Virulence Factors Genes and Transcription

At present, there are two databases of P. brasiliensis expression sequences that differ in the isolate used, fungal phase, status, and growth conditions. The transcriptome reported by Goldman et al. (2003) analyzed mRNA from Pb18 (S1 species) yeasts, which had been recovered from the spleen of Balb/c mice infected intraperito-neally and then grown under aerobic conditions in rich medium. The authors obtained 13,490 EST sequences from both 5' and 3'ends (http://143.107.203.68/ pbver2/default.html), among which 5,121 singlets and 1,397 contigs have been identified from a total of 4,692 expressed sequences. Felipe et al. (2003, 2005) obtained a total of 19,718 EST sequences from both mycelium and yeast phases of in vitro-adapted Pb01 isolate grown in semisolid rich medium. These sequences yielded 6,022 assembled sequences from 2,655 contigs and 3,367 singlets that are deposited in the http://www.biomol.unb.br/Pb database. The details concerning bioinformatics of this project can be found in Brigido et al. (2005), including the subtraction of yeast and mycelium ESTs that was carried out to determine differentially expressed genes. The information obtained with the Pb01 EST sequencing project was discussed in a series of reviews on the annotated transcripts following gene categories related to RNA biogenesis (Albuquerque et al., 2005), therapeutic targets (Amaral et al., 2005), metabolism (Arraes et al., 2005), hydrolytic enzymes (Benoliel et al., 2005; Parente et al., 2005), oxidative stress (Campos et al., 2005), GPI-anchored proteins (Castro et al., 2005), transporters and drug resistance (Costa et al., 2005), cell signaling (Fernandes et al., 2005), molecular chaperones (Nicola et al., 2005), DNA-related events (Reis et al., 2005), translation and protein fate (Souza et al., 2005), virulence (Tavares et al., 2005), and cell wall (Tomazett et al., 2005).

By random sequencing seven loci ranging between 4,439 and 11,093 kb from P. brasiliensis Venezuelan isolate IVICPb73, Reinoso et al. (2005) found 20 ORFs at an estimated density of one gene per 3.0-3.7 kb, which would average 9,166 expressed genes for an estimated genome size between 25 and 30 Mb. Therefore, more than 50% of the expressed genes by P. brasiliensis may be represented in each EST database described above. The complete genome of P. brasiliensis will probably be available in a couple of years, following the efforts of the Broad Institute, with support of the Dimorphic Fungal Genomes Consortium, that is currently developing a comparative genomics project on dimorphic fungal pathogens. The project is centered on C. immitis, whose genome will be compared with that of closely related P. brasiliensis, H. capsulatum, B. dermatitidis, and L. loboi. This study will allow the identification, among many other features, of shared and individual genetic determinants of pathogenicity and virulence in dimorphic fungi. The P. brasiliensis isolates included in this study are Pb18, representing major S1 group and virulence, Pb3 from phylogenetic cryptic species PS2, and Pb01 as a molecular model. It has recently been shown that Pb01 alone belongs to a phylogenetic group distinct from those previously identified.

The P. brasiliensis EST databases were screened for clusters similar to virulence genes from C. albicans and other pathogenic fungi (Goldman et al., 2003; Felipe et al., 2005; Tavares et al., 2005). Although C. albicans might not be the best choice for comparison with P. brasiliensis in terms of fungal biology and disease outcome, it is still the best-studied fungal pathogen, with the relevance of many genes being assessed using gene-directed mutagenesis. Virulence gene homologs have been recognized in groups of metabolic, cell wall, signal transduction, detoxification, and other genes encoding secreted proteins, suggesting that they could be important for P. brasiliensis pathogenesis as well. Another approach to finding virulence-related genes involved detection of differentially expressed transcripts in the yeast phase and during mycelium-to-yeast transition. Large-scale strategies to achieve that purpose included Pb01 database subtraction, macro and microarray hybridization, notably using P. brasiliensis biochip carrying 4,962 expressed genes from the Pb18 database (Marques et al., 2004; Felipe et al., 2005; Nunes et al., 2005; Ferreira et al., 2006). Genes with increased mRNA expression in the yeast phase have originally been identified using differential display (Venancio et al., 2002) and later, by high-throughput suppression subtraction hybridization (SSH) analysis, where yeast mRNA was the tester and mycelium mRNA was the driver population (Marques et al., 2004). The latter study added 163 new Pb18 clusters to the EST database, while 20 gene homologs were more expressed in the yeast phase of P. brasiliensis grown in three different culture media. Since temperature shift is used to promote the dimorphic transition in all these studies, genes that are exclusively committed to phase transition are hardly distinguishable. Fungal isolate, growth phase, aeration, and culture media are other features that could change the metabolic pattern independently from phase specificity, hence results that do not test these points should be cautiously interpreted. Bailao et al. (2006) aimed at detecting P. brasiliensis genes differentially expressed by Pb01 in host conditions using SSH (or RDA for representational difference analysis). The authors used as tester mRNA

from yeasts either briefly cultured in vitro after isolation from mouse liver or incubated for 10 and 60 min in human blood. The cDNA population obtained from P. brasiliensis yeasts exposed to host conditions was subtracted from that isolated from in vitro-adapted yeasts grown in solid rich medium. A total of 490 ESTs were identified in the library representing survival in liver and 417 ESTs represented early fungal response upon contact with human blood, but new genes were scarce. In all the studies mentioned above, selected genes were validated for differential expression using Northern blotting, semiquantitative RT-PCR and/or real time RT-PCR.

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

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