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1
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Zhang N, Jiang N, Chen Q. Key Regulators of Parasite Biology Viewed Through a Post-Translational Modification Repertoire. Proteomics 2024:e202400120. [PMID: 39690890 DOI: 10.1002/pmic.202400120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2024] [Revised: 11/29/2024] [Accepted: 12/04/2024] [Indexed: 12/19/2024]
Abstract
Parasites are the leading causes of morbidity and mortality in both humans and animals, imposing substantial socioeconomic burdens worldwide. Controlling parasitic diseases has become one of the key issues in achieving "One Health". Most parasites have sophisticated life cycles exhibiting progressive developmental stages, morphologies, and host-switching, which are controlled by various regulatory machineries including protein post-translational modifications (PTMs). PTMs have emerged as a key mechanism by which parasites modulate their virulence, developmental transitions, and environmental adaptations. PTMs are enzyme-mediated additions or removals of chemical groups that dynamically regulate the stability and functions of proteins and confer novel properties, playing vital roles in a variety of biological processes and cellular functions. In this review, we circumscribe how parasites utilize various PTMs to regulate their intricate lives, with a focus on the biological role of PTMs in parasite biology and pathogenesis.
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Affiliation(s)
- Naiwen Zhang
- Key Laboratory of Livestock Infectious Diseases, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China
- The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, Shenyang, China
| | - Ning Jiang
- Key Laboratory of Livestock Infectious Diseases, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China
- The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, Shenyang, China
| | - Qijun Chen
- Key Laboratory of Livestock Infectious Diseases, Ministry of Education, Key Laboratory of Zoonosis, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China
- The Research Unit for Pathogenic Mechanisms of Zoonotic Parasites, Chinese Academy of Medical Sciences, Shenyang, China
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2
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Stadler KA, Ortiz-Joya LJ, Singh Sahrawat A, Buhlheller C, Gruber K, Pavkov-Keller T, O'Hagan TB, Guarné A, Pulido S, Marín-Villa M, Zangger K, Gubensäk N. Structural investigation of Trypanosoma cruzi Akt-like kinase as drug target against Chagas disease. Sci Rep 2024; 14:10039. [PMID: 38693166 PMCID: PMC11063076 DOI: 10.1038/s41598-024-59654-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Accepted: 04/12/2024] [Indexed: 05/03/2024] Open
Abstract
According to the World Health Organization, Chagas disease (CD) is the most prevalent poverty-promoting neglected tropical disease. Alarmingly, climate change is accelerating the geographical spreading of CD causative parasite, Trypanosoma cruzi, which additionally increases infection rates. Still, CD treatment remains challenging due to a lack of safe and efficient drugs. In this work, we analyze the viability of T. cruzi Akt-like kinase (TcAkt) as drug target against CD including primary structural and functional information about a parasitic Akt protein. Nuclear Magnetic Resonance derived information in combination with Molecular Dynamics simulations offer detailed insights into structural properties of the pleckstrin homology (PH) domain of TcAkt and its binding to phosphatidylinositol phosphate ligands (PIP). Experimental data combined with Alpha Fold proposes a model for the mechanism of action of TcAkt involving a PIP-induced disruption of the intramolecular interface between the kinase and the PH domain resulting in an open conformation enabling TcAkt kinase activity. Further docking experiments reveal that TcAkt is recognized by human inhibitors PIT-1 and capivasertib, and TcAkt inhibition by UBMC-4 and UBMC-6 is achieved via binding to TcAkt kinase domain. Our in-depth structural analysis of TcAkt reveals potential sites for drug development against CD, located at activity essential regions.
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Affiliation(s)
- Karina A Stadler
- Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Graz, Austria
| | - Lesly J Ortiz-Joya
- Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Graz, Austria
- Programa de Estudio y Control de Enfermedades Tropicales (PECET), Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
- Department of Biochemistry, McGill University, Montreal, Canada
| | - Amit Singh Sahrawat
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
- Innophore GmbH, Graz, Austria
| | | | - Karl Gruber
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
- Innophore GmbH, Graz, Austria
- Field of Excellence BioHealth, University of Graz, Graz, Austria
- BioTechMed-Graz, Graz, Austria
| | - Tea Pavkov-Keller
- Institute of Molecular Biosciences, University of Graz, Graz, Austria
- Field of Excellence BioHealth, University of Graz, Graz, Austria
- BioTechMed-Graz, Graz, Austria
| | | | - Alba Guarné
- Department of Biochemistry, McGill University, Montreal, Canada
| | - Sergio Pulido
- Programa de Estudio y Control de Enfermedades Tropicales (PECET), Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
- LifeFactors ZF SAS, Rionegro, Colombia
| | - Marcel Marín-Villa
- Programa de Estudio y Control de Enfermedades Tropicales (PECET), Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
| | - Klaus Zangger
- Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Graz, Austria.
- Field of Excellence BioHealth, University of Graz, Graz, Austria.
- BioTechMed-Graz, Graz, Austria.
| | - Nina Gubensäk
- Institute of Molecular Biosciences, University of Graz, Graz, Austria.
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3
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Maldonado E, Canobra P, Oyarce M, Urbina F, Miralles VJ, Tapia JC, Castillo C, Solari A. In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes. Microorganisms 2024; 12:907. [PMID: 38792752 PMCID: PMC11124317 DOI: 10.3390/microorganisms12050907] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2024] [Revised: 04/06/2024] [Accepted: 04/17/2024] [Indexed: 05/26/2024] Open
Abstract
Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in T. cruzi epimastigotes.
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Affiliation(s)
- Edio Maldonado
- Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile; (P.C.); (M.O.); (F.U.); (J.C.T.)
| | - Paz Canobra
- Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile; (P.C.); (M.O.); (F.U.); (J.C.T.)
| | - Matías Oyarce
- Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile; (P.C.); (M.O.); (F.U.); (J.C.T.)
| | - Fabiola Urbina
- Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile; (P.C.); (M.O.); (F.U.); (J.C.T.)
| | - Vicente J. Miralles
- Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, 46110 Valencia, Spain;
| | - Julio C. Tapia
- Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile; (P.C.); (M.O.); (F.U.); (J.C.T.)
| | - Christian Castillo
- Programa de Anatomía y Biología del Desarrollo, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile;
| | - Aldo Solari
- Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago 8380453, Chile; (P.C.); (M.O.); (F.U.); (J.C.T.)
