AFMP2 encodes a novel immunogenic protein of the antigenic mannoprotein superfamily in Aspergillus fumigatus

Abstract

We cloned the Aspergillus fumigatus mannoprotein 2 (AFMP2) gene, which encodes a novel immunogenic protein (Afmp2p) of the antigenic mannoprotein superfamily, in A. fumigatus. Sequence analysis revealed that Afmp2p has 510 amino acid residues, with a predicted molecular mass of 51.5 kDa. Afmp2p has a putative N-terminal signal peptide, a putative C-terminal glycosylphosphatidylinositol membrane attachment signal sequence, and an upstream GAA cleavage site commonly used for cytoplasmic membrane attachment and implicated in fungal cell wall assembly. Upstream of the GAA cleavage site, Afmp2p contains a 302-amino-acid serine- and threonine-rich region as a site for potential O-glycosylation. Within this serine- and threonine-rich region, 13 repeats of ETSTPCE(T)(n) were observed. Western blot analysis of Afmp2p in A. fumigatus fungal cell lysate and culture supernatant and immunogold staining and electron microscopy showed that Afmp2p is predominantly secreted into the culture supernatant, whereas only minimal amounts can be detected in the cell lysate and cell wall. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. fumigatus develop a specific antibody response against recombinant Afmp2p. The abundance of Afmp2p in secreted form, its minimal cross-reactivity with Afmp1p, and the presence of an antibody response against Afmp2p in patients with A. fumigatus infections suggest that Afmp2p is a good candidate for complementing Afmp1p in serodiagnosis of A. fumigatus infections.

FIG. 1.

Western blot analysis of purified Afmp2p and Afmp1p of A. fumigatus with guinea pig sera. When purified Afmp2p was used as the antigen, a strong antigen-antibody interaction was detected with serum from a guinea pig immunized with A. fumigatus (lane 1) or Afmp2p (lane 2), but not with preimmune guinea pig serum (lane 3) or serum from a guinea pig immunized with Afmp1p (lane 4). When purified Afmp1p was used as the antigen, a strong antigen-antibody interaction was detected with serum from a guinea pig immunized with A. fumigatus (lane 5) or Afmp1p (lane 8), but not with preimmune guinea pig serum (lane 7) or serum from a guinea pig immunized with Afmp2p (lane 6).

FIG. 2.

Western blot analysis of Afmp1p and Afmp2p in sonicated fungal cell lysate and concentrated culture supernatant of A. fumigatus with guinea pig sera. A band of about 60 kDa was detected when serum from a guinea pig immunized with Afmp1p reacted with concentrated culture supernatant (lane 1). A band of about 45 kDa (probably due to core glycosylation) and a broad band of about 60 to 100 kDa (probably due to various forms of glycosylation) were detected when the serum reacted with sonicated fungal cell lysate (lane 2). A band of about 200 kDa was detected when serum from a guinea pig immunized with Afmp2p reacted with concentrated culture supernatant (lane 3), and a very faint band of the same size was detected when the serum reacted with sonicated fungal cell lysate (lane 4). No bands were detected with preimmune guinea pig serum (lanes 5 and 6).

FIG. 3.

Immunoelectron microscopy of A. fumigatus hyphae stained with preimmune rabbit serum (A), rabbit anti-Afmp1p antibody (B), and rabbit anti-Afmp2p antibody (C). Magnification, ×28,000.

FIG. 4.

Western blot analysis of purified Afmp2p of A. fumigatus with human sera. Strong antigen-antibody interaction was detected with the serum from 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 A. flavus (lanes 11 and 12), Candida albicans (lanes 13 and 14), Cryptococcus neoformans (lanes 15 and 16), and P. marneffei (lanes 17 and 18).