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Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

     Department of Biology, Rhodes College, Memphis, Tennessee 38112

Darlene M. Loprete

     Department of Chemistry, Rhodes College, Memphis, Tennessee 38112

Michelle Momany
Youngsil Ha

     Department of Plant Biology, The University of Georgia, Athens, Georgia 30602

Lisa M. Harsch
Jennifer A. Livesay
Amit Mirchandani
Jeremy J. Murdock
Michael J. Vaughan
Mridula B. Watt

     Departments of Biology and Chemistry, Rhodes College, Memphis, Tennessee 38112

	ABSTRACT

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As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.

Key words: Calcofluor hypersensitivity, cell wall mutants, swollen cell phenotype

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The cell wall is a highly complex assembly of cross-linked polysaccharides and glycoproteins, which at maturity provides the rigidity needed to define the shape of each cell and to contain the outwardly directed force of turgor pressure (Beauvais and Latgé 2001, Duran and Nombela 2004). The wall is not only a product of the complex developmental programs of the fungal cell, but it is also an essential participant in probably all aspects of morphogenesis (Wendland 2001, Borkovich et al 2004). A great deal of interest exists in the potential clinical and plant pathological consequences of fungal cell wall structure and metabolism. Fungal walls are elicitors of host defensive responses in both plants and animals (Hoffmann et al 1999, Selitrennikov 2001), and components of the wall are associated directly with colonization of host tissues and tissue damage (Rementeria et al 2005). Because the fungal wall has no equivalent in animal hosts and differs substantially from the cell wall of plant hosts, it is expected that fungal wall metabolism will provide unique sites of action for antifungal drugs (Beauvais and Latgé 2001). The echinocandin and Nikkomycin classes of antibiotics are based upon this principle.

Despite this interest and activity, much remains to be learned about the architecture of the wall, about how that architecture changes during growth and development and about the mechanisms behind wall synthesis and modification. The shortfall of understanding is greater for the developmentally more complex filamentous fungi than for yeasts. In principle, it should be possible to identify novel processes of wall metabolism by screening genetic mutants for phenotypes that are observed frequently amongst known wall mutants. One promising strategy, which has been employed successfully with yeasts, is to screen cells for hypersensitivity to wall-compromising agents, especially to Calcofluor White (Ram et al 1994, Lussier et al 1997, Carnero et al 2000). Calcofluor (CFW) binds to ß-glycan polymers, including the glucan and chitin components of yeast and hyphal walls (Maeda and Ishida 1967). Binding interferes with the crystallization step of microfibril assembly in growing walls (Elorza 1983), causing reduction in growth rate, lysis of hyphal tips (Roncero and Duran 1985), and altered incorporation of mannoproteins into walls (Murgui et al 1985). CFW-induced wall damage activates the cell wall integrity (maintenance and repair) pathway in Saccharomyces (reviewed in Levin 2005) as well as its presumed equivalent in filamentous fungi (Damveld et al 2005). No penetration of the plasma membrane or alternative mode of toxicity has been observed. Using this method, Lussier et al (1997) identified yeast genes affecting synthesis of several known wall constituents, as well as genes affecting pathways of signal transduction and ones whose roles in wall metabolism are not yet clear. Many of these CFW-hypersensitive yeast strains showed no other phenotype that might have hinted at a defect in cell wall metabolism.

In this report we describe the use of CFW hypersensitivity as a primary screening step in filamentous fungi, resulting in the identification of 10 mutant strains in A. nidulans, designated cal for Calcofluor hypersensitivity. The conclusion that the cal mutants suffer from defects in wall structure is supported by a high incidence of morphological and sporulation defects and by heightened sensitivities to further agents known to interfere with wall metabolism or to adversely affect growth of cell wall mutants.

	MATERIALS AND METHODS

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Aspergillus strains and media.— – Strains used in this study are listed (TABLE I). Complete medium (CM) consisted of 1% glucose, 0.2% peptone, 0.1% yeast extract, 0.1% casamino acids, 5% nitrate salts, 1% trace elements, 0.1% vitamin mix, 1.2 mM L-arginine, 10 mM uracil and 5 mM uridine. Vitamin mix and nitrate salts are described in the appendix of Kafer (1977). Trace element solution is described in Hill and Kafer (2001). For gelatin medium, 1% gelatin was substituted for glucose, peptone, casamino acids and yeast extract in the preceding formula. Minimal medium (MM) consisted of 1% glucose, 5% nitrate salts, 1% trace elements, 0.001% thiamine hydrochloride and 25 ppb biotin. The effect of increased osmotic strength was investigated using media supplemented with 1 M sucrose, 0.6 M KCl, or 0.6 M NaCl. Solid media contained 1.5% agar. All media also contained 50 µg/ mL ampicillin.

