MALDI-TOF ICMS: a proteomic method for identification of clinical Sporothrix complex isolates

Publicado em "Biological resource centres : closing the gap between science and society : abstracts book...". ISBN 978-972-97916-5-9Sporotrichosis is a subcutaneous mycosis of worldwide distribution. However, Latin America, South Africa, India and Japan are areas of high endemicity. Recently, a combination of phenotypic and genotypic features suggested that Sporothrix schenckii should not be considered as a single taxon causing sporotrichosis as 3 new species, S. brasiliensis, S. globosa and S. mexicana have recently been described. Sporothrix mexicana was related with environmental samples and apparently restricted to Mexico. However, our Research Group has recently described the first case of human sporotrichosis caused by S. mexicana in Portugal [1]. An identification key for the Sporothrix complex species has now been proposed which includes macro- and micro-morphology and auxonogram analyses using raffinose and sucrose as carbon sources. Nevertheless, identification based on this methodology could be ambiguous due to phenotypic variability within these species [2]. In addition, conclusive species identification is reached only after partial calmodulin gene (CAL) sequence analysis. In order to show the potential of the Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) technique on the identification of Sporothrix complex species the aim of this study was to optimise the MALDI-TOF ICMS methodology for the 4 available Sporothrix isolates related with human sporotrichosis. For that proposal the type strain S. brasiliensis IPEC16490 (CBS 120339) and the reference strains S. globosa IPEC27135, S. schenckii IPEC27722 and S. mexicana MUM11.02 were used. Also were compared this isolates in two morphologic phases. The analysis demonstrated that optimal spectra and statistical clustering were obtained when the microbial cells were analysed on the yeast phase. The present methodology is simple, reliable, and rapid making it an ideal routine identification system for clinical mycology laboratories and culture collections

Implementating a methodology for identification of Sporothrix complex isolates by MALDI-TOF ICMS

Sporotrichosis is a subcutaneous mycosis worldwide distributed. However, Latin America, South Africa, India and Japan are areas of high endemicity. Recently, the combination of phenotypic and genotypic features suggested that Sporothrix schenckii should not be considered as a single taxon causing sporotrichosis once the 3 new species S. brasiliensis, S. globosa and S. mexicana have recently been described for the Science. Sporothrix mexicana was related with environmental samples and apparently restricted to Mexico. However, our Research Group has recently described the first case of human sporotrichosis caused by S. mexicana in Portugal [1]. A key identification for the Sporothrix complex species has now been proposed. It includes macro- and micro-morphology and auxonogram analyses using raffinose and sucrose as carbon sources. Nevertheless, identification based on this methodology could be inconclusive due to phenotypic variability within these species. In addition, conclusive species identification is reached only after partial calmodulin gene (CAL) sequence analysis. In order to show the potentiality of Matrix-Assisted Laser Desorption⁄Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) technique on the identification of Sporothrix complex species the aim of this study was to optimise the MALDI-TOF ICMS methodology for the 4 available Sporothrix isolates related with human sporotrichosis. For that proposal the type strain S. brasiliensis IPEC16490 (CBS 120339) and the reference strains S. globosa IPEC27135, S. schenckii IPEC27722 and S. mexicana MUM11.02 were used. Data obtained from the MALDI-TOF ICMS analyses show that the best spectral profiles and statistic clustering were obtained when the microbial cell were analysed on the yeast phase.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Biological Function and Molecular Mapping of M Antigen in Yeast Phase of Histoplasma capsulatum

Histoplasmosis, due to the intracellular fungus Histoplasma capsulatum, can be diagnosed by demonstrating the presence of antibodies specific to the immunodominant M antigen. However, the role of this protein in the pathogenesis of histoplasmosis has not been elucidated. We sought to structurally and immunologically characterize the protein, determine yeast cell surface expression, and confirm catalase activity. A 3D-rendering of the M antigen by homology modeling revealed that the structures and domains closely resemble characterized fungal catalases. We generated monoclonal antibodies (mAbs) to the protein and determined that the M antigen is present on the yeast cell surface and in cell wall/cell membrane preparations. Similarly, we found that the majority of catalase activity was in extracts containing fungal surface antigens and that the M antigen is not significantly secreted by live yeast cells. The mAbs also identified unique epitopes on the M antigen. The localization of the M antigen to the cell surface of H. capsulatum yeast and the characterization of the protein's major epitopes have important implications since it demonstrates that although the protein may participate in protecting the fungus against oxidative stress it is also accessible to host immune cells and antibody

Mucus extravasation and retention phenomena: a 24-year study

<p>Abstract</p> <p>Background</p> <p>Mucoceles are benign lesions related to the minor salivary glands and their respective ducts frequently affecting oral structures which are generally asymptomatic. Mucoceles are generally characterized by swollen nodular lesions preferentially located on the lower lip and differ from the so-called ranulas, which are lesions located on the floor of the mouth and related to the sublingual or submandibular glands.</p> <p>Methods</p> <p>The objective of the present study was to analyze data such as age, gender, race and site of the lesion of 173 mucocele cases diagnosed at the Discipline of Stomatology, São José dos Campos Dental School, UNESP, over a period of 24 years (April 1980 to February 2003).</p> <p>Results</p> <p>Of the 173 cases analyzed, 104 (60.12%) were females and 69 (39.88%) were males. Age ranged from 4 to 70 years (mean ± SD: 17 ± 9.53) and most patients were in the second decade of life (n = 86, 49.42%); white (n = 124, 71.68%). The lower lip was the site most frequently affected by the lesions (n = 135, 78.03%), whereas the lowest prevalence was observed for the soft palate, buccal mucosa, and lingual frenum.</p> <p>Conclusion</p> <p>In this study, mucoceles predominated in white female subjects in the second decade of life, with the lower lip being the most frequently affected site.</p

The Homeostasis of Iron, Copper, and Zinc in Paracoccidioides Brasiliensis, Cryptococcus Neoformans Var. Grubii, and Cryptococcus Gattii: A Comparative Analysis

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways

Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence