Heterolougous expression and antigenic characterization of Plgp43 from Paracoccidioides lutzii

• Grant number: 10/03193-6
• Support type: Scholarships in Brazil - Master
• Effective date (Start): August 01, 2010
• Effective date (End): July 31, 2012
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Rosana Puccia
• Grantee: Natanael Pinheiro Leitão Júnior
• Home Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 06/05095-6 - Analysis of exported vesicular structures and of secreted molecules by the pathogenic fungi Paracoccidioides brasiliensis e Cryptococcus neoformans, AP.TEM

Abstract

The gp43 antigen is the main diagnosis antigen of paracoccidioidomycosis (PCM), a systemic mycosis caused by Paracoccidioides species. Our laboratory has recombinant isoforms of the molecule, produced in Pichia pastoris, which are useful in diagnostic assays of patients infected with phylogenetic groups S1, PS2 and PS3 of P. brasiliensis. The group of Pb01-like isolates have recently been considered a new species called P. lutzii. The predicted Plgp43 ortholog from P. lutzii preserves only 80% of identity with gp43, and it does not show gp43 main characteristics: a glycosylation site, conserved T-cell epitope P10 and an inactive glucanase active site. This may have consequences not only in diagnosis, but in the interaction of fungus and host. The aim of this work is the heterologous expression of the Plgp43 gene from Pb01 in both bacteria and P. pastoris. The recombinant molecule will be used to produce rabbit polyclonal antibodies; tests with monoclonal and polyclonal antibody anti-gp43 will be performed; a large number of patients sera with PCM from the Southeast and Midwestern Brazil will be analyzed. These analyzes will be performed mainly by immunoblot, but also by passive immunodiffusion and dot blot if a soluble form of purified protein is obtained. The purified soluble recombinant molecule will be compared with other gp43 isoforms by circular dichroism and stability by fluorescence spectroscopy. In parallel, attempts of the expression of gp43r isoforms with mutation at the glycosylation site using cDNA already available in our laboratory will be revisited. Additionally, the expression and purification of recombinant gp43r3 isoform for large scale trials aiming at multicenter diagnosis studies and crystallization will be carried out.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

LEITAO, JR., NATANAEL P.; VALLEJO, MILENE C.; CONCEICAO, PALLOMA M.; CAMARGO, ZOILO P.; HAHN, ROSANE; PUCCIA, ROSANA. Paracoccidioides lutzii Plp43 Is an Active Glucanase with Partial Antigenic Identity with P. brasiliensis gp43. PLoS Neglected Tropical Diseases, v. 8, n. 8 AUG 2014. Web of Science Citations: 11.