1535-9778/10/$12.00 doi:10.1128/EC.00362-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Aspergillus fumigatus
Catalytic Glucokinase and Hexokinase:
Expression Analysis and Importance for Germination,
Growth, and Conidiation
䌤
†
Christian B. Fleck and Matthias Brock*
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Microbial Biochemistry and Physiology, Beutenbergstr. 11a, 07745 Jena, Germany
Received 7 December 2009/Accepted 30 April 2010
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 fumigatusand 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, theglkA hxkAdeletion 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 inA. fumigatusand are indispensable for growth under all nutritional conditions.
Aspergillus fumigatus is an opportunistic human pathogen and is able to cause life-threatening invasive aspergillosis mainly in immunocompromised patients (34). Only a limited number of antifungals are currently available to combat fungal infections. Nutrition assimilation is a prerequisite for infection, and a better understanding of the metabolic processes during infection may help to identify new antifungal drug targets. However, since infection is a dynamic process, high metabolic versatility is assumed to favor adaptation to rapidly changing environmental conditions within a host (4).
Glucose is highly abundant in some sites within the human body, and the concentration in the bloodstream ranges be-tween 6 and 8 mM (12). Additionally, the brain of vertebrates contains high glucose and low protein levels, and investigations
of a diploid Saccharomyces cerevisiae hexokinase 2 mutant
(hex2⌬/hex2⌬) showed that Hex2p is essential for successful
survival within murine brains (29). Although the glucose con-centration within tissues may be much lower, a role for glucose metabolism in pathogenesis is conceivable. The utilization of glucose could be beneficial for pathogenic fungi, because it
allows an easy acquisition of building block precursors for biomass formation, reducing equivalents and energy. The ini-tial step in sugar utilization is its uptake and subsequent acti-vation to a sugar phosphate. Bacteria frequently use a coupled phosphoenolpyruvate-dependent carbohydrate:phosphotrans-ferase system for sensing, uptake, and activation of sugars (30). In contrast, fungi seem to possess specific hexose transporters and activate sugars by soluble hexokinases via the consumption of ATP (6).
The activation of hexoses, especially of glucose and fructose,
in relatives of A. fumigatus, that is, Aspergillus nidulans and
Aspergillus niger, has previously been studied. Previous inves-tigations revealed that both fungi contain at least two catalyt-ically active hexose kinases called glucokinase and hexokinase (19, 35, 36, 41). Several other enzymes with high sequence similarity to hexose kinases have been detected by genome analyses, but these enzymes seem to possess regulatory func-tions in carbon sensing rather than contributing directly to the activation of hexoses to sugar phosphates (3, 18, 28).
Glucokinase and hexokinase fromA. nigerhave been
puri-fied and biochemically characterized by homologous overpro-duction and subsequent purification of the enzymes (35, 36). Comparison of the catalytic properties of both enzymes showed that glucokinase possesses a very high specificity for
glucose, with a specific activity of 233 U/mg and aKmvalue of
63M. The activation of fructose was not assumed to occur
naturally, because theKmfor fructose was approximated to be
120 mM. Additionally, it was shown previously that the activity
* Corresponding author. Mailing address: Leibniz Institute for Nat-ural Product Research and Infection Biology, Hans Knoell Institute, Microbial Biochemistry and Physiology, Beutenbergstr. 11a, 07745 Jena, Germany. Phone: 49 (0)3641 532 1710. Fax: 49 (0)3641 532 0809. E-mail: Matthias.brock@hki-jena.de.
† Supplemental material for this article may be found at http://ec .asm.org/.
䌤Published ahead of print on 7 May 2010.
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of the glucokinase was not significantly inhibited by the addi-tion of the hexokinase inhibitor trehalose-6-phosphate (T6P)
(35). In contrast, purifiedA. nigerhexokinase showed a specific
activity of 220 U/mg for fructose and aKmof 2 mM but was
also significantly active with glucose (specific activity⫽ ⬃20
U/mg;Km⫽0.35 mM). Fructose phosphorylation activity was
inhibited by trehalose-6-phosphate in a concentration-depen-dent manner, which allowed the discrimination of glucokinase and hexokinase activities in cell extracts (36). Those investiga-tions implied that glucokinase might be mainly responsible for glucose metabolism whereas the main function of hexokinase is the activation of fructose. However, none of the respective
genes had been deleted inA. niger, and thein vivocontribution
of each enzyme to sugar metabolism remained speculative. Although a detailed biochemical characterization of these
two enzymes in the model organismA. nidulanshas not been
performed, mutants with defective hexokinase (frA1) (38) and
glucokinase (glkA4) as well as a double mutant (19) have been
constructed and phenotypically analyzed. Those authors inves-tigated the contribution of both enzymes to sugar metabolism and their impact on carbon catabolite repression. That analysis
revealed no visible growth defect for theglkA mutant, which
implied that the function was completely compensated for by the hexokinase. In contrast, the hexokinase mutant was no longer able to grow on fructose as the sole carbon source, confirming that glucokinase is indeed unable to perform
fruc-tose phosphorylationin vivo. However, on glucose as the sole
carbon source, no phenotype was observed for the hexokinase mutant. As expected, the double mutant was unable to grow on glucose or fructose. In addition, this double mutant displayed deregulated carbon catabolite repression, which was not ob-served for the single mutants (19). However, the mutants in-vestigated in that study were generated by UV mutagenesis, and it might be possible, although unlikely, that one enzyme still possessed low catalytic activity or some regulatory
func-tion. In contrast to investigations ofA. nidulans, the deletion of
the hexokinase in the fruit-pathogenic fungusBotrytis cinerea
revealed a pleiotropic growth defect on various carbon sources,
whereas a glucokinase mutant, in agreement with data forA.
nidulans, displayed no altered phenotype. Furthermore, forB. cinerea, attempts to generate a double-deletion strain were unsuccessful, implying the synthetic essentiality of the two genes (40).
Due to these discrepancies betweenA. nidulansandB.
ci-nerea, we aimed to elucidate the impact of glucokinase and
hexokinase on the growth and development ofA. fumigatus. Of
special interest was the investigation of the physiological role
of glucokinase, since the deletion of this enzyme in bothA.
nidulansandB. cinereadid not alter their phenotypes. For this purpose, we performed recombinant protein productions with
Escherichia coliand recorded the biochemical parameters of
both enzymesin vitro. These data were used to predict the role
of both enzymesin vivo. Additionally, we generated deletion
mutants and studied their phenotypes at different developmen-tal stages and during growth on various carbon sources. Our investigations revealed that glucokinase is associated mainly with resting conidia and seems to be required for glucose activation from storage sugars such as trehalose. Additionally, glucokinase might contribute to glucose phosphorylation un-der conditions of low glucose concentrations in the
environ-ment. In contrast, hexokinase is associated mainly with the mycelium and plays a major role in sugar utilization during vegetative growth. Additionally, severe phenotypes of a dou-ble-deletion mutant on glycolytic and gluconeogenic carbon sources demonstrated that at least one enzyme must be present to allow normal growth, conidiation, and cell wall integrity.
