Abstract
Chitin is an important fungal cell wall component that is cross-linked to β-glucan for structural integrity. Acquisition of chitin to glucan cross-links has previously been shown to be performed by transglycosylation enzymes in Saccharomyces cerevisiae, called Congo Red hypersensitive (Crh) enzymes. Here, we characterized the impact of deleting all seven members of the crh gene family (crhA-G) in Aspergillus niger on cell wall integrity, cell wall composition and genome-wide gene expression. In this study, we show that the seven-fold crh knockout strain shows slightly compact growth on plates, but no increased sensitivity to cell wall perturbing compounds. Additionally, we found that the cell wall composition of this knockout strain was virtually identical to that of the wild type. In congruence with these data, genome-wide expression analysis revealed very limited changes in gene expression and no signs of activation of the cell wall integrity response pathway. However, deleting the entire crh gene family in cell wall mutants that are deficient in either galactofuranose or α-glucan, mainly α-1,3-glucan, resulted in a synthetic growth defect and an increased sensitivity towards Congo Red compared to the parental strains, respectively. Altogether, these results indicate that loss of the crh gene family in A. niger does not trigger the cell wall integrity response, but does play an important role in ensuring cell wall integrity in mutant strains with reduced galactofuranose or α-glucan.
Highlights
The fungal cell wall is a rigid, yet dynamic structure that dictates cell shape, provides protection from other microbes, helps to colonize new environments and is required for virulence in pathogenic fungi
In line with the results observed for A. fumigatus, we showed that single knockouts strains of either crhA, crhB, crhC, crhD, crhE, crhF or crhG, as well as a double, triple, quintuple and seven-fold knockouts strains of crhA-G (TLF39), were unaffected by the presence of cell wall disturbing compounds Congo Red (CR) and Calcofluor White (CFW)
As β-1,3-glucan has been reported to become alkali insoluble due to its covalent linkage with chitin in the wild type cell wall (Hartland et al, 1994; Mol and Wessels, 1987; Sietsma and Wessels, 1979), we looked in detail whether deletion of the crh genes resulted in a shift of the amount of β1,3-glucan in the alkali-insoluble fraction (AIF) to the ASFs of the cell wall, as shown before in yeast (Rodríguez-Peña et al, 2000)
Summary
The fungal cell wall is a rigid, yet dynamic structure that dictates cell shape, provides protection from other microbes, helps to colonize new environments and is required for virulence in pathogenic fungi. Polymers are extruded into the periplasmic space, where chitin becomes cross-linked to glucan by GPI-anchored Crh transglycosylases (Arroyo et al, 2016). Crh enzymes exhibit both chitinase and transglycosylation activity, effectively hydrolyzing a chitin donor molecule followed by subsequent (re-) attachment to acceptor substrates. Crh enzymes can be classified as part of either of the fungal specific GH16_18/19 subfamilies (Viborg et al, 2019) These chitin to β-1,3-glucan cross-linking enzymes have been most extensively studied in Saccharomyces cerevisiae on both functional and structural level (Blanco et al, 2015; Cabib et al, 2008, 2007; Rodríguez-Peña et al, 2000). Overexpression of either CRH1 or UTR2 in C. albicans led to protection against osmotic shock as a result of reduced cell wall elasticity, whereas a triple deletion resulted in increased osmo-sensitivity (Ene et al, 2015)
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