Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms

Journalof Medical& VeterinaryMycology1997,35, 13-18 Accepted 8 June 1996 Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms M. C. S O U Z A , * J.-L. GESZTESI, t A. R. S O U Z A , * J. Z. MORAES,* J. D. LOPES* & Z. P. C A M A R G O * *Department of Microbiology, Immunology and Parasitology, Federal University of $6o Paulo (UNIFESP), SCmPaulo, SP, Brazil; and tDepartment of Immunology, State University of Sao Paulo, (USP), S(soPaulo, SP, Brazil Keywords gp43 isoforms, P. brasiliensis, reactive sera Introduction Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. This mycosis is found throughout South and Central America, where PCM is endemic in many areas [1]. According to McEwen et al. [2], it is believed that approximately 10 million people are presently infected by this fungus, of which 2% will develop the disease. A conclusive diagnosis of PCM includes direct observation of the characteristic multiple-budding yeast forms in biological fluids and tissue or by isolating the fungus from clinical materials. Because P. brasiliensis is not always found by direct examination, several serological tests have been developed to detect antibodies against the fungus to *Correspondence: Dr Z. P. Camargo: Universal Federal de $5.o Paulo, Disciplina de Biologia Celular, 04023-062, Rua Botucatu, 862, 8o. andar, Silo Paulo, SP. Fax: 55-11-5715877; E-mail: . © 1997ISHAM establish the diagnosis [3]. It is now well known that the secreted glycoprotein gp43 from P. brasiliensis is the main antigen in PCM because all patients produce antibodies against this molecule [3-5]. We have recently developed a capture immunoassay using a monoclonal antibody (MAb 17c) raised against gp43, which showed a higher sensitivity when compared with other serological tests [3]. It was also shown by our group that secreted gp43 from different strains of P. brasiliensis presents, at least, four different isoform profiles as seen by isoelectrofocusing [6]. In each of those gp43 isoform profiles a major band could be identified with a distinct pI. Accordingly, isoforms secreted by strains such as B-339 with pls of 6'0, 6-2, 6'6 (main component) and 7.0 and were assigned as profile A. Other strains such as S.S., 1925 and 19 produce gp43 isoforms which were classified as profile B (pls of 6"4, 6.8 and 7.2, with 7.2 as the major component), C (pI > 8-5) and D (pls of 5.8, 6.2 and 6.6, with the latter being the dominant secreted component), respectively. The glycoprotein gp43 from Paracoccidioides brasiliensis is the main antigenic component in paracoccidioidomycosis (PCM) because it is recognized by 100% of PCM patients. It has also been shown that different fungal strains produce gp43 with at least four isoform profiles. In this study, different isoform profiles from gp43, with pls ranging from 5.8 to 8.5, were affinity purified from various P. brasiliensis (B-339, S.S., 1925 and I9) exoantigens. Because of the isoform heterogeneity, we questioned whether those isoform profiles could be similarly recognized by acute or chronic PCM patients. By using a specific and sensitive method for detection of human lgG anti-gp43 antibodies, the monoclonal antibody capture immunoassay, we report that not all gp43 isoform profiles are equally recognized in PCM sera when anti-gp43 MAb 17c was employed as capturing antibody. Our results showed that recognition of pl 8.5 gp43 isoform was significantly lower for both acute (56%) and chronic patients (71%), compared with gp43 isoforms from the standard strain B-339. On the other hand, when anti-gp43 MAb 8a, which recognizes a different antigenic epitope was used to capture the different gp43 isoform profiles, all patient's sera reacted similarly. The results described suggest that not all the antigenic epitopes expressed by gp43 are equally present in all P. brasiliensis strains. ~L~ Souzaet al. Materials and methods Fungal strains and production of exoantigens Four isolates of P. brasiliensis (B-339, an isolate from a patient with the chronic form of PCM, was kindly provided by Dr A Restrepo, Corporaci6n de Investigaciones Biologicas, Medellin, Colombia; S.S., an isolate from a patient with the acute form of PCM fi'om Giofinia, Goifis, Brazil; 1925, provided by Dr Maria B de Albornoz, Instituto de Biomedicina-Secci6n de Micologia, Caracas, Venezuela and 19, from a patient with the chronic form of the disease, by Dr E. Castafieda, lnstituto Nacional de Salud, Bogotfi, Colombia) were grown for 7 days in peptone medium and exoantigens were obtained as previously described [3]. Sera and monoclonal antibody Thirty PCM sera were obtained from Hospital $5.o Paulo, UNIFESP, from both acute (ten sera) and chronic PCM patients (20 sera), who had the presence of P. brasiliensis in their clinical specimens confirmed by direct examination. Anti-gp43 monoclonal antibodies (MAb 17c and 8a) were obtained in our laboratory and purified from ascitic fluids in Sepharose-Protein A columns, as described [3,4,8]. Both MAbs were characterized as IgG 2b with k light chains and were shown to recognize peptidic sequences of the antigen [8]. Binding inhibition assays of peroxidase-labelled MAb 17c to gp43 was carried out in presence of MAb 8a. The results obtained showed that MAb 8a clearly recognized a different epitope in the molecule [8]. The concentrations of affinity purified MAbs 17c and 8a were always estimated spectrophotometrically at 280 nm. Immunodiffusion tests This assay was performed as described previously [9]. Each PCM serum was individually tested against an exoantigen preparation from P. brasiliensis strain B-339 and the titre of each serum was determined [9]. Purification of gp43 isoform profiles Purification of each gp43 isoform profile A, B, C and D (respectively, from exoantigens from 7 day cultures of P. brasiliensis strains B-339, S.S., 1925 and I9) was performed by affinity chromatography using CNBrSepharose (Pharmacia) coupled with anti-gp43 MAb 17c, prepared according to the manufacturer's instructions [3,8]. The protein content of eluted material was determined by the Bradford method [10]. Gp43 purification was always monitored by sodium-polyacrylamide gel electrophoresis (SDS-PAGE) [11] and by isoelectric focusing, as described [6], and protein bands were visualized by silver staining [12]. Deglycosylation of gp43 isoform profiles Affinity purified gp43 (10#g) from profiles A, B, C and D was treated with recombinant PGNase (New England, BioLabs) in phosphate-buffered saline for 24 h at 37 °C, following the manufacturer's instructions. Reaction products were analyzed by 10% polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were visualized by silver staining [12]. Partial proteolytic digestion of gp43 isoform profiles with papain Affinity purified gp43 (50 #g) from profiles A, B, C and D was treated with 2 #g of papain (E.C.C.C 3.4.22.2, Sigma) in 0.1 M pho (...truncated)