MATERIALS AND METHODS
Media, growth conditions, and preparation of cell extracts.To obtain conidial suspensions, all strains were inoculated on malt extract agar slants (Fluka, Taufkirchen, Germany) and incubated for 5 to 7 days at room temperature. Strains with delayed growth and conidiation were incubated at 30°C. Media were overlaid with phosphate-buffered saline (PBS) plus 0.1% Tween 20 (PBS-Tween), and conidia were scraped from the mycelium. Conidial suspensions were filtered through 40-m cell strainers (BD Biosciences, CA) to remove clumps and mycelial fragments. Conidial suspensions were stored for a maximum of 7 days at 4°C without a significant loss of viability. For testing of growth phenotypes on different carbon sources,Aspergillusminimal media were prepared as de-scribed by the Fungal Genetic Stock Center (http://www.fgsc.net/Aspergillus /protocols/MediaForAspergillus.pdf), with the pH adjusted to 6.5. For solid media, 2% agar was added prior to sterilization. Carbon sources were either malt extract (Fluka), potato dextrose broth (Sigma), Sabouraud medium (Sigma), peptone (1%), Casamino Acids (1%), bovine serum albumin (1%), starch (1%), lecithin from egg yolk (1%; Fluka), glucose (50 mM, if not indicated otherwise), ribose (50 mM), mannose (50 mM), galactose (50 mM), trehalose (25 mM), lactose (25 mM), saccharose (25 mM), fructose (50 mM), sorbose (50 mM), glucosamine (50 mM), acetate (100 mM), or ethanol or glycerol (each 100, 50, or 10 mM). Incubations were performed at 37°C, and liquid cultures were agitated at 210 rpm on a rotary shaker. For the preparation of cell extracts from mycelia, liquid cultures were filtered through Miracloth filter gauze (Merck, Darmstadt, Germany). The retained mycelium was washed once with water and pressed dry. Cells were disrupted under liquid nitrogen in a mortar, and the powdered mycelium was suspended in an appropriate buffer for subsequent enzyme activity determinations. For the preparation of cell extracts from conidia, fresh conidial suspensions were washed once with an appropriate buffer, resuspended as a thick paste, and mixed in 0.5-ml screw-cap vials with zirconia beads (diameter, 0.5 mm; Carl Roth GmbH, Karlsruhe, Germany). Conidia (ca. 1⫻109
conidia in 400l) were disrupted in a Speed Mill (Analytik Jena, Jena, Germany) three times for 2 min, with 2 min on ice after each cycle. All crude extracts were centrifuged at 15,000⫻gfor 5 min, and the cell-free supernatants were collected. Protein concentrations were determined by using Bradford protein assay concentrate as described by the manufacturer (Bio-Rad, Munich, Germany).
Germination analysis. To determine the germination rates of differentA. fumigatusisolates, freshly harvested conidia were labeled with fluorescein iso-thiocyanate (FITC) as described previously (26). After three washes of labeled conidia with PBS-Tween, the number of conidia in suspension was counted with a hemocytometer, and 100,000 conidia were used to inoculate 1 ml of the respective medium added to wells of four-well LabTek chamber slides (Nalgene Nunc, New York, NY). Slides were incubated at 37°C in a humid incubator in the presence of 5% CO2for the times indicated in Results. Germination was
termi-nated by removing the medium and fixing the cells with 1% paraformaldehyde (PFA) for 9 min at room temperature. PFA was removed, and slides were washed with 50 mM (NH4)2SO4and subsequently incubated for 9 min in the
presence of 50 mM (NH4)2SO4. After a washing step with PBS the chambers
were removed, and cells were embedded in a DAPI (4⬘ ,6-diamidino-2-phenylin-dole)-containing mounting solution (ProLong Gold Antifade reagent with DAPI; Invitrogen). Slides were sealed and incubated for at least 12 to 24 h at 4°C prior to fluorescence microscopy with an AxioImager fluorescence microscope (Zeiss, Jena, Germany). Bright-field, FITC fluorescence, and DAPI fluorescence pictures were recorded at a⫻40 magnification, and pictures were overlaid by using the MetaMorph software package (version 6.1; Molecular Devices, CA). Overlays were magnified with PowerPoint (Microsoft Office; Microsoft Corpo-ration, WA), and hard-copy printouts were used to evaluate each single cell. For each condition, at least six independent pictures were evaluated, resulting in more than 300 individual cells evaluated for each strain and culturing condition. Germination was classified by a numbering system (score of 1 to 5), as explained in more detail in Results. In parallel, the viability of conidia was tested by incubating conidia for 7 to 8 h in Sabouraud complete medium. Due to the poor growth of the⌬glkA⌬hxkAdouble mutant on Sabouraud medium, 1% peptone-containing medium was used for viability control. Evaluation of at least 1,000 conidia was performed directly with a fluorescence microscope using FITC
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labeling of conidia as a readout system. Resting conidia appeared as small but brightly fluorescent spheres, whereas swollen conidia were three to four times larger, and the fluorescence of conidia was interrupted at the site of germ tube formation. For microscopic evaluation of germination and colony formation of the double-deletion strain, glass slides were coated with minimal medium con-taining 1.5% agarose. Between 200 and 1,000 conidia were plated onto the slides and incubated in a humid chamber for various time points. For microscopic analysis the medium was overlaid with 20l of PBS, and a coverslip was applied. Cells were observed in bright field using a⫻10 to⫻40 magnification.
Recombinant production and purification of GlkA, HxkA, and HxkB. Se-quences of all oligonucleotides used in this study are listed in Table SA1 in the supplemental material. For the amplification of coding regions of glucokinase and hexokinase, cDNA was used as a template. The RNA for cDNA synthesis was derived fromA. fumigatuscultures grown for 20 h at 37°C on liquid medium containing 50 mM glucose and 0.5% (wt/vol) peptone. RNA extraction and reverse transcription were performed as described previously (5). The open reading frame ofglkAwas amplified from cDNA with the sequence-specific oligonucleotides cDNAglkAAfBam_up and cDNAglkAAfHind_d,hxkAwas am-plified with oligonucleotides HxkAfNco_up and HxkAfHind_down, andhxkB
was amplified by using oligonucleotides HxkBcDNABgl_f and HxkBcDNA Hind_r. All amplifications were performed with Phusion proofreading polymer-ase (New England Biolabs, Frankfurt am Main, Germany). PCR fragments were cloned into the pJet1.2/blunt vector (MBI Fermentas, St. Leon-Rot, Germany) and transferred intoE. coliDH5␣cells (Invitrogen, Karlsruhe, Germany). Plas-mid DNA was isolated with the NucleoSpin kit (Machery-Nagel, Du¨ren, Ger-many). Three plasmids each, containing either theglkA, thehxkA, or thehxkB
gene, were selected for sequencing to confirm the predicted intron positions and sequences of the genes. TheglkAgene was excised by BamHI and HindIII restriction, thehxkBgene was excised by BglII and HindIII restriction, and the
hxkAgene was excised by NcoI and HindIII restriction. All fragments were subsequently cloned into a modified pET43.1a expression vector containing an N-terminal His tag (22). After the transformation ofE. coliDH5␣cells, plasmid DNA was isolated, andE. coliBL21(DE3) Rosetta 2 cells (Merck/Novagen) were transformed. Positive clones were transferred into 10 ml of Overnight Express Instant TB medium (Merck/Novagen) and incubated for 22 h at 30°C. For HxkB purification, incubation at 18°C for 48 h was required to avoid the formation of inclusion bodies. Cells were harvested by centrifugation, resus-pended in 4 ml HEPES buffer (50 mM [pH 7.5]) (buffer A), and disrupted by sonication (Sonoplus; Bandelin, Berlin, Germany). After the removal of cell debris by centrifugation, the clear supernatant was loaded onto a Ni-nitrilotri-acetic acid (NTA) agarose column (1-ml bed volume) previously equilibrated by gravity flow with buffer A. The column was rinsed with 8 ml buffer A containing 30 mM imidazole and 100 mM NaCl. Enzymes were eluted with buffer A containing 200 mM imidazole and 150 mM NaCl. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad) with bovine serum albumin as a standard. The purity of fractions was determined by sodium dodecyl sulfate (SDS)-PAGE analysis on NuPage Bis-Tris 4 to 12% gradient gels using an MES (morpholineethanesulfonic acid)-buffered running system (Invitrogen). Pure fractions were combined and desalted against buffer A by using centrifugal filter devices with a molecular cutoff of 30 kDa (Millipore, Schwalbach, Germany). Glycerol was added to a final concentration of 50%, and enzymes were stored at
⫺20°C. Under these conditions all enzyme preparations remained stable for at least 6 months.
