Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: Representational difference analysis identifies candidate genes associated with fungal pathogenesis

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans. (c) 2006 Elsevier Masson SAS. All rights reserved.

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Host-parasite relationships in paracoccidioidomycosis

A viewpoint of host-parasite relationships in paracoccidioidomycosis is presented. The characteristics of the fungus which are important to the host-parasite interaction are discussed. Aspects of inhibition of mycelium-to-yeast transformation by estrogens acting at receptors on the fungal wall and in the cytoplasm, and the role of polysaccharide components of the cell wall in virulence are reviewed. The natural mechanisms of host defense are also examined, including phagocytosis, complement system, natural-killer cells and genetic control of resistance and susceptibility. Finally, a discussion of granuloma morphogenesis and its relationship to the humoral and cellular anti-P. brasiliensis immune response is presented.

Antigen distribution in mucocutaneous biopsies of human paracoccidiomycosis

The authors studied the distribution of Paracoccidioides brasiliensis antigen(s) in human skin and oral mucosa. In biopsies obtained from untreated patients showing the chronic form of the disease, the authors demonstrated the P. brasiliensis antigen using two polyclonal immune sera raised in rabbits, one against the exoantigens of P. brasiliensis and the other against a 43-kDa glycoprotein. Langerhans' cells were detected through double immunolabeling using an anti-S100 protein monoclonal antibody. Double labeling immunohistochemistry showed that both of the immune sera labeled the yeast cells in the center of the granuloma and those transmigrating through the epithelial layer equally well. Granulomas exhibited the P. brasiliensis antigen permeating cells, mainly at the periphery of the granulomatous inflammation. The P. brasiliensis antigen(s) accumulated in the macrophages but not in the Langerhans' cells. P. brasiliensis antigens, detected by antiserum against parasite exoantigens, were also deposited between basal keratinocytes, but not in the granular cells, in 47% of the biopsies. P. brasiliensis antigens, as assessed by immunoelectron microscopic techniques, are present in the cytoplasm of the yeast cells in the host tissues. Antigens are transported to the cell membrane and later excreted through the cell wall. Antigenic deposits are also seen at the fungus-host interface.

Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Antibiotic 26-deoxylaidlomycin isolated from Streptomyces sp. Ar386 from Brazilian soil

An actinomycete strain (Ar386) was isolated from the soil of the Araraquara regio, SP, Brazil. The strain, named Streptomyces jacareensis, formed irregular rayed, rugose, grayish-white mycelium with sinuous, branched hyphae carrying rare isolated spores; assimilated glucose, galactose, inositol, ribose, maltose, sucrose, melibiose and starch but not mannitol, rhamnose, arabinose, xylose, lactose and raffinose; and contained LL- diaminopimelic acid in its cell wall. An antibiotic active against Gram- positive bacteria, which was characterized as being 26-deoxylaidlomycin and which may have application against poultry coccidiosis, was isolated from cultures of the strain. This was the first isolation of this antibiotic from a microorganism of the genus Streptomyces and also the first isolation of this antibiotic in Brazil.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Evidence for involvement of Saccharomyces cerevisiae protein kinase C in glucose induction of HXT genes and derepression of SUC2

The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Δ mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V max of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Δ mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Δ mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Effects of soil water deficit and nitrogen nutrition on water relations and photosynthesis of pot-grown Coffea canephora Pierre

Coffea canephora plants (clone INCAPER-99) were submitted to low N (LN) or high N (HN) applications and two watering regimes (daily irrigation and irrigation every 5 days for a month). Although water potential was not altered significantly by N, HN plants showed higher relative water content than did LN plants under water deficit. Only HN plants exhibited some ability for osmotic adjustment. Plants from both N treatments increased their cell wall rigidity under drought, with a more pronounced augmentation in HN plants. In well-watered plants, carbon assimilation rate increased with increasing N while stomatal conductance did not respond to N supply. Under drought conditions, carbon assimilation decreased by 68-80% compared to well-watered plants, whereas stomatal conductance and transpiration rate declined by 35% irrespective of the N applications. Stable carbon isotope analysis, combined with leaf gas exchange measurements, indicated that regardless of the watering treatments, N increased the long-term water use efficiency through changes in carbon assimilation with little or no effect on stomatal behaviour.