Genetic manipulations.— – Strain construction and genetic analysis followed standard procedures (Kafer 1977, Kaminskyj 2001). CFW hypersensitive strains were crossed separately to strain AH12 and to strain GR5, each of which shows wild type growth in the presence of 10 µg/mL CFW, and randomly selected haploid progeny were tested for CFW hypersensitivity via toothpick transfer. Diploid strains were isolated from the respective heterokaryotic cultures; diploidy was confirmed by benomyl-induced sectoring. Eleven strains showing 1 : 1 segregation of hypersensitivity in crosses to both GR5 and AH12 and whose respective diploids also showed wild type growth with CFW (recessiveness of hypersensitivity) were selected for further study. Complementation groups were determined by constructing all possible combinations of diploids between each pair of mutations (including self-diploids), and the CFW sensitivity of each diploid was tested at 30 C and 42 C, as was morphology at 42 C for those strains with temperature-induced growth abnormalities. Gene designations were assigned according to Clutterbuck and Arst (1995).

Additional phenotype tests.— – All phenotypic comparisons were performed using strains with cal alleles in the GR5 genetic background (pyrG89; wA3; pyroA4). Sensitivity to potential inhibitors was tested by culturing 5-µL drops of freshly collected conidia (2000 conidia per microliter) for 48 h on CM agar plates to which one of the following supplements was added: CFW, 1–100 µg/mL; Caspofungin acetate, 10 µg/mL; Nikkomycin, 12.5 µg/ mL; sodium dodecyl sulfate (SDS), 35 or 50 µg/mL; Congo red (CR), 50 µg/mL; Caffeine 5–20 mM; and Tunicamycin, 5–20 µg/mL. The respective stock solutions were: CFW, 1% in 25 mM KOH; Caspofungin acetate, 10 mg/mL in 50% ethanol; Nikkomycin, 5 mg/ mL; SDS, 50 mg/mL; CR, 10 mg/mL; caffeine, 200 mM in CM liquid; and Tunicamycin, 5 mg/mL in 0.1 N NaOH. SDS and CR were sterilized by autoclaving. CFW and caffeine were filter sterilized. Caspofungin acetate was a gift of Merck & Co. Inc., Rahway, New Jersey; all other biochemicals were purchased from Sigma Chemical Company, St. Louis, Missouri.

For assessment of microscopic characters, agar cultures were established with 5-µL drops containing a total of 500 to 2000 spores. After 15 to 24 h of growth, germlings were covered with liquid medium of matching solute composition and observed under a cover slip with Nomarski DIC optics. Measurements were made from photographs taken with the 40x objective and displayed at 1167x magnification. Statistical differences between treatments were determined by using ANOVA and a Tukey HSD a posteriori test at the 0.05 level of significance, based on observation of at least 25 randomly selected germlings for each condition.

To compare the effects of mutations on sporulation, horizontal CM agar surfaces (3 mL of medium in 15-mL conical centrifuge tubes) were inoculated in triplicate with approximately 10 000 conidia and incubated 4 d at 30 C or 42 C. Preliminary tests showed that all strains reached maximum sporulation within that time. Conidia were harvested in 1 mL of sterile water using a glass rod and counted with a hemacytometer. The morphology of conidiating structures was determined after growth in slide cultures (Raper and Fennell 1965). Slides were stained with lactophenol acid fuchsin (Raper and Fennel 1965) and observed under the microscope.

Cell wall carbohydrate analysis.— – A. nidulans GR5 and cal mutants were grown with rotary shaking at 30 C for 48 h in 100-mL volumes of liquid CM, inoculated at a spore density of 1 x 10⁷ spores per mL. Cell walls were isolated and lyophilized as previously described (Guest and Momany 2000). Dried cell wall material was prepared for analysis by methanolysis and re-N-acetylation as previously described (York et al 1985). Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis at the Complex Carbohydrate Research Center at the University of Georgia. GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 mass selective detector, using a Supelco DB-1 fused silica capillary column (30 m x 0.25 mm ID).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Before screening strains from the Harris et al (1994) mutant collection, we first determined the effect of CFW on colony growth (measured as increase in colony diameter) in strains A28 (background strain of the Harris collection), A4, AH12 and GR5. (These strains are collectively referred to as "wild type" in this report.) A concentration of 10 µg/mL was the highest CFW level that produced no more than a 10% decrease in growth rate of wild type strains at either 30 C or 42 C (data not shown). Approximately 1100 strains from the Harris collection were screened at this CFW concentration using the toothpick transfer technique, and 64 strains were initially judged to be CFW hypersensitive at one or both temperatures.

With further observations it became clear that most of the selected strains produce colonies with reduced asexual sporulation and that even wild type strains have difficulty establishing growth on CFW if a young, not yet well sporulating colony is used as the inoculum. Therefore we reinvestigated the CFW sensitivity of all 64 strains using a standardized spore inoculum of approximately 10 000 conidia, applied in a 5-µl drop to the surface of CM agar containing 10 µg/mL CFW. At this density, GR5 (TABLE II, FIG. 1) and the other wild type strains (data not shown) produce significant colonies within 2 d on media containing up to 30 µg/mL (30 C) and 50 µg/mL CFW (42 C). As a result of this more rigorous screening, only 23 of the original 64 strains proved to be CFW-hypersensitive at one or both temperatures.

View larger version (48K): [in this window] [in a new window]   FIG. 1. Effect of temperature and medium osmotic strength on Calcofluor sensitivity in cal mutant strains and wild type strain GR5. Cultures were grown for 2 d at the indicated temperatures. Beginning from the upper left, strains are GR5 (wild type), calA8, calB1, calC2, calD4, calE3, calE10, calF7, calG5, calH6, and calH9.

Isolation of diploids from pairwise crosses between strains bearing the 10 cal alleles established that the alleles belong to eight complementation groups (TABLE I). Because of strong similarities between the calH phenotype and the swoA phenotype reported by Momany et al (1999), we produced diploids between swoA strain AGA7 and calH strains bearing reciprocal auxotrophies, confirming that the calH and swoA loci are identical. Strains bearing the cal alleles in a GR5 genetic background were selected for carrying out phenotypic comparisons.

The CFW sensitivities of the ten cal mutant strains are compared (TABLE II). All mutant strains show elevated sensitivity to CFW at both tested temperatures, and for each strain there is at least one temperature at which its sensitivity to CFW is at least 7.5x greater than that of the wild type. In all cases the inhibitory effect of CFW is reduced by adding 1.0 M sucrose to the medium (FIG. 1). This osmotic remediability helps eliminate the possibility that CFW hypersensitivity might result from some unrecognized toxicity, unrelated to wall binding.

Sensitivities to the glycan-binding agent Congo red, the ionic detergent SDS, and the ß-1,3-glucan synthesis inhibitor Caspofungin acetate are shown (TABLE III). The different SDS levels used at 30 C and 42 C represent the approximate maximum SDS levels having little or no effect upon colony growth of strain GR5 at the respective temperatures. All of the tested chemicals prevent colony growth of at least some of the mutants at one or both temperatures, under conditions where the wild type still produces a colony. Only the calF7 and calG5 mutants show wild type sensitivity to all tested chemicals. No strain was more resistant than wild type when exposed to higher inhibitor levels (data not shown). Caffeine, which is inhibitory to many cell wall mutants in yeast (Lussier et al 1997, deGroot et al 2001), as well as Tunicamycin and Nikkomycin, had no differential effects on colony growth of any cal mutant at either temperature (data not shown).

The Harris et al (1994) collection consists of strains that were selected originally for having growth defects at 42 C, and five of the eight cal mutants isolated from this collection show vacuolated swollen-cell phenotypes in germlings grown on CM at 42 C (FIGS. 3–7, TABLE V). Of these only calD4 also shows a similar, albeit lesser, morphological aberration at 30 C (FIG. 4, left panel). In most cases swelling manifests as excessive isotropic growth of the spore during germination, defined in this study as swelling to at least 150% of the diameter attained by wild type spores under identical conditions, which corresponds to a roughly three-fold increase in volume. Strains bearing either of the two alleles of calE show no temperature-induced swelling on CM, but do show swelling and vacuolation of hyphae (though not of spores) after growth for 24 h at 42 C on a medium containing gelatin as the sole carbon source (FIGS. 8, 9, right panels). Growth on gelatin medium does not affect the phenotypes of any other strains. (The original purpose for testing growth on gelatin medium was to test for differences in protein secretion. No clear differences were observed.) Temperature-induced swelling of spores and hyphae is osmotically remediated in strains bearing the calB1, calG5, calH6, and calH9 alleles but not in the strain bearing the calD4 allele (TABLE V). Swelling in strains bearing the calE3 and calE10 alleles on gelatin medium is also osmotically remediated (data not shown).

