Trichodina heterodentata (Ciliophora) infestation on Prochilodus lineatus larvae: a host-parasite relationship study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Interaction of biologically-relevant peptides with membrane model systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização e identificação de moléculas de superfície presentes em Paracoccidioides spp

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoramento da expressão gênica de Xanthomonas axonopodis pv. citri em diferentes condições ambientais

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Haematology and histopathology of Nile tilapia parasitised by Epistylis sp., an emerging pathogen in South America

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Funcionalidade das proteínas EF-Tu e 14-3-3 na virulência de Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The genome sequence of the plant pathogen Xylella fastidiosa: The Xylella fastidiosa consortium of the organization for nucleotide sequencing and analysis, Sao Paulo, Brazil

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis - a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and 'two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

An introductory informatics theoretical framework, based on an integrable lattice model using quantized function algebras, for studying DNA-ionized gas interaction system

Usually we observe that Bio-physical systems or Bio-chemical systems are many a time based on nanoscale phenomenon in different host environments, which involve many particles can often not be solved explicitly. Instead a physicist, biologist or a chemist has to rely either on approximate or numerical methods. For a certain type of systems, called integrable in nature, there exist particular mathematical structures and symmetries which facilitate the exact and explicit description. Most integrable systems, we come across are low-dimensional, for instance, a one-dimensional chain of coupled atoms in DNA molecular system with a particular direction or exist as a vector in the environment. This theoretical research paper aims at bringing one of the pioneering ‘Reaction-Diffusion’ aspects of the DNA-plasma material system based on an integrable lattice model approach utilizing quantized functional algebras, to disseminate the new developments, initiate novel computational and design paradigms.

THE GLYCOPROTEINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND THEIR ROLE IN INFECTION AND IMMUNITY

The porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen of swine and is known to cause abortion and infertility in pregnant sows and respiratory distress in piglets. PRRSV contains a major glycoprotein (GP5) and three minor glycoproteins (GP2a, GP3, and GP4) on the virion envelope, all of which are required for infectious virus production. To study their interactions amongst each other and with a cellular receptor for PRRSV, CD163, I cloned each of the viral glycoproteins and CD163 in various expression vectors. My studies have shown that while the GP2a, GP3, and GP4 are co-translationally glycosylated, the GP5 is post-translationally glycosylated. By using co-immunoprecipitation (co-IP) assays, strong interaction was demonstrated between GP4 and GP5 proteins, although weak interactions among the other envelope glycoproteins were also detected. Further, GP4 was found to mediate interactions leading to formation of multiprotein glycoprotein complex. My results also show that GP2a and GP4 proteins are the only two GPs that specifically interact with the CD163 molecule and that glycosylation of these GPs is required for efficient interaction. Based on these studies, I have developed an interactome map of the viral GPs and CD163 and have proposed a model of the viral glycoprotein complex and its interaction with CD163. Studies reported here also show that glycan addition at residue 184 (N184) of GP2a, and residues N42, N50, and N131 of GP3 is essential for recovery of infectious virus. Although single site glycosylation mutants of GP4 had no effect on infectious virus production, introduction of double mutations was lethal. The loss of glycan moieties of GP2a, GP3, and GP4 proteins had no effect on host neutralizing antibody production. Overall, I conclude that the PRRSV glycoproteins are co-translationally and post-translationally glycosylated, the GP4 protein is central to mediating interglycoprotein interactions, and along with GP2a, serves as the viral attachment protein that is responsible for interactions with the viral receptor, CD163. Further, glycosylation of GP2a, GP3, and GP4 proteins is required for infectious virus production, efficient interaction with CD163, but does not play any role in neutralizing antibody response in infected animals.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Novel aryl beta-aminocarbonyl derivatives as inhibitors of Trypanosoma cruzi trypanothione reductase: binding mode revised by docking and GRIND2-based 3D-QSAR procedures

Trypanothione reductase has long been investigated as a promising target for chemotherapeutic intervention in Chagas disease, since it is an enzyme of a unique metabolic pathway that is exclusively present in the pathogen but not in the human host, which has the analog Glutathione reductase. In spite of the present data-set includes a small number of compounds, a combined use of flexible docking, pharmacophore perception, ligand binding site prediction, and Grid-Independent Descriptors GRIND2-based 3D-Quantitative Structure-Activity Relationships (QSAR) procedures allowed us to rationalize the different biological activities of a series of 11 aryl beta-aminocarbonyl derivatives, which are inhibitors of Trypanosoma cruzi trypanothione reductase (TcTR). Three QSAR models were built and validated using different alignments, which are based on docking with the TcTR crystal structure, pharmacophore, and molecular interaction fields. The high statistical significance of the models thus obtained assures the robustness of this second generation of GRIND descriptors here used, which were able to detect the most important residues of such enzyme for binding the aryl beta-aminocarbonyl derivatives, besides to rationalize distances among them. Finally, a revised binding mode has been proposed for our inhibitors and independently supported by the different methodologies here used, allowing further optimization of the lead compounds with such combined structure- and ligand-based approaches in the fight against the Chagas disease.

Biological and behavioral parameters of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae) are altered by the pathogen Nosema sp (Microsporidia: Nosematidae)

A major issue for mass rearing of insects concerns sanitary conditions and disease. Microsporidian infection (Nosema sp.) in laboratory colonies of Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae), used in producing the parasitoid. Cotesia flavipes Cameron (Hymenoptera: Braconidae), is representative of the problems faced by growers and industry. Although C. flavipes has been produced for several years in Brazil for biological control of D. saccharalis, we have only recently observed that the parasitoid becomes infected when developing inside hosts infected with Nosema sp. We assessed the effects of Nosema sp. on C. flavipes, including the ability to locate and select hosts, and evaluated pathogen transmission. Third instar larvae of D. saccharalis were inoculated with Nosema sp. spores at different concentrations and were parasitized when larvae reached fifth instar. Heavily infected D. saccharalis larvae did not support parasitism. Parasitoids that developed in infected D. saccharalis larvae exhibited increased duration of larval and pupal stages, decreased adult longevity and number of offspring, and reduced tibia size compared to parasitoids developing in uninfected D. saccharalis larvae. Infection by Nosema sp. reduced the ability of the C. flavipes parasitoid to distinguish between volatiles released by the sugarcane infested by healthy larvae and pure air. Uninfected parasitoids preferred plants infested with uninfected hosts. But infected C. flavipes did not differentiate between uninfected hosts and those infected with Nosema sp. The pathogen is transmitted from host to parasitoids and parasitoids to hosts. Pathogenic effects of the microsporidium in C. flavipes are sufficiently severe to justify disease management efforts, particularly considering the importance of C. flavipes as a biological control agent in sugarcane. (C) 2012 Elsevier Inc. All rights reserved.

Dissecting Phaseolus vulgaris Innate Immune System against Colletotrichum lindemuthianum Infection

Background: The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. Methodology and Principal Findings: As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. Conclusions/Significance: We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-alpha, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this beta-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.