Rac1/WAVE2 and Cdc42/N-WASP Participation in Actin-Dependent Host Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi

This study evaluated the participation of host cell Rho-family GTPases and their effector proteins in the actin-dependent invasion by Trypanosoma cruzi extracellular amastigotes (EAs). We observed that all proteins were recruited and colocalized with actin at EA invasion sites in live or fixed cells. EA internalization was inhibited in cells depleted in Rac1, N-WASP, and WAVE2. Time-lapse experiments with Rac1, N-WASP and WAVE2 depleted cells revealed that EA internalization kinetics is delayed even though no differences were observed in the proportion of EA-induced actin recruitment in these groups. Overexpression of constitutively active constructs of Rac1 and RhoA altered the morphology of actin recruitments to EA invasion sites. Additionally, EA internalization was increased in cells overexpressing CA-Rac1 but inhibited in cells overexpressing CA-RhoA. WT-Cdc42 expression increased EA internalization, but curiously, CA-Cdc42 inhibited it. Altogether, these results corroborate the hypothesis of EA internalization in non-phagocytic cells by a phagocytosis-like mechanism and present Rac1 as the key Rho-family GTPase in this process.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e TecnologicoUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilFAPESP: 2012/21335-8, 2011/51475-3CNPq: 302068/2016-3Web of Scienc

Reactivity of MEST-1 (antigalactofuranose) with Trypanosoma cruzi, glycosylinositol phosphorylceramides (GIPCs): Immunolocalization of GIPCs in acidic vesicles of epimastigotes

Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various Trypanosoma cruzi forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. in epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilWeb of Scienc

Amastigote Synapse: The Tricks of Trypanosoma cruzi Extracellular Amastigotes

To complete its life cycle within the mammalian host, Trypanosoma cruzi, the agent of Chagas’ disease, must enter cells. Trypomastigotes originating from the insect vector (metacyclic) or from infected cells (bloodstream/tissue culture-derived) are the classical infective forms of the parasite and enter mammalian cells in an actin-independent manner. By contrast, amastigotes originating from the premature rupture of infected cells or transformed from swimming trypomastigotes (designated extracellular amastigotes, EAs) require functional intact microfilaments to invade non-phagocytic host cells. Earlier work disclosed the key features of EA-HeLa cell interplay: actin-rich protrusions called ‘cups’ are formed at EA invasion sites on the host cell membrane that are also enriched in actin-binding proteins, integrins and extracellular matrix elements. In the past decades we described the participation of membrane components and secreted factors from EAs as well as the actin-regulating proteins of host cells involved in what we propose to be a phagocytic-like mechanism of parasite uptake. Thus, regarding this new perspective herein we present previously described EA-induced ‘cups’ as parasitic synapse since they can play a role beyond its architecture function. In this review, we focus on recent findings that shed light on the intricate interaction between extracellular amastigotes and non-phagocytic HeLa cells

Parasite-mediated remodeling of the host microfilament cytoskeleton enables rapid egress of Trypanosoma cruzi following membrane rupture

Chagas’ disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this patho-gen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for mainte-nance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell’s actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages—a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to pre-serve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomasti-gotes may trigger sudden egress

The Repetitive Cytoskeletal Protein H49 of Trypanosoma cruzi Is a Calpain-Like Protein Located at the Flagellum Attachment Zone

Background: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. in the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins.Methods and Findings: All of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. the H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. the H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body.Conclusions: H49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of ensuring that the cell body remains attached to the flagellum by connecting the subpellicular microtubule array to it.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Beca Presidente de la Republica-ChileUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilUniv Antofagasta, Lab Bioquim, Dept Biomed, Antofagasta, ChileUniv Bandeirante São Paulo, São Paulo, BrazilUniv Brasilia, Dept Biol Celular, Inst Biol, Brasilia, DF, BrazilFiocruz MS, Ctr Pesquisa Rene Rachou CPqRR, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilWeb of Scienc

Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion

Trypanosoma cruzi takes advantage of a sphingomyelinase-dependent plasma membrane repair pathway to gain access to host cells

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.Fundacao de Amparo a Pesquisa do Estado de Minas Gerais [APQ-00621-11]Fundacao de Amparo a Pesquisa do Estado de Minas GeraisFundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao PauloCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior [23038005295/2011-40]Coordenacao de Aperfeicoamento de Pessoal de Nivel SuperiorConselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Cientifico e Tecnologic

Fusion between Leishmania amazonensis and Leishmania major Parasitophorous Vacuoles: Live Imaging of Coinfected Macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes - which were destroyed - differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation – a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs