High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis

Introduction: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. Methods: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. Results: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson’s correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. Conclusions: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients. Key-words: Paracoccidioidomycosis. Soluble antigen. Capture ELISA. Monoclonal antibodies. IgG. RESUMO Introdução: Diferentes níveis sorológicos de IgG/IgE contra a fração de alta massa molecular (hMM) (~366kDa) de Paracoccidioides brasiliensis têm sido encontrados na PCM aguda e crônica. Considerando a inexistência de investigação sobre esta fração em isolados clínicos de P. brasiliensis, o objetivo deste estudo foi investigar a presença da fração hMM (~366kDa) no preparado livre de células (CFA) de P. brasiliensis obtidos de isolados clínicos. Métodos: CFA de 10 isolados e de cepa de referência (Pb18) foram submetidas à eletroforese em gel de SDS-poliacrilamida (SDS-PAGE) seguida de captura de imagem e análise por densitometria. Adicionalmente, CFA de 20 isolados e de Pb18 foram analisados por ELISA captura (cELISA) utilizando anticorpos policlonal (polAb) ou monoclonal (mAb) para fração hMM. Resultados: A presença do componente de hMM foi observada em todas as amostras analisadas por SDS-PAGE/densitometria e por cELISA. Adicionalmente, o teste de correlação de Pearson demonstrou forte relação entre os níveis de fração hMM usando pAb e mAb (R = 0.853) no cELISA. Conclusões: Conclui-se que a fração hMM está presente em todos os isolados clínicos de P. brasiliensis analisados e no isolado referencial, sugerindo a possibilidade dos mesmos serem utilizados como fonte desta fração antigênica. Este trabalho também introduz pela primeira vez o método de cELISA para detecção da fração hMM de P. brasiliensis, sugerindo que detecção utilizando anticorpos polAb ou mAb é viável e essa metodologia poderá ser útil para investigação futura desta fração solúvel (~366kDa) em soros de pacientes com PCM. Palavras-chaves: Paracoccidioidomicose. Antígeno solúvel. ELISA de captura. Anticorpos monoclonais. IgG. Paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis, which develops as yeast at body temperature and as mycelium at room temperature. P. brasiliensis causes natural infections by inhalation of conidia or mycelial elements1. Most exposed subjects develop an asymptomatic infection, although some individuals present clinical manifestations that can vary from benign and localized to severe and disseminated forms2. Two forms of the disease are distinguished: the acute or subacute and chronic form. The acute form is more severe and rare, while the chronic form occurs more frequently and mostly affects adult males3,4. Usually, PCM diagnosis is inferred from indirect evidence obtained via serological tests and clinically relevant antigens have been identified and adapted for use in immunoassays for the detection of specific antibodies5. For this purpose, several fungal components have already been identified. The antigens most frequently identified in PCM patient sera are the glycoproteins of 43kDa (97-100%)6-8, the main PCM diagnostic antigen6,7,9,10, 160kDa (78%) and 70kDa (60%)8. The other alternative laboratory approach for diagnosis of PCM is the detection of circulating P. brasiliensis antigens. The inhibition ELISA methodology (inh-ELISA) is able to detect gp43 in 96.3% of PCM patients, mainly in those with the acute form of the disease (100%)11. Gp70 has also been detected in the urine or in cerebrospinal fluid (CSF) of PCM patients12,13. Gómez et al14,15 reported the use of inh-ELISA and detected gp87 circulating antigen in sera from patients with active disease. Puccia et al9 demonstrated polydispersed highmolecular mass glycoprotein, with heterogeneous electrophoresis migration. From this heterogeneous electrophoresis migration Marquez et al16 isolated fractions ranging from 278kDa to 466kDa (mean approximately 366kDa) obtained from sonicated

Paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis, which develops as yeast at body temperature and as mycelium at room temperature.P. brasiliensis causes natural infections by inhalation of conidia or mycelial elements 1 .Most exposed subjects develop an asymptomatic infection, although some individuals present clinical manifestations that can vary from benign and localized to severe and disseminated forms 2 .Two forms of the disease are distinguished: the acute or subacute and chronic form.The acute form is more severe and rare, while the chronic form occurs more frequently and mostly affects adult males 3,4 .
Usually, PCM diagnosis is inferred from indirect evidence obtained via serological tests and clinically relevant antigens have been identified and adapted for use in immunoassays for the detection of specific antibodies 5 .For this purpose, several fungal components have already been identified.The antigens most frequently identified in PCM patient sera are the glycoproteins of 43kDa (97-100%) [6][7][8] , the main PCM diagnostic antigen 6,7,9,10 , 160kDa (78%) and 70kDa (60%) 8 .
The other alternative laboratory approach for diagnosis of PCM is the detection of circulating P. brasiliensis antigens.The inhibition ELISA methodology (inh-ELISA) is able to detect gp43 in 96.3% of PCM patients, mainly in those with the acute form of the disease (100%) 11 .Gp70 has also been detected in the urine or in cerebrospinal fluid (CSF) of PCM patients 12,13 .Gómez et al 14,15 reported the use of inh-ELISA and detected gp87 circulating antigen in sera from patients with active disease.
Puccia et al 9 demonstrated polydispersed highmolecular mass glycoprotein, with heterogeneous electrophoresis migration.From this heterogeneous electrophoresis migration Marquez et al 16 isolated fractions ranging from 278kDa to 466kDa (mean approximately 366kDa) obtained from sonicated

RESULTS
or cell free antigens (CFA) preparations.Moreover, different serum levels of the IgG/IgE to the hMM fraction was verified in sera of acute and chronic PCM patients and the authors suggested the analysis as a new characteristic to differentiate between these two clinical forms of the disease 16 .
Taking into account the lack of data regarding the soluble hMM fraction (~366kDa) in P. brasiliensis clinical isolates and the immunological methodology for identifying it, in the present study, the hMMAg antigen was investigated in different clinical isolates and capture ELISA (cELISA) was introduced.In principle, observation verified that all the P. brasiliensis samples analyzed produced the hMM fraction (~366kDa).In addition, this hMM fraction was detected by cELISA using monoclonal or polyclonal antibodies to the hMM fraction.

Fungal isolates
Clinical isolates of P. brasiliensis of the chronic form of PCM disease were obtained: 17 isolates (LDR1 to LDR17) from Londrina State University (MOOI/CCS, HC, HC, Londrina State University, Londrina, Paraná) patients (2000 to 2006); two isolates (RC-Wang and RC-Hori) from Chiba University, Chiba, Japan; one isolate (EPM-01) and reference strain (Pb18) from UNIFESP, São Paulo, Brazil.P. brasiliensis strains were maintained on potato dextrose agar (Difco Laboratories, MI, USA) slants at room temperature.Prior to experiments, samples of the isolates were inoculated onto a slant of brain heart infusion agar (BHI, Difco Laboratories) supplemented with 1% dextrose and cultured at 35ºC to produce the yeast form and maintained by subculturing at 35ºC at 5-day intervals on Sabouraud agar (Micromed, Rio de Janeiro, RJ, Brazil).

