Glycosylphosphatidylinositol-anchored Ecm33p influences conidial cell wall biosynthesis in Aspergillus fumigatus

Abstract

ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long-term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils.

FIG. 1.

Disruption of the AfECM33 gene (black arrow) by double-crossover events. (a) Restriction maps of the AfECM33 deletion constructs and cloning steps during AfECM33 gene deletion. Restriction enzyme abbreviation: B (BamHI), N (NcoI), E (EcoRI), X (XbaI), H (HindIII), and Bg (BglII). See Materials and Methods for the amplification of PCR hi and jk fragments, and ab and hi probes. (b) Restriction map after the integration of the HPH gene marker (gray box) at the AfECM33 locus.

FIG. 2.

Dendrogram composed of A. fumigatus Ecm33p and six yeast orthologs: the four members of the SPS2 family in S. cerevisiae (Sps2p, Pst1p, Ecm33p, and YCL048wp) and the two homologs (Meu10p and SPAC23H4.19p) of S. pombe.

FIG. 3.

Southern and Western blot analyses of the AfECM33 gene and protein. (a) Southern blot of the Afecm33 mutant (m) and WT strains. Genomic DNA of each strain was digested with BamHI, HindIII, or NcoI and hybridized with the radioactively labeled hi probe (see Fig. 1 for the identification of the probe). (b) Western blots of the IC and CW fractions obtained after 4,000 × g centrifugation from the WT strain and the Afecm33 mutant (m) after incubation of electrotransferred proteins with an anti-AfEcm33p hyperimmune antiserum.

FIG. 4.

Light microscopy of 6-day-old A. fumigatus WT and Afecm33 mutant (m) conidia under phase-contrast (a) or fluorescence imaging microscopy after labeling of the conidia with CFW (b). Note that the conidia of the mutant are larger, less refractile (a), and more labeled by CFW (b) than the conidia of the wild-type strain. Bars, 2 μm.

FIG. 5.

Measurements of the resting (T = 0) and swollen (after 5 h 30 min of incubation at 37°C in Sabouraud medium) (T = 5h30) conidia of the parental strain (WT), the ecm33 mutant (Δ), and the ecm33::ECM33 revertant (R). Means ± standard errors are indicated. The asterisk indicates that the conidium diameter of the Afecm33 mutant is significantly different from the diameters of the conidia of the parental and revertant strains.

FIG. 6.

Electron microscopy images of 6-day-old conidia from A. fumigatus WT and the Afecm33 mutant (m1 to m3) showing the incomplete separation of the conidia apparently caused by melanin bridging between adjacent conidia. The conidia of the revertant looked like those of the wild-type strain (data not shown). Bars, 2 μm.

FIG. 7.

Sensitivity of the WT, the ecm33 mutant (Δ), and the ecm33::ECM33 revertant (R) strains to phagocytes. Killing of resting conidia (Co) and mycelium (Myc) by mouse AM and human PMNs was estimated as percent germination and percent mycelial-growth inhibition using the XTT assay. Values are expressed as means plus standard errors. The asterisks indicate that the values obtained with the ecm33 mutant were significantly different from the ones obtained with the parental and revertant strains.