Differential roles of the ChiB chitinase in autolysis and cell death of Aspergillus nidulans

Abstract

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (DeltaflbA) mutant autolyzed completely after a 3-day incubation, the DeltaflbA DeltachiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the DeltaflbA-induced autolysis. However, importantly, both DeltaflbA and DeltaflbA DeltachiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the DeltaflbA and DeltaflbA DeltachiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, beta-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.

FIG. 1.

Phenotypes of WT (FGSC26), OEchiB (TNJ13), ΔflbA (RNJ3.1), ΔchiB (RNJ1.8), and ΔflbA ΔchiB (RNJ3.5) strains on solid medium. The conidia of each strain were point inoculated in the center of solid MMG and incubated at 37°C for 3 days. Photographs of the top and bottom views of the entire colonies and close-up views of the center and edge of individual colonies (bar, 200 μm) are shown. Note that the ΔflbA ΔchiB mutant colony exhibits reduced levels of hyphal disintegration compared to the ΔflbA colony. The centers of the two colonies (marked by black and white arrows) show a clear difference in the remaining of undifferentiated hyphae and the autolytic zones.

FIG. 2.

Phenotypes and mycelial dry weights of WT (FGSC26), OEchiB (TNJ13), ΔflbA (RNJ3.1), ΔchiB (RNJ1.8), and ΔflbA ΔchiB (RNJ3.5) strains in liquid MMG submerged culture. The mycelium of ΔflbA strain (RNJ3.1) autolyzed completely upon 4 days of incubation, whereas ΔflbA ΔchiB strain (RNJ3.5) maintained abnormal cottonlike mycelial pellets even at day 8. The photomicrographs of the mycelial pellets of individual strains (bar, 1.0 mm) were taken at day 3. Note that, while ΔflbA strain shows fragmented mycelial pellets due to accelerated autolysis, ΔflbA ΔchiB strain exhibits nonautolyzed cottonlike mycelial pellets.

FIG. 3.

AB reduction analyses of five A. nidulans strains. Note that the color of ΔflbA and ΔflbA ΔchiB strains began to turn into blue as early as day 3 of incubation.

FIG. 4.

SDS-PAGE images of intracellular proteins isolated from WT, ΔflbA, and ΔflbA ΔchiB strains grown in liquid MMG. Protein portions of ca. 100 μg were loaded onto each lane.

FIG. 5.

Cell viability of WT, ΔdppV, ΔAN2395.2, ΔinoB, and ΔnagA strains determined by AB reduction rates. Note the color difference of the ΔnagA mutant cell extract at day 2. The mycelial pellets of the ΔAN2395.2 and ΔinoB strains were somewhat disintegrated and reduced in size at day 2 (bar, 1.0 mm).

FIG. 6.

Working model linking G protein signaling and autolysis and/or cell death in A. nidulans. ChiB and NagA may play differential roles in FadA-mediated autolysis and cell death signaling (see the text). A potential role of ChiB in cell death is indicated by a dotted arrow.