A Phosphoproteomic Approach towards the Understanding of the Role of TGF-β in Trypanosoma cruzi Biology

Transforming growth factor beta (TGF-β) plays a pivotal role in Chagas disease, not only in the development of chagasic cardiomyopathy, but also in many stages of the T. cruzi life cycle and survival in the host cell environment. The intracellular signaling pathways utilized by T. cruzi to regulate these mechanisms remain unknown. To identify parasite proteins involved in the TGF-β response, we utilized a combined approach of two-dimensional gel electrophoresis (2DE) analysis and mass spectrometry (MS) protein identification. Signaling via TGF-β is dependent on events of phosphorylation, which is one of the most relevant and ubiquitous post-translational modifications for the regulation of gene expression, and especially in trypanosomatids, since they lack several transcriptional control mechanisms. Here we show a kinetic view of T. cruzi epimastigotes (Y strain) incubated with TGF-β for 1, 5, 30 and 60 minutes, which promoted a remodeling of the parasite phosphorylation network and protein expression pattern. The altered molecules are involved in a variety of cellular processes, such as proteolysis, metabolism, heat shock response, cytoskeleton arrangement, oxidative stress regulation, translation and signal transduction. A total of 75 protein spots were up- or down-regulated more than twofold after TGF-β treatment, and from these, 42 were identified by mass spectrometry, including cruzipain–the major T. cruzi papain-like cysteine proteinase that plays an important role in invasion and participates in the escape mechanisms used by the parasite to evade the host immune system. In our study, we observed that TGF-β addition favored epimastigote proliferation, corroborating 2DE data in which proteins previously described to be involved in this process were positively stimulated by TGF-β

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

Artigo disponível em acesso aberto no link da Editora.Submitted by Repositório Arca ([email protected]) on 2019-03-07T16:42:11Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) F83-1481999a.pdf: 399135 bytes, checksum: c78054a1227902e3a657c5fde27c4759 (MD5)Approved for entry into archive by monique santos ([email protected]) on 2019-03-08T15:39:22Z (GMT) No. of bitstreams: 2 F83-1481999a.pdf: 399135 bytes, checksum: c78054a1227902e3a657c5fde27c4759 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-03-08T15:39:22Z (GMT). No. of bitstreams: 2 F83-1481999a.pdf: 399135 bytes, checksum: c78054a1227902e3a657c5fde27c4759 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Universidade Federal de Pelotas. Centro de Biotecnologia. Pelotas, RS, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade de Surrey. Escola de Biológica Ciências. Guildford, Surrey, UK.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Mycobacterium vaccae represents an alternative mycobacterial cloning host that has been largely overlooked to date. The main reason for this may be the reported non-transformability of this species, specifically the so-called Stanford strain (NCTC 11659), with expression vectors that use kanamycin resistance as a selection method. However, this strain can be transformed using hygromycin resistance as an alternative selectable phenotype. The present study has shown that in contrast to previous reports, M. vaccae (ATCC 15483) is capable of being transformed with a range of vectors encoding kanamycin resistance as the selectable marker. Thereafter, the expression of the lacZ reporter gene in M. vaccae, Mycobacterium bovis BCG and Mycobacterium smegmatis mc(2)155 was evaluated using a range of characterized mycobacterial promoter sequences (hsp60, hsp70, PAN, 18kDa and 16S rRNA) cloned in the same promoter probe vector. In general, the promoters showed similar levels of activity in the three species, demonstrating that existing expression systems can readily be employed with M. vaccae (ATCC 15483). This was further confirmed by the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those obtained with BCG and M. smegmatis. Analysis of structural and functional stability of a range of vectors demonstrated that the incidence of instability noted for M. vaccae was lower than that recorded for M. smegmatis. Taken together, the results indicate that M. vaccae is an additional cloning host which may prove useful for specific aspects of mycobacterial biology and provide increased flexibility to the field of recombinant protein technology for mycobacteria

Dengue virus–specific antibodies enhance brazilian Zika vrus infection

Anti-Flavivirus antibodies are highly cross-reactive and may facilitate Zika virus (ZIKV) infection through the antibody-dependent enhancement (ADE) mechanism. We demonstrate that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line. In addition, we demonstrate that serum samples from dengue-immune pregnant women enhanced ZIKV infection. These findings highlight the need for epidemiological studies and animal models to further confirm the role of ADE in the development of congenital and neurological complications associated with ZIKV infections

