Wangiella (Exophiala) dermatitidis WdChs5p, a Class V ChitinSynthase, Is Essential for Sustained Cell Growth at
Temperature of InfectionHongbo Liu,1 Sarah Kauffman,2 Jeffrey M. Becker,2 and Paul J. Szaniszlo1*
Section of Molecular Genetics and Microbiology, School of Biological Science and Institute of Cellularand Molecular Biology, The University of Texas at Austin, Austin, Texas 78712,1 and Microbiology
Department, University of Tennessee, Knoxville, Tennessee 379192
Received 2 October 2003/Accepted 4 December 2003
The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humansWangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its5� end and a chitin synthase (class V)-encoding region at its 3� end. Northern blotting showed that WdCHS5is expressed at high levels under conditions of stress. Analysis of the 5� upstream region of WdCHS5 fused toa reporter gene indicated that one or more of the potential regulatory elements present may have contributedto the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25°C but havesevere growth and cellular abnormalities at 37°C. Osmotic stabilizers, such as sorbitol and sucrose, rescued thewild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animalsurvival tests with a mouse model of acute infection showed that all wdchs5� mutants are less virulent than theparental strain. Reintroduction of the WdCHS5 gene into the wdchs5� mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required forthe sustained growth of W. dermatitidis at 37°C and is of critical importance to its virulence.
Wangiella (Exophiala) dermatitidis is a polymorphic, dema-tiaceous (melanized) fungal pathogen of humans which existspredominantly as a yeast form in vitro but can be easily ma-nipulated to undergo morphological transitions to produceisotropically enlarged yeast, multicellular forms and varioustypes of hyphae (10, 11, 17, 44, 45, 54). In vivo, this fungus alsoproduces various morphological forms, such as budding yeast,pseudohyphae, true hyphae, isotropically enlarged yeast cells,and sclerotic bodies (18, 21, 31). Although considered a para-digm for phaeohyphomycosis, because of its increasing detec-tion in cutaneous, subcutaneous, and central nervous systeminfections (18, 21, 22), it is better known as a model for themore than 100 other dematiaceous agents of mycoses (11, 44,45). The well-defined polymorphic features and well-docu-mented cell wall structure of W. dermatitidis, together with itsincreasing molecular tractability, make it also an excellent sys-tem for studies of fungal cell wall biosynthesis, as well as fungalcell wall-related virulence factors (27, 44). To date, both mel-anin and chitin have been shown to have relevance to the fullvirulence of this fungus (12, 19, 44, 50)
Chitin, the �(1,4)-linked homopolymer of N-acetylglu-cosamine, is an essential structural component of fungal cellwalls and plays an important role in fungal morphogenesis (8,35, 37). Chitin synthases (UDP-N-acetyl-D-glucosamine:chitin4-�-N-acetylglucosamine transferase; EC 2.4.1.16), which aremembrane-bound proteins, are responsible for the synthesisand deposition of this chitin (28, 30, 35). The presence ofdistinct classes of chitin synthases was initially indicated by the
identification and analysis of multiple chitin synthase genes invarious fungi, which have more recently been distributed be-tween two families (3, 4, 28, 32, 35, 37). Extensive study of thechitin synthases of Saccharomyces cerevisiae (ScChsp) has doc-umented that a specific function can be assigned to each of itsthree chitin synthases (8, 33). Briefly, ScChs1p (class I) isresponsible for the synthesis of chitin after cell separation andcounterbalances any chitinase activity: it is thus considered tobe a repair enzyme (5, 6). ScChs2p (class II) is responsible forchitin deposition in the primary septum (40, 41). Finally,ScChs3p (class IV) is responsible for chitin deposition in thering and lateral cell wall and contributes to the synthesis ofmost of the cell wall chitin during vegetative growth (36, 40).
Unlike the situation in S. cerevisiae, there is no general rulefor defining the specific function of each chitin synthase amongother fungi. To the contrary, it has been suggested that chitinsynthases of the same class may play different roles in differentfungi (28). Furthermore, the complicated life cycles of filamen-tous fungi may require them to have more chitin synthasesthan yeast, and isozymes of as many as six classes have beenidentified in some aspergilli (4, 7, 13, 24, 42). By use of genedisruption technologies, many of these chitin synthases havebeen proven to contribute directly to cell wall integrity and cellmorphogenesis (28). In pathogenic fungi, some have also beenshown to contribute directly or indirectly to virulence (28, 50).