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4
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Macedo-da-Silva J, Mule SN, Rosa-Fernandes L, Palmisano G. A computational pipeline elucidating functions of conserved hypothetical Trypanosoma cruzi proteins based on public proteomic data. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2024; 138:401-428. [PMID: 38220431 DOI: 10.1016/bs.apcsb.2023.07.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/16/2024]
Abstract
The proteome is complex, dynamic, and functionally diverse. Functional proteomics aims to characterize the functions of proteins in biological systems. However, there is a delay in annotating the function of proteins, even in model organisms. This gap is even greater in other organisms, including Trypanosoma cruzi, the causative agent of the parasitic, systemic, and sometimes fatal disease called Chagas disease. About 99.8% of Trypanosoma cruzi proteome is not manually annotated (unreviewed), among which>25% are conserved hypothetical proteins (CHPs), calling attention to the knowledge gap on the protein content of this organism. CHPs are conserved proteins among different species of various evolutionary lineages; however, they lack functional validation. This study describes a bioinformatics pipeline applied to public proteomic data to infer possible biological functions of conserved hypothetical Trypanosoma cruzi proteins. Here, the adopted strategy consisted of collecting differentially expressed proteins between the epimastigote and metacyclic trypomastigotes stages of Trypanosoma cruzi; followed by the functional characterization of these CHPs applying a manifold learning technique for dimension reduction and 3D structure homology analysis (Spalog). We found a panel of 25 and 26 upregulated proteins in the epimastigote and metacyclic trypomastigote stages, respectively; among these, 18 CHPs (8 in the epimastigote stage and 10 in the metacyclic stage) were characterized. The data generated corroborate the literature and complement the functional analyses of differentially regulated proteins at each stage, as they attribute potential functions to CHPs, which are frequently identified in Trypanosoma cruzi proteomics studies. However, it is important to point out that experimental validation is required to deepen our understanding of the CHPs.
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Affiliation(s)
- Janaina Macedo-da-Silva
- GlycoProteomics Laboratory, Department of Parasitology, ICB, University of São Paulo, Sao Paulo, Brazil
| | - Simon Ngao Mule
- GlycoProteomics Laboratory, Department of Parasitology, ICB, University of São Paulo, Sao Paulo, Brazil
| | - Livia Rosa-Fernandes
- GlycoProteomics Laboratory, Department of Parasitology, ICB, University of São Paulo, Sao Paulo, Brazil; Centre for Motor Neuron Disease Research, Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, Sydney, NSW, Australia
| | - Giuseppe Palmisano
- GlycoProteomics Laboratory, Department of Parasitology, ICB, University of São Paulo, Sao Paulo, Brazil; School of Natural Sciences, Macquarie University, Sydney, NSW, Australia.
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5
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Desale H, Herrera C, Dumonteil E. Trypanosoma cruzi amastigote transcriptome analysis reveals heterogenous populations with replicating and dormant parasites. Microbes Infect 2024; 26:105240. [PMID: 37866547 DOI: 10.1016/j.micinf.2023.105240] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2023] [Revised: 10/13/2023] [Accepted: 10/17/2023] [Indexed: 10/24/2023]
Abstract
Trypanosoma cruzi is a protozoan parasite causing Chagas disease, with a complex life cycle involving different stages in insect vectors and mammalian hosts. Amastigotes are an intracellular form that replicates in the cytoplasm of host cells, and recent studies suggested that dormant forms may be contributing to parasite persistence, suggesting cellular heterogeneity among amastigotes. We investigated here if a transcriptomic approach could identify some heterogeneity in intracellular amastigotes and identify a dormant population. We used gene expression data derived from bulk RNA-sequencing of T. cruzi infection of human fibroblasts for deconvolution using CDSeq, which allows to simultaneously estimate amastigote cell-type proportions and cell-type-specific expression profiles. Six amastigote subpopulations were identified, confirming intracellular amastigotes heterogeneity, and one population presented characteristics of non-replicative dormant parasites, based on replication markers and TcRAD51 expression. Transcriptomic approaches appear to be powerful to understand T. cruzi cell differentiation and expansion of these studies could provide further insight on the role different cell types in parasite persistence and Chagas disease pathogenesis.
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Affiliation(s)
- Hans Desale
- Department of Tropical Medicine, School of Public Health and Tropical Medicine, and Vector-Borne and Infectious Disease Research Center, Tulane University, New Orleans, LA, USA
| | - Claudia Herrera
- Department of Tropical Medicine, School of Public Health and Tropical Medicine, and Vector-Borne and Infectious Disease Research Center, Tulane University, New Orleans, LA, USA
| | - Eric Dumonteil
- Department of Tropical Medicine, School of Public Health and Tropical Medicine, and Vector-Borne and Infectious Disease Research Center, Tulane University, New Orleans, LA, USA.
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6
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Marcelino TDP, Fala AM, da Silva MM, Souza-Melo N, Malvezzi AM, Klippel AH, Zoltner M, Padilla-Mejia N, Kosto S, Field MC, Burle-Caldas GDA, Teixeira SMR, Couñago RM, Massirer KB, Schenkman S. Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation. J Biol Chem 2023; 299:104857. [PMID: 37230387 PMCID: PMC10300260 DOI: 10.1016/j.jbc.2023.104857] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2023] [Revised: 05/02/2023] [Accepted: 05/05/2023] [Indexed: 05/27/2023] Open
Abstract
The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.
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Affiliation(s)
- Tiago de Paula Marcelino
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Angela Maria Fala
- Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, Campinas, SP, Brazil
| | - Matheus Monteiro da Silva
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Normanda Souza-Melo
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Amaranta Muniz Malvezzi
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil
| | - Angélica Hollunder Klippel
- Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, Campinas, SP, Brazil; Departamento de Ciências Biológicas da Faculdade de Ciências Farmacêuticas da Universidade Estadual Paulista "Júlio de Mesquita Filho"-Unesp, Araraquara, SP, Brazil
| | - Martin Zoltner
- Drug Discovery and Evaluation Unit, Department of Parasitology, Faculty of Science, Charles University in Prague, BIOCEV, Vestec, Czech Republic
| | | | - Samantha Kosto
- School of Life Sciences, University of Dundee, Dundee, UK
| | - Mark C Field
- School of Life Sciences, University of Dundee, Dundee, UK; Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Ceske Budejovice, Czech Republic
| | | | | | - Rafael Miguez Couñago
- Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, Campinas, SP, Brazil
| | - Katlin Brauer Massirer
- Center for Molecular Biology and Genetic Engineering - CBMEG, Center of Medicinal Chemistry - CQMED, Structural Genomics Consortium - SGC, University of Campinas - UNICAMP, Campinas, SP, Brazil.
| | - Sergio Schenkman
- Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
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7
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Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation. Biochem J 2022; 479:225-243. [PMID: 35015072 PMCID: PMC8883493 DOI: 10.1042/bcj20210313] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Revised: 12/15/2021] [Accepted: 01/11/2022] [Indexed: 11/17/2022]
Abstract
The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.
