Detection of cell wall mannoprotein Mp1p in culture supernatants of Penicillium marneffei and in sera of penicilliosis patients

Abstract

Mannoproteins are important and abundant structural components of fungal cell walls. The MP1 gene encodes a cell wall mannoprotein of the pathogenic fungus Penicillium marneffei. In the present study, we show that Mp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Mp1p was capable of detecting this protein from the cell culture supernatant of P. marneffei at 10(4) cells/ml. The anti-Mp1p antibody is specific since it fails to react with any protein-form lysates of Candida albicans, Histoplasma capsulatum, or Cryptococcus neoformans by Western blotting. In addition, this Mp1p antigen-based ELISA is also specific for P. marneffei since the cell culture supernatants of the other three fungi gave negative results. Finally, a clinical evaluation of sera from penicilliosis patients indicates that 17 of 26 (65%) patients are Mp1p antigen test positive. Furthermore, a Mp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for P. marneffei carry a sensitive of 88% (23 of 26), with a positive predictive value of 100% and a negative predictive value of 96%. The specificities of the tests are high since none of the 85 control sera was positive by either test.

FIG. 1

Western blot analysis of Mp1p in the culture supernatant of P. marneffei. Lane 1, 20 μg of a cell lysate of P. marneffei; lane 2, 5 μl of a concentrated culture supernatant; lane 3, 10 μl of a concentrated culture supernatant.

FIG. 2

Mp1p is specific for P. marneffei cells. Approximately 20 μg of each of the cell lysates from P. marneffei (lane 1), C. albicans (lane 2), C. neoformans (lane 3), and H. capsulatum (lane 4) was loaded into each lane for Western blot analysis with a specific rabbit anti-Mp1p antibody.

FIG. 3

Standard curve for Mp1p and determination of Mp1p concentration in culture supernatants of P. marneffei. The standard curve of the antigen ELISA was determined with a purified recombinant Mp1p protein. Two culture supernatants of P. marneffei with final densities of 2 × 10⁶ (a) and 8.6 × 10⁵ (b) cells/ml were diluted 1 to 9 and were subjected to the antigen ELISA. The OD₄₀₅ values obtained by ELISA were plotted to determine the Mp1p protein concentrations in the two supernatants (3.8 and 3.4 ng/ml, respectively).

FIG. 4

Mp1p antigen ELISA is specific for P. marneffei. Cell culture supernatants were obtained from cultures of P. marneffei, C. albicans, C. neoformans, and H. capsulatum grown to densities of about 1 × 10⁶ to 2 × 10⁶ cells/ml and were subjected to the Mp1p antigen ELISA. The positive ELISA values are restricted to the supernatants of P. marneffei cells. Notice that the largest dilution for the P. marneffei culture supernatant with a positive OD₄₀₅ value is 1:125.

FIG. 5

Evaluation of sensitivity and specificity of the P. marneffei antigen test for the detection of penicilliosis in patients.