Deletion ofglkAandhxkAfromA. fumigatus.For the deletion of the coding regions ofglkAandhxkA, upstream and downstream fragments containing an overlapping NotI restriction site were amplified, which allowed the assembly of both fragments by subsequent PCR as described previously (17). TheglkA up-stream fragment was amplified with oligonucleotides Bam-215glkAAf_up (P1) and NotGlkAAfup_r (P2), and the downstream fragment was amplified with NotGlkAAfdown_f (P3) and BamGlkAAfdown_r (P4). For thehxkAdeletion, the upstream fragment was amplified with oligonucleotides BamHxkAAFup_f (P1) and NotHxkAAFup_r (P2), and the downstream fragment was amplified with NotHxkAAFdown_f (P3) and BamHxkAAFdown_r (P4). All fragments were gel purified, and aliquots of the respective up- and downstream fragments were mixed, denatured, annealed, and amplified by PCR for five cycles prior to the addition of oligonucleotides P1 and P4 and further amplification for 30 cycles. The assembled up- and downstream fragments were gel purified and subcloned into the pJet1.2/blunt vector. Fragments were excised by BamHI restriction and subcloned into a BamHI-restricted and -dephosphorylated pUC19 vector (Fermentas). The resulting plasmids, ⌬glkAAf_pUC and
⌬hxkAAf_pUC, were restricted with NotI and dephosphorylated. The pyrithia-mine resistance cassette (ptrA) was excised with NotI from plasmid ptrA-pJET1, and the hygromycin resistance cassette (hph) was excised from plasmid
hph-pCRIV.ptrAwas cloned into⌬hxkAAf_pUC, yielding⌬hxkAAf_pUC/ptrA, and
hphwas cloned into⌬glkAAf_pUC, yielding⌬glkAAf_pUC/hph. For the trans-formation of theA. fumigatus⌬akuB pyrG⫹strain (7) containing a deletion of theakuBgene, which is required for the nonhomologous end-joining repair mechanism, the deletion cassettes were excised by BamHI restriction. Fungal transformation was carried out according to standard procedures (48) by using 0.1g pyrithiamine/ml as selection marker for thehxkAdeletion and 240g hygromycin B/ml as a selection marker for theglkAdeletion. Transformants were purified by the repeated streaking of conidia onto selective medium, and genomic DNA was isolated from glucose-grown cultures by a yeast DNA puri-fication kit (Biozym, Hess. Oldendorf, Germany). Probes for Southern blot analysis were labeled by PCR with digoxigenin-11-dUTPs (Roche Diagnostics, Mannheim, Germany). The probe forglkAdeletion strains was directed against the downstream region, and the 900-bp probe was amplified with oligonucleo-tides NotGlkAAf_down_f and BamGlkAAf_down_r. ForhxkAdeletion strains the 800-bp probe was directed against the upstream region and amplified with oligonucleotides BamHxkAAFup_f and NotHxkAAFup_r. From each deletion approach, eight independent transformants were randomly selected for genomic DNA isolation. The DNA forglkAdeletion strains was restricted with HindIII, and a shift of the wild-type fragment from 4.9 kb to 7.7 kb was expected. ForhxkA
the genomic DNAs were restricted with BamHI, and a shift of the wild-type frag-ment from 3.1 kb to 6.0 kb was expected. Bands were visualized by CDPStar
lumi-nescence after hybridization with␣-digoxigenin Fab fragments conjugated with al-kaline phosphatase according to the manufacturer’s protocol (Roche Diagnostics).
Generation ofglkA hxkAdouble-deletion mutants.To investigate the pheno-type of a mutant strain devoid of any catalytically active hexose kinase, the hexokinase was deleted in the glucokinase deletion background. For this pur-pose, one of the glucokinase deletion strains was selected and transformed with the construct⌬hxkAAf_pUC/ptrA for thehxkAdeletion. Transformants were selected on medium containing glycerol as the sole carbon source and pyrithia-mine as a selectable marker. Small colonies appeared approximately 7 days after transformation, and these colonies were streaked repeatedly onto glycerol-pyri-thiamine medium. Simultaneously, the strains were checked for their resistance to hygromycin, which is indicative of the⌬glkAbackground. Genomic DNA of the wild type, 10 transformants, the⌬glkAstrain, and an⌬hxkAstrain were restricted with EcoRI, and Southern blotting was performed with probes against theglkAdownstream region and thehxkAupstream region. For theglkA dele-tion, a shift of the hybridizing fragment from 10.9 to 4.4 kb was expected, whereas forhxkA, the wild-type fragment was expected to shift from 4.9 to 3.2 kb.
Complementation ofglkAandhxkAdeletion mutants.For the complementa-tion ofglkAandhxkAdeletion mutants, one representative clone each was selected. Since theglkAdeletion did not result in a severe growth phenotype, we used the pyrithiamine resistance cassette to screen for complemented strains. Due to the fact that thehxkAdeletion resulted in an inability to use fructose as a substrate, no additional selection marker was required for complementation, and transformants were selected on fructose-containing regeneration plates. However, to distinguish complemented mutants from wild-type contaminations, we introduced an artificial HindIII restriction site into the downstream noncod-ing region ofhxkA. For both complementation approaches, we expected a ho-mologous integration of the construct at the original genomic locus. ForglkA
complementation a 250-bp upstream fragment together with the whole coding sequence and a 190-bp downstream region were amplified with oligonucleotides Hind250glkAAf_up and GlkAAfNotdo_r. An additional downstream fragment of approximately 700 bp, overlapping with its NotI restriction site to the down-stream fragment of the former PCR product, was amplified with oligonucleotides GlkAAfNotdo_f and HindglkAdo_r2. As described above, PCR fragments were gel purified, mixed, and fused by PCR using oligonucleotides Hind250glkAAf_up and HindglkAdo_r2. The fragment was first cloned into the pJet1.2 cloning vector, excised by HindIII restriction, and subcloned into pUC19. The vector was linearized with NotI, and theptrAresistance cassette was inserted. The complete complementation cassette was excised by HindIII restriction and used for the trans-formation of theA. fumigatus⌬glkAmutant. Clones were selected by pyrithiamine resistance and subsequently by the loss of hygromycin resistance, which was indic-ative of recombination at the original locus. Transformants were checked by South-ern analysis using a 720-bp probe directed against the downstream region ofglkA, which was amplified with oligonucleotides GlkAAfNotdo_f and HindGlkAdo_r. Genomic DNA was restricted with BamHI, leading to the expected fragment size of 8.8 kb for the wild type and 7.7 and 6.3 kb, respectively, for the complemented strains, depending on the orientation of theptrAcassette in the two independent plasmids used for transformation.
For the complementation of thehxkAmutant, an 870-bp upstream fragment together with the coding region and a 180-bp downstream region were amplified with oligonucleotides CompHxkAAfKpnF and HindCompHxkAAfR.