View larger version (154K): [in this window] [in a new window]   FIGS. 2–9. Swollen cell morphologies in selected cal strains and wild type strain GR5 grown at 30 C (left panel of each pair) and 42 C (right panel of each pair). 2–7. Growth on CM for 15–20 h. 2. GR5 (wild type). 3. calB1. 4. calD4. 5. calG5. 6. calH6. 7. calH9. 8–9. Growth on gelatin medium for 24 h. 8. calE3. 9. calE10. Scale bar = 25 µm.

The effect of the cal mutations on asexual sporulation is shown (TABLE VI). Several mutations cause significant (in some cases severe) reduction in sporulation at 30 C on CM, and all mutations severely reduce sporulation at 42 C. In most cases these impairments are at least partially remediated by growth on media with 0.6 M KCl. Reduced sporulation at 30 C is not accompanied by notable developmental alterations in conidiophore or phialide morphology (data not shown)—reduced sporulation correlates instead with a lower number or smaller size of conidiophores. At 42 C several strains produce virtually no conidia—occasional rudimentary conidiophores with a few phialides and lacking swollen vesicles (data not shown) can be found in these cultures with careful searching.

	DISCUSSION

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In a transposon-mutagenized collection of 82 CFW-hypersensitive yeast strains (Lussier et al 1997), some 58% of the strains had major alterations in the balance of wall sugars. We had thus expected to find similar major changes in at least some of the cal mutants; however, the slight reduction in glucose content notwithstanding, this did not prove to be the case. We conclude that any wall defects present in these 10 cal mutants will prove to be of a relatively subtle nature, perhaps involving protein glycosylation or the linkages between major wall components. The absence of conspicuous changes in cell wall composition does, however, obligate us to present additional independent evidence that our screen has identified strains with wall defects. This evidence is provided in part by the mutants’ range of sensitivities toward several inhibitory agents known to perturb wall synthesis or inhibit protein glycosylation (TABLE III). Congo red (CR) is a polysaccharide-binding dye with a similar mode of action to that of CFW but with a greater binding affinity toward ß-1,3-glucans (Wood 1980). Like CFW, CR interferes with growth and causes morphological aberrations in filamentous fungi (Pancaldi et al 1984, Matsuoka et al 1985), and a wide range of cell wall mutants show hypersensitivity to the compound (e.g. Horiuchi et al 1999, Müller et al 2002, Oka et al 2004, Yamada et al 2005). Several cal mutant strains also show sensitivity to CR, though six of the10 are resistant to CR at one or both temperatures. We are unaware of other mutants that combine hypersensitivity to CFW with resistance to CR, though the differing structures of the two compounds, as well as their demonstrated differential affinity for ß-1,3-glucans (Wood 1980), argue that such a situation ought not to come as a surprise.

Echinocandins, of which Caspofungin is an example, inhibit ß-1,3-glucan synthesis in yeasts and filamentous fungi (Beauvais and Latgé 2001), causing growth defects in sufficient concentration (Bowman et al 2002). In yeasts, elevated sensitivity to echinocandins is shown by a number of cell wall mutants (e.g. Ram et al 1994, Carnero et al 2000, Martín et al 2003, Markovich et al 2004)—in the less studied filamentous fungi, the only report of which we are aware that correlates echinocandin sensitivity to a presumed wall defect is in a rho deletion strain of A. nidulans, which is also CFW-hypersensitive (Guest et al 2004). In yeasts, not all wall mutant strains that are CFW sensitive are also sensitive to echinocandins (Carnero et al 2000, Bates et al 2005), also seen amongst the cal mutants (TABLE III). Growth inhibition by SDS is another character commonly associated with wall structure defects in yeasts (deGroot et al 2001), where the effect has been correlated with increases in wall porosity, possibly allowing for easier solubilization of detergent-extractable cell wall proteins (deGroot et al 2005). In filamentous fungi, SDS, like CFW and Caspofungin, triggers the presumed cell wall integrity pathway of A. niger (Damveld et al 2005), indicating that its effect is at least partly on the wall. Elevated sensitivity to SDS has been shown by an A. nidulans double deletion strain lacking the chitin synthases chsA and chsC, which also shows hypersensitivity to CFW along with reduced sporulation (Fujiwara et al 2000).