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Coutinho JVP, Rosa-Fernandes L, Mule SN, de Oliveira GS, Manchola NC, Santiago VF, Colli W, Wrenger C, Alves MJM, Palmisano G. The thermal proteome stability profile of Trypanosoma cruzi in epimastigote and trypomastigote life stages. J Proteomics 2021; 248:104339. [PMID: 34352427 DOI: 10.1016/j.jprot.2021.104339] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Revised: 06/24/2021] [Accepted: 07/28/2021] [Indexed: 12/18/2022]
Abstract
Trypanosoma cruzi is a flagellate protozoa being the etiological agent of Chagas disease, a neglected tropical disease, which still poses a public health problem worldwide. The intricate molecular changes during T. cruzi-host interaction have been explored using different largescale omics techniques. However, protein stability is largely unknown. Thermal proteome profiling (TPP) methodology has the potential to characterize proteome-wide stability highlighting key proteins during T. cruzi infection and life stage transition from the invertebrate to the mammalian host. In the present work, T. cruzi epimastigotes and trypomastigotes cell lysates were subjected to TPP workflow and analyzed by quantitative large-scale mass spectrometry-based proteomics to fit a melting profile for each protein. A total of 2884 proteins were identified and associated to 1741 melting curves being 1370 in trypomastigotes (TmAVG 53.53 °C) and 1279 in epimastigotes (TmAVG 50.89 °C). A total of 453 proteins were identified with statistically different melting profiles between the two life stages. Proteins associated to pathogenesis and intracellular transport had regulated melting temperatures. Membrane and glycosylated proteins had a higher average Tm in trypomastigotes compared to epimastigotes. This study represents the first large-scale comparison of parasite protein stability between life stages. SIGNIFICANCE: Trypanosoma cruzi, a unicellular flagellate parasite, is the etiological agent of Chagas disease, endemic in South America and affecting more that 7 million people worldwide. There is an intense research to identify novel chemotherapeutic and diagnostic targets of Chagas disease. Proteomic approaches have helped in elucidating the quantitative proteome and PTMs changes of T. cruzi during life cycle transition and upon different biotic and abiotic stimuli. However, a comprehensive knowledge of the protein-protein interaction and protein conformation is still missing. In order to fill this gap, this manuscript elucidates the T. cruzi Y strain proteome-wide thermal stability map in the epimastigote and trypomastigote life stages. Comparison between life stages showed a higher average melting temperature stability for trypomastigotes than epimastigotes indicating a host temperature adaptation. Both presented a selective thermal stability shift for cellular compartments, molecular functions and biological processes based on the T. cruzi life stage. Membrane and glycosylated proteins presented a higher thermal stability in trypomastigotes when compared to the epimastigotes.
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Affiliation(s)
- Joao V P Coutinho
- GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
| | - Livia Rosa-Fernandes
- GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
| | - Simon Ngao Mule
- GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
| | - Gilberto Santos de Oliveira
- GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
| | | | - Veronica Feijoli Santiago
- GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
| | - Walter Colli
- Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Brazil
| | - Carsten Wrenger
- Unit for Drug Discovery, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
| | | | - Giuseppe Palmisano
- GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil.
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9
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Lander N, Chiurillo MA, Docampo R. Signaling pathways involved in environmental sensing in Trypanosoma cruzi. Mol Microbiol 2021; 115:819-828. [PMID: 33034088 PMCID: PMC8032824 DOI: 10.1111/mmi.14621] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2020] [Revised: 09/28/2020] [Accepted: 09/30/2020] [Indexed: 12/18/2022]
Abstract
Trypanosoma cruzi is a unicellular parasite and the etiologic agent of Chagas disease. The parasite has a digenetic life cycle alternating between mammalian and insect hosts, where it faces a variety of environmental conditions to which it must adapt in order to survive. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Major environmental changes include temperature, nutrient availability, ionic composition, pH, osmolarity, oxidative stress, contact with host cells and tissues, host immune response, and intracellular life. Some of the signaling pathways and second messengers potentially involved in the response to these changes have been elucidated in recent years and will be the subject of this review.
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Affiliation(s)
- Noelia Lander
- Center for Tropical and Emerging Global Diseases, and Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA
| | - Miguel A. Chiurillo
- Center for Tropical and Emerging Global Diseases, and Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA
| | - Roberto Docampo
- Center for Tropical and Emerging Global Diseases, and Department of Cellular Biology, University of Georgia, Athens, GA, 30602, USA
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10
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Oliveira LN, Lima PDS, Araújo DS, Portis IG, Santos Júnior ADCMD, Coelho ASG, de Sousa MV, Ricart CAO, Fontes W, Soares CMDA. iTRAQ-based proteomic analysis of Paracoccidioides brasiliensis in response to hypoxia. Microbiol Res 2021; 247:126730. [PMID: 33662850 DOI: 10.1016/j.micres.2021.126730] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 01/29/2021] [Accepted: 02/13/2021] [Indexed: 02/06/2023]
Abstract
Aerobic organisms require oxygen for energy. In the course of the infection, adaptation to hypoxia is crucial for survival of human pathogenic fungi. Members of the Paracoccidioides complex face decreased oxygen tensions during the life cycle stages. In Paracoccidioides brasiliensis proteomic responses to hypoxia have not been investigated and the regulation of the adaptive process is still unknown, and this approach allowed the identification of 216 differentially expressed proteins in hypoxia using iTRAQ-labelling. Data suggest that P. brasiliensis reprograms its metabolism when submitted to hypoxia. The fungus reduces its basal metabolism and general transport proteins. Energy and general metabolism were more representative and up regulated. Glucose is apparently directed towards glycolysis or the production of cell wall polymers. Plasma membrane/cell wall are modulated by increasing ergosterol and glucan, respectively. In addition, molecules such as ethanol and acetate are produced by this fungus indicating that alternative carbon sources probably are activated to obtain energy. Also, detoxification mechanisms are activated. The results were compared with label free proteomics data from Paracoccidioides lutzii. Biochemical pathways involved with acetyl-CoA, pyruvate and ergosterol synthesis were up-regulated in both fungi. On the other hand, proteins from TCA, transcription, protein fate/degradation, cellular transport, signal transduction and cell defense/virulence processes presented different profiles between species. Particularly, proteins related to methylcitrate cycle and those involved with acetate and ethanol synthesis were increased in P. brasiliensis proteome, whereas GABA shunt were accumulated only in P. lutzii. The results emphasize metabolic adaptation processes for distinct Paracoccidioides species.
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Affiliation(s)
- Lucas Nojosa Oliveira
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICB II, Campus II, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
| | - Patrícia de Sousa Lima
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICB II, Campus II, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
| | - Danielle Silva Araújo
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICB II, Campus II, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
| | - Igor Godinho Portis
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICB II, Campus II, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
| | | | | | - Marcelo Valle de Sousa
- Departmento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Campus Darcy Ribeiro, Asa Norte, 70910-900, Brasília, DF, Brazil.
| | - Carlos André Ornelas Ricart
- Departmento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Campus Darcy Ribeiro, Asa Norte, 70910-900, Brasília, DF, Brazil.
| | - Wagner Fontes
- Departmento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Campus Darcy Ribeiro, Asa Norte, 70910-900, Brasília, DF, Brazil.
| | - Célia Maria de Almeida Soares
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICB II, Campus II, Universidade Federal de Goiás, 74001-970, Goiânia, Goiás, Brazil.
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11
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Santos Júnior ADCMD, Melo RM, Ferreira BVG, Pontes AH, Lima CMRD, Fontes W, Sousa MVD, Lima BDD, Ricart CAO. Quantitative proteomics and phosphoproteomics of Trypanosoma cruzi epimastigote cell cycle. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2021; 1869:140619. [PMID: 33561577 DOI: 10.1016/j.bbapap.2021.140619] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Revised: 01/19/2021] [Accepted: 02/01/2021] [Indexed: 11/30/2022]
Abstract
The protozoan Trypanosoma cruzi is the causative agent of the neglected infectious illness Chagas disease. During its life cycle it differentiates into replicative and non-replicative life stages. So far, T. cruzi cell division has been investigated by transcriptomics but not by proteomics approaches. Here we show the first quantitative proteome analysis of T. cruzi cell division. T. cruzi epimastigote cultures were subject to synchronization with hydroxyurea and harvested at different time points. Analysis by flow cytometry, bright field and fluorescence microscopy indicated that samples collected at 0 h, 2 h, 6 h and 14 h overrepresented G1, G1-S, S and M cell cycle phases, respectively. After trypsin digestion of these samples, the resulting peptides were labelled with iTRAQ and subjected to LC-MS/MS. Also, iTRAQ-labelled phosphopeptides were enriched with TiO2 to access the phosphoproteome. Overall, 597 protein groups and 94 phosphopeptides presented regulation with the most remarkable variation in abundance at 6 h (S-phase). Comparison of our proteomic data to previous transcriptome-wise analysis of epimastigote cell cycle showed 16 sequence entries in common, with the highest mRNA/protein correlation observed in transcripts with peak abundance in G1-phase. Our data revealed regulated proteins and phosphopeptides which play important roles in the control of cell division in other organisms and some of them were previously detected in the nucleus or associated with T. cruzi chromatin.