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ally, a 560-bp downstream fragment was amplified with oligonucleotides Hind CompHxkAAfF and CompHxkAAfKpnR, whereby the forward primer contain-ing a HindIII restriction site was complementary to HindCompHxkAAfR. As described above, both fragments were fused by PCR, amplified with oligonucle-otides CompHxkAAfKpnF and CompHxkAAfKpnR, and subcloned into the pJet1.2 vector. The complementation cassette was removed by KpnI restriction and used directly for the transformation of the⌬hxkAmutant. After the selection of transformants on fructose medium, strains were checked for the loss of pyrithiamine resistance, which was indicative of homologous integration at the
hxkAlocus. Genomic DNA of transformants was restricted with HindIII, and the probe directed against the upstream region ofhxkAwas used for Southern analysis. A shift of the wild-type fragment from 7.2 to 4.6 kb was expected.
qRT-PCR for determination ofglkAandhxkAexpression.To isolate RNA from conidia and mycelium, the respective cell type was either disrupted by using a Speed Mill or by grinding under liquid nitrogen as described above. For RNA extraction the RNeasy plant minikit (Qiagen, Hilden, Germany) with a DNase Zero treatment (Biozym) was applied. The absence of contaminating DNA was checked by PCR on the isolated total RNA with primers against the citrate synthase gene as described previously (24). An aliquot of the RNA was reverse transcribed by using SuperScript III reverse transcriptase and an anchored oligo(dT)20primer (both from Invitrogen) as described previously (24).
Quan-titative real-time PCR (qRT-PCR) was performed with a Rotor-Gene 6000 system (Qiagen) with the DyNAmo Flash SYBR green quantitative PCR kit (New England Biolabs). Each set of oligonucleotides was tested for its efficiency to amplify the respective partial gene. Final data analysis was performed by using the efficiency values incorporated in the 2⫺⌬⌬CTmethod (31, 37). Actin (primer set ACT1-for and ACT1-rev) and -tubulin (primer set RT_Tub_f and RT_Tub_r) served as housekeeping genes for data normalization. Transcript levels forhxkAwere determined with the primer set qRT_hxkAAf_for and qRT_ hxkAAf_rev, whereasglkAtranscript levels were determined by using the oligo-nucleotides qRT_glkAAf_for and qRT_glkAAf_rev. Comparative analysis be-tween actin and-tubulin showed that the latter was not constitutively expressed in resting conidia, because transcript levels for this gene showed extremely high sample-to-sample variation. Therefore, only the data fromact1expression were used for the normalization ofglkAandhxkAtranscript levels.
Semiquantitative determination ofglkAandhxkAtranscript levels from in-fected mouse lungs.Lungs of mice were obtained from an independent study to investigate the time-dependent fungal burden under different immunosuppres-sion regimens (25). In brief, 6- to 8-week-old male BALB/c-J mice were immu-nosuppressed either with the cytostatic drug cyclophosphamide (200 mg/kg body weight at day⫺4 and day⫺1) or with the corticosteroid cortisone acetate (1 mg/g body weight at day⫺3 and at the day of infection). Mice were infected intrana-sally with suspensions containing 2⫻106
conidia ofA. fumigatuswild-type strain CBS144.89. This strain is the parental strain of the⌬akuBstrain that was used to create theglkAandhxkAdeletion mutants. Two mice from each immunosup-pression regimen were sacrificed 24 h after infection, and two additional mice were sacrificed 72 h after infection. Lungs were removed, frozen in liquid nitro-gen, and ground to a fine powder. RNA was isolated and transcribed into cDNA as described elsewhere previously (24). Due to unspecific amplification and very low transcript levels ofglkAandhxkA, a quantitative evaluation by qRT-PCR approaches was not possible. Therefore, a semiquantitative approach was cho-sen, in which different amounts of cDNA served as templates for amplification by standard PCR with a SpeedCycler (Analytik Jena AG, Jena, Germany) using GoTaq polymerase and the same primers as those described above. After 35 amplification cycles the PCR products were loaded on an ethidium bromide-stained 2% agarose gel. Bands were visualized by UV illumination, and the intensities of the bands specific forglkAandhxkAwere quantified by using the TraceQuantity method of the QuantityOne software package (Bio-Rad Labora-tories GmbH, Munich, Germany). From these quantifications, the ratio between
glkAandhxkAtranscript levels in each individual sample was calculated.
Enzyme assays and biochemical characterization of hexose kinases.The ATP-dependent phosphorylation of sugars was determined at pH 7.5, as described elsewhere previously (36), by utilizing a coupled assay system with pyruvate kinase and lactate dehydrogenase to monitor ADP formation during the reac-tion. Glucose phosphorylation was independently determined via glucose-6-phosphate dehydrogenase (35) but yielded the same specific activities and was therefore not monitored further. Glucose, fructose, mannose, galactose, gluco-samine, 2-deoxyglucose (2-DG), ribose, lactose, trehalose, and sorbose were tested as substrates.Kmvalues were determined by varying the concentration of
the sugars while keeping all other components constant. TheKmvalues were
calculated by double-reciprocal plotting of the activity versus the substrate con-centration. By adding different amounts of trehalose-6-phosphate to the standard assay mixture, the inhibition of phosphorylating activity was tested. At least three
activity determinations were performed under each condition tested, and data represent mean values with standard deviations.
Isocitrate lyase activity was determined, as previously described (11), by using a phenylhydrazine-based system to measure the release of glyoxylate from isoci-trate.
Sensitivity ofglkAandhxkAdeletion strains to cell wall-stressing compounds.
Several different cell wall-stressing agents were used to investigate the cell wall integrity ofA. fumigatusmutant strains in comparison to the wild type (45). All strains except the double-deletion strain were tested for sensitivity on agar plates containing either 50 mM glucose or 100 mM glycerol minimal medium. Plates were supplemented with one of the following compounds: calcofluor white (150 and 300g/ml), sodium dodecyl sulfate (0.01 and 0.02%), caffeine (5 and 10 mM), or Congo red (20 and 40g/ml). For inclusion of the ⌬glkA⌬hxkA
double-deletion mutant, 1%-peptone-containing agar plates were supplemented with 100g calcofluor white, 0.01% sodium dodecyl sulfate, or 40g/ml Congo red. Serial 10-fold dilutions (range of between 1⫻105and 1⫻101conidia in 5
l) were prepared and spotted onto the plates. All plates were incubated at 37°C for up to 144 h.
RESULTS
Identification, purification, and determination of initial
ac-tivity of GlkA, HxkA, and HxkB. For the identification of
glucokinase and hexokinase coding sequences, we used the
respective A. niger protein sequences in a BLAST search
against the genome of sequencedA. fumigatusstrain A1163.
TheA. fumigatusglucokinase GlkA was identified by usingA. nigerGlkA (GenBank accession number Q92407 for the
pro-tein) as a template, and the resulting A. fumigatus enzyme
(AFUB_096090; accession number EDP47757) showed 82%
identity over the whole sequence. TheA. fumigatushexokinase
HxkA was identified by using theA. niger hexokinase HxkA
(accession number CAA08922) as a template, and the resulting
A. fumigatus protein (AFUB_022950; accession number EDP54242) showed 88% sequence identity. A third enzyme, named HxkB (AFUB_094300; accession number EDP47586), was also selected since it showed 48% and 33% sequence
identities toA. fumigatusHxkA and GlkA, respectively. Two
additional putative hexose kinases (AFUB_089570 [accession number EDP48243] and AFUB_031350 [accession number EDP55074]) were annotated previously (18), but their se-quence identity to GlkA and HxkA is below 25% (see Table SA2 in the supplemental material). These enzyme most likely belong to the regulatory hexose kinases, and we therefore did not focus on these additional enzymes.
