Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of Aspergillus nidulans

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 (ΔflbA) mutant autolyzed completely after a 3-day incubation, the ΔflbA ΔchiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the ΔflbA-induced autolysis. However, importantly, both ΔflbA and ΔflbA ΔchiB 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 ΔflbA and ΔflbA ΔchiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, β-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.Autolysis can be described as enzymatic self-degradation of the cells. It involves the activation of several key enzyme classes, resulting in the catabolism of macromolecules within the cell (11, 12, 23). Autolysis is observed in most filamentous fungi at the late stages of cultures and is affected by aging, programmed cell death, development, nutrient limitation, and numerous other factors (39). Despite its fundamental importance in growth, differentiation, secondary metabolism, and heterologous protein production, autolysis is a poorly understood feature of fungal biology (26, 39). It is anticipated that the elucidation of the molecular mechanisms governing fungal autolysis would have great impacts on both fundamental and applied aspects of filamentous fungi.The Aspergillus nidulans ΔflbA mutant exhibits autolysis as a predominant phenotype (18). FlbA is a regulator of G protein signaling (RGS) that negatively controls vegetative growth signaling, primarily mediated by a heterotrimeric G protein composed of FadA (Gα) and SfaD::GpgA (Gβγ) (31, 33, 34, 42, 43). Loss of flbA function causes prolonged activation of G protein signaling, which results in uncontrolled proliferation of hyphal mass, the blockage of sporulation, and hyphal disintegration (autolysis). Because nearly the entire mycelium disappears during autolysis of ΔflbA mutant strains, some component of this phenomenon is hypothesized to involve enzymatic degradation as observed during autolysis of aging fungal cultures (11, 12, 23).In A. nidulans, the last step of autolysis is thought to be the degradation of the cell wall of empty hyphae, which is associated with increased proteinase and chitinase activities (10). There are 15 potential chitinase open reading frames (ORFs) in the genome of A. nidulans. Among these, the class V endochitinase ChiB was shown to play an important role in autolysis. The deletion of chiB considerably reduced the intracellular and extracellular chitinase activities, and the levels of ChiB significantly increased when the fungal cells were starved for carbon sources, an induced condition for hyphal autolysis of A. nidulans (41).In the present study we addressed two primary questions: (i) does ChiB function in the accelerated autolysis and/or cell death caused by loss of FlbA and (ii) are there other hydrolytic enzymes involved in autolysis and/or cell death in A. nidulans? In order to investigate a hypothetical connection between FlbA-controlled autolysis and the ChiB activity, we carried out genetic, biochemical, and cellular studies using the ΔchiB, ΔflbA, ΔflbA ΔchiB (double deletion), and chiB overexpression mutants. We found that, while ChiB plays a crucial role in ΔflbA-induced autolysis, ChiB is dispensable for cell death, corroborating the idea that cell death and autolysis are independent processes in A. nidulans (8). We further present the identification and partial functional characterization of four gene encoding the proteins accumulate at high level in ΔflbA and/or ΔflbA ΔchiB strains and propose a working model linking G protein signaling and autolysis.