Cell free antigens preparation
Yeast cells were collected and the CFA samples were obtained according to Camargo et al 17 , modified by the addition of PMSF protease inhibitor at 2.5mM to the supernatant.The protein concentration was determined by the Lowry method 18 , adjusted to 3mg/mL and stored in -80ºC freezer until ready to use.
Capture ELISA for IgG-hMM fraction ELISA immunoplates sensitized with rabbit IgG anti-hMM fraction (25µg/ml) were incubated with CFA samples at 30μg/ml at 37ºC for 1h and then with polyclonal mouse IgG anti-hMM (IgG pAb) (30μg/ml), anti-mouse IgG peroxidase conjugate and OPD (100μL well).The absorbance was read at 492nm.Additionally, cELISA for hMM fraction level determination was performed as described, substituting polyclonal mouse IgG anti-hMM fraction for monoclonal IgG anti-hMM fraction (IgG mAb) as secondary antibodies.Polyclonal rabbit and mouse antibodies were produced by using the hMM fractions obtained, according to Pavanelli et al 19 for immunizations.A monoclonal antibody was obtained by spleen cells (from immunized BALB/c mice with the hMM fraction) fused with P3U1 cell line by using PEG.Hybridomas were screened by ELISA with hMM fraction (~366kDa) and cloned by limiting dilutions.Pristane-primed BALB/c mice were injected i.p. with hybridoma and IgG purified (Sepharose-G protein column) from ascite fluid.The cutoff was determined as the mean plus 2 standard deviations of the absorbance obtained with control: primary rabbit IgG anti-hMM, secondary mouse pAb or mAb IgG and peroxidase conjugate without the CFA sample.

Statistical analysis
Statistical comparisons were performed by analysis of variance (ANOVA) and by the Tukey test.All values are reported as the mean ± SD of the mean, with significance assumed in the range of p<0.05.Pearson's correlation was applied between hMM fraction levels using IgG pAb and mAb anti-hMM fraction and a significant correlation was considered when r ≥ 0.50.

Ethical
This study was approved by the Internal Scientific Commission and the Bioethics in Research Committee of the State University of Londrina (Londrina, PR, Brazil).

CFA analysis by SDS-PAGE
The presence of the hMM fraction was observed in CFA from all the clinical isolates and reference Pb18 by SDS-PAGE.The SDS-PAGE was submitted to densitometry in an attempt to quantify the hMM fraction.The results show that 20.5 ± 5.7% (clinical isolates) and 16.7% (Pb18) from CFA correspond to the hMM fraction (Figure 1).This study indicates that this antigen is present in different clinical isolates from chronic patients and could be used as source of the hMM fraction.
Capture ELISA for hMM fraction levels in CFA samples Various researchers have attempted to detect circulating antigens in PCM patients using polyvalent antigens or antibodies in different assays.For this purpose, the presence of the hMM fraction in CFA samples was determined using cELISA expressed as optical density units (OD).Analysis of the result verified the presence of the hMM fraction in Pb18, a strain characterised as highly virulent, and in all the clinical isolates of P. brasiliensis, with the use of polAb or mAb to the hMM fraction (Table 1).All values greater than the values obtained in the control systems of polyclonal/polyclonal or monoclonal/monoclonal with free samples of CFA plus 2 x standard (0.118 ± 0.008 and 0.086 ± 0.005, respectively) were considered as positive.

Correlation analysis
Analysis of the results confirmed a strong correlation between hMM fraction levels with IgG pAb or mAb anti-hMM as the secondary antibody (r = 0.853) (Figure 2).This result demonstrates that the hMM fraction can be detected by cELISA using monoclonal or polyclonal antibodies to hMM, as a secondary antibody.ELISA immunoplates sensitized with rabbit IgG anti-hMM were incubated with CFA samples and then with polyclonal mouse IgG anti-hMM (pAb-hMM) or with monoclonal mouse IgG anti-hMM (mAb-hMM).After incubation with anti-mouse IgG peroxidase conjugate, absorbance was read at 492nm.All values higher than the controls: polyclonal/polyclonal (0.118 ± 0.008) or polyclonal/monoclonal (0.086 ± 0.005), with free CFA sample, plus 2 x standard were considered positive.