To determine the biochemical properties of all three
se-lected A. fumigatus hexose kinases, we recombinantly
pro-duced the enzymes inE. coli. The open reading frames of the
respective hexose kinases were amplified from total cDNA, sequenced, and subcloned into expression vectors with an N-terminal His tag. In all cases, cDNA sequencing confirmed the
open reading frames predicted forglkA,hxkA, andhxkB. The
overproduction of all enzymes was performed with BL21(DE3) Rosetta 2 cells using OvernightExpress Instant TB medium, whereby GlkA and HxkA were produced at 30°C and HxkB was produced at 18°C. At higher incubation temperatures, the overproduction of HxkB led to the formation of inclusion bodies, whereas at 18°C, most of the protein was soluble. All proteins were purified to near homogeneity by chromatogra-phy over Ni-NTA Sepharose (Fig. 1). Purified HxkB displayed no activity with glucose, 2-deoxyglucose, fructose, mannose, galactose, ribose, or trehalose, implying that this hexokinase-like protein either is not catalytically active and may act as a
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regulatory hexokinase or was not produced in an active form. Therefore, we were not able to provide further details on the biochemical characteristics of HxkB. In contrast, purified GlkA and HxkA showed catalytic activities and were analyzed in more detail.
Substrate specificity of GlkA and HxkA and inhibition by
trehalose-6-phosphate.We first determined the catalytic
activ-ities and substrate affinactiv-ities of GlkA and HxkA with glucose and fructose. With glucose, GlkA showed a specific activity of
52 U/mg (substrate turnover number⫽ 47.2 s⫺1) and aKm
value of 25M, leading to a catalytic efficiency of 1.89⫻106
M⫺1s⫺1. Although GlkA still showed a specific activity of 38
U/mg with fructose (substrate turnover number⫽34.5 s⫺1),
theKmvalue for fructose was 31.2 mM, leading to a catalytic
efficiency of 1.11⫻103M⫺1s⫺1. Therefore, the
phosphoryla-tion of glucose by GlkA is approximately 1,700 times more efficient than that of fructose, and we concluded that GlkA
does not support fructose phosphorylation underin vivo
con-ditions.
HxkA displayed a specific activity of 7.8 U/mg (substrate
turnover number⫽7.04 s⫺1) and aKmvalue of 0.23 mM with
glucose, leading to a catalytic efficiency of 3.06⫻104M⫺1s⫺1,
which is 62 times less efficient than the efficiency determined for GlkA. However, with fructose, HxkA displayed a specific
activity of 21.6 U/mg (substrate turnover number⫽19.5 s⫺1)
and aKmvalue of 2.1 mM, leading to a catalytic efficiency of
9.29⫻103M⫺1s⫺1. Although this efficiency with fructose is
only 8.4 times higher than the efficiency for fructose activation
by GlkA, the 15-times-lowerKmvalue is likely sufficient for
catalyzing fructose activation underin vivo conditions. These
determinations additionally revealed that HxkA also displayed a higher efficiency for glucose phosphorylation than for fruc-tose activation (factor of 3.3), in agreement with a previously
reported biochemical characterization of hexokinase fromA.
niger(36).
We then determined activities with other sugars by using different substrate concentrations (1 mM, 10 mM, and 100 mM) in the test assay mixture. The resulting activities were compared with the activity of GlkA with glucose or the activity of HxkA with fructose, and the respective activities were set as 100% (Table 1). Besides the activities with glucose and fruc-tose, both enzymes were also able to activate mannose and 2-DG, whereby GlkA showed a higher affinity for both sub-strates than did HxkA. Glucosamine, although a substrate for both enzymes, inhibited its own phosphorylation when applied at concentrations above 10 mM. Neither enzyme phosphory-lated the other tested sugars.
Trehalose-6-phosphate (T6P) has been described to be an inhibitor of hexokinase but not glucokinase activity (19, 35, 36).
To confirm this finding for theA. fumigatusenzymes, we tested
the inhibition of glucose and fructose activation by GlkA and HxkA in the presence of different inhibitor concentrations. GlkA was not affected in sugar phosphorylation, confirming
the results for A. niger and A. nidulans. In contrast, HxkA
activity was inhibited by T6P, depending on the substrate and inhibitor concentration. Glucose phosphorylation was inhib-ited only when 1 mM, but not 10 mM, glucose was applied to the assay mixture, and an equal concentration of 1 mM glucose and 1 mM T6P inhibited HxkA by 96%. Fructose phosphory-lation by HxkA was always reduced. At 1 mM fructose, the
FIG. 1. SDS-PAGE analysis of purified recombinant GlkA, HxkA, and HxkB. Between 2 g (HxkB) and 3g (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 vari-ation in mobility within the gel, all purified proteins met their expected molecular masses.
TABLE 1. Substrate spectrum and activity with respect to the substrate concentration of recombinant GlkA and HxkA fromA. fumigatus
Substrate
% activity
GlkA HxkA
100 mM 10 mM 1 mM 100 mM 10 mM 1 mM
Glucose 100 100 96 79 66 57
Fructose 15 2 0 100 95 26
Mannose 58 54 54 15 13 13
2-Deoxyglucose 70 68 60 70 68 27
Glucosamine 3 18 17 35 43 19
Lactose 0 0 0 0 0 0
Galactose 0 0 0 0 0 0
Ribose 0 0 0 0 0 0
Trehalose 0 0 0 0 0 0
Sorbose 0 0 0 0 0 0
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addition of 0.1 mM T6P inhibited the activity by 93%, and the addition of 1 mM T6P inhibited the enzyme by 98%. In the presence of 10 mM fructose, 0.1 mM T6P inhibited the activity by 35%, and 1 mM T6P reduced the activity by 90%. These results show that the mode of inhibition seems competitive with respect to the affinity for the natural substrate. It further-more shows that T6P inhibition can be used to discriminate between GlkA- and HxkA-derived activities in cell extracts.
Single and double deletions of glkA and hxkAfrom A.
fu-migatus.To study the impact of GlkA and HxkA at different developmental stages and on different carbon sources, we
aimed to delete the coding sequence from A. fumigatus by
utilizing a parental strain (⌬akuB) with a defective
nonho-mologous end-joining repair mechanism, which significantly increases the rate of homologous recombination (7). The
de-letion ofglkAproved problematic. First, the sequence of the
5⬘-untranslated region of AFUB_096090 from strain A1163,
resembling the sameA. fumigatussubspecies used in this study,
contained a large sequence gap. Therefore, we utilized the
5⬘-untranslated region annotated for strain Af293 (locus tag
AFUA_6G02230) for primer design. Generally, we use an 800-to 1,000-nucleotide flanking region for homologous recombi-nation. However, it turned out that although we obtained
spe-cific PCR products, these fragments were not amplified inE.
coli. We therefore reduced the size of the upstream fragment
to 215 bp, which was usable for the construction of theglkA
deletion construct. Despite this small upstream region we ob-tained several transformants and selected eight of them for Southern blot analysis. All eight transformants showed a single
homologous integration into theglkAlocus (see Fig. SA1A in
the supplemental material).
