Oxidative stress response in Paracoccidioides brasiliensis

Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.

Transporters in the Paracoccidioides brasiliensis transcriptome: insights on drug resistance

In the struggle for life, the capacity of microorganisms to synthesize and secrete toxic compounds (inhibiting competitors) plays an important role in successful survival of these species. This ability must come together with the capability of being unaffected by these same compounds. Several mechanisms are thought to avoid the toxic effects. One of them is toxin extrusion from the intracellular environment to the outside vicinity, using special transmembrane proteins, referred to as transporters. These proteins are also important for other reasons, since most of them are involved in nutrient uptake and cellular excretion. In cancer cells and in pathogens, and particularly in fungi, some of these proteins have been pointed out as responsible for an important phenotype known as multidrug resistance (MDR). In the present study, we tried to identify in the Paracoccidioides brasiliensis transcriptome, transporter-ortholog genes from the two major classes: ATP binding cassette and major facilitator superfamily transporter. We found 22 groups with good similarity with other fungal ATP binding cassette transporters, and four Paracoccidioides brasilienses assembled expressed sequence tags that probably code for major facilitator superfamily proteins. We also focused on fungicide resistance orthologs already characterized in other pathogenic fungi. We were able to find homologs to C. albicans CDR1, CDR2, and MDR1, Saccharomyces cerevisiae PDR5 and Aspergillus AtrF genes, all of them related to azole resistance. As current treatment for paracoccidioidomycosis mainly uses azole derivatives, the presence of these genes can be postulated to play a similar role in P. brasiliensis, warning us for the possibility of resistant isolate emergence.

Nucleotide sequence of a variant protein A of Staphylococcus aureus suggests molecular heterogeneity among strains.

Protein A is a cell wall linked protein of Staphylococcus aureus that binds mammalian IgG. Although protein A displays high size heterogeneity among strains, cloning and sequencing of its gene from two strains had not shown a large difference in size. Here we report a third protein A gene sequence that shows a size variation relative to these two, due to deletions on both one IgG binding domain and a cell wall binding domain (region X). By analysis of the three sequences we were able to delineate a hypothetic model for region X domain evolution and discussed the origin of genetic variability within and without strains.

Are synthetic peptides suitable for the detection of continuous epitopes only?

Present-day methods for the definition of antibody binding sites on antigenic polypeptides show a strong bias toward identification of sequential epitopes. We have employed anti-sequence monoclonal antibodies raised against short synthetic peptides to screen a random-primed cDNA library constructed in an expression vector. Sequence data analysis performed on three clones thus isolated showed that the clones did not cross-hybridize and that the antigenic peptide sequence was found in none of them. Our findings suggest therefore that sequential epitopes may be preferentially identified because of an inherent bias in commonly used epitope mapping protocols which masks available conformational determinants.