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. 2001 Nov;39(11):3830-7.
doi: 10.1128/JCM.39.11.3830-3837.2001.

Characterization of AFMP1: a novel target for serodiagnosis of aspergillosis

K Y Yuen  1 , C M Chan, K M Chan, P C Woo, X Y Che, A S Leung, L Cao
Affiliations

Characterization of AFMP1: a novel target for serodiagnosis of aspergillosis

K Y Yuen et al. J Clin Microbiol. 2001 Nov.

Abstract

We cloned the AFMP1 gene, which encodes the first antigenic cell wall galactomannoprotein in Aspergillus fumigatus. AFMP1 codes for a protein, Afmp1p, of 284 amino acid residues, with a few sequence features that are present in Mp1p, the antigenic cell wall mannoprotein in Penicillium marneffei that we described previously, as well as several other cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidyl inositol attachment signal sequence. Specific anti-Afmp1p antibody was generated with recombinant Afmp1p protein purified from Escherichia coli to allow further characterization of Afmp1p. Afmp1p has a high affinity for Galanthus nivalis agglutinin, a characteristic indicative of a mannoprotein. Furthermore, it was recognized by a rat monoclonal antibody against the galactofuran side chain of galactomannan, indicating that it is a galactomannoprotein. Ultrastructural analysis by immunogold staining indicated that Afmp1p is present in the cell walls of the hyphae and conidia of A. fumigatus. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. fumigatus develop a specific antibody response against Afmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.

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Figures

FIG. 1
FIG. 1
DNA and amino acid sequences of Afmp1p. AFMP1 cDNA contains a single open reading frame that encodes 284 amino acid residues and that has a predicted molecular mass of 31.4 kDa. The N-terminal cleavable signal peptide of 17 amino acids is underlined. The C-terminal cleavable glycosylphosphatidyl inositol signal peptide of 18 amino acids is double underlined. An 84-amino-acid serine- and threonine-rich region is indicated in italics, indicating that this protein may have many O-glycosylation sites. The asterisk indicates the stop codon, and the arrow indicates a potential cleavage site.
FIG. 2
FIG. 2
In vitro translation of AFMP1. AFMP1 was translated in vitro with Promega's rabbit reticulocyte lysate, and a band of about 32 kDa could be detected (lane 1). Afmp1p was immunoprecipitated with guinea pig immune serum against A. fumigatus cells (lane 3) but not with preimmune serum (lane 2).
FIG. 3
FIG. 3
Detection of mannoprotein after immunoprecipitation of two protein fractions, a cell lysate obtained by sonication and a lyticase wash extract, with guinea pig preimmune serum, serum after immunization with A. fumigatus whole cells, and serum after immunization with GST-Afmp1p. Carboxypeptidase was used as the mannoprotein positive control (lane 1). A band of about 32 kDa could be detected when a cell lysate obtained by sonication or a lyticase wash extract was immunoprecipitated with guinea pig serum after immunization with A. fumigatus whole cells (lanes 3 and 6) or serum after immunization with GST-Afmp1p (lanes 4 and 7) but not with preimmune serum (lanes 2 and 5).
FIG. 4
FIG. 4
Detection of galactomannan after immunoprecipitation of whole-cell A. fumigatus lysate with serum obtained from a guinea pig before immunization or after immunization with GST-Afmp1p. Purified galactomannan extracted from A. fumigatus was used as the positive control (lane 1). A band of about 32 kDa could be detected with an antigalactomannan monoclonal antibody (lane 2) or antibody against purified Afmp1p (lane 4) when A. fumigatus lysate was immunoprecipitated with guinea pig serum after immunization with GST-Afmp1p but not with guinea pig preimmune serum (lanes 3 and 5).
FIG. 5
FIG. 5
Immunoelectron micrographs of an A. fumigatus hyphal septum stained with preimmune rabbit serum (A) and a hyphal septum (B), a tip of a hypha (C), and a conidiospore (D) stained with rabbit anti-Afmp1p antibody.
FIG. 6
FIG. 6
Specific immunoprecipitation of Afmp1p by sera of patients with A. fumigatus infections. A band of about 32 kDa (arrow) was the Afmp1p translated in vitro (lane 1). Positive control sera (guinea pig immune serum, lanes 2 and 12), a negative control serum (preimmune guinea pig immune serum (lane 11), sera from patients positive for Afmp1p protein antibodies (two patients with aspergilloma [lanes 3 and 4] and two patients with invasive aspergillosis [lanes 5 and 6]), sera from healthy blood donors (lanes 7 to 10 and 17 to 20), and sera from patients infected with C. albicans (lanes 13 and 14) and P. marneffei (lanes 15 and 16) were tested.
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