The generation of thehxkAdeletion cassette was performed
by standard procedures, and no problems arose in the cloning of the upstream and downstream fragments. Interestingly, transformants showed a rather delayed conidiation on glucose minimal medium. Eight transformants were selected for Southern analysis, and all clones showed a single homologous
integration into thehxkAlocus (see Fig. SA2A in the
supple-mental material). One representative glkA mutant and one
hxkA mutant were selected for complementation. Initial
growth tests revealed no altered phenotype forglkA on agar
plates, whereas the hxkA mutant grew poorly and did not
sporulate on fructose. Therefore, the complementation ofglkA
required the use of the pyrithiamine resistance cassette as a
selectable marker during transformation, whereas hxkA was
complemented by selecting transformants for their abilities to grow on fructose (Fig. SA1B and SA2B).
We next attempted to generate aglkA hxkAdouble mutant.
Initial attempts in which either the⌬glkAor the⌬hxkAstrain
served as an acceptor and different media were used for re-generation were unsuccessful. However, the transformation of
the⌬glkAstrain with the⌬hxkAdeletion construct, with
trans-formant regeneration in the presence of glycerol, yielded sev-eral small colonies 7 days after transformation. All transfor-mants continued to grow poorly following subculture on fresh glycerol medium, but all transformants were resistant to hy-gromycin and pyrithiamine, as expected for a double-deletion strain. Southern blot analysis with probes visualizing the glu-cokinase and the hexokinase deletion confirmed that all 10 selected transformants showed the expected shifts of
hybridiz-ing fragments compared to the shybridiz-ingle-deletion strains (see Fig. SA3 in the supplemental material). Therefore, it can be con-cluded that viable strains devoid of both catalytic hexose ki-nases can be generated, but their growth phenotypes are more severe than that described previously for a respective
double-mutant fromA. nidulans, which was described only to fail to
grow on glucose and fructose (19).
Analysis of growth of mutants on different carbon sources. A previous investigation of glucokinase and hexokinase
mu-tants from A. nidulans indicated that only the mutation of
hexokinase, but not glucokinase, leads to a defect in sugar utilization, whereby the defect of the hexokinase mutant was restricted to an inability to grow on fructose (19). Interestingly,
the respective mutants of Botrytis cinerea showed a similar
unaltered phenotype for a glucokinase mutant, but a hexoki-nase mutant was severely affected on various sugars and also some gluconeogenic carbon sources such as acetate and
glyc-erol (40). Since the mutation in theA. nidulans glkA4mutant
was not analyzed by sequencing, it remains unclear whether the mutated enzyme still retained some residual activity. To test
whether theA. fumigatusorthologues had similar functions, we
inoculated solid medium containing different sugars or glu-coneogenic carbon sources with conidia of the wild type; the
glkA, the hxkA, and the glkA hxkA deletion strains; and the
complemented strains (Fig. 2).
As described for the other fungi, theglkAdeletion did not
overtly affect the phenotype on the different carbon sources. The only minor differences in comparison to the wild type and the complemented strain were observed with trehalose, in
which theglkAmutant colonies always appeared to be
some-what delayed in biomass formation. However, with complete medium as well as all other sugars and nonfermentable carbon sources, no difference in colony formation was observed, im-plying that GlkA function was completely complemented by
HxkA. Therefore, theglkAphenotype was in agreement with
previously reported observations ofA. nidulansandB. cinerea
(19, 40). In contrast, thehxkAdeletion strain showed
pleiotro-pic growth phenotypes similar to those ofB. cinereabut notA.
nidulans(19, 40). In general, conidium formation of the⌬hxkA
mutant was delayed on nearly all carbon sources tested, which was visualized by the delayed appearance of the gray-greenish color on top of the colonies. Additionally, and as expected, this mutant was strongly inhibited in growth on fructose. This find-ing confirmed the results from activity determinations, in
which HxkA showed a much lowerKmfor fructose than did
GlkA. However, the mutant was also unable to grow on glu-cosamine and exhibited an extreme delay in growth on sorbose. The former was unexpected, because both GlkA and HxkA were able to activate glucosamine, whereby HxkA was signifi-cantly more active with this substrate than GlkA. The strong effect on sorbose was unexpected, because neither enzyme used this sugar as a substrate. An additional strong delay was observed with the disaccharide saccharose, which can be ex-plained by its composition of glucose and fructose. Also, on mannose, which was an excellent substrate for the glucokinase, delayed growth and conidiation were observed. Interestingly, reduced growth was also observed for some gluconeogenic carbon sources, especially ethanol and acetate but also glyc-erol. This indicates that hexokinase may also be involved in the activation of glucose derived from gluconeogenesis.
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The most severe phenotypes were observed for the double-deletion strain. This mutant failed to grow on glucose, fructose, mannose, glucosamine, sorbose, and saccharose. Some growth on galactose and lactose (galactose containing) was observed, which may be due mainly to the Leloir pathway, which gener-ates glucose-6-phosphate from galactose-1-phosphate in sev-eral steps (44). Some very minor growth on starch was also observed. The degradation of starch yields not only free glucose units but also some glucose-1-phosphate by the glycogen/starch
phosphorylase, which may be encoded by AFUA_1G12920 inA.
fumigatus. Interestingly, growth was also strongly retarded with complete medium containing glucose and amino acids/peptides, although growth on peptone and Casamino Acids was only slightly affected. Despite the relatively strong growth on peptone, this mutant failed to produce substantial quantities of conidia under these conditions (even after 2 weeks of incubation [not shown]). Interestingly, on bovine serum albumin, which requires
protease secretion for growth, the colony formation of the mutant was also strongly retarded. Some biomass formation on ribose, lecithin, ethanol, acetate, and glycerol was also observed, but growth was strongly delayed, and significant conidiation was not observed.
This phenotypic analysis indicates that (i) HxkA and GlkA are the main catalytic hexose kinases; (ii) both HxkA and GlkA are required for normal conidiation, but HxkA plays a major role; (iii) even on gluconeogenic carbon sources, at least one of the hexose kinases is required for normal biomass formation; (iv) both en-zymes seem to be present in mycelium, but HxkA has a greater impact on sugar metabolism; and (v) GlkA does not significantly contribute to the metabolism of fructose, galactose, glucosamine,
and sorbose, since the⌬hxkAstrain and the double mutant
ex-hibited very similar phenotypes under these conditions.
Cell wall integrity of hexose kinase mutants. We
addition-ally tested the hexose kinase mutants for their sensitivities to
FIG. 2. Growth analysis ofglkAandhxkAmutants. All plates were spot inoculated with 1⫻103conidia and incubated for 44, 48, or 76 h, as
indicated by the number of asterisks. WT, wild type;⌬glkA,glkAdeletion mutant; C⌬glkA, complementedglkAdeletion mutant;⌬hxkA,hxkA
deletion mutant; C⌬hxkA, complementedhxkAdeletion mutant;⌬glkA⌬hxkA, 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.
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the cell wall stress-inducing compounds calcofluor white, so-dium dodecyl sulfate, caffeine, and Congo red. In a first screen on glucose- and glycerol-containing media, the double-deletion strain was omitted due to its severe growth defect on these media. This analysis revealed that both single mutants dis-played no or only a very minor increase in the sensitivity to cell wall-stressing agents compared to the parental wild-type strain (data not shown). However, since both mutants seemed, in principle, to be able to phosphorylate various sugars required for the synthesis of cell wall components and glycoproteins, we included the double-deletion strain. Here, we used peptone medium as a nutrient source, because it was shown to
contrib-ute best to the biomass formation of the⌬glkA⌬hxkAmutant
(Fig. 2). With this medium the effects of calcofluor white, Congo red, and sodium dodecyl sulfate were tested. As shown in Fig. 3, the double-deletion strain showed colony formation, although slightly delayed in growth and without conidiation, at all dilutions when spotted onto peptone medium. However, the addition of each cell wall stress-inducing agent severely af-fected biomass formation, and sensitivity was strongly in-creased compared to those of the wild type and the single-deletion mutants (shown only for 0.01% SDS). Therefore, the presence of at least one catalytically active hexose kinase is essential for the full integrity of the fungal cell wall.