The Transcriptional and Protein Profile From Human Infected Neuroprogenitor Cells Is Strongly Correlated to Zika Virus Microcephaly Cytokines Phenotype Evidencing a Persistent Inflammation in the CNS

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-09-27T17:34:17Z No. of bitstreams: 1 Lima, C. M The Transcriptional....pdf: 3509801 bytes, checksum: 454f6b43052365e7fcc8eec4efcf6676 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-09-27T17:57:48Z (GMT) No. of bitstreams: 1 Lima, C. M The Transcriptional....pdf: 3509801 bytes, checksum: 454f6b43052365e7fcc8eec4efcf6676 (MD5)Made available in DSpace on 2019-09-27T17:57:48Z (GMT). No. of bitstreams: 1 Lima, C. M The Transcriptional....pdf: 3509801 bytes, checksum: 454f6b43052365e7fcc8eec4efcf6676 (MD5) Previous issue date: 2019Fundação de Amparo à Ciência e Tecnologia de Pernambuco/FACEPE, grant agreement nos. APQ-0055.2.11/16 and APQ-0044.2.11/16, and Conselho Nacional de Desenvolvimento Científico e Tecnológico/CNPq grant agreement 439975/2016-6 under RF coordination and responsibility. UK Medical Research Council Grants MC_UU_12014/8 and MR/N017552/1 under AK’s coordination. VN received grant support from the Stanley Medical Research Institute (07R-1712), National Institutes of Health (MH63480 and D43 TW009114) and Cura Zika—University of Pittsburgh.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.University of Liverpool. Institute of Infection and Global Health. Liverpool, United Kingdom.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.University of Pittsburgh. School of Medicine. Department of Psychiatry. Pittsburgh, PA, United States.University of Pittsburgh. School of Medicine. Department of Psychiatry. Pittsburgh, PA, United States.University of Pittsburgh. School of Medicine. Department of Psychiatry. Pittsburgh, PA, United States.University of Pittsburgh. School of Medicine. Department of Psychiatry. Pittsburgh, PA, United States.University of Liverpool. Institute of Infection and Global Health. Liverpool, United Kingdom.Federal University of Pernambuco. Recife, PE, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil / University of Pittsburgh. Center for Vaccine Research. Pittsburgh, PA, United States.University of Pittsburgh. Center for Vaccine Research. Pittsburgh, PA, United States.University of Glasgow Centre for Virus Research. Glasgow, United Kingdom.University of Glasgow Centre for Virus Research. Glasgow, United Kingdom.University of Pittsburgh. School of Medicine. Department of Psychiatry. Pittsburgh, PA, United States / University of Pittsburgh. Graduate School of Public Health. Department of Human Genetics. Pittsburgh, PA, United States.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Zika virus (ZIKV) infection during pregnancy is associated with microcephaly, a congenital malformation resulting from neuroinflammation and direct effects of virus replication on the developing central nervous system (CNS). However, the exact changes in the affected CNS remain unknown. Here, we show by transcriptome analysis (at 48 h post-infection) and multiplex immune profiling that human induced-neuroprogenitor stem cells (hiNPCs) respond to ZIKV infection with a strong induction of type-I interferons (IFNs) and several type-I IFNs stimulated genes (ISGs), notably cytokines and the pro-apoptotic chemokines CXCL9 and CXCL10. By comparing the inflammatory profile induced by a ZIKV Brazilian strain with an ancestral strain isolated from Cambodia in 2010, we observed that the response magnitude differs among them. Compared to ZIKV/Cambodia, the experimental infection of hiNPCs with ZIKV/Brazil resulted in a diminished induction of ISGs and lower induction of several cytokines (IFN-α, IL-1α/β, IL-6, IL-8, and IL-15), consequently favoring virus replication. From ZIKV-confirmed infant microcephaly cases, we detected a similar profile characterized by the presence of IFN-α, CXCL10, and CXCL9 in cerebrospinal fluid (CSF) samples collected after birth, evidencing a sustained CNS inflammation. Altogether, our data suggest that the CNS may be directly affected due to an unbalanced and chronic local inflammatory response, elicited by ZIKV infection, which contributes to damage to the fetal brain