Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: Identification of putative homologues of Candida albicans virulence and pathogenicity genes

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. the first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.Univ São Paulo, Dept Ciencias Farmaceut, Fac Ciencias Farmaceut Ribeirao Preto, BR-14040903 Ribeirao Preto, SP, BrazilUniv São Paulo, Fac Filosofia Ciencias & Letras Ribeirao Pret, BR-14040903 Ribeirao Preto, SP, BrazilInst Pasteur, Unite Genet Mol Levures, Paris, FranceUniv Vale do Paraiba, UNIVAP, Vale Do Paraiba, BrazilUniv Mogi das Cruzes, Nucleo Integrado Biotecnol, Mogi Das Cruzes, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Gα (Gpa1–3), Gβ (Gpb1) and Gγ (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130 +/- 102 and 1200 +/- 97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35 % of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels. (c) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.The State of Sao Paulo Research Foundation (FAPESP)[03/07702-9]FAPES

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -3

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -5

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -2

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p>nd prey vectors were simultaneously transformed into yeast strain AH109 and plated out on SD/–Leu/–Trp for 3 days. Yeast colonies that grew on SD/–Leu/Trp were restreaked on SD/–Ade/–His/–Leu/–Trp and incubated for a further 3 days. The results growths indicate that pGAD- and pGAD- could, but pGAD- and pGAD- could not, directly interact with pGBK-. In a series of negative controls, pGBK- could not interact with pGAD-, pGADT7-T, pGAD- and pGAD-. B. Pull-down assays to demonstrate that Gpa1 and Gpb1 both interact with Gpb1. GST and GST-Cyr were purified from bacteria, loaded onto glutathione sepharose beads before incubation with translated S-Gpa1 and 10 mM nucleotide. After washing the beads, the proteins were eluted by the addition of 4× NuPAGE LDS sample buffer, followed by boiling at 90°C for 5 min, and separated on a 4–12% NuPAGE gel under denaturing conditions. Bound Gpa1 was detected as a gel band by autoradiography. Lanes 2, 4 and 6 establish that Gpa1 binds to immobilized Cyr1, but there was little difference in apparent affinity after incubation with GTP (lane 2), no nucleotide (lane 4) or GDP (lane 6). Negative controls, using immobilized GST, are shown in lanes 3, 5, 7, 10 and 13. A control using immobilized GST-Gpb1 to pull-down S-Gpa1, in the presence of 10 mM GDP, shows a more intense band, suggesting that Gpa1-GDP is bound with higher affinity to Gpb1 than to Cyr. Using translated S-Cyr (lane 11), an interaction with immobilized GST-Gpb1 (lane 12) was detected

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -1

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -0

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p