DISCUSSION
In this work, the hMM fraction was detected in all the P. brasiliensis clinical isolates and the reference strain Pb18 by SDS-PAGE, suggesting that all these P. brasiliensis could be used as a source of this fraction.
For diagnostic purposes, the presence of the target antigen in all isolates of the P. brasiliensis is important.The present study indicates that this antigen is present in different clinical isolates from chronic patients as the common P. brasiliensis antigen.However, further studies involving larger sample of isolates are required to confirm these findings.
Panunto-Castelo et al 8 identified hMM antigens with 172 or 160kDa in exoAg from three P. brasiliensis isolates (DGO, C-9 and BAT).In this study, the considered hMM band identified in CFA from the P. brasiliensis isolates presented ~366kDa and is, therefore, a different hMM antigen.
The detection of circulating antigens is a useful approach for serodiagnosis for monitoring PCM treatment.The gp43 glycoprotein, one of the most important immunodominant antigens of P. brasiliensis, has been extensively investigated, particularly in relation to diagnosis [20][21][22] .Gp43 has been detected at higher levels in PCM patient sera as circulating antigens 22 and in CSF and bronchoalveolar lavage (BAL) fluid samples from PCM patients 11 .However, recently the existence of a P. brasiliensis isolate presenting differences in this major antigen coding gene gp43 23,24 has been demonstrated and speculation of the possibility of a new species in the genus Paracoccidioides has been noted.Considering genetic differences in gp43 according to P. brasiliensis isolate, it has become important to investigate other P. brasiliensis antigens for diagnosis.Besides gp43 11 , the 87-kDa molecule 15 , gp70 13 and the high molecular mass antigen with 160kDa 25 were also introduced as potential candidates for diagnosis and/or for follow-up of patients with PCM.
Panuto-Castelo et al 8 demonstrated that the hMM antigens (172 or 160kDa) are highly reactive with serum IgG of patients with acute or chronic PCM, indicating their potential application in the diagnosis and follow-up of the disease.In addition, Coltri et al 25 characterized this antigen as a protein of 160kDa, designated paracoccin, with selective binding to immobilized GlcNAc and able to interact with laminin.
In this study, the association between SDS-PAGE and densitometry analysis shows that approximately 16-20% of P. brasiliensis CFA antigens correspond to the hMM fraction.Considering the proportion and as a soluble antigen present in CFA, we speculate that its presence may also be observed as a soluble antigen during infection, similar to other antigens, such as gp43 or gp70, present in CFA and in the serum 11,13,22,26 , with the potential for diagnosis and follow-up of the PCM patients.Thus, the hMM fraction is also important when considering the distinct isotypic humoral immune response to hMM antigens with ~336kDa observed in the acute and chronic forms of the PCM disease, which suggest that it has potential as a new biomarker for differentiating these two clinical forms 16 .
In this study cELISA was introduced to detect the hMM fraction in clinical isolates and the reference Pb18 strain was introduced and the presence of the hMM fraction was detected in all the samples analyzed, in agreement with SDS-PAGE.This study is qualitative and the results are expressed as optical density.cELISA has the advantage of being able to process large numbers of samples at the same time and presents high sensitivity and a high specificity.The analysis was performed using polAb or mAb to the hMM fraction as a secondary antibody and the results showed a stronger correlation between hMM fraction levels obtained using pAb or mAb anti-hMM.cELISA with specific polAb and mAb was used in previous studies by our group to determine circulating soluble gp43 levels in PCM 22 and determining plasmatic hMM fraction levels in PCM patients by cELISA will be the object of future investigations.
Unexpectedly, the results of ELISA showed homogeneity.We believe that regardless of differences in isolates or strains, these components are better preserved and produced more homogeneously, as observed with other antigens reported in the literature 8 .The same isolates showed less homogeneity in relation to gp43 determined by capture ELISA (data not shown) and the heterogeneity observed in the percentage of the hMM fraction in the electrophoresis was due to variations in the other components present in CFA (data not shown).
In conclusion, the soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as source of this fraction.The work also introduced for first time the capture ELISA method for P. brasiliensis hMM fraction (~366kDa) detection.This work was supported financial by the Financiadora de Estudos e Projetos (FINEP), the Secretaria de Estado da Ciência, Tecnologia e Ensino Superior (SETI/PR), the Fundação Araucaria/PR, Próreitoria de Extensão (PROEX/UEL), the Pró-reitoria de Pesquisa (PROPPG/UEL) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).