Major Antigenic Molecules of P brasiliensis

The 43 kDa glycoprotein (gp43) is the major diagnostic antigen of P. brasiliensis. Discovered in 1986 (Puccia et al., 1986), this glycoprotein represents approximately 80% of the exoantigen protein as examined by SDS-PAGE (reviewed in Camargo & Franco, 2000). By using Western blotting, anti-gp43 antibodies from patients in recovery after chemotherapy had lower reactivity in contrast to patients with relapsing disease who showed higher reactivity relative to the patients with installed disease. A decrease of anti-gp43 IgG, IgA, and IgM correlated well with clinical improvement (Giannini et al., 1990). Since anti-gp70 in parallel with anti-gp43 antibodies also decreased in patients undergoing antimycotic therapy, both molecules are markers of human PCM (Camargo et al., 1989). Different isoforms of the gp43 with pIs ranging from 5.8 to 8.5 were not all recognized by patients' sera in capture immunoassays with bound anti-gp43 monoclonal antibodies. The pI 8.5 gp43 was the least recognizable isoform by both sera from acute and chronic patients (Souza et al., 1997). We determined early in 1991 that epitopes in gp43 that elicited a strong antibody response were peptidic in nature and therefore the patients' sera reacted intensely with the deglycosylated molecule (Puccia & Travassos, 1991).

Heat-shock proteins have also been shown to elicit antibody responses in patients with PCM. Recombinant hsp60 was recognized by sera from 72/75 patients. Overall the sensitivity and specificity of assays with hsp60 were high (97.3% and 92.5%, respectively) (Cunha et al., 2002). Also, sera from PCM patients recognized recombinant HSP70 from P. brasiliensis (Bisio et al., 2005). The 87 kDa antigen can be detected in the sera of infected patients. It is homologous with heat-shock proteins and a mAb raised against H. capsulatum 80 kDa hsp cross-reacted with purified 87 kDa, predominantly yeast-form antigen (Diez et al., 2002). A 45 kDa formamidase detected in both P. brasiliensis yeast and mycelium reacted with sera from PCM patients and not with sera from healthy individuals (Borges et al., 2005). Another recombinant antigen which also reacted with sera from patients with 87.5% specificity, although at 58.7% compared to patients with other mycoses, is the 27 kDa protein used at 1 ng/well. Cross-reactivity with sera from patients with aspergillosis and histoplasmosis was observed (Ortiz et al., 1998).

Gp43 is regularly isolated from yeast culture supernatants by affinity chromatography using monoclonal antibody (mAb) 17c. Intracellularly this antigen is found in cytoplasmic vacuoles and within lomasomes connected to the plasma membrane (Straus et al., 1996). It then diffuses in the cell wall but is secreted at restricted sites as drop-like aggregates. Therefore gp43 is responsible in part for the surface reactivity of yeast cells in vitro and in vivo and, as a secreted product, for the interaction with macrophages and B cells, and with specific antibodies. Yeast cells adhered to laminin-1 from mouse sarcoma and the interaction seemingly involved gp43. In vitro, laminin-1 bound to gp43 with a Kd of 3.7 nM. In vivo, laminin-coated yeast cells had increased virulence in hamsters challenged by testicular injection (Vicentini et al., 1994). Different anti-gp43 mAbs, recognizing different epitopes in the gp43, also showed different effects in the hamster infection model. Some mAbs were protective, inhibited granuloma formation and tissue destruction whereas another mAb did not control infection and increased histopathogenicity (Gesztesi et al., 1996). Laminin-1-coated yeast cells were also administered intratracheally in mice. No differences in CFUs were found in the lungs, livers, and spleens of these animals (Andre et al., 2004) in comparison with untreated yeasts. Apparently, the internal basement lamina containing laminin is less accessible for homotypic binding to externally present laminin-coated yeasts. The latter interact with the alveolar epithelium cells and macrophages and are equally infective compared to uncoated cells. Moreover, P. brasiliensis produces a serine-thiol proteinase that degrades laminin, fibronectin, collagen IV, and proteoglycan (Carmona et al., 1995; Puccia et al., 1998) which can be construed as a virulence factor allowing fungal cells to degrade the basal membrane, thus favoring tissue invasion and hematogenous dissemination. The gp43 mediated phagocytosis of yeast cells and anti-gp43 F(ab) fragments inhibited this activity (Almeida et al., 1998b). The interaction of gp43 with macrophages involved in part the mannose/fucose receptor which agrees with the single high-mannose N-linked Hex13GlcNAc2 oligosaccharide chain identified in the glycoprotein (Almeida et al., 1996). Gp43 can be presented by dendritic cells or by B cells resulting in Th1 or Th2-type immune responses, respectively, with the corresponding major cytokines and implications in the outcome of P. brasiliensis infection (Almeida et al., 1998a; Ferreira et al., 2003).

