Two KTR Mannosyltransferases Are Responsible for the Biosynthesis of Cell Wall Mannans and Control Polarized Growth in Aspergillus fumigatus

Abstract

Fungal cell wall mannans are complex carbohydrate polysaccharides with different structures in yeasts and molds. In contrast to yeasts, their biosynthetic pathway has been poorly investigated in filamentous fungi. In Aspergillus fumigatus, the major mannan structure is a galactomannan that is cross-linked to the β-1,3-glucan-chitin cell wall core. This polymer is composed of a linear mannan with a repeating unit composed of four α1,6-linked and α1,2-linked mannoses with side chains of galactofuran. Despite its use as a biomarker to diagnose invasive aspergillosis, its biosynthesis and biological function were unknown. Here, we have investigated the function of three members of the Ktr (also named Kre2/Mnt1) family (Ktr1, Ktr4, and Ktr7) in A. fumigatus and show that two of them are required for the biosynthesis of galactomannan. In particular, we describe a newly discovered form of α-1,2-mannosyltransferase activity encoded by the KTR4 gene. Biochemical analyses showed that deletion of the KTR4 gene or the KTR7 gene leads to the absence of cell wall galactomannan. In comparison to parental strains, the Δktr4 and Δktr7 mutants showed a severe growth phenotype with defects in polarized growth and in conidiation, marked alteration of the conidial viability, and reduced virulence in a mouse model of invasive aspergillosis. In yeast, the KTR proteins are involved in protein 0- and N-glycosylation. This study provided another confirmation that orthologous genes can code for proteins that have very different biological functions in yeasts and filamentous fungi. Moreover, in A. fumigatus, cell wall mannans are as important structurally as β-glucans and chitin.IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It allows fungal pathogens to survive environmental challenge posed by nutrient stress and host defenses, and it also is central to polarized growth. The cell wall is mainly composed of polysaccharides organized in a three-dimensional network. Aspergillus fumigatus produces a cell wall galactomannan whose biosynthetic pathway and biological functions remain poorly defined. Here, we described two new mannosyltransferases essential to the synthesis of the cell wall galactomannan. Their absence leads to a growth defect with misregulation of polarization and altered conidiation, with conidia which are bigger and more permeable than the conidia of the parental strain. This study showed that in spite of its low concentration in the cell wall, this polysaccharide is absolutely required for cell wall stability, for apical growth, and for the full virulence of A. fumigatus.

Keywords: Aspergillus fumigatus; KTR; biosynthesis; cell wall; galactomannan; mannosyltransferase.

FIG 1

Phylogenetical tree of KTR genes in A. fumigatus and S. cerevisiae.

FIG 2

Growth of the parental Δku80 strain, Δktr1, Δktr4 and Δktr7 mutants, and Δktr4::KTR4 and Δktr7::KTR7 revertant strains on solid media. Strains were grown on either Sabouraud or minimum (MM) solid medium. A total of 10³ conidia of each strain were spotted on media and incubated at 37°C or 50°C for 48 h.

FIG 3

Growth of parental Δku80 strain, Δktr1, Δktr4, and Δktr7 mutants, and Δktr4::KTR4 and Δktr7::KTR7 revertant strains in liquid media. Strains were grown in Sabouraud liquid media. Flasks (50 ml) were inoculated with 10⁶ conidia and incubated under shaking conditions (150 rpm/min) at 37°C for 24 h. (A) Mycelium biomass. The biomass/flask was quantified by the weight of the mycelium after drying at 80°C. Each value of mycelium dry weight represents the average of data from three independent replicates (*, P < 0.05; error bars represent standard deviations). (B) Gross morphology of mycelium.

FIG 4

Morphology of germinated conidia of parental Δku80 strain, Δktr4 and Δktr7 mutants, and Δktr4::KTR4 and Δktr7::KTR7 revertant strains after 20 ± 2 h of incubation in MM liquid medium at 37°C. White bars represent 100 µm.

FIG 5

Conidial germination of parental Δku80 strain, Δktr1, Δktr4, and Δktr7 mutants, and Δktr4::KTR4 and Δktr7::KTR7 revertant strains. (A) Germination. Conidia (5 µl of 2.10⁶ conidia/ml suspension) were spotted on a slide of Sabouraud agar medium and incubated at 37°C under conditions of a humid atmosphere. Percentage of germination was quantified by bright-field counting of germinated and nongerminated conidia under a microscope. Each value represents the average of data from three independent replicates. Differences in the percentages of germination of Δktr4 and Δktr7 mutants were significant at 4, 5, and 6 h (P < 0.0001; error bars represent standard deviations). (B and C) The size of germinated conidia (B) and the number of germ tubes/conidium (C) were determined after 16 h of incubation in liquid MM medium at 37°C. Each value represents an average of data resulting from the counting of hundred conidia. (*, P < 0.001; error bars represent standard deviations).

FIG 6

Monosaccharide composition of the alkali-insoluble fraction of the cell wall of the parental Δku80 strain, Δktr1, Δktr4, and Δktr7 mutants, and Δktr4::KTR4 and Δktr7::KTR7 revertant strains. The cell wall alkali-insoluble fraction (AI) was purified from mycelium grown for 24 h in liquid Sabouraud medium at 37°C (expressed as a percentage of total hexoses plus hexosamines). Monosaccharides were quantified after total acid hydrolysis and chromatography analysis. Each value represents an average of data from three independent triplicates. Statistical differences between parental and mutant strains are indicated as follows: ns, not significant; *, p < 0.01; error bars represent standard deviations. Man, mannose; Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine.

FIG 7

Ktr4p enzyme activity. (A) HPLC analysis of the products obtained after incubation of recombinant ktr4p with GDP-mannose and mannobiose (α-1,6-mannobiose or α-1,2-mannobiose). HPLC profiles were obtained after incubation of nontreated (Ktr4p) or heat-inactivated (Control) Ktr4p with mannobiose and GDP-mannose. (B) HPLC analysis of products submitted to exo-α-mannosidase digestions (PED, pulsed electrochemical detection; JB-α-Mannanase, Jack bean α-mannosidase; α2M, α-1,2-mannobiose; α6M, α-1,6-mannobiose; Man, mannose; *, contaminant from buffer; P6, trisaccharide obtained in the presence of α-1,6-mannobiose; P2, trisaccharide obtained in the presence of α-1,2-mannobiose). Jack bean α-mannosidase fully degraded both acceptors (α2M and α6M) and products (P2 and P6) to release mannose residue, showing the α configuration of all mannose residues. α-1,6-Mannosidase degraded the α6M but not the P6. α-1,2-Mannosidase degraded α2M and P2 into mannose and P6 into α6M and mannose.