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Affiliation(s)
- Agenor de Castro Moreira Dos Santos Júnior
- Laboratory Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil; Laboratory of Gene Biology, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil
| | - Reynaldo Magalhães Melo
- Laboratory Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil
| | | | - Arthur Henriques Pontes
- Laboratory Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil
| | | | - Wagner Fontes
- Laboratory Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil
| | - Marcelo Valle de Sousa
- Laboratory Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil
| | - Beatriz Dolabela de Lima
- Laboratory of Gene Biology, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil
| | - Carlos André Ornelas Ricart
- Laboratory Protein Chemistry and Biochemistry, Department of Cell Biology, University of Brasília, Brasília, DF, Brazil.
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12
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Stryiński R, Łopieńska-Biernat E, Carrera M. Proteomic Insights into the Biology of the Most Important Foodborne Parasites in Europe. Foods 2020; 9:E1403. [PMID: 33022912 PMCID: PMC7601233 DOI: 10.3390/foods9101403] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 09/24/2020] [Accepted: 09/27/2020] [Indexed: 02/07/2023] Open
Abstract
Foodborne parasitoses compared with bacterial and viral-caused diseases seem to be neglected, and their unrecognition is a serious issue. Parasitic diseases transmitted by food are currently becoming more common. Constantly changing eating habits, new culinary trends, and easier access to food make foodborne parasites' transmission effortless, and the increase in the diagnosis of foodborne parasitic diseases in noted worldwide. This work presents the applications of numerous proteomic methods into the studies on foodborne parasites and their possible use in targeted diagnostics. Potential directions for the future are also provided.
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Affiliation(s)
- Robert Stryiński
- Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland;
| | - Elżbieta Łopieńska-Biernat
- Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland;
| | - Mónica Carrera
- Department of Food Technology, Marine Research Institute (IIM), Spanish National Research Council (CSIC), 36-208 Vigo, Spain
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13
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Azeredo CM, Saraiva MF, de Oliveira MR, Barbosa G, de Almeida MV, de Souza MVN, Soares MJ. The terpenic diamine GIB24 inhibits the growth of Trypanosoma cruzi epimastigotes and intracellular amastigotes, with proteomic analysis of drug-resistant epimastigotes. Chem Biol Interact 2020; 330:109165. [PMID: 32771326 DOI: 10.1016/j.cbi.2020.109165] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 05/18/2020] [Accepted: 06/05/2020] [Indexed: 10/23/2022]
Abstract
The effect of N-geranyl-ethane-1,2-diamine dihydochloride (GIB24), a synthetic diamine, was assayed against different developmental forms of the parasitic protozoan Trypanosoma cruzi (strain Dm28c). The compound was effective against culture epimastigote forms (IC50/24h = 5.64 μM; SI = 16.4) and intracellular amastigotes (IC50/24h = 12.89 μM; SI = 7.18), as detected by the MTT methodology and by cell counting, respectively. Incubation of epimastigotes for 6h with 6 μM GIB24 (IC50/24h value) resulted in significant dissipation of the mitochondrial membrane potential, prior to permeabilization of the plasma membrane. Rounded epimastigotes with cell size reduction were observed by scanning electron microscopy. These morpho-physiological changes induced by GIB24 suggest an incidental death process. Treatment of infected Vero cells did not prevent the intracellular amastigotes from completing the intracellular cycle. However, there was a decrease in the number of released parasites, increasing the ratio amastigotes/trypomastigotes. Proteomic analysis of 15 μM GIB24 resistant epimastigotes indicated that the compound acts mainly on mitochondrial components involved in the Krebs cycle and in maintaining the oxidative homeostasis of the parasites. Our data suggest that GIB24 is active against the main morphological forms of T. cruzi.
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Affiliation(s)
- Camila Maria Azeredo
- Laboratory of Cell Biology, Carlos Chagas Institute/Fiocruz-PR, Curitiba, PR, Brazil
| | | | | | - Gisele Barbosa
- Department of Chemistry, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil
| | | | | | - Maurilio José Soares
- Laboratory of Cell Biology, Carlos Chagas Institute/Fiocruz-PR, Curitiba, PR, Brazil
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14
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Petito G, de Curcio JS, Pereira M, Bailão AM, Paccez JD, Tristão GB, de Morais COB, de Souza MV, de Castro Moreira Santos A, Fontes W, Ricart CAO, de Almeida Soares CM. Metabolic Adaptation of Paracoccidioides brasiliensis in Response to in vitro Copper Deprivation. Front Microbiol 2020; 11:1834. [PMID: 32849434 PMCID: PMC7430155 DOI: 10.3389/fmicb.2020.01834] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Accepted: 07/13/2020] [Indexed: 02/06/2023] Open
Abstract
Copper is an essential micronutrient for the performance of important biochemical processes such as respiration detoxification, and uptake of metals like iron. Studies have shown that copper deprivation is a strategy used by the host against pathogenic fungi such as Cryptoccocus neoformans and Candida albicans during growth and development of infections in the lungs and kidneys. Although there are some studies, little is known about the impact of copper deprivation in members of the Paracoccidioides genus. Therefore, using isobaric tag labeling (iTRAQ)-Based proteomic approach and LC-MS/MS, we analyzed the impact of in vitro copper deprivation in the metabolism of Paracoccidioides brasiliensis. One hundred and sixty-four (164) differentially abundant proteins were identified when yeast cells were deprived of copper, which affected cellular respiration and detoxification processes. Changes in cellular metabolism such as increased beta oxidation and cell wall remodeling were described.
