Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections.
Although α-1,3-glucan is a major cell wall polysaccharide in filamentous fungi, its biological functions remain unclear, except that it acts as a virulence factor in animal and plant pathogenic fungi: it conceals cell wall β-glucan on the fungal cell surface to circumvent recognition by hosts. However, cell wall α-1,3-glucan is also present in many of non-pathogenic fungi. Recently, the universal function of α-1,3-glucan as an aggregation factor has been demonstrated. Applications of fungi with modified cell wall α-1,3-glucan in the fermentation industry and of in vitro enzymatically-synthesized α-1,3-glucan in bio-plastics have been developed. This review focuses on the recent progress in our understanding of the biological functions and biosynthetic mechanism of cell wall α-1,3-glucan in fungi. We briefly consider the history of studies on α-1,3-glucan, overview its biological functions and biosynthesis, and finally consider the industrial applications of fungi deficient in α-1,3-glucan.
α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE) or agsB (agsBOE), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE, 41.6 ± 5.8 kDa; agsBOE, 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain–fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans.
Bisphosphonates (BPs) are clinically used for the treatment of bone metabolic abnormalities because they are powerful inhibitors of bone resorption. Osteonecrosis of the jaw has been observed after tooth extraction in a considerable number of BP-treated cancer patients, but the reason for this is not known. We studied the effects of BP on extraction socket healing in rats that were pretreated with BP prior to tooth extraction. Male Wistar rats (approximately 5 weeks old) were divided into experimental (BP) and control groups. In both groups, maxillary right second molars were extracted under general anesthesia. BP group rats were injected with 50 microl (1.0 mg/kg) alendronate into the right buccal alveolar bone every 4 days for 14 days, starting 2 days before tooth extraction. Control group rats were injected with physiological saline instead of alendronate. Rats were euthanized 3, 7, 10 or 14 days after tooth extraction, and maxillary bones were collected. Bone morphometric analysis using microfocus X-ray CT images and calculation of bone-resorption parameters based on hematoxylin and eosin or TRAP-stained pathological sections of the molar region showed that new bone formation in the extraction socket was delayed in the BP group relative to the control group during the first 7 days after extraction. A subsequent increase in new bone formation showed that bone resorption in the BP rats was eventually inhibited. This delay in initial healing may explain the jaw osteonecrosis observed in some BP-treated cancer patients.
Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.
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