Identification of Paracoccidioides brasiliensis highly virulent isolate ligands peptides by phage display with fungicidal activity in vitro
Abstract number: P732
Kioshima E.S., Aliperti F., Villa L.L., Lopes J.D.
Introduction: Paracoccidioidomycosis is a human systemic granulomatous disease, prevalent in South America, caused by a thermodimorphic fungus, Paracoccidioides brasiliensis. This fungus presents complex antigenic structure and some of these components have been related with its pathogenicity, of which little is known. Objective: To isolate molecules expressed in the surface of highly virulent but not in non-virulent isolated yeast cells of P. brasiliensis by phage display techonology, thus identifing possible virulence factors.
Methods: A phage display library containing the insert CX7C was screened to search high-virulent isolate of yeast cells of P. brasiliensis ligands by Biopanning and Rapid Analysis of Selective Interactive Ligands method. After three rounds of selection, the DNA corresponding to peptide inserts of randomly chosen phage clones were sequenced. Peptide sequences were then analysed according to their enrichment and by Clustal W sequence alignment. Selected motifs were used to search nonredundant protein databanks (National Center for Biotechnology Information [NCBI] BLAST.
Result: The B10.A mice were inoculated with yeasts of different isolates of P. brasiliensis. Animals that received non-virulent isolates Pb18 and Pb265 groups behaved as the control group which received only PBS, but the group that was infected with highly virulent isolate Pb18 died 90 days after injection. We identified three peptides (CGSYGFNAC, CGLRLESTC and CGLRLESTC) that bound to the surface of the high-virulent but did not or did less in non-virulent isolate of P. brasiliensis in all experimental conditions tested (P < 0.01). Other experiment showed that phage-cells binding was dose-dependent. By EIA assay with synthetic peptides, a significant binding of these peptides to gp43 (the majority molecule) was not observed. These peptides exhibited fungicidal activity in vitro against P. brasiliensis by inhibition of colony forming units.
Conclusion: The peptides CGSYGFNAC, CGLRLESTC and CGLRLESTC may represent ligands of molecules that can be involved in the virulence and pathogenicity of this fungus. Financial support: FAPESP.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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