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Affiliation(s)
- Guilherme Petito
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Brazil
- Programa de Pós-graduação em Genética e Biologia Molecular, Universidade Federal de Goiás, Goiânia, Brazil
| | - Juliana Santana de Curcio
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Brazil
| | - Maristela Pereira
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Brazil
| | - Alexandre Melo Bailão
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Brazil
| | - Juliano Domiraci Paccez
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Brazil
| | - Gabriel Brum Tristão
- Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Brazil
| | | | - Marcelo Valle de Souza
- Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Brasília, Brazil
| | | | - Wagner Fontes
- Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Brasília, Brazil
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15
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Parthasarathy A, Kalesh K. Defeating the trypanosomatid trio: proteomics of the protozoan parasites causing neglected tropical diseases. RSC Med Chem 2020; 11:625-645. [PMID: 33479664 PMCID: PMC7549140 DOI: 10.1039/d0md00122h] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Accepted: 05/12/2020] [Indexed: 12/20/2022] Open
Abstract
Mass spectrometry-based proteomics enables accurate measurement of the modulations of proteins on a large scale upon perturbation and facilitates the understanding of the functional roles of proteins in biological systems. It is a particularly relevant methodology for studying Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei, as the gene expression in these parasites is primarily regulated by posttranscriptional mechanisms. Large-scale proteomics studies have revealed a plethora of information regarding modulated proteins and their molecular interactions during various life processes of the protozoans, including stress adaptation, life cycle changes and interactions with the host. Important molecular processes within the parasite that regulate the activity and subcellular localisation of its proteins, including several co- and post-translational modifications, are also accurately captured by modern proteomics mass spectrometry techniques. Finally, in combination with synthetic chemistry, proteomic techniques facilitate unbiased profiling of targets and off-targets of pharmacologically active compounds in the parasites. This provides important data sets for their mechanism of action studies, thereby aiding drug development programmes.
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Affiliation(s)
- Anutthaman Parthasarathy
- Rochester Institute of Technology , Thomas H. Gosnell School of Life Sciences , 85 Lomb Memorial Dr , Rochester , NY 14623 , USA
| | - Karunakaran Kalesh
- Department of Chemistry , Durham University , Lower Mount Joy, South Road , Durham DH1 3LE , UK .
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16
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Wozniak JM, Silva TA, Thomas D, Siqueira-Neto JL, McKerrow JH, Gonzalez DJ, Calvet CM. Molecular dissection of Chagas induced cardiomyopathy reveals central disease associated and druggable signaling pathways. PLoS Negl Trop Dis 2020; 14:e0007980. [PMID: 32433643 PMCID: PMC7279607 DOI: 10.1371/journal.pntd.0007980] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2019] [Revised: 06/08/2020] [Accepted: 03/25/2020] [Indexed: 12/13/2022] Open
Abstract
Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.
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Affiliation(s)
- Jacob M. Wozniak
- Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America
- Department of Pharmacology; University of California San Diego; La Jolla, CA, United States of America
| | - Tatiana Araújo Silva
- Cellular Ultrastructure Laboratory; Oswaldo Cruz Institute, FIOCRUZ; Rio de Janeiro, RJ, Brazil
| | - Diane Thomas
- Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America
| | - Jair L. Siqueira-Neto
- Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America
| | - James H. McKerrow
- Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America
| | - David J. Gonzalez
- Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America
- Department of Pharmacology; University of California San Diego; La Jolla, CA, United States of America
- * E-mail: (DJG); (CMC)
| | - Claudia M. Calvet
- Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America
- Cellular Ultrastructure Laboratory; Oswaldo Cruz Institute, FIOCRUZ; Rio de Janeiro, RJ, Brazil
- * E-mail: (DJG); (CMC)
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17
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Wang T, Ma G, Ang CS, Korhonen PK, Stroehlein AJ, Young ND, Hofmann A, Chang BCH, Williamson NA, Gasser RB. The developmental phosphoproteome of Haemonchus contortus. J Proteomics 2019; 213:103615. [PMID: 31846766 DOI: 10.1016/j.jprot.2019.103615] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2019] [Revised: 11/22/2019] [Accepted: 12/13/2019] [Indexed: 12/22/2022]
Abstract
Protein phosphorylation plays essential roles in many cellular processes. Despite recent progress in the genomics, transcriptomics and proteomics of socioeconomically important parasitic nematodes, there is scant phosphoproteomic data to underpin molecular biological discovery. Here, using the phosphopeptide enrichment-based LC-MS/MS and data-independent acquisition (DIA) quantitation, we characterised the first developmental phosphoproteome of the parasitic nematode Haemonchus contortus - one of the most pathogenic parasites of ruminant livestock. Totally, 1804 phosphorylated proteins with 4406 phosphorylation sites ('phosphosites') from different developmental stages/sexes were identified. Bioinformatic analyses of quantified 'phosphosites' exhibited distinctive stage- and sex-specific patterns during development, and identified a subset of phosphoproteins proposed to play crucial roles in processes such as spindle positioning, signal transduction and kinase activity. A sequence-based comparison of the phosphoproteome of H. contortus with those of two free-living nematode species (Caenorhabditis elegans and Pristionchus pacificus) suggested a limited number of common protein phosphorylation events among these species. Our findings infer active roles for protein phosphorylation in the adaptation of a parasitic nematode to a constantly changing external environment. The phosphoproteomic data set for H. contortus provides a basis to better understand phosphorylation and associated biological processes (e.g., regulation of signal transduction), and might enable the discovery of novel anthelmintic targets. SIGNIFICANCE: Here, we report the first phosphoproteome for a socioeconomically parasitic nematode (Haemonchus contortus). This phosphoproteome exhibits distinctive patterns during development, suggesting active roles of post-translational modification in the parasite's adaptation to changing environments within and outside of the host animal. This work sheds a light on the developmental phosphorylation in a parasitic nematode, and could enable the discovery of novel interventions against major pathogens.
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Affiliation(s)
- Tao Wang
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Guangxu Ma
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Ching-Seng Ang
- Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Pasi K Korhonen
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Andreas J Stroehlein
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Neil D Young
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Andreas Hofmann
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Bill C H Chang
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Nicholas A Williamson
- Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Robin B Gasser
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
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18
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Mandacaru SC, Queiroz RML, Alborghetti MR, de Oliveira LS, de Lima CMR, Bastos IMD, Santana JM, Roepstorff P, Ricart CAO, Charneau S. Exoproteome profiling of Trypanosoma cruzi during amastigogenesis early stages. PLoS One 2019; 14:e0225386. [PMID: 31756194 PMCID: PMC6874342 DOI: 10.1371/journal.pone.0225386] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2019] [Accepted: 11/04/2019] [Indexed: 11/20/2022] Open
Abstract
Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2–4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.
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Affiliation(s)
- Samuel C. Mandacaru
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Rayner M. L. Queiroz
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
| | - Marcos R. Alborghetti
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Lucas S. de Oliveira
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Consuelo M. R. de Lima
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Izabela M. D. Bastos
- Pathogen-Host Interface Laboratory, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Jaime M. Santana
- Pathogen-Host Interface Laboratory, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Peter Roepstorff
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
| | - Carlos André O. Ricart
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
| | - Sébastien Charneau
- Laboratory of Protein Chemistry and Biochemistry, Department of Cell Biology, Institute of Biology, University of Brasilia, Brasilia, Brazil
- * E-mail:
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19
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The Novel Serine/Threonine Protein Kinase LmjF.22.0810 from Leishmania major may be Involved in the Resistance to Drugs such as Paromomycin. Biomolecules 2019; 9:biom9110723. [PMID: 31718000 PMCID: PMC6920834 DOI: 10.3390/biom9110723] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Revised: 11/01/2019] [Accepted: 11/05/2019] [Indexed: 12/13/2022] Open
Abstract
The identification and clarification of the mechanisms of action of drugs used against leishmaniasis may improve their administration regimens and prevent the development of resistant strains. Herein, for the first time, we describe the structure of the putatively essential Ser/Thr kinase LmjF.22.0810 from Leishmania major. Molecular dynamics simulations were performed to assess the stability of the kinase model. The analysis of its sequence and structure revealed two druggable sites on the protein. Furthermore, in silico docking of small molecules showed that aminoglycosides preferentially bind to the phosphorylation site of the protein. Given that transgenic LmjF.22.0810-overexpressing parasites displayed less sensitivity to aminoglycosides such as paromomycin, our predicted models support the idea that the mechanism of drug resistance observed in those transgenic parasites is the tight binding of such compounds to LmjF.22.0810 associated with its overexpression. These results may be helpful to understand the complex machinery of drug response in Leishmania.