Sporulation defects such as those shown by all cal mutants (TABLE VI) are frequently observed consequences of wall mutations, including several that involve defects in chitin synthesis (e.g. Aufauvre-Brown et al 1997, Fujiwara et al 2000, Müller et al 2002, Yamada et al 2005). Conidiation is also reduced in an A. nidulans strain deleted for the O-mannosyl-transferase gene pmtA/swoA (Oka et al 2005), the same gene as that mutated in calH. These sporulation defects are frequently osmotically remediable in the same manner as most cal mutants (TABLE VI). Osmotic remediability usually is considered to be strong evidence of a wall defect (d’Enfert 1997). The association is not absolute, however. Some apparent wall defects are not osmotically remediable (e.g. Yanai et al 1994, Aufauvre-Brown et al 1997, Müller et al 2002), and remediability has been observed in some mutants for which no theoretical connection to wall metabolism exists (e.g. Martin and Debusk 1975). Nevertheless, this property has been correlated with a sufficiently wide range of wall mutants to merit the conclusion that osmotically remediable characters are most likely due to wall defects, especially when they are accompanied by independent supporting phenotypes.

Among the most commonly observed secondary morphological phenotypes associated with cell wall defects is excessive swelling of spores and hyphal compartments (Momany 2005), although spore swelling also can result from genetic defects having no immediately obvious connection to wall metabolism (e.g., Shaw and Upadhyay 2005). With the exception of the calA8 strain, all cal strains reported in this study could be induced to show abnormal swelling through some combination of growth conditions that do not produce swelling in the wild type (TABLE V; FIGS. 2–9), and in all but one case (temperature-induced swelling of calD4) the phenotype is osmotically remediable. In those cases where a wall defect is thought to be involved, swelling is thought to result from the inability of a weakened wall to resist the uniform outwardly directed force of turgor. The inhibitors that induce swelling in cal mutants presumably do so by further weakening a wall that is already damaged. The probable mode of action of Nikkomycin and Caspofungin is intuitively straightforward. Tunicamycin, which blocks N-glycosylation (Tkacz and Lampen 1975) and interferes with protein folding (Mulder et al 2004), might reduce wall strength by preventing transport or incorporation of any of the several glycosylated proteins found in the wall (Kapteyn et al 1999, de Groot 2005). There was no correlation between the effects of these three agents on spore swelling (TABLE V) and their inhibitory effects on colony growth (TABLE III). Neither Tunicamycin nor Nikkomycin had a differential effect on growth at either temperature (data not shown), though each induced spore swelling in some cal mutants. (Tunicamycin’s effects on spore swelling at 42 C are not reported because germlings of all strains were so deformed that the swollen spore could not be reliably identified.) We conclude that the wall defects in some cal mutants are more pronounced during isotropic growth (spore swelling), while in others the defect more greatly affects polarized growth (colony development). This differential developmental effect extends even to the level of merely allelic differences. Caspofungin induces spore swelling at 42 C in calE3 but not in calE10, and at 30 C in calH6 but not in calH9.

The demonstration that Nikkomycin can induce a distinctly temperature-sensitive (ts) spore swelling in wild type strains A4, A28, AH12, and GR5 (TABLE V) indicates that the A. nidulans wall at 42 C is in some way different from the wall at 30 C. The recent demonstration that a heat shock transcription factor mediates cell wall remodeling in S. cerevisiae may be a relevant model in this regard (Imazu and Sakurai 2005). This observation may need to be considered in interpreting the mode of action of ts developmental mutations in A. nidulans. The standard assumption has been that ts phenotypes result from temperature-induced instability of proteins that fold normally at lower temperatures (Bainbridge et al 1979, King et al 2002). However we cannot now exclude the possibility that some ts developmental phenotypes in A. nidulans may result from alleles whose products function the same at both permissive and restrictive temperatures, but whose interactions with the wall are different at the two temperatures because the wall itself is different. It will be interesting to discover the nature of this intrinsic temperature-sensitive alteration of wall metabolism in A. nidulans and to understand how it relates to the effects of Nikkomycin and to the steps governed by those alleles whose defects cause similar phenotypes at 42 C.

In conclusion, we have isolated 10 A. nidulans mutants using CFW hypersensitivity as a primary screening strategy, and we document through the nature and the range of their secondary phenotypes the high probability that these mutations are in genes affecting cell wall metabolism. We are currently working to clone and characterize these genes.

	ACKNOWLEDGMENTS

 
A.J. Clutterbuck helped with advice about genetic nomenclature and osmotic remediation of non-wall-related phenotypes, and S. G. W. Kaminskyj helped with discussions about Mendelian methods. A. G. Teepe was a critical reader of the manuscript. This research was supported by NSF grant C-RUI-0211600 to TWH and DML, by DOE Biosciences grant DE-FG02-97ER20275 to MM, by a Merck Company Foundation grant to Rhodes College, and by funds from Rhodes College in support of undergraduate research.

	FOOTNOTES

 
Accepted for publication March 7, 2006.

¹ Corresponding author. E-mail: hill{at}rhodes.edu

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