Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis

Background: the transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our understanding of the synthesis and intracellular trafficking of GP82 and GP90 during metacyclogenesis is still limited. Therefore, we decided to determine whether GP82 and GP90 are expressed only in fully differentiated metacyclic forms or they start to be expressed in intermediate forms undergoing differentiation.Methods: Parasite populations enriched in intermediate forms undergoing differentiation were analyzed by quantitative real-time PCR, Western blot, flow cytometry and immunofluorescence to assess GP82 and GP90 expression.Results: We found that GP82 and GP90 mRNAs and proteins are expressed in intermediate forms and reach higher levels in fully differentiated metacyclic forms. Surprisingly, GP82 and GP90 presented distinct cellular localizations in intermediate forms compared to metacyclic trypomastigotes. in intermediate forms, GP82 is localized in organelles at the posterior region and colocalizes with cruzipain, while GP90 is localized at the flagellar pocket region.Conclusions: This study discloses new aspects of protein expression and trafficking during T. cruzi differentiation by showing that the machinery involved in GP82 and GP90 gene expression starts to operate early in the differentiation process and that different secretion pathways are responsible for delivering these glycoproteins toward the cell surface.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21949900 Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

Bacteria-Killing Type IV Secretion Systems

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I–IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors

Distribution, function and regulation of type 6 secretion systems of xanthomonadales

Members of the Xanthomonadales order include several plant pathogens of significant economic and agricultural impact, such as Xanthomonas spp. Type 6 secretion systems (T6SSs) are contractile nanomachines used by many bacterial species to inject protein effectors into target prokaryotic and eukaryotic cells and provide a competitive advantage for bacteria in different environments. Effectors with antibacterial properties include peptidoglycan hydrolases, lipases and phospholipases that break down structural components of the cell envelope, promoting target-cell lysis; and RNases, DNAses, and NADases that affect target-cell metabolism, arresting growth. Effectors with anti-eukaryotic properties are functionally more diverse. The T6SS of Xanthomonas citri is the only example experimentally characterized so far within the Xanthomonadales order and displays anti-eukaryotic function by providing resistance to predation by amoeba. This T6SS is regulated at the transcriptional level by a signaling cascade involving a Ser/Thr kinase and an extracytoplasmic function (ECF) sigma factor. In this review, we performed in silico analyses of 35 genomes of Xanthomonadales and showed that T6SSs are widely distributed and phylogenetically classified into three major groups. In silico predictions identified a series of proteins with known toxic domains as putative T6SS effectors, suggesting that the T6SSs of Xanthomonadales display both anti-prokaryotic and anti-eukaryotic properties depending on the phylogenetic group and bacterial species10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação2017/02178-2; 2017/17303-7; 2018/01852-4; 2018/04553-8; 2017/02318-

TRENDS ON TRYPANOSOMA (HERPETOSOMA) RANGELI RESEARCH

Trypanosoma rangeli is a hemoflagellate protozoan parasite presenting an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, in a wide geographical area in Latin America. Despite considered as non-pathogenic for man, T. rangeli shares several characteristics with T. cruzi such as vertebrate and invertebrate reservoirs, vectors and approximately half of the soluble antigenic determinants. Despite the importance of specific detection, little is know about T. rangeli in comparison to T. cruzi; several questions lack proper answers, including the controversies concerning T. rangeli´s taxonomic position. In this context, this short review attempted to congregate current aspects on the research of this parasite, approaching several subjects as life cycle, vector suscepbility, specific genes studies and genomic data

Cerâmicas medievais do Museu de Francisco Tavares Proença Jr.

BACKGROUND: To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. METHODS AND FINDINGS: Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. CONCLUSIONS: This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization

Characterization of the small RNA content of Trypanosoma cruzi extracellular vesicles

A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether Trypanosoma cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16-40 nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. in addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells. (C) 2014 the Authors. Published by Elsevier B.V.National Institutes of Health (NIH)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Biomolecule Analysis and Genomic Analysis Core Facilities at BBRC/UTEPUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USAFundacao Oswaldo Cruz, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilNational Institutes of Health (NIH): R0lAI070655-A5National Institutes of Health (NIH): R01AI070655-A5S1National Institutes of Health (NIH): 5G12MD007592FAPESP: 2011/51475-3Biomolecule Analysis and Genomic Analysis Core Facilities at BBRC/UTEP: 5G12MD007592Web of Scienc

The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing.

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings

Cell adhesion and lysosome exocytosis-inducing properties of SAP-CE associated with <i>T. cruzi</i> metacyclic trypomastigote internalization.

<p>(A) Increasing amounts of the purified recombinant protein SAP-CE or GST were added to 96-well plates covered with HeLa cells. After fixation and washes in PBS, the cells were incubated with MAb-SAP (diluted 1∶100) and with anti-mouse IgG peroxidase conjugate. The bound protein was revealed by <i>o</i>-phenylenediamine. Values are the means ± standard deviations of triplicates. (B) HeLa cells were incubated for 30 min with or without the recombinant protein SAP-CE or GST (40 μg/mL) and then incubated with metacyclic forms. After incubation for 1 h, cells were washed in PBS, fixed, and stained with Giemsa. The number of internalized parasites was counted in 500 cells. The values represent the means ± standard deviations of three independent experiments performed in duplicate. SAP-CE significantly inhibited parasite invasion (*p<0.05). (C) Semi-confluent HeLa cell monolayers were incubated in absence or in the presence of GST or the purified recombinant protein SAP-CE (20 μg/mL) for 60 min. The supernatant was collected and the release of β-hexosaminidase measured. Exocytosis was expressed as a percentage of the total β-hexosaminidase activity (supernatant + cell extract). Values are the means ± standard deviations of four independent experiments performed in duplicate. β-hexosaminidase activity was significantly higher in the presence of SAP-CE (*p<0.05). (D) HeLa cells were incubated with or without the purified recombinant protein SAP-CE (20 μg/mL) and processed for indirect immunofluorescence using anti-Lamp-2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA. Scale bar, 10 µm.</p