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20
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Lucena ACR, Amorim JC, de Paula Lima CV, Batista M, Krieger MA, de Godoy LMF, Marchini FK. Quantitative phosphoproteome and proteome analyses emphasize the influence of phosphorylation events during the nutritional stress of Trypanosoma cruzi: the initial moments of in vitro metacyclogenesis. Cell Stress Chaperones 2019; 24:927-936. [PMID: 31368045 PMCID: PMC6717228 DOI: 10.1007/s12192-019-01018-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2018] [Revised: 05/15/2019] [Accepted: 06/14/2019] [Indexed: 12/16/2022] Open
Abstract
Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.
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Affiliation(s)
- Aline Castro Rodrigues Lucena
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
| | - Juliana Carolina Amorim
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
| | - Carla Vanessa de Paula Lima
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
| | - Michel Batista
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
- Mass Spectrometry Facility RPT02H, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
| | - Marco Aurelio Krieger
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
| | - Lyris Martins Franco de Godoy
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil
| | - Fabricio Klerynton Marchini
- Laboratory of Applied Science and Technologies in Health, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil.
- Mass Spectrometry Facility RPT02H, Carlos Chagas Institute, Fiocruz, Curitiba, Parana, Brazil.
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21
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Lobo M, Balouz V, Melli L, Carlevaro G, Cortina ME, Cámara MDLM, Cánepa GE, Carmona SJ, Altcheh J, Campetella O, Ciocchini AE, Agüero F, Mucci J, Buscaglia CA. Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins. PLoS Negl Trop Dis 2019; 13:e0007245. [PMID: 30870417 PMCID: PMC6435186 DOI: 10.1371/journal.pntd.0007245] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Revised: 03/26/2019] [Accepted: 02/14/2019] [Indexed: 01/02/2023] Open
Abstract
BACKGROUND TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. METHODS/PRINCIPLE FINDINGS Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. CONCLUSION/SIGNIFICANCE This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.
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Affiliation(s)
- Maite Lobo
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Virginia Balouz
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Luciano Melli
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Giannina Carlevaro
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - María E Cortina
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - María de Los Milagros Cámara
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Gaspar E Cánepa
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Santiago J Carmona
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Jaime Altcheh
- Servicio de Parasitología-Chagas, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina
| | - Oscar Campetella
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Andrés E Ciocchini
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Fernán Agüero
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Juan Mucci
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
| | - Carlos A Buscaglia
- Instituto de Investigaciones Biotecnológicas "Dr Rodolfo Ugalde" (IIB-INTECh, Universidad Nacional de San Martín and CONICET), Buenos Aires, Argentina
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22
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Mattos EC, Canuto G, Manchola NC, Magalhães RDM, Crozier TWM, Lamont DJ, Tavares MFM, Colli W, Ferguson MAJ, Alves MJM. Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix. PLoS Negl Trop Dis 2019; 13:e0007103. [PMID: 30726203 PMCID: PMC6380580 DOI: 10.1371/journal.pntd.0007103] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Revised: 02/19/2019] [Accepted: 12/20/2018] [Indexed: 12/11/2022] Open
Abstract
Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.
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Affiliation(s)
- Eliciane C. Mattos
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Gisele Canuto
- Departamento de Química Analítica, Instituto de Química, Universidade Federal da Bahia, Salvador, BA, Brazil
| | - Nubia C. Manchola
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Rubens D. M. Magalhães
- Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
| | - Thomas W. M. Crozier
- Wellcome Centre for Anti-Infectives Research, School of Life Science, University of Dundee, Dundee, United Kingdom
| | - Douglas J. Lamont
- Fingerprints Proteomics Facility, School of Life Sciences, University of Dundee, Dundee, United Kingdom
| | - Marina F. M. Tavares
- Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Walter Colli
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Michael A. J. Ferguson
- Wellcome Centre for Anti-Infectives Research, School of Life Science, University of Dundee, Dundee, United Kingdom
| | - Maria Júlia M. Alves
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
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23
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Garcia CH, Depoix D, Queiroz RM, Souza JM, Fontes W, de Sousa MV, Santos MD, Carvalho PC, Grellier P, Charneau S. Dynamic molecular events associated to Plasmodium berghei gametogenesis through proteomic approach. J Proteomics 2018; 180:88-98. [DOI: 10.1016/j.jprot.2017.11.009] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2017] [Revised: 11/08/2017] [Accepted: 11/14/2017] [Indexed: 10/18/2022]
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24
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Belew AT, Junqueira C, Rodrigues-Luiz GF, Valente BM, Oliveira AER, Polidoro RB, Zuccherato LW, Bartholomeu DC, Schenkman S, Gazzinelli RT, Burleigh BA, El-Sayed NM, Teixeira SMR. Comparative transcriptome profiling of virulent and non-virulent Trypanosoma cruzi underlines the role of surface proteins during infection. PLoS Pathog 2017; 13:e1006767. [PMID: 29240831 PMCID: PMC5746284 DOI: 10.1371/journal.ppat.1006767] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2017] [Revised: 12/28/2017] [Accepted: 11/22/2017] [Indexed: 01/23/2023] Open
Abstract
Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving several morphologically and biochemically distinct stages that establish intricate interactions with various insect and mammalian hosts. It has also a heterogeneous population structure comprising strains with distinct properties such as virulence, sensitivity to drugs, antigenic profile and tissue tropism. We present a comparative transcriptome analysis of two cloned T. cruzi strains that display contrasting virulence phenotypes in animal models of infection: CL Brener is a virulent clone and CL-14 is a clone that is neither infective nor pathogenic in in vivo models of infection. Gene expression analysis of trypomastigotes and intracellular amastigotes harvested at 60 and 96 hours post-infection (hpi) of human fibroblasts revealed large differences that reflect the parasite’s adaptation to distinct environments during the infection of mammalian cells, including changes in energy sources, oxidative stress responses, cell cycle control and cell surface components. While extensive transcriptome remodeling was observed when trypomastigotes of both strains were compared to 60 hpi amastigotes, differences in gene expression were much less pronounced when 96 hpi amastigotes and trypomastigotes of CL Brener were compared. In contrast, the differentiation of the avirulent CL-14 from 96 hpi amastigotes to extracellular trypomastigotes was associated with considerable changes in gene expression, particularly in gene families encoding surface proteins such as trans-sialidases, mucins and the mucin associated surface proteins (MASPs). Thus, our comparative transcriptome analysis indicates that the avirulent phenotype of CL-14 may be due, at least in part, to a reduced or delayed expression of genes encoding surface proteins that are associated with the transition of amastigotes to trypomastigotes, an essential step in the establishment of the infection in the mammalian host. Confirming the role of members of the trans-sialidase family of surface proteins for parasite differentiation, transfected CL-14 constitutively expressing a trans-sialidase gene displayed faster kinetics of trypomastigote release in the supernatant of infected cells compared to wild type CL-14. Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an infection that occurs in several Latin American countries, resulting in a mild illness or in severe damage of the heart and intestinal tract. Such a broad spectrum of clinical manifestations observed in Chagas disease patients is likely due to differences in host susceptibility as well as to a large heterogeneity among T. cruzi isolates. The identification of virulence factors that are differentially expressed in the parasite population is a valuable strategy for understanding of the distinct interactions that occur between this pathogen and its host, which may or may not lead to pathogenesis. By comparing the gene expression profiles of two T. cruzi strains that display contrasting virulence phenotypes in animal models of infection, we identified a central role for genes encoding surface proteins that is associated with the differentiation from intracellular replicative amastigotes to infective trypomastigotes. We showed that the expression of these genes occurs differentially within the two strains and this difference may be a factor that impacts parasite survival and dissemination in the mammalian host.
