Clathrin expression in Trypanosoma cruzi

Kalb, Ligia Cristina; Frederico, Yohana Camila Antunes; Batista, Cassiano Martin; Eger, Iriane; Fragoso, Stênio Perdigão; Soares, Maurílio J.

doi:10.1186/1471-2121-15-23

Cited by 18 publications

(16 citation statements)

References 36 publications

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“…These studies highlighted the broad specificity of the SPC by showing that it was capable of shuttling BSA, IgG, Transferrin, and CFSE labeled host protein through this endocytic pathway. Surprisingly, we failed to observe any endocytosis of cargo, including albumin (BSA), via the flagellar pocket in our assays suggesting that the SPC may be the exclusive endocytic route for whole protein uptake in this parasite (Soares and de Souza, 1991;de Figueiredo and Soares, 2000;Kalb et al, 2014).…”

Section: Discussionmentioning

confidence: 67%

“…It is notable that we only observed endocytosis via the SPC and failed to detect protein uptake through the flagellar pocket. This is in stark contrast to T. brucei which endocytoses exclusively through its flagellar pocket and to previously published reports of T. cruzi (de Figueiredo and Soares, 2000;Kalb et al, 2014). In order to evaluate endocytosis in intracellular amastigotes, we stained host cells first with the free-amine labeling fluorescent dye CFSE followed by infection with trypomastigotes from the CP1-Ty tagged line.…”

Section: Cp1 Labels the Protein Endocytic Pathway Of The Cytostome-cymentioning

confidence: 95%

“…CP3-mNeon-Ty also localized to the SPC in epimastigotes, but additionally localized diffusely to disparate areas of the cell which may be due to overexpression (Figure 4F). The dispersed labeling of CP3 suggests a potential minor affinity for cytoskeletal microtubules when overexpressed (Angelopoulos, 1970) however because the most intense labeling is along the SPC it suggests that CP3 may be labeling one or both of the microtubule root fibers associated with the endocytic structure that can also be seen in Supplementary Figure 4 ( Kalb et al, 2014;Alcantara et al, 2017). To demonstrate the co-localization of CP2 and CP3 fusions to the SPC in amastigotes, we generated a polyclonal mouse antibody against a 240 amino acid antigenic region of CP1 recombinantly expressed in E. coli (Supplementary Figure 5A) (α-CP1).…”

Section: Identification Of Two Additional Cytostome-cytopharynx Protementioning

confidence: 99%

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Identification and Localization of the First Known Proteins of the Trypanosoma cruzi Cytostome Cytopharynx Endocytic Complex

Chasen

Coppens

Etheridge

2020

Front. Cell. Infect. Microbiol.

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The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 7 million people in the Americas, with an at-risk population of 70 million. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease continues to receive insufficient attention and resources in order to be effectively combatted. Unlike the other parasitic trypanosomatids that infect humans (Trypanosoma brucei and Leishmania spp.), T. cruzi retains an ancestral mode of phagotrophic feeding via an endocytic organelle known as the cytostome-cytopharynx complex (SPC). How this tubular invagination of the plasma membrane functions to bring in nutrients is poorly understood at a mechanistic level, partially due to a lack of knowledge of the protein machinery specifically targeted to this structure. Using a combination of CRISPR/Cas9 mediated endogenous tagging, fluorescently labeled overexpression constructs and endocytic assays, we have identified the first known SPC targeted protein (CP1). The CP1 labeled structure co-localizes with endocytosed protein and undergoes disassembly in infectious forms and reconstitution in replicative forms. Additionally, through the use of immunoprecipitation and mass spectrometry techniques, we have identified two additional CP1-associated proteins (CP2 and CP3) that also target to this endocytic organelle. Our localization studies using fluorescently tagged proteins and surface lectin staining have also allowed us, for the first time, to specifically define the location of the intriguing pre-oral ridge (POR) surface prominence at the SPC entrance through the use of super-resolution light microscopy. This work is a first glimpse into the proteome of the SPC and provides the tools for further characterization of this enigmatic endocytic organelle. A better understanding of how this deadly pathogen acquires nutrients from its host will potentially direct us toward new therapeutic targets to combat infection.

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Section: Discussionmentioning

confidence: 67%

Section: Cp1 Labels the Protein Endocytic Pathway Of The Cytostome-cymentioning

confidence: 95%

Section: Identification Of Two Additional Cytostome-cytopharynx Protementioning

confidence: 99%

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Identification and Localization of the First Known Proteins of the Trypanosoma cruzi Cytostome Cytopharynx Endocytic Complex

Chasen

Coppens

Etheridge

2020

Front. Cell. Infect. Microbiol.

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“…Two different tracers were used: transferrin (mostly internalized via the cytostome) and albumin [41]. …”

Section: Resultsmentioning

confidence: 99%

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

et al. 2018

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BackgroundThe palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease.ResultsThe effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 μM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity.ConclusionOur results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.Electronic supplementary materialThe online version of this article (10.1186/s12860-018-0170-3) contains supplementary material, which is available to authorized users.

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“…Despite the fact that this parasite has clathrin that could participate in endocytosis [ 54 , 55 ], morphological studies have demonstrated that Tf internalization is carried out through a clathrin-independent and cholesterol-dependent endocytosis pathway. This pathway was identified by the utilization of specific inhibitors of endocytic pathways.…”

Section: Tfr In Protozoan Parasitic Organismsmentioning

confidence: 99%

Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa

Reyes-López

Piña-Vázquez

Serrano-Luna

2015

BioMed Research International

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Iron is the fourth most abundant element on Earth and the most abundant metal in the human body. This element is crucial for life because almost all organisms need iron for several biological activities. This is the case with pathogenic organisms, which are at the vanguard in the battle with the human host for iron. The latest regulates Fe concentration through several iron-containing proteins, such as transferrin. The transferrin receptor transports iron to each cell that needs it and maintains it away from pathogens. Parasites have developed several strategies to obtain iron as the expression of specific transferrin receptors localized on plasma membrane, internalized through endocytosis. Signal transduction pathways related to the activation of the receptor have functional importance in proliferation. The study of transferrin receptors and other proteins with action in the signaling networks is important because these proteins could be used as therapeutic targets due to their specificity or to differences with the human counterpart. In this work, we describe proteins that participate in signal transduction processes, especially those that involve transferrin endocytosis, and we compare these processes with those found in T. brucei, T. cruzi, Leishmania spp., and E. histolytica parasites.

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Clathrin expression in Trypanosoma cruzi

Cited by 18 publications

References 36 publications

Identification and Localization of the First Known Proteins of the Trypanosoma cruzi Cytostome Cytopharynx Endocytic Complex

Identification and Localization of the First Known Proteins of the Trypanosoma cruzi Cytostome Cytopharynx Endocytic Complex

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa

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