Aspergillus fumigatus catalytic glucokinase and hexokinase: expression analysis and importance for germination, growth, and conidiation

Abstract

Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.

Fig. 1.

SDS-PAGE analysis of purified recombinant GlkA, HxkA, and HxkB. Between 2 μg (HxkB) and 3 μg (GlkA and HxkA) of protein was loaded, and the gel was stained with Coomassie blue. Lane M, molecular mass marker with masses in kDa indicated at the right. Expected masses with the N-terminal His tag were 55.8 kDa for GlkA, 55.6 kDa for HxkA, and 54.0 kDa for HxkB. Allowing for some variation in mobility within the gel, all purified proteins met their expected molecular masses.

Fig. 2.

Growth analysis of glkA and hxkA mutants. All plates were spot inoculated with 1 × 10³ conidia and incubated for 44, 48, or 76 h, as indicated by the number of asterisks. WT, wild type; ΔglkA, glkA deletion mutant; CΔglkA, complemented glkA deletion mutant; ΔhxkA, hxkA deletion mutant; CΔhxkA, complemented hxkA deletion mutant; ΔglkA ΔhxkA, double-deletion mutant. The complete media malt extract agar (Malt), Sabouraud medium, and potato dextrose agar (PDA) were prepared as recommended by the manufacturers. All monosaccharides were used at a final concentration of 50 mM, and disaccharides were used at a concentration of 25 mM. Starch, peptone, Casamino Acids (CAS-AA), bovine serum albumin (BSA), and lecithin were added to a final concentration of 1% (wt/vol). Glycerol, ethanol, and acetate were used at a concentration of 100 mM. For a detailed explanation of growth phenotypes, refer to the text.

Fig. 3.

Effect of cell wall stress-inducing agents on growth of hexose kinase mutants. WT, wild type; ΔglkA, glkA deletion mutant; ΔhxkA, hxkA deletion mutant; ΔglkA ΔhxkA, double-deletion mutant. Media contained 1% (wt/vol) peptone as a nutrient source. Only the effect of 0.01% SDS is shown. Plates were photographed after 60 h of incubation at 37°C.

Fig. 4.

Analysis of conidium germination in the presence of different carbon sources. (A) Classification of the germination state. The pictures show overlays of bright-field images with fluorescent photographs to visualize FITC-labeled conidia (green) and DAPI-stained nuclei (blue). These pictures were used to classify each single cell from germination assays. Classification was as follows: 1, resting conidium with bright FITC fluorescence and nuclei not or barely visible; 2, swollen conidium and germ tube sometimes visible, with a brightly stained nucleus; 3, germinated conidium with a single nucleus, sometimes migrating toward the germ tube; 4, germinated conidium with at least two nuclei but without branching hyphae; 5, germinated conidium with three or more nuclei, with hyphae sometimes starting to branch or a germ tube at a second site. (B) Analysis of germination in the presence of 0.1 mM glucose. (C) Microcolony formation of a ΔglkA ΔhxkA double-deletion mutant grown for 163 h on a glass slide covered with 50 mM glucose minimal medium. Magnifications are indicated. At a ×40 magnification the short septated hyphae (arrowheads) and the hyperbranching phenotype are visible. (D) Analysis of germination in the presence of 10 mM ethanol. (E) Analysis of germination in the presence of 10 mM glycerol. For each analysis, the wild-type, deletion mutant, and complemented strains were incubated on the same chamber slide. The variations observed for wild-type germination between ΔglkA, ΔhxkA, and ΔglkA ΔhxkA analyses are due to slight variations in the incubation times but did not influence the overall results (as determined from time response studies). Incubation times were 7 to 8 h for 0.1 mM glucose, ca. 11 h for 10 mM glycerol, and ca. 13 h for 10 mM ethanol. To obtain representative data, at least six microscopic fields from each strain and each condition with 50 or more cells each were evaluated, and data show the mean values with standard deviations. Significance was calculated by a Student's t test, and two stars indicate P values of ≤0.01.

Fig. 5.

Transcript analysis of glkA and hxkA from different developmental states by quantitative real-time PCR. Data shown represent mean values of data from two biological replicates, each measured in three technical replications, and error bars give standard deviations. Conidia 0 h, resting conidia; conidia 4 h and conidia 8 h, incubation for 4 h and 8 h, respectively, in 50 mM glucose minimal medium; hyphae glucose, hyphae fructose, and hyphae ethanol, mycelium grown for 18 to 21 h on the respective carbon sources. Glucose and fructose concentrations were 50 mM, and the ethanol concentration was 100 mM. (A) Transcript levels of glkA normalized against actin. High transcript levels were obtained from resting conidia, which steadily declined during germination. In vegetative mycelium, a constantly low level of expression was observed. (B) Transcript levels of hxkA normalized against actin. The expression of hxkA is strongly induced during germination and constantly expressed in vegetative mycelium. (C) Direct comparison of the ratio of hxkA transcript levels to glkA transcript levels. Levels of glkA were used for normalization. In resting conidia, levels of glkA transcripts exceeded those of hxkA. This ratio inverts during early germination and remains in favor of hxkA in vegetative mycelia.

Fig. 6.

Determination of the ratio of hxkA transcript levels to glkA transcript levels by semiquantitative analysis of PCR products. After separation on a 2% agarose gel, band intensities were determined by using the TraceQuantity method from the QuantityOne software package (Bio-Rad). Black bars show the transcript ratios from resting conidia (Conidia) and glucose-grown mycelium (Mycelium). Light gray bars show transcript levels determined from lungs of infected mice immunosuppressed with cyclophosphamide (Cyclo) and sacrificed 1 or 3 days after infection. Dark gray bars show the same analysis as the light gray bars, but mice were immunosuppressed with cortisone acetate (Corti). For each analysis the mean values from two independent biological probes measured in replicates were used, and standard deviations are indicated.

Fig. 7.

Determination of isocitrate lyase activity from mycelia grown in the presence of glucose and ethanol. A mixture of 50 mM glucose and 100 mM ethanol was used as the carbon source. Activity was determined in two independent biological replicates, each measured in three technical replications. Mean values with standard deviations are shown. WT, wild type; ΔglkA, glkA deletion mutant; CΔglkA, complemented glkA deletion mutant; ΔhxkA, hxkA deletion mutant; CΔhxkA, complemented hxkA deletion mutant; ΔglkA ΔhxkA, double-deletion mutant. In comparison to the wild type, the activities increased in the ΔglkA strain by a factor of 12, in the ΔhxkA strain by a factor of 5, and in the double-deletion strain by a factor of 22.