Mônica Santiago Barbosa

Glyceraldehyde-3-Phosphate Dehydrogenase of Paracoccidioides brasiliensis Is a Cell Surface Protein Involved in Fungal Adhesion to Extracellular Matrix Proteins and Interaction with Cells

ABSTRACT The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis.

Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus–host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome‐PTS‐1‐targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen‐dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis. Copyright

Characterization of Paracoccidioides brasiliensis PbDfg5p, a cell-wall protein implicated in filamentous growth.

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a Dfg5p (defective for filamentous growth) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Saccharomyces cerevisiae. The protein, the cDNA and genomic sequences were analysed. The cloned cDNA was expressed in Escherichia coli and the purified rPbDfg5p was used to obtain polyclonal antibodies. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbDfg5p in the fungal cell wall. Enzymatic treatments identified PbDfg5p as a β‐glucan linked protein that undergoes N‐glycosylation. The rPbDfg5p bound to extracellular matrix components, indicating that those interactions could be important for initial steps leading to P. brasiliensis attachment and colonization of host tissues. The P. brasiliensis dfg5 nucleotide and deduced protein, PbDfg5p, sequences reported in this paper had been submitted to the GenBank database under Accession Nos AY307855 (cDNA) and DQ534495 (genomic). Copyright

Characterization of a secreted aspartyl protease of the fungal pathogen Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis, a human systemic disease prevalent in Latin America. Proteases have been described as playing an important role in the host invasion process in many pathogenic microorganisms. Here we describe the identification and characterization of a secreted aspartyl protease (PbSAP), isolated from a cDNA library constructed with RNAs of mycelia transitioning to yeast cells. Recombinant PbSAP was produced in Escherichia coli, and the purified protein was used to develop a polyclonal antibody that was able to detect a 66 kDa protein in the P. brasiliensis proteome. PbSAP was detected in culture supernatants of P. brasiliensis and this data strongly suggest that it is a secreted molecule. The protein was located in the yeast cell wall, as determined by immunoelectron microscopy. In vitro deglycosylation assays with endoglycosidase H, and in vivo inhibition of the glycosylation by tunicamycin demonstrated N-glycosylation of the PbSAP molecule. Zymogram assays indicated the presence of aspartyl protease gelatinolytic activity in yeast cells and culture supernatant.

Detection of a homotetrameric structure and protein–protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism.

Molecular cloning, expression and insulin reduction activity of a thioredoxin 1 homologue (TRX1) from the pathogenic fungus Paracoccidioides lutzii

The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. The Paracoccidioides lutzii response to oxidative stress is largely unexplored. Thioredoxins (TRX) are involved in the regulation of the redox environment in the cell, responding to oxidative stress in several organisms. In this study, we describe the isolation and characterization of a cDNA encoding a thioredoxin 1 from yeast cells from P. lutzii. The cDNA codes for a 12kDa protein containing the characteristic thioredoxin active site. The thioredoxin 1 gene was expressed in Escherichia coli and the isolated thioredoxin 1 recombinant protein as the native PlTRX1 from yeast cells showed insulin reduction activity in vitro. We also showed by semi-quantitative RT-PCR analysis that the expression of thioredoxin 1 gene was induced in response to H2O2 and may exert an antioxidant activity in vivo. Our results suggest that the thioredoxin 1 may play an important role in controlling the redox status in P. lutzii which may contribute to this organisms virulence.

Cultivable microbiome and its resistance to antimicrobials isolated from Zaprionus indianus

The objective of this study was to identify Z. indianus in PEJC and PESCAN. Listed are the following methods: to isolate bacteria from the integument of the Z. indianus species collected and to check the resistance of microorganisms to antibiotics. Collections of Z. indianus were performed in four seasons of the two parks. The results obtained suggest that the low amount of Z. indianus collected can be justified by environmental factors such as high average temperature and low average humidity. It is noted that there is a predominance of bacteria of the Enterobacteriaceae family found in both the PEJC and the PESCAN. The antibiogram performed for the isolated PEJC bacteria shows statistical significance when comparing the edge and inside values of the park. Studies with fungi were also carried out, and it was evidenced that Trichophyton spp. was the genus that most inhabited the two environments studied. The ability of fluconazole and ketoconazole to inhibit fungal growth was also investigated, and considering the concentration tested may suggest that they have good action spectra. Plasmid profile data show that 60% of antibiotic-resistant bacteria have plasmids. The values found show that Z. indianus can act as vectors of microorganisms that affect the healthy animals and humans and that these organisms may be influenced by seasons.

