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

Co-infection with Trypanosoma cruzi protects mice against early death by neurological or pulmonary disorders induced by Plasmodium berghei ANKA

Objective: the objective of this study was to investigate whether the infection of C57BL/ 6 mice by P. berghei ANKA, which causes severe malaria, was modulated by co-infection with Trypanosoma cruzi.Methods: Groups of C57BL/ 6 mice were infected either with P. berghei ANKA, T. cruzi strain G, or with both parasites. the presence of parasites was checked by microscopic examination of blood samples. Symptoms of neurological or respiratory disorders, as well as mortality, were registered. Breakdown of the blood brain barrier was determined by injecting the dye Evans blue. Histological sections of the lung were prepared and stained with hematoxilin-eosin.Results: All mice infected only with P. berghei ANKA died within 7-11 days post-infection, either with symptoms of cerebral malaria or with respiratory abnormalities. the animals co- infected with T. cruzi strain G survived longer, without any of the referred to symptoms. Protection against the early death by severe malaria was effective when mice were given T. cruzi 15 days before P. berghei inoculation. Breakdown of the blood brain barrier and extensive pulmonary oedema, caused by malaria parasites, were much less pronounced in co- infected mice. the degree of protection to severe malaria and early death, conferred by co- infection with T. cruzi, was comparable to that conferred by treatment with anti-CD8 antibodies.Conclusion: Co-infection with T. cruzi protects C57BL/ 6 against the early death by malaria infection, by partially preventing either the breakdown of the blood brain, and cerebral malaria as a consequence, or the pulmonary oedema.Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol, São Paulo, BrazilUniv Estadual Campinas, Inst Biol, Dept Parasitol, Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol, São Paulo, BrazilWeb of Scienc

Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection

Propranolol inibe a invasão da célula hospedeira por formas metacíclicas do trypanosoma cruzi

The invasion of the host cell is a crucial step for the establishment of Trypanosoma cruzi infection, the protozoan parasite that causes Chagas disease. The mechanisms involved in the entry of T. cruzi in to the cells are only partially known. The available information indicates that the interaction of metacyclic trypomastigotes, form of the parasite found in the insect vector and responsible for initiating infection in the mammalian host, target cells through gp82, a surface molecule of the parasite. It triggers signaling cascades in the host cells resulting in mobilization and exocytosis of lysosomes, an event that requires the participation of microtubules.It is known that host cell surface components are involved in this process. During experiments in which different ligands of cell receptors were tested, we observed that pretreatment with propranolol, an antagonist of ?-adrenergic receptor, is able to significantly inhibit the entry of metacyclic forms (CL strain) in HeLa cells, whereas isoproterenol, an ?-adrenergic receptor agonist, showed no effect. The inhibitory activity of propranolol was detected at 10 µM, and was even higher at 50 µM. We observed that propranolol acts predominantly on the host cell, as preincubation of cells for 30 minutes with propranolol led to a reduction in the levels of internalized parasites. The same effect was not seen pre-treating the parasites before invasion. To determine whether propranolol affected the mobilization of lysosomes induced by gp 82 and required for invasion, HeLa cells were treated for 30 minutes with 10 µM of the drug, washed, and incubated in PBS++ a nutritionally depleted medium that induces lysosomal exocytosis. Immunofluorescence analysis showed that this pretreatment interfered with the mobilization of lysosomes, causing retention of these organelles in the perinuclear region. Similarly, propranolol blocked the lysosome mobilization caused by gp82. These results indicate that propranolol interferes with cell signaling promoted by gp82 when inducing exocytosis of lysosomes, a process required for cellular invasion of metacyclic forms.A invasão de células no hospedeiro mamífero é uma etapa crucial para o estabelecimento da infecção pelo Trypanosoma cruzi, o protozoário parasita causador da doença de Chagas. Os mecanismos envolvidos na entrada do T. cruzi nas células são apenas parcialmente conhecidos. No que se refere aos tripomastigotas metacíclicos, formas do parasita encontradas no inseto vetor e responsáveis por iniciar a infecção no hospedeiro mamífero, as informações disponíveis indicam que a interação dos parasitas com a célula alvo é mediada pela sua molécula de superfície gp82 que dispara cascatas de sinalização que resultam na mobilização de lisossomos e exocitose, eventos que requerem a participação de microtúbulos. Não se sabe que componentes de superfície da célula hospedeira estão envolvidas neste processo. Durante experimentos em que diferentes ligantes de receptores celulares foram testados, observamos que o pré-tratamento com propranolol, um antagonista do receptor ?-adrenérgico, é capaz de inibir significativamente a entrada das formas metacíclicas (cepa CL) em células HeLa, enquanto o isoproterenol, agonista do receptor ?-adrenérgico, não mostrou nenhum efeito. A atividade inibitória do propranolol foi detectada a 10 ?M, e foi ainda maior a 50 ?M. Verificamos que propranolol age predominantemente sobre a célula hospedeira, pois a pré-incubação de 30 minutos com 10 ?M propranolol levou à redução nos níveis de parasitas internalizados, sendo o efeito maior à concentração de 50 ?M. O mesmo efeito não foi visto pré-tratando os parasitas antes da invasão. Para determinar se o propranolol afeta a mobilização de lisossomos, necessário para a internalização dos parasitas e causada pela proteína gp82, as células HeLa foram tratadas por 30 minutos com 10 ?M da droga, lavadas e incubadas em PBS++, um meio nutricionalmente empobrecido que induz a exocitose lisossomal. Análise das células processadas para imunofluorescência mostrou que este pré-tratamento interferiu com a mobilização de lisossomos, ocasionando a retenção dessas organelas na região perinuclear. Da mesma forma, o propranolol inibiu a mobilização dos lisossomos causada pela gp82. Esses resultados indicam que propranolol interfere com a sinalização celular e a exocitose de lisossomos promovida pela gp82 e requeridas para a invasão celular de formas metacíclicas.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Lack of requirement of gp82 in G strain MT invasion.

