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.

Virulence insights from the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.

Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients

Osteosarcoma is the commonest type of primary malignant bone tumor, frequently found in adolescents at sites of rapid bone growth. Despite current management protocols, up to half of the patients succumb to this disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. Since phage display methodology allows the selection of human antibody fragments with potential use in clinical applications, we applied this procedure to construct a recombinant Fab (antigen binding fragment) library from patients with osteosarcoma. We used peripheral blood lymphocyte total RNA from 11 osteosarcoma patients and cloned recombinant Fab representing the micro, gamma and kappa chain antibody repertoires of these individuals. The resulting library was cloned in the pComb3X vector and attained 1.45 x 10(8) different functional forms. BstO I fingerprinting and DNA sequencing analysis of randomly selected clones revealed the diversity of the library, demonstrating that Fab harbors Vkappa chains from subgroups I to V, biased towards the A27 fragment, as normally reported for the human repertoire. Analysis of the VH repertoire revealed that our library has a slight bias towards the VH4 family, instead of the usually reported VH3. This is the first description of a phage display library from osteosarcoma patients. We believe these human Fab fragments will provide a valuable tool for the study of this neoplasia and could also contribute to improvements in the diagnosis of this disease.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.