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Affiliation(s)
- A. Trey Belew
- Department of Cell Biology and Molecular Genetics and Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America
| | - Caroline Junqueira
- Centro de Pesquisas Rene Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil
| | - Gabriela F. Rodrigues-Luiz
- Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
| | - Bruna M. Valente
- Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
| | - Antonio Edson R. Oliveira
- Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
| | - Rafael B. Polidoro
- Departmento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Luciana W. Zuccherato
- Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
| | - Daniella C. Bartholomeu
- Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
| | - Sergio Schenkman
- Departmento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil
| | - Ricardo T. Gazzinelli
- Centro de Pesquisas Rene Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, Brazil
- Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
| | - Barbara A. Burleigh
- Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, United States of America
| | - Najib M. El-Sayed
- Department of Cell Biology and Molecular Genetics and Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America
- * E-mail: (SMRT); (NES)
| | - Santuza M. R. Teixeira
- Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
- * E-mail: (SMRT); (NES)
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25
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Cámara MDLM, Cánepa GE, Lantos AB, Balouz V, Yu H, Chen X, Campetella O, Mucci J, Buscaglia CA. The Trypomastigote Small Surface Antigen (TSSA) regulates Trypanosoma cruzi infectivity and differentiation. PLoS Negl Trop Dis 2017; 11:e0005856. [PMID: 28800609 PMCID: PMC5568413 DOI: 10.1371/journal.pntd.0005856] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2017] [Revised: 08/23/2017] [Accepted: 08/05/2017] [Indexed: 02/06/2023] Open
Abstract
Background TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties. Methods/Principle findings Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the ‘adhesive’ TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the ‘non-adhesive’ TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. Conclusion/Significance In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains. Infection with Trypanosoma cruzi produces a chronic and debilitating infectious disease known as Chagas disease, of major significance in Latin America and an emergent threat to global public health. In the absence of vaccines and/or appropriate chemotherapies, the search for parasite effectors that support infection of mammalian cells is a focus of significant interest. One such candidate is the Trypomastigote Small Surface Antigen (TSSA), a polymorphic molecule expressed on the surface coat of infective trypomastigote forms. Previous data indicated that recombinant versions of two different TSSA variants (termed TSSA-CL and TSSA-Sy) encoded by parasite strains belonging to extant phylogenetic groups exhibited contrasting host cell binding and signaling abilities. Here, we generated genetically modified strains of T. cruzi over-expressing different TSSAs to address this issue. Trypomastigotes over-expressing TSSA-CL, the ‘adhesive variant’, displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. In addition, and irrespective of the protein variant, TSSA over-expression enhanced trypomastigote-to-amastigote conversion. Overall, our data strongly suggest that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by different strains of this parasite. These data, together with the fact that TSSA recalls a strong and likely protective humoral response during human infections, support this molecule as an excellent candidate for molecular intervention and/or vaccine development in Chagas disease.
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Affiliation(s)
- María de los Milagros Cámara
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Instituto de Tecnología, Universidad Argentina de la Empresa (UADE), Buenos Aires, Argentina
| | - Gaspar E. Cánepa
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Andrés B. Lantos
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Virginia Balouz
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Hai Yu
- Department of Chemistry, University of California, Davis, Davis, California, United States of America
| | - Xi Chen
- Department of Chemistry, University of California, Davis, Davis, California, United States of America
| | - Oscar Campetella
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Juan Mucci
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Carlos A. Buscaglia
- Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECh), Universidad Nacional de San Martín (UNSAM) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- * E-mail:
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26
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Queiroz RML, Ricart CAO, Machado MO, Bastos IMD, de Santana JM, de Sousa MV, Roepstorff P, Charneau S. Insight into the Exoproteome of the Tissue-Derived Trypomastigote form of Trypanosoma cruzi. Front Chem 2016; 4:42. [PMID: 27872839 PMCID: PMC5097913 DOI: 10.3389/fchem.2016.00042] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Accepted: 10/19/2016] [Indexed: 01/07/2023] Open
Abstract
The protozoan parasite Trypanosoma cruzi causes Chagas disease, one of the major neglected infectious diseases. It has the potential to infect any nucleated mammalian cell. The secreted/excreted protein repertoire released by T. cruzi trypomastigotes is crucial in host-pathogen interactions. In this study, mammalian tissue culture-derived trypomastigotes (Y strain) were used to characterize the exoproteome of the infective bloodstream life form. Proteins released into the serum-free culture medium after 3 h of incubation were harvested and digested with trypsin. NanoLC-MS/MS analysis resulted in the identification of 540 proteins, the largest set of released proteins identified to date in Trypanosoma spp. Bioinformatic analysis predicted most identified proteins as secreted, predominantly by non-classical pathways, and involved in host-cell infection. Some proteins possess predicted GPI-anchor signals, these being mostly trans-sialidases, mucin associated surface proteins and surface glycoproteins. Moreover, we enriched phosphopeptides and glycopeptides from tryptic digests. The majority of identified glycoproteins are trans-sialidases and surface glycoproteins involved in host-parasite interaction. Conversely, most identified phosphoproteins have no Gene Ontology classification. The existence of various proteins related to similar functions in the exoproteome likely reflects this parasite's enhanced mechanisms for adhesion, invasion, and internalization of different host-cell types, and escape from immune defenses.
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Affiliation(s)
- Rayner M L Queiroz
- Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia, Brasilia, Brazil; Department of Biochemistry and Molecular Biology, University of Southern DenmarkOdense, Denmark
| | - Carlos A O Ricart
- Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia , Brasilia, Brazil
| | - Mara O Machado
- Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia , Brasilia, Brazil
| | - Izabela M D Bastos
- Laboratory of Host-Pathogen Interaction, Department of Cell Biology, University of Brasilia Brasilia, Brazil
| | - Jaime M de Santana
- Laboratory of Host-Pathogen Interaction, Department of Cell Biology, University of Brasilia Brasilia, Brazil
| | - Marcelo V de Sousa
- Laboratory of Host-Pathogen Interaction, Department of Cell Biology, University of Brasilia Brasilia, Brazil
| | - Peter Roepstorff
- Department of Biochemistry and Molecular Biology, University of Southern Denmark Odense, Denmark
| | - Sébastien Charneau
- Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology, University of Brasilia , Brasilia, Brazil
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27
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Parsons M, Myler PJ. Illuminating Parasite Protein Production by Ribosome Profiling. Trends Parasitol 2016; 32:446-457. [PMID: 27061497 DOI: 10.1016/j.pt.2016.03.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2015] [Revised: 02/25/2016] [Accepted: 03/04/2016] [Indexed: 12/29/2022]
Abstract
While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low-abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites including trypanosomes and Plasmodium. It has been used to identify new protein-coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation.