HeLa cells were incubated for 1 h with: (A) MT pretreated for 30 min with non purified anti-gp82 mAb 3F6 or mAb 5E7, or (B) purified mAb 3F6 at indicated concentrations, (C) MT in absence or in the presence of recombinant gp82 protein or GST, and processed for intracellular parasite quantification. MT invasion was significantly increased by treatment with non purified mAb 3F6 or by purified antibody at 200 μg/ml (*PD) HeLa cells were incubated for 30 min in absence or in the presence of MT. After washings, the cell extracts were analyzed by western blotting, using antibody directed to phosphorylated PKC or ERK1/2. Note that incubation with MT did not alter the phosphorylation levels of PKC or ERK1/2.</p

Inhibition of G strain MT invasion by pretreatment of host cells with sucrose.

(A) HeLa cells were treated for 30 min with 0.45 M sucrose in serum-free medium, washed and incubated with: (A) G strain MT in PBS++ or (B) G strain EA in R10. After 1 h, the cells were processed for intracellular parasite quantification. Values are the means ± five independent assays. HeLa cells pretreated with sucrose were significantly more resistant to invasion by MT (*PC) Untreated and sucrose-treated HeLa cells were incubated for 30 min with MT and processed for immunofluorescence microscopy, using anti-clathrin antibody and mAb 10D8. The images show clathrin (red), parasite mucins (green) and nucleus (blue). Scale bar = 30 μm.</p

Increased resistance of annexin A2-depleted cells to G strain MT invasion.

(A) HeLa cells were submitted to lentiviral transduction for annexin A2 knockdown (kd) and analyzed by western blotting. Note the depletion of annexin A2 in two independent cell lines. (B) HeLa cells depleted in annexin A2 and WT cells were incubated for 1 h with G strain MT. The amounts of intracellular parasites are shown as means ± SD of three independent assays performed in duplicate. MT invasion was significantly diminished in cells deficient in annexin A2 (*PC) Non infected annexin A2-deficient and WT cells were analyzed by immunofluorescence, to visualize actin (red), lysosome (green) and nucleus (blue). Scale bar = 30 μm. Note the altered morphology of annexin-kd1 cells, as compared to WT cells, and the distinct lysosome distribution in annexin-kd2 cells.</p

Colocalization of invading G strain MT and released gp35/50 mucins with host cell clathrin.

(A) HeLa cells were incubated for 30 min with parasites and processed for immunofluorescence microscopy, using anti-clathrin antibody and mAb 10D8. The images show clathrin (red), parasite mucins (green) and nucleus (blue). Scale bar = 30 μm. Note the colocalization of MT with clathrin. (B) A single cell with invading parasites is depicted to show the colocalization of clathrin with MT and also with shed gp35/50 mucins at the cell membrane (white arrows). Scale bar = 5 μm.</p