Immunoblotting of soluble antigens in Paracoccidioides brasiliensis culture

Authors


ABSTRACT

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice-enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.

List of Abbreviations
CFAg

cell-free antigens

ExoAg

exoantigens

gp43

43 kDa glycoprotein

gp70

70 kDa glycoprotein

hMM

high-molecular mass

MM

molecular mass

Pb339

P. brasiliensis strain 339

PCM

paracoccidioidomycosis

S-BHI

Sabouraud dextrose agar plus BHI

TOM

tomato juice-enriched complex medium

The thermally dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, an important systemic mycosis in Latin America [1]. The main soluble P. brasiliensis antigens are 43 kDa (gp43) and 70 kDa (gp70) glycoproteins and hMM antigens; these are important for diagnosis and investigating pathogenesis, virulence and vaccines [2-6]. The present study investigated the culture conditions for optimizing soluble antigen production by P. brasiliensis yeast cells. Pb339 was grown in individual tubes at 35 °C for 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days in Sabouraud dextrose agar at pH 5.6 and 8.5 and S-BHI and CFAgs were produced according to the method described by Camargo et al. [7], with some modifications. Briefly, the fungal masses were diluted in PBS (0.15 M, pH 7.4) with phenylmethylsulfonyl fluoride protease inhibitors (1 mM) and 0.02% thimerosal and then vortexed and centrifuged. In addition, Pb339 was cultured by stirring at 35 °C in TOM [8]. Samples were collected every 3 days from the same culture and the ExoAgs obtained by centrifugation. The protein concentration was determined by the Folin method [9]. The CFAgs and ExoAgs were analyzed by immunoblotting (5–20% polyacrylamide gel electrophoresis with SDS, transferred to a nitrocellulose membrane) and incubated with a pool of PCM patient serum (1:10, mixture of sera from six patients with chronic PCM displaying high reactivity to ExoAg by ELISA and anti-human IgG-peroxidase conjugate (A-8775; Sigma, St Louis, MO, USA). The reaction was evidenced by using 3,3′,5,5′-tetramethylbenzidine substrate. The immunoblotting results for P. brasiliensis cultured in Sabouraud dextrose agar pH 5.6, pH 8.5, S-BHI and TOM are shown in Fig. 1a,b,c and d, respectively.

Figure 1.

Cell-free antigens immunoblotted from P. brasiliensis (Pb339) grown on (a) Sabouraud dextrose agar medium at pH 5.6, (b) pH 8.5, (c) Sabouraud dextrose agar plus BHI and (d) exoantigens from TOM medium for 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days.

Polyacrylamide gel electrophoresis (5–20%) was transferred to nitrocellulose membrane and incubated with polyclonal antibodies (mixture of sera from six patients with chronic PCM displaying high reactivity by indirect ELISA, 1:10) and a peroxidase-conjugated anti-human IgG. MW, molecular weight standard.

The most intense bands were observed in the early stage of solid culture, the greatest antigen diversity being detected when cultured in S-BHI (Fig. 1a–c). According to immunoblotting, the greatest protein concentration was also observed in the first three periods in all CFA preparations (pH 5.6, pH 8.5 and S-BHI), particularly with S-BHI (data not shown). Given that the fungal mass ratio was the same for all CFAg preparations, the immunoblotting results suggest that major P. brasiliensis antigens are produced in the initial stage of growth in these solid media.

However, more evident bands were detected in the later period of culture (12–18 days) in ExoAg (Fig. 1d), the highest protein concentration being observed on day 12 (data not shown). These differences may be attributable to different growth conditions and growth rates in a liquid medium, making it difficult to compare with solid media. Also, an unknown component in the free fungal TOM medium reacted with Folin–Ciocalteu reagent; subtracting the value attributable to this reaction would not reliably provide a value for concentration of protein produced by the fungus. Due to the complexity of the TOM medium, further studies will be undertaken using a defined medium such as RPMI-1640.

In the later phase (24 days) the fungal masses increased in all culture media (data not shown) and less contaminated gp43 were detected. Therefore, older cultures have advantages for this antigen.

In conclusion, optimizing the source of gp43, gp70 and hMM requires consideration of both time and growth conditions. Although preliminary data require further studies using other Paracoccidioides species and cultivation media, these results may contribute to obtaining the main soluble antigens of P. brasiliensis.

ACKNOWLEDGMENTS

We are grateful for funding received from the Ministry of Education/University Extension Program, Research Program for the Health System(PPSUS)/Araucária Foundation and State University of Londrina.

DISCLOSURE

The authors have no financial conflict of interest.

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