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Affiliation(s)
- Marilyn Parsons
- Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), 307 Westlake Avenue North STE 500, Seattle, WA 98109 USA; Department of Global Health, Box 357965, University of Washington, Seattle, WA 98195, USA.
| | - Peter J Myler
- Center for Infectious Disease Research (formerly Seattle Biomedical Research Institute), 307 Westlake Avenue North STE 500, Seattle, WA 98109 USA; Department of Global Health, Box 357965, University of Washington, Seattle, WA 98195, USA; Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, WA 98195, USA
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Li Y, Shah-Simpson S, Okrah K, Belew AT, Choi J, Caradonna KL, Padmanabhan P, Ndegwa DM, Temanni MR, Corrada Bravo H, El-Sayed NM, Burleigh BA. Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection. PLoS Pathog 2016; 12:e1005511. [PMID: 27046031 PMCID: PMC4821583 DOI: 10.1371/journal.ppat.1005511] [Citation(s) in RCA: 116] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2015] [Accepted: 02/28/2016] [Indexed: 01/22/2023] Open
Abstract
Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts. In-depth knowledge of the functional processes governing host colonization and transmission of pathogenic microorganisms is essential for the advancement of effective intervention strategies. This study focuses on Trypanosoma cruzi, the vector-borne protozoan parasite responsible for human Chagas disease and the leading cause of infectious myocarditis worldwide. To gain global insights into the biology of this parasite and its interaction with mammalian host cells, we have exploited a deep-sequencing approach to generate comprehensive, high-resolution transcriptomic maps for mammalian-infective stages of T. cruzi with the simultaneous interrogation of the human host cell transcriptome across an infection time course. We demonstrate that the establishment of intracellular T. cruzi infection in mammalian host cells is accompanied by extensive remodeling of the parasite and host cell transcriptomes. Despite the lack of transcriptional control mechanisms in trypanosomatids, our analyses identified functionally-enriched processes within sets of developmentally-regulated transcripts in T. cruzi that align with known or predicted biological features of the parasite. The novel insights into the biology of intracellular T. cruzi infection and the regulation of amastigote development gained in this study establish a unique foundation for functional network analyses that will be instrumental in providing functional links between parasite dependencies and host functional pathways that have the potential to be exploited for intervention.
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Affiliation(s)
- Yuan Li
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America
| | - Sheena Shah-Simpson
- Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
| | - Kwame Okrah
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America
| | - A Trey Belew
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America
| | - Jungmin Choi
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America
| | - Kacey L Caradonna
- Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
| | - Prasad Padmanabhan
- Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
| | - David M Ndegwa
- Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
| | - M Ramzi Temanni
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America
| | - Héctor Corrada Bravo
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America
| | - Najib M El-Sayed
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, United States of America.,Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America
| | - Barbara A Burleigh
- Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America
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29
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Romaniuk MA, Cervini G, Cassola A. Regulation of RNA binding proteins in trypanosomatid protozoan parasites. World J Biol Chem 2016; 7:146-157. [PMID: 26981203 PMCID: PMC4768119 DOI: 10.4331/wjbc.v7.i1.146] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/04/2015] [Accepted: 01/29/2016] [Indexed: 02/05/2023] Open
Abstract
Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.
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30
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Peloso EF, Dias L, Queiroz RML, Leme AFPP, Pereira CN, Carnielli CM, Werneck CC, Sousa MV, Ricart CAO, Gadelha FR. Trypanosoma cruzi mitochondrial tryparedoxin peroxidase is located throughout the cell and its pull down provides one step towards the understanding of its mechanism of action. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2015; 1864:1-10. [PMID: 26527457 DOI: 10.1016/j.bbapap.2015.10.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Revised: 09/23/2015] [Accepted: 10/16/2015] [Indexed: 01/11/2023]
Abstract
Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several proteins that differ in many aspects from host proteins. Although extensive research has been performed examining hydroperoxide cytosolic antioxidant defense centered on trypanothione, the mechanisms of mitochondrial antioxidant defense are not yet known. The aim of this study was to elucidate the partners of TcMPx antioxidant pathway and to determine the influence of the cellular context (physiological versus oxidative stress). Through co-precipitation coupled with a mass spectrometry approach, a variety of proteins were detected under physiological and oxidative stress conditions. Interestingly, functional category analysis of the proteins identified under physiological conditions showed that they were involved in the stress response, oxidoreduction, thiol transfer, and metabolic processes; this profile is distinct under oxidative stress conditions likely due to structural alterations. Our findings help to elucidate the reactions involving TcMPx and most importantly also reveal that this protein is present throughout the cell and that its interaction partners change following oxidative stress exposure. The involvement and significance of the proteins found to interact with TcMPx and other possible functions for this protein are discussed widening our knowledge regarding T. cruzi mitochondrial antioxidant defenses.
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Affiliation(s)
- E F Peloso
- Departamento de Bioquímica e Biologia Tecidual, UNICAMP, Campinas, SP, Brazil
| | - L Dias
- Departamento de Bioquímica e Biologia Tecidual, UNICAMP, Campinas, SP, Brazil
| | - R M L Queiroz
- Laboratório de Bioquímica e Química de Proteínas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil
| | - A F P Paes Leme
- Centro Nacional de Pesquisa em Energia e Materiais, Laboratório Nacional de Biociências, Campinas, SP, Brazil
| | - C N Pereira
- Departamento de Bioquímica e Biologia Tecidual, UNICAMP, Campinas, SP, Brazil
| | - C M Carnielli
- Centro Nacional de Pesquisa em Energia e Materiais, Laboratório Nacional de Biociências, Campinas, SP, Brazil
| | - C C Werneck
- Departamento de Bioquímica e Biologia Tecidual, UNICAMP, Campinas, SP, Brazil
| | - M V Sousa
- Laboratório de Bioquímica e Química de Proteínas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil
| | - C A O Ricart
- Laboratório de Bioquímica e Química de Proteínas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil
| | - F R Gadelha
- Departamento de Bioquímica e Biologia Tecidual, UNICAMP, Campinas, SP, Brazil.
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Abstract
Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions.
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Hashimoto M, Morales J, Uemura H, Mikoshiba K, Nara T. A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor. PLoS One 2015; 10:e0135726. [PMID: 26267656 PMCID: PMC4534300 DOI: 10.1371/journal.pone.0135726] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Accepted: 07/24/2015] [Indexed: 12/04/2022] Open
Abstract
Background Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro. Methodology/Principal Findings Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R)—an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite—is important for the transformation process. Conclusion/Significance Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.
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Affiliation(s)
- Muneaki Hashimoto
- Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan
- * E-mail:
| | - Jorge Morales
- Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan
| | - Haruki Uemura
- Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852–8523, Japan
| | - Katsuhiko Mikoshiba
- Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama 351–0198, Japan
- Calcium Oscillation Project, International Cooperative Research Project and Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332–0012, Japan
| | - Takeshi Nara
- Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan
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