Molecular technique for detection and identification of Helicobacter pylori in clinical specimens: a comparison with the classical diagnostic method

Introduction: Helicobacter pylori is a bacterium found in human epithelial cells of the gastrointestinal tract. Its infection is related to different diseases, such as chronic gastritis, peptic ulcers, gastric lymphoma and adenocarcinoma. The infection by H. pylori is present in more than a half of the world population. Objectives: To detect H. pylori and to compare the diagnostic methods of the rapid urease test (RUT) and polymerase chain reaction (PCR). Materials and methods: The study was conducted between April and July, 2015. For such, three biopsies were collected from each patient. Two were used for PCR and one for RUT. Results: A total of 85 samples were collected from patients undergoing endoscopy, with 56 (65.88%) females and 29 (34.11%) males. From the total samples subjected to RUT, 15 (17.64%) were positive and 70 (82.35%), negative. In PCR for detection of gene 16S ribosomal ribonucleic acid (rRNA) of H. pylori, 66 (77.64%) presented positive results and 19 (22.35%), negative results. For the analysis of the presence of UreA gene in all samples, positive results were found in 70 (82.35%), and negative in 15 (17.64%). According to the results, RUT and the molecular test presented statistical difference. Conclusion: PCR is a useful method in the laboratorial routine to detect the presence of H. pylori in the stomach tissue, due to high sensitivity and specificity, but it requires a more careful analysis and standardization.

A produção de insulina artificial através da tecnologia do DNA recombinante para o tratamento de diabetes mellitus doi: http://dx.doi.org/10.5892/ruvrv.2012.101.234245

Genetic Engineering has provided advances in medicine and the pharmaceutical industry with the creation of recombinant DNA technology (DNA from the combination of different sequences of DNAs belonging different organisms). For example, this technology is used in the production of artificial insulin through the bacterium Escherichia coli, that is genetically modified so that there is capable of synthesizing this hormone. The application of insulin is the primary method used in the treatment of patients with Diabetes mellitus (DM), a chronic disease that occurs when the pancreas does not produce enough insulin or when the body does not effectively use the insulin produced. With this technology in artificial insulin is produced in less time and on a larger scale, will benefit people with diabetes, so this way can lead a normal life.

A ENGENHARIA GENÉTICA APLICADA NO MELHORAMENTO DA CANA-DE-AÇÚCAR: UMA NOVA ALTERNATIVA PARA A PRODUÇÃO DE BIODIESEL DE SEGUNDA GERAÇÃO http://dx.doi.org/10.5892/ruvrv.2011.92.0323

A biotecnologia tem proporcionado grandes avancos ao melhoramento genetico. Neste sentido, objetivou-se abordar contextos atuais das dinâmicas institucional, organizacionais e tecnologicas nas quais os Organismos Geneticamente Modificados se inserem. A preocupacao inicial foi relatar as tecnologias utilizadas para producao sustentavel, e as modificacoes geneticas da cana-de-acucar para a producao de biodiesel. Para isso, foi utilizada a tecnologia do DNA recombinante, que permite isolar, preparar, manipular e estudar pequenos segmentos de DNA, e tem sido amplamente usada para tornar resistentes aos herbicidas, certas culturas comercialmente importantes. A transformacao genica permite transferir o gene de interesse atraves de metodos naturais, como a Agrobacterium tumefaciens, ou metodos fisicos e diretos como a eletroporacao e a biobalistica. A producao do biodiesel e uma opcao alternativa que permitiria contornar a escassez de petroleo, pois e constituido de carbono neutro. No entanto, utilizar a biotecnologia para aperfeicoar o processo de producao de biodiesel, requer atencao visando o controle de possiveis riscos para o ambiente e o equilibrio biologico. Com base nessa revisao da literatura, podemos concluir que ainda existem poucos estudos conclusivos, sobre o melhoramento genetico da cana-de-acucar na producao de biodiesel, e que estudos futuros sao necessarios para validar o processo.