Valor nutritivo de macrófitas aquáticas flutuantes (Eichhornia crassipes, Pistia stratiotes e Salvinia molesta) utilizadas no tratamento de efluentes de aqüicultura

The aim of this study was to evaluate the nutritive value of free-floating aquatic macrophytes, Eichhornia crassipes (Mart.) Solms (Pontederiaceae), Pistia stratiotes (L.) (Araceae) and Salvinia molesta (Mitchell) (Salviniaceae) used in a Nile tilapia (Oreochromis niloticus) waste treatment, and these species biomass potential uses. The vegetal biomass samples were collected from 0.25 m 2 floating squares and divided in aerial and submerse parts, to determine the concentrations of cell wall fraction, soluble carbohydrates, polyphenols, lipids, crude protein and total phosphorus. The higher nutritive value was observed in E. crassipes and S. molesta aerial parts, and in P. stratiotes total biomass, due to their lower cell wall fraction mean rates (60.7; 64.2 and 56.9 % dry mass, respectively) and to the higher rates of: crude protein (10.1; 9.1 and 8.8 % dry mass, respectively), soluble carbohydrates (26.6; 18.7 and 12.4 mg.g -1 dry mass, respectively) and lipids (7.6; 4.5 and 4.4% dry mass, respectively). It may be concluded that P. stratiotes total biomass, and E. crassipes and S. molesta aerial biomass have nutritive values with potential use for ruminant feeding or as ration ingredients.

Levedura íntegra e derivados do seu processamento em rações para tilápia do Nilo: Aspectos hematológicos e histológicos

The effects of inclusion of whole yeast, autolyzed yeast and yeast cell wall on hematological parameters and gut villus perimeter were evaluated in juvenile Nile tilapia, after 80 experimental days. Isoproteic (32.0% DP) and isoenergetic (3200 kcal DE kg -1) practical diets were supplemented with three levels of whole yeast or autolyzed yeast (1.0, 2.0 and 3.0%) and three levels of yeast cell wall (0.1, 0.2 and 0.3%), plus a control diet (with no test microingredients). Red blood cell count, hemoglobin concentration, total plasmatic protein, hematocrit percentage, mean corpuscular volume, mean corpuscular hemoglobin concentration and gut villus perimeter were evaluated. Variations on hematological parameters in animals fed diets with whole yeast; autolyzed yeast and yeast cell wall were observed to be within normal ranges for this species. There was significant influence (p<0.05) of different levels of yeast and derivatives on intestinal villus perimeter. Results showed that experimental period and proposed levels of whole yeast, autolyzed yeast and yeast cell wall do not provide undesirable alterations on standard hematological parameters to Nile tilapia and can be safely used to compound diets for this species. Results also showed that supplement of yeast cell wall provide higher intestinal villus perimeter.

Vascular hyporeactivity to angiotensin II induced by Escherichia coli endotoxin is reversed by Nω-Nitro-L-Arginine, an inhibitor of nitric oxide synthase

