS⁺⁺–induced lysosome spreading and host cell invasion by MT.

<p>A) HeLa cells were incubated for 1 h in PBS⁺⁺ with G strain MT in absence or in the presence of G-CM generated in D10 alone, or preincubated with anti-gp90 mAb 5E7 or unrelated mAb 2C2. After fixation and Giemsa-staining, the number of intracellular parasites was counted in 250 cells. Values are the means ± SD of three independent assays performed in duplicate. The inhibitory effect of G-CM was significantly reverted by mAb 5E7 (*P<0.005). B) HeLa cells were incubated for 30 min in PBS⁺⁺ in absence (-) or in the presence of G-CM and processed for confocal immunofluorescence analysis using anti-LAMP2 antibody, Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, with 63X objective. Scale bar: 15 μm. C) Schematic representation of gp90 and the recombinant protein containing the C-terminal domain of gp90 fused to GST (r-gp90). D) HeLa cell-coated microtiter plates were used for binding assays of native or r-gp90 and binding was detected in ELISA using anti-gp90 monoclonal antibody. A representative result of assays performed in triplicates, whose variation was <5%, is shown. E) HeLa cells were incubated for 30 min in PBS⁺⁺, in absence or in the presence of 20 μg/ml r-gp90 or GST, and then processed for confocal immunofluorescence analysis as in (B). Scale bar: 15 μm. F) HeLa cells were incubated for 1 h with G strain MT in PBS⁺⁺, in absence or in the presence of r-gp90 or GST at various concentrations, and processed as in A) for parasite counting. Values are the means ± SD of three independent assays performed in duplicate. Parasite invasion was significantly diminished in the presence of r-gp90 (*P<0.05, **P<0.0005). G) HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of r-gp90 or GST at 40 μg/ml, and processed as in (A). Values are the means ± SD of three independent assays performed in duplicate. Parasite invasion was significantly diminished in the presence of r-gp90 (*P<0.0005).</p

Effect of <i>T</i>. <i>cruzi</i> conditioned medium containing high gp90 and gp82 amounts on host cell invasion by metacyclic forms.

<p>A) Western blot of detergent-soluble MT extract (E) of G and CL strains and the corresponding conditioned medium (CM) generated at 15, 30 and 60 min incubation of MT in D10, was probed with anti-gp90 mAb 5E7 or anti-gp82 mAb 3F6. B) HeLa cells were incubated for 1 h with G or CL strain MT, in PBS⁺⁺ or in D10. After fixation and staining with Giemsa, the number of intracellular parasites was counted in a total of 250 cells. Values are the means ± SD of five independent assays performed in duplicate. MT invasion rate was significantly higher in PBS⁺⁺ than in D10 (*<i>P</i>< 0.<i>05</i>, **<i>P</i><0.0001). C) HeLa cells were incubated for 30 min in D10 or in PBS⁺⁺, and then processed for immunofluorescence analysis using anti-LAMP2 antibody, Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA. Epifluorescence microscope, with 100X objective, was used. Scale bar: 15 μm. Note the lysosome scattering and LAMP2 accumulation (arrow) at the edges of cells incubated in PBS⁺⁺. D) HeLa cells were incubated for 1 h with G or CL strain MT, in PBS⁺⁺ or in D10, in absence or in the presence of G-CM generated as in (A), at 1:50 dilution. After fixation and staining with Giemsa, the number of intracellular parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. G-CM significantly inhibited cell invasion by G strain MT (*<i>P</i><0.01, ** <i>P</i> = 0.0005, *** <i>P</i><0.0001) and by CL strain MT (*<i>P</i><0.05, ** <i>P</i><0.01). E) HeLa cells were incubated for 1 h with G or CL strain MT, in PBS⁺⁺ or in D10, in absence of in the presence of G-CM or CL-CM generated after 1 h MT incubation in D10, and processed for parasite counting. Values are the means ± SD of four independent assays performed in duplicate. Invasion by both strains was significantly inhibited by G-CM (*<i>P</i><0.005) but not by CL-CM. F) HeLa cells were incubated for the indicated times with G or CL strain MT in D10 and processed for intracellular parasite counting. Invasion by CL strain, but not by G strain, increased proportional to the incubation times.</p