Impact of GlkA and HxkA on conidium germination.Our
growth analyses implied that GlkA is functionally redundant in the presence of intact HxkA. Therefore, we asked why this enzyme, which seems to be conserved in nearly all fungal species but has not resulted in a severe phenotype when de-leted, was maintained during evolution. Furthermore, al-though GlkA showed a very high affinity for glucose and had the highest specific activity for this substrate, it only partially
complemented the phenotype of anhxkAmutant. The minor
growth defect on trehalose, however, provided a clue to thein
vivofunction of GlkA.
Trehalose is one of the main storage compounds within conidia of different fungal species. This sugar protects conidia from oxidative and heat stress (16) and is degraded for energy
metabolism in the germinating conidium. Studies ofA.
nidu-lanshave shown that trehalose breakdown in conidia is
depen-dent mainly on TreB, a neutral trehalase. The deletion of the
treBgene resulted in no visible germination defect in the
pres-ence of high glucose concentrations, but germination was sig-nificantly delayed when only trace concentrations of glucose were present in the medium (9). Since glucose released from trehalose needs to be activated to glucose-6-phosphate and, furthermore, the initial glucose concentration may be far below
theKmof the hexokinase, we assumed that GlkA might be of
major importance during early conidial germination. To test this hypothesis, we performed germination experiments with liquid media containing either different concentrations of glu-cose (0.1 to 10 mM) or the gluconeogenic carbon source eth-anol or glycerol at a fixed concentration of 10 mM. Using glucose, germination was stopped after 7 to 8 h, whereas this time was prolonged to approximately 11 h with glycerol and to 13 h with ethanol. Since germination on minimal medium is not synchronized, we evaluated at least six microscopic fields
(ⱖ300 individual cells) under each condition and classified
them in a numbering system between 1 and 5 (for a detailed classification, see the legend of Fig. 4A). The sum of the numbers of all cells was calculated and divided by the number of cells counted, leading to a minimal value of 1.0 (all cells resting) or a maximum of 5.0 (all cells germinated, branched, and containing multiple nuclei). The incubation of conidia of all strains except the double-deletion strain for 7 to 8 h in complete Sabouraud medium and evaluation of approximately 1,000 individual cells independently were performed to test the viability of the conidial preparations. All conidial preparations displayed a viability of between 93% and 98%.
Evaluation of the different growth conditions confirmed our hypothesis. At 0.1 mM glucose, the wild-type and comple-mented strains showed no significant difference in germination
speed, whereas the germination of theglkAmutant was
signif-icantly delayed (Fig. 4B). Increasing the concentration of glu-cose to 2 or 10 mM generally increased the germination speed
but also reduced the germination delay between theglkA
mu-tant and the wild type (not shown). In contrast, the deletion of
hxkAresulted in no significant delay in germination at either
glucose concentration, implying that GlkA but not HxkA is the major glucose-activating enzyme in germinating conidia.
How-ever, due to the minor germination delay of theglkAmutant at
higher glucose concentrations, this enzyme appears to be es-pecially important at extremely low glucose concentrations. Germination analysis of the double-deletion mutant was prob-lematic. Using peptone as a viability control we found that more than 60% of conidia remained in the resting state after 8 and 13 h of incubation. This may indicate that the double-deletion strain either has a strongly reduced viability of conidia or germinates extremely asynchronously. Nevertheless, germi-nation analyses were performed with glucose, glycerol, and ethanol. As expected from the viability control, most conidia remained in the resting state on glucose, with only a small proportion of conidia, which showed some swelling within the 8-h observation period (Fig. 4B). Therefore, germination on glucose requires at least one active hexose kinase. To confirm this assumption, we microscopically determined the germina-tion and colony formagermina-tion of the double mutant from glass slides covered with glucose minimal medium. After 20 h some swollen but no germinating conidia were detected (not shown). However, 163 h after inoculation, some microcolonies were observed, which showed a hyperbranching phenotype and did
FIG. 3. Effect of cell wall stress-inducing agents on growth of hex-ose kinase mutants. WT, wild type; ⌬glkA, glkA deletion mutant; ⌬hxkA,hxkAdeletion mutant;⌬glkA⌬hxkA, 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.
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not develop further (Fig. 4C). Interestingly, on the gluconeo-genic carbon sources glycerol and ethanol, GlkA and HxkA had similar impacts on germination (Fig. 4D and E). Both deletions caused a retardation of germination on ethanol but had little effect on germination on glycerol. The deletion of both hexose kinases led to an additional delay in germination, confirming the contribution of both enzymes to germination and growth on gluconeogenic carbon sources.
Due to the more pronounced effect of GlkA on germination in the presence of low glucose concentrations, this enzyme
could be responsible for the activation of glucose from stored trehalose. The degradation of stored trehalose reserves is pre-dicted to release only small amounts of free glucose, which requires an enzyme with a high glucose affinity. In contrast, HxkA appears to be involved mainly in sugar activation during vegetative growth regardless of the carbon source applied to the growth medium.
Growth inhibition by 2-DG.To test whether GlkA is
gener-ally required in the presence of low glucose concentrations, we performed growth analyses in the presence of 2-deoxyglucose (2-DG). The catalytic parameters of GlkA and HxkA indicated that both enzymes activated 2-DG in a manner similar to that for the activation of glucose. GlkA activated 2-DG at nearly maximum rates at 1 mM, whereas HxkA was significantly less
effective at low 2-DG concentrations (Table 1). The in vivo
activation of 2-DG leads to 2-DG-6-phosphate, which cannot be further metabolized and thereby inhibits growth (6). Solid media that contained glycerol as the major carbon source were prepared and were supplemented with 2-DG at concentrations
ranging from 25g/ml (0.15 mM) to 100g/ml (0.6 mM). All
strains were affected in growth and sporulation by the addition of small amounts of 2-DG (see Fig. SA4 in the supplemental
material). However, theglkAdeletion strain was more resistant
to the addition of 2-DG, whereas thehxkAmutant was most
strongly affected. Therefore, it can be concluded that the high-affinity enzyme GlkA is mainly involved in the activation of 2-DG when present at low concentrations.
Presence of GlkA and HxkA activities in conidia and
myce-lium. For GlkA to phosphorylate trehalose-derived glucose
early during germination, this enzyme must be present in rest-ing conidia. Furthermore, assumrest-ing that HxkA is the major sugar-activating enzyme during vegetative growth, the pres-ence of a significant proportion of this enzyme during hyphal growth was expected. To discriminate the different activities, we utilized the above-described inhibition of HxkA by T6P, whereby this inhibitor did not affect GlkA activity. To deter-mine activities for glucose and fructose phosphorylation from both enzymes, we always utilized the respective substrate at 10 mM and added 1 mM T6P for the inhibition of HxkA activity when fructose was the substrate. This inhibitor concentration was shown to strongly decrease the fructose-activating activity of HxkA.
