SummaryThe molecular mechanisms of host cell invasion by T. cruzi metacyclic trypomastigotes (MT), the developmental forms that initiate infection in the mammalian host, are only partially understood. Here we aimed at further identifying the target cell components involved in signalling cascades leading to MT internalization, and demonstrate for the first time the participation of mammalian target of rapamycin (mTOR). Treatment of human epithelial HeLa cells with mTOR inhibitor rapamycin reduced lysosomal exocytosis and MT invasion. Downregulation of phosphatidylinositol 3-kinase and protein kinase C also impaired exocytosis and MT internalization. The recombinant protein based on gp82, the MT surface molecule that mediates cell adhesion/invasion, induced exocytosis in HeLa cells. Such an effect has not previously been attributed to any T. cruzi surface molecule. Rapamycin treatment diminished gp82 binding as well. Cell invasion assays under conditions that promoted lysosome exocytosis, such as 1 h incubation in starvation medium PBS ++ , increased MT invasion, whereas pre-starvation of cells for 1-2 h had an opposite effect. In contrast to MT, invasion of tissue culture trypomastigotes (TCT) increased upon host cell pre-starvation or treatment with rapamycin, a novel finding that discloses quite distinctive features of the two infective forms in a key process for infection.

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.

BackgroundOutbreaks 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.MethodsTrypanosoma 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.ResultsAll 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1455-z) contains supplementary material, which is available to authorized users.

BackgroundDiversity of T. cruzi strains is a central problem in Chagas disease research because of its correlation with the wide range of clinical manifestations and the biogeographical parasite distribution. The role played by parasite microdiversity in Chagas disease epidemiology is still debatable. Also awaits clarification whether such diversity is associated with the outcome of oral T. cruzi infection, responsible for frequent outbreaks of acute Chagas disease.Methods and FindingsWe addressed the impact of microdiversity in oral T. cruzi infection, by comparative analysis of two strains, Y30 and Y82, both derived from Y strain, a widely used experimental model. Network genealogies of four nuclear genes (SSU rDNA, actin, DHFR-TS, EF1α) revealed that Y30 is closely related to Discrete Typing Unit TcII while Y82 is more closely related to TcVI, a group containing hybrid strains. Nevertheless, excepting one A-G transition at position 1463, Y30 and Y82 SSU rDNAs were identical. Y82 strain, expressing the surface molecule gp82, infected mice orally more efficiently than Y30, which expresses a related gp30 molecule. Both molecules are involved in lysosome exocytosis-dependent host cell invasion, but exhibit differential gastric mucin-binding capacity, a property critical for parasite migration toward the gastric mucosal epithelium. Upon oral infection of mice, the number of Y30 and Y82 parasites in gastric epithelial cells differed widely.ConclusionsWe conclude that metacyclic forms of gp82-expressing Y82 strain, closely related to TcVI, are better adapted than Y30 strain (TcII) to traverse the stomach mucous layer and establish oral route infection. The efficiency to infect target cell is the same because gp82 and gp30 strains have similar invasion-promoting properties. Unknown is whether differences in Y30 and Y82 are natural parasite adaptations or a product of lab-induced evolution by differential selection along the 60 years elapsed since the Y strain isolation.

The surface molecule gp82 of metacyclic trypomastigote (MT) forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca2+ signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca2+-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca2+ from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.