Gp43 gene was cloned, sequenced, and expressed in Escherichia coli as a recombinant fusion protein (Cisalpino et al., 1996). It encodes a polypeptide of 416 amino acids with a leader sequence of 35 residues and is present in one copy per genome as shown by Southern blotting and chromosomal mega-restriction analysis. The open-reading frame has two exons and one intron. As mentioned, the mature protein has a single N-glycosylation site. Sequences containing B cell epitopes are still under study chiefly by examining the reactivity of anti-gp43 mAbs. The T cell epitope responsible for delayed-type hypersensitivity reactions, and CD4+ T cell proliferation, has been mapped to a 15-mer peptide called P10 with the sequence: QTLIAIHTLAIRYAN (Taborda et al., 1998). The hexapeptide HTLAIR with varying flanking regions is essential for the immune cellular response. Both gp43 and P10 protect mice against the i.t. challenge with virulent P. brasiliensis. Gp43 has 54-60% homology and 50% identity with the exo-P-1, 3-D-glucanase sequences from different fungi: Aspergillus oryzae, Blumeria graminis, Schizosaccharomyces pombe, Pichia augusta, Saccharomyces cerevisae, and Candida albicans. The sequences in these proteins that could contain homologous peptides to P10 or gp43 (181-195) showed 60% identity of P10 with peptide NTLRAIQALAERYAP from P-1, 3-glucanase of B. graminis (Genbank accession number Q96V64). For comparison, the identity of the corresponding sequences from P-1,3-glucanases of dimorphic fungi related to P. brasiliensis, H. capsulatum and B. dermatitidis, and of A. nidulans (hypothetical protein with endoglucanase region) and C. albicans to gp43 (179-199) that contains P10, was of 57.1%, 57.1%, 42.8%, and 28.5%, respectively (Blast, NCBI assessments XP661656, P29717 and website http://genome.wustl.edu/). Gp43 has a mutated catalytic peptide (NKP instead of NEP) which is essential for glucanase activity (Figure 11.2).

Polymorphism in gp43 was suggested owing to the identification of isoforms with distinct isoelectric points (Souza et al., 1997). The gp43 precursor genes of 17 P. brasiliensis isolates were aligned and compared with a consensus sequence. The genotypic types showed 1-4 or 14-15 informative substitution sites, localized between 578 and 1166 bp (Morais et al., 2000). Nucleotide differences were consistent with a second isoelectric point for the deduced protein. The most polymorphic sequences encoded basic gp43 isoforms. The nucleotides encoding the P10 sequence were conserved, reinforcing the possibility of a peptide based vaccine (Travassos et al., 2004a, b).

The promoter region and exon 2 of the PbGP43 locus have been included in the multilocus studies of Matute et al. (2006). Nucleotide polymorphism in the PbGP43 gene characterized by Morais et al. (2000) has later been analyzed for the promoter region as well (Carvalho et al., 2005). The authors observed the existence of six genotypes by comparing sequences of two cloned PCR fragments of the whole gene, where substitutions were concentrated on exon 2 and tended to lead to amino acid changes. The core of P10 containing the T cell epitope and glycosylation site in gp43 are conserved. The sequences from isolates Pb2, Pb3, and Pb4, recovered from adult patients with PCM, were highly polymorphic, hence phylogenetically

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Figure 11.2 Sequence alignment of fragments of P. brasiliensis gp43 containing P10 and the tripeptide NKP, with homologous sequences of fungal (3-1, 3-glucanases or hypothetical proteins with endoglucanase domains, showing the catalytic NEP motiv distant from the others. Similar sequences were later found in an armadillo (T10F1) and another clinical (Bt84) sample (Hebeler-Barbosa et al., 2003), where the translated gp43 has basic isoelectric point in contrast with the neutral to acidic protein resulting from the other genotypes. Not coincidentally, the isolates bearing highly polymorphic PbGP43 sequences are in the PS2 group defined by Matute et al. (2006a). Therefore, the PbGP43 genotypes are reflecting global differences in genetic background. Although there was no association between PbGP43 and PCM clinical forms (Morais et al., 2000), differences in the outcome of experimental PCM have been observed in animals inoculated with isolates from the S1 and PS2 species. Using the susceptible B10.A mouse model and organ-recovered yeasts from i.t. and intravenous (i.v.) infections, Carvalho et al. (2005, unpublished data) observed that Pb2, Pb3, and Pb4 (phylogenetic group PS2) evoked few deaths and were recovered from the lungs at significantly fewer colony forming units (CFU) than five isolates from the S1 species, including the highly virulent Pb18 strain. Infection was followed for 30, 60, and 120 days. Anti-gp43 antibody responses elicited by i.t.-inoculated Pb2, Pb3, and Pb4 were rich in IgG2a, IgG2b, and IgG3, while the other sera were richer in IgG1 and IgA. Further i.t. infections, with representative isolates that were adapted in vivo before inoculation, showed that mice infected with Pb3, whose CFU in the lungs declined after 120 days infection, secreted increasing amounts of IFN-y, whereas IL-10 could only be detected after 30 days of infection. The animals infected with S1 isolates had progressive infection and higher CFU counts by day 120, when IFN-y was undetectable. In these animals, IL-10 was detected at all time points. It is relevant to point out that a reliable correlation between genetic background and virulence should be made using comparable animal models, inoculation routes, inoculum size, and fungal growth status and conditions. In that respect, armadillo isolate T10F1 from species PS2 has been reported as very aggressive in the hamster testicular model (Hebeler-Barbosa et al., 2003), in contrast with the results mentioned above for other PS2 members in the mouse model.

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