Glucan synthase complex of Aspergillus fumigatus

Abstract

The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.

FIG. 1

Expression of IntF in E. coli (A) Coomassie blue staining of proteins after SDS-PAGE with a 10% separating gel. Lane 1, IntF-GST fusion protein; lane 2, IntF polypeptide after thrombin digestion (note that the digestion was incomplete with two polypeptides corresponding to the undigested IntF-GST and IngF). (B) Immunolabeling of IntF-GST and IntF with anti-IntF antibodies (1/1,000 dilution). Lanes 1 and 2 correspond to lanes 1 and 2 of panel A. No labeling was seen when lanes 1 and 2 of panel A were incubated with preimmune sera (not shown). The numbers at the left of each panel are molecular sizes in kDa.

FIG. 2

Phylogenetic dendrogram of Rho proteins of A. fumigatus (AfRho1, AfRho2, AfRho3, AfRho4), S. cerevisiae (ScRho1, ScRho2, ScRho3, ScRho4), C. albicans (CaRho1), and S. pombe (SpRho1, SpRho2).

FIG. 3

Alignment of Rho1 proteins of A. fumigatus (AfRho1), S. pombe (SpRho1), S. cerevisiae (ScRho1), and C. albicans (CaRho1). GTP binding and GTP hydrolysis domains are indicated by stars and dots, respectively. Geranyl-geranylation domains of each Rho1p are underlined.

FIG. 4

Recombinant Rho1p (rRho2p). P. pastoris proteins were separated by SDS-PAGE with a 12% separating gel followed by Coomassie blue staining of molecular size standards in kDa (lane 1), cell lysate of control P. pastoris without rRho1p (lane 2), cell lysate of P. pastoris expressing rRho1p after methanol induction (lane 3), and purified rRho1p from Rho1Sup (lane 4) and Rho1PSol (lane 5) fractions of recombinant P. pastoris. Lanes 6 and 7, immunolabeling of rRho1p from lanes 3 and 4, respectively, with the antiserum against the recombinant AfRho1p (1/15,000 dilution). No labeling was seen when the lysate shown in lane 2 was incubated with anti-AfrRho1p antibodies (not shown).

FIG. 5

Analysis of the β(1-3) glucan synthase complex after product entrapment. (A) Coomassie blue staining of proteins (15 μg of protein per lane) separated by SDS-PAGE with an 8% separating gel of molecular size standards in kDa (lane 1), MMF (lane 2), W1-solubilized MMF (lane 3), and PE (lane 4). Inserts show immunoblotting of PE (lane 5) and MMF (lane 6) with anti-IntF (1/1,000 dilution) and PE (lane 7) with an anti-β(1-3) glucan antibody (1/1,000 dilution). MMF remained negative after incubation with anti-IntF diluted 1/40 (not shown). (B) Immunoblotting with anti-AfrRho1p antibodies (1/15,000 dilution) of MMF (lane 1), solubilized MMF (lane 2), and PE (lane 3). A total of 2.6 μg of protein per lane after separation of the proteins by SDS-PAGE with a 12% separating gel was used.

FIG. 6

ADP-ribosylation of Rho1p in A. fumigatus. Lane 1, molecular size standards in kDa; lane 2, Coomassie blue staining of CHAPS-solubilized MMF proteins separated by SDS-PAGE with a 12% separating gel; lane 3, autoradiography of CHAPS-solubilized MMF incubated with 7.5 μM [³²P]NAD⁺ and 15 ng of C₃ exoenzyme for 1 h at 37°C followed by SDS-PAGE (12% separating gel).

FIG. 7

(A and B) Immunofluorescence of a germ tube of A. fumigatus labeled with anti-IntF antibodies and goat anti-IgG fluorescein. (A) Anti-IntF antiserum, arrow indicates the apex of the germ tube; (B) preimmune serum. (C and D) Labeling of a germ tube of A. fumigatus with aniline blue fluorochrome. (C) Epifluorescence; (D) transmitted light.