AFLMP1 encodes an antigenic cel wall protein in Aspergillus flavus

Abstract

We have previously reported the cloning and characterization of the MP1 gene in Penicillium marneffei and the AFMP1 gene in Aspergillus fumigatus and their use for serodiagnosis of penicilliosis and aspergilloma and invasive aspergillosis, respectively. In this study, we describe the cloning of the AFLMP1 gene, which encodes the homologous antigenic cell wall protein in Aspergillus flavus, the most common Aspergillus species associated with human disease in our locality and in other Asian countries and the second most common Aspergillus species associated with human disease in Western countries. AFLMP1 codes for a protein, Aflmp1p, of 273 amino acid residues, with a few sequence features that are present in Mp1p and Afmp1p, the homologous antigenic cell wall proteins in P. marneffei and A. fumigatus, respectively, 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 glycosylphosphatidylinositol attachment signal sequence. Specific anti-Aflmp1p antibody was generated with recombinant Aflmp1p protein purified from Escherichia coli to allow further characterization of Aflmp1p. Indirect immunofluorescence analysis indicated that Aflmp1p is present on the surface of the hyphae of A. flavus. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. flavus develop a specific antibody response against Aflmp1p. 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.

FIG. 1.

DNA and amino acid sequences of Aflmp1p. AFLMP1 cDNA contains a single open reading frame encoding 273 amino acid residues with a predicted molecular mass of 26.3 kDa. The N-terminal cleavable signal peptide of 17 amino acids is underlined. The C-terminal cleavable GPI signal peptide of 20 amino acids is double underlined. A 75-amino-acid serine- and threonine-rich region is in italics, indicating that this protein may have many O glycosylation sites. The 141-amino-acid region CR4, showing homology to CR1 and CR2 of Mp1p in P. marneffei, CR3 of Afmp1p in A. fumigatus, and CR5 of the homologous protein in A. nidulans, is shaded in gray.

FIG. 2.

(A) Diagrammatic representation of Mp1p of P. marneffei, Afmp1p of A. fumigatus, Aflmp1p of A. flavus, and the homologous protein of A. nidulans, showing the regions representing the signal peptide, internal homologous regions (CR1 to CR5), potential O glycosylation regions, and GPI signal peptide. (B) Phylogenetic tree based on the amino acid sequences of CR1 and CR2 in P. marneffei, CR3 in A. fumigatus, CR4 in A. flavus, and CR5 in A. nidulans. The tree was inferred from amino acid data by the neighbor-joining method. The scale bar indicates the estimated number of substitutions per 50 amino acids by using the Jukes-Cantor correction.

FIG. 3.

Western blot analysis of purified Aflmp1p of A. flavus by using guinea pig sera. Strong antigen-antibody interaction was detected with sera of guinea pigs immunized with A. flavus (lane 1) or Aflmp1p (lane 2), but not with preimmune guinea pig serum (lane 3).

FIG. 4.

Western blot analysis of Aflmp1p in sonicated A. flavus cell lysate by using guinea pig sera. A band of about 70 kDa (arrow) was detected with the sera of guinea pigs immunized with Aflmp1p (lane 1) but not with preimmune guinea pig serum (lane 2).

FIG. 5.

Indirect immunofluorescence staining of A. flavus hyphae with preimmune guinea pig serum (a), guinea pig anti-A. flavus whole-cell antibody (b), and guinea pig anti-Afmp1p antibody (c). Apple green fluorescence indicates a positive signal for antigen-antibody interaction.

FIG. 6.

Western blot analysis of purified Aflmp1p of A. flavus by using human sera. Strong antigen-antibody interaction was detected with the sera of two patients with aspergilloma (lanes 1 and 2) and two patients with invasive aspergillosis (lanes 3 and 4). Controls were sera from healthy blood donors (lanes 5 to 10) and from patients infected with C. albicans (lanes 11 and 12) and P. marneffei (lanes 13 and 14).