We first determined activities from resting conidia of the wild type and the two mutant strains (Table 2). As expected, high levels of glucose- and fructose-activating activities were found for the wild-type extract. However, since activity with fructose made up only one-third of the glucose activity, a significant proportion of the activity was assumed to be derived
from GlkA. In agreement with this assumption, aglkAmutant
showed low glucose-activating activity (reduced by a factor of 7 in comparison to the wild type) and relatively high fructose-activating activity, with the latter being sensitive to T6P
inhi-bition. In contrast, an hxkA mutant displayed high levels of
glucose-phosphorylating activity (reduced by a factor of 1.3 in comparison to the wild type), and the low, but significant, activity with fructose was insensitive to inhibition. Therefore, we conclude that, indeed, high concentrations of GlkA and only a minor proportion of HxkA are present within conidia, supporting the idea of the proposed role of GlkA in activating trehalose-derived glucose. It furthermore implies that GlkA
FIG. 4. Analysis of conidium germination in the presence of dif-ferent carbon sources. (A) Classification of the germination state. The pictures show overlays of bright-field images with fluorescent photo-graphs 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, some-times 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⌬glkA
⌬hxkAdouble-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 cham-ber slide. The variations observed for wild-type germination between ⌬glkA,⌬hxkA, and⌬glkA⌬hxkAanalyses are due to slight variations in the incubation times but did not influence the overall results (as de-termined 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 devia-tions. Significance was calculated by a Student’sttest, and two stars indicatePvalues ofⱕ0.01.
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and HxkA seem to be the only major glucose- and fructose-activating enzymes, since the sum of the activities derived from the mutants approximately met the levels determined for the wild type. Due to difficulties in obtaining sufficient amounts of conidia of the double-deletion strain for the preparation of crude extracts, the hexose-phosphorylating activity was not de-termined. However, the fact that only a minor proportion of conidia from the double mutant germinated within 24 h in the presence of glucose additionally supports the assumption that no other enzyme with significant hexose-phosphorylating ac-tivity is present in conidia.
We then tried to confirm that HxkA contributes mainly to sugar activation in vegetative mycelium (Table 2). In glucose-grown wild-type mycelia, the level of fructose-phosphorylating activity exceeded that of the glucose-phosphorylating activity and was highly sensitive to T6P inhibition, indicating high
levels of HxkA. In the glkA mutant, the level of activity for
glucose phosphorylation was reduced, but activity for fructose stayed at high levels and was sensitive to T6P inhibition. In
contrast, thehxkAmutant displayed only low levels of
fructose-phosphorylating activity, and this activity was only slightly af-fected by T6P. Using ethanol as a carbon source, the levels of hexose-phosphorylating activities of the wild type were always lower than those with glucose, but, as observed with glucose, the activities for glucose and fructose phosphorylation were in a similar range, and fructose phosphorylation was highly sen-sitive to T6P inhibition. Unexpectedly, investigations of the two mutants implied that for each mutant, the level of the
respective residual enzyme (HxkA in the glkA mutant and
GlkA in thehxkAmutant) increased to some extent, since the
sum of the activities from both mutants exceeded that deter-mined for the wild type.
Determinations of hexose kinase activity from the double-deletion strain grown on ethanol revealed values below the detection limit of 10 mU/mg in crude cell-free preparations. This finding indicates that no other major hexose-phosphory-lating enzyme is induced on gluconeogenic carbon sources.
Therefore, our results show that both enzymes are present in conidia and in vegetative mycelia regardless of the growth condition applied. However, HxkA is the major sugar-activat-ing enzyme dursugar-activat-ing vegetative growth, whereas GlkA seems to be the dominant enzyme in resting conidia.
Determination of glkA andhxkAtranscript levels by
qRT-PCR.Our activity determinations revealed that GlkA, but not
HxkA, is highly abundant in resting conidia. In contrast, HxkA seems to constitute the major enzyme during vegetative growth. Due to the very early presence of GlkA activity, we next tested whether this was reflected by the mRNA level. Therefore, we isolated mRNA from resting and swollen conidia (0 and 4 h), early germ tubes (8 h), and glucose-grown mycelia (15 h). In addition, we analyzed fructose- and
ethanol-grown mycelia. For the standardization ofglkAandhxkA
tran-script levels, data were normalized against the trantran-script levels
of the actin gene act1. Our analysis showed that glkA
tran-scripts were extremely abundant in resting conidia but that levels steadily declined during the germination process (Fig.
5A). In contrast,hxkAtranscripts were less abundant in resting
conidia, but expression was strongly induced during early ger-mination (4 and 8 h) and stayed at a rather high level in the mycelium (Fig. 5B) regardless of the carbon source present in the medium.
We additionally determined the ratio between levels ofglkA
andhxkAtranscripts to visualize the switch ofglkAandhxkA
gene expression patterns. Here, theglkAtranscript levels were
used for normalization. This analysis confirmed thatglkA
tran-scripts were approximately six times more abundant in resting conidia (Fig. 5C), whereas this ratio was inverted early during
germination. Mycelia contained hxkA transcript levels that
were approximately two times higher thanglkAtranscript
lev-els. Interestingly, this ratio in the mycelium was not affected by the carbon source, since the same ratios were obtained from glucose-, fructose-, and ethanol-grown mycelia. Therefore, qRT-PCR independently confirmed our activity determina-tions showing that GlkA is the major conidium-associated sug-ar-phosphorylating enzyme, whereas HxkA is mainly mycelium associated.
Expression ofglkAandhxkAin lungs of infected mice.
Phe-notypic analysis of the double-deletion strain implied thatglkA
andhxkAmay be expressed and may play a role during lung
infection and the manifestation of invasive aspergillosis. Therefore, we investigated the expressions of both genes from the lungs of mice, which were immunosuppressed with either cortisone acetate or the cytostatic drug cyclophosphamide, which renders mice neutropenic. In addition, lungs of mice sacrificed 1 or 3 days after infection were investigated.
Previous studies of the growth behavior ofA. fumigatuswith
both immunosuppression regimens have shown that conidia germinate within 1 day when mice were treated with cortico-steroids (5, 25), but at later time points, neutrophils were recruited and attacked outgrowing hyphae, reducing the num-ber of living fungal cells at the cost of severe tissue destruction. In contrast, the germination of conidia is delayed in neutro-penic animals, but, once germinated, the mycelium can grow unrestricted through the lung parenchyma (25). In a first
ap-proach we attempted to quantify the expression levels ofglkA
and hxkA by qRT-PCR using SYBR green as a fluorescent
marker for gene amplification. However, we noticed that the
TABLE 2. Hexokinase and glucokinase activities determined from cell extracts of differentA. fumigatusstrainsa
Source of extracts MeanAspec(mU/mg)⫾SD (% inhibition) Glucose Fructose Fructose⫹T6P
Conidia
WT 435.8⫾41 141.3⫾15.4 24.5⫾12.6 (82.7) ⌬glkA 60.8⫾10 130.1⫾14.2 37.0⫾14.5 (71.6) ⌬hxkA 330.2⫾34 62.0⫾13.4 59.4⫾9.3 (4.2)
Mycelium
(glucose grown)
WT 297.8⫾41 305.9⫾41 63.6⫾14 (79.2) ⌬glkA 204.7⫾26 304.2⫾31 76.7⫾7 (74.8) ⌬hxkA 149.5⫾9 44.7⫾9 40.6⫾3 (9.2)
Mycelium
(ethanol grown)
WT 189.1⫾48 183.2⫾33 59.0⫾5 (67.8) ⌬glkA 184.4⫾39 182.9⫾47 82.3⫾24 (55.0) ⌬hxkA 188.8⫾23 61.8⫾9 61.0⫾10 (1.3) ⌬glkA⌬hxkA ⬍10 ⬍10 ND
a
Substrates were used at a final concentration of 10 mM. For inhibition, 1 mM T6P was added.Aspec, specific activity. WT, wild type; ND, not determined.