Septic shock or sepsis is reported to be one of the major causes of death when followed by systemic infectious trauma in humans and other mammals. Its development leads to a large drop in blood pressure and a reduction in vascular responsiveness to physiological vasoconstrictors which, if not contained, can lead to death. It is proposed that this vascular response is due to the action of bacterial cell wall products released into the bloodstream by the vascular endothelium and is considered a normal response of the body's defenses against infection. A reduction in vascular reactivity to epinephrine and norepinephrine is observed under these conditions. In the present study in rats, the aim was to assess whether those effects of hypotension and hyporeactivity are also related to another endogenous vasoconstrictor, angiotensin II (AII). We evaluated the variation in the power of this vasoconstrictor over the mean arterial pressure in anesthetized rats, before and after the establishment of hypotension by Escherichia coli endotoxin (Etx). Our results show that in this model of septic shock, there is a reduction in vascular reactivity to AII and this reduction can be reversed by the inhibitor of nitric oxide synthase, Nω-Nitro-L- Arginine (NωNLA). Our results also suggest that other endogenous factors (not yet fully known) are involved in the protection of rats against septic shock, in addition to the L-arginine NO pathway.

Selective factors involved in oil flotation isolation of black yeasts from the environment

The oil flotation isolation technique has been successfully applied to recover chaetothyrialean black yeasts and relatives from the environment. The selective mechanisms playing a role in isolation are unknown. The fungi concerned are supposed to occupy specialized microniches in nature, taking advantage of (1) oligotrophism. Mineral oil as a main selective agent may be based on (2) hydrophobicity or on (3) assimilation. All three hypotheses are tested in this paper. Results show that cell wall hydrophobicity is unlikely to be a selective factor. Incubation under poor nutrient conditions provides competitive advantage for black yeasts, especially for Exophiala strains, which are subsequently enriched by mineral oil which enhances growth in this group of fungi. Incubation under mineral media and mineral oil can be used as selective factor.

Polissacarídeos de parede celular fúngica: purificação e caracterização

The cell wall is a rigid structure essential for the survival of fungi. A knowledge of its composition is therefore useful for the development of novel anti-fungal drugs. In this context, polysaccharides as main components of the fungal cell wall have been the subject of intense scientific study over the years. The information gained from the knowledge of the structure of these macrobiomolecules could therefore be valuable in elucidating the mechanisms of their biosynthesis in the cell walls of pathogenic fungi infecting plants and animals alike. Determination of the chemical structures of these polysaccharides (endo) is preceded by their extraction and purification. The extractions, generally lead to neutral and/ or alkaline soluble biopolymers in groups according to their solubilities. Mixtures of polysaccharides in these extracts can then be purified by a combination of chemical and chromatographic methods. Following purification, the polysaccharides, considered homogeneous, can be characterized structurally using conventional techniques of carbohydrate chemistry, such as hydrolysis, methylation analysis, and FT-IR, 13C- and 1H- NMR spectroscopy. This review surveys the main scientific literature that characterizes polysaccharides constituting the fungal cell wall.

Massa seca e composição bromatológica de quatro espécies de braquiárias semeadas na linha ou a lanço, em consórcio com milho no sistema plantio direto na palha

The objective of this research was to evaluate the dry mass yield and chemical composition of four Brachiaria species in different options for sowing, exclusively or in intercrop with corn crop, under a no-tillage system. The experiment was carried out during the growing seasons of 2006 at FEPE (FE/Unesp, Ilha Solteira Campus) located in Selvíria, Mato Grosso do Sul State, Brazil. The soil of the experimental area was classified as distroferric Red Latosol (Oxisol). The experimental design was in randomized blocks, in a factorial scheme (4 × 4), with five replications. The treatments consisted of four Brachiaria species (Brachiaria brizantha cv. Marandu, Brachiaria decumbens, Brachiaria ruziziensis and Mulato II grass) grown in rows and spread on total area, exclusively or intercropped simultaneously with corn crop sowing. The study evaluated the dry mass yield and total digestible nutrients, crude protein, ash, neutral detergent fiber, acid detergent fiber, hemicelluloses, cellulose and lignin content of forage. The spread on total area intercrop of forages with corn crop proved to be viable by presenting similar dry mass yield to exclusive sowing arrangements, conversely to what happened with intercrop in row of corn crop, which decreased such yield. Brachiaria ruziziensis showed superior chemical composition and the intercrops increased energy and crude protein contents, and decreased cell wall components.