In W. dermatitidis, chitin is found throughout the cell wall inhyphae and isotropic forms, but the primary site of chitinlocalization in yeast cells is in septal regions (9, 15, 23). Ini-tially, four different chitin synthase structural genes (WdCHS)were identified in this fungus; each of these encodes a memberof a different chitin synthase (WdChsp) class, as follows:WdCHS2, class I; WdCHS1, class II; WdCHS3, class III;WdCHS4, class IV (44). Characterization of mutants with each
* Corresponding author. Mailing address: Section of Molecular Ge-netics and Microbiology, University of Texas at Austin, Austin, TX78712-1095. Phone: (512) 471-3384. Fax: (512) 471-7088. E-mail:[email protected].
of these four WdCHS genes disrupted singly or in all possibledouble-mutant combinations revealed that no single WdChspis essential for the viability of W. dermatitidis at 25°C (44).However, double mutants with both WdCHS1 and WdCHS2disrupted grow poorly at 25°C, are incapable of growth at 37°C,and are avirulent (44, 48), whereas mutants with both WdCHS2and WdCHS3 disrupted have reduced virulence but grow nor-mally at both 25 and 37°C (50). In this report, we characterizea fifth chitin synthase structural gene (WdCHS5) in W. derma-titidis; this gene encodes a class V chitin synthase with a myosinmotor-like domain at its N-terminal region. We also reportthat disruption of this single gene produced mutants that werenormal at 25°C but could not sustain growth at 37°C and wereless virulent. Our discovery of WdCHS5 broadens the range ofthis kind of gene to a polymorphic fungus, which we think willeventually allow a more definitive functional characterizationof a class V isozyme with relevance to human pathogenicity.
MATERIALS AND METHODS
Strains, media, and growth conditions. The W. dermatitidis strains used in thiswork are listed in Table 1. The wild-type W. dermatitidis strain 8656 (ATCC34100 [E. dermatitidis CBS525.76]) and the temperature-sensitive mutants Mc3(wdcdc2; ATCC 38716) and Hf1 have been described in detail previously (10, 49,54). Routine propagation of these strains was carried out in the rich mediumYPD (2% peptone, 1% yeast extract, 2% dextrose) as reported previously (48,56). The synthetic defined (SD) medium (0.17% yeast nitrogen base withoutamino acids and ammonium sulfate, 0.2% ammonium nitrate, 0.1% asparagine,1% glucose) and the media for inducing development of the sclerotic or hyphalmorphologies were prepared as described previously (49). Methods for transfor-mation by electroporation of intact yeast cells have also been described previ-ously (48, 56). Drug selection plates for isolating W. dermatitidis transformantswere made by adding agar (1.5%, wt/vol) and hygromycin B (HmB; Sigma, St.Louis, Mo.) to YPD for selection for resistance conferred by the hygromycinphosphotransferase (hph) gene at a final concentration of 50 �g/ml or by addingchlorimuron ethyl (provided by J. Sweigard, Dupont Co., Wilmington, Del.) toSD for detection of resistance conferred by the sulfonyl urea resistance (sur)gene at a final concentration of 20 �g/ml. Escherichia coli XL-1 Blue (Stratagene,La Jolla, Calif.), which was used for subcloning and plasmid preparation, wasgrown in Luria-Bertani medium supplemented with ampicillin (100 �g/ml) orchloramphenicol (25 �g/ml). Growth rates of the wild type and the wdchs5�mutants were determined by three different methods, using cultures inoculatedwith 106 cells/ml in 50 ml of YPD. Samples from cultures grown at 25 and 37°Cwere collected at 4- or 8-h intervals and subjected to spectrophotometric, hema-cytometric, and plate viable counting procedures. Three independent measure-ments were performed, and the average at each time was used for the growthplots.
Nucleic acid manipulations and plasmids. W. dermatitidis genomic DNA wasisolated either as described previously (48, 56) by spheroplasting with Zymolyase20T (ICB Biomedicals, Inc., Aurora, Ohio) followed by detergent lysis or byusing a glass bead method adapted from reference 2. Briefly, the latter method
involved pelleting and washing cells with distilled, deionized water by centrifu-gation, adding breaking buffer (2% Triton X-100, 1% sodium dodecyl sulfate,100 mM NaCl, 10 mM Tris-Cl [pH 8.0], 1 mM EDTA), glass beads (diameter,400 to 520 �m; Thomas Scientific, Swedesboro, N.J.), and phenol-chloroform,and then vortexing vigorously using a Multi-Tube Vortexer (VWR International,West Chester, Pa.) for 3 min prior to the addition of Tris-EDTA (pH 8.0). Afterbrief vortexing and centrifugation, the genomic DNA was precipitated using100% ethanol, then washed with 75% ethanol and treated with RNase (RocheApplied Science, Indianapolis, Ind.). Methods for the isolation of total RNA andfor Southern and Northern blot analyses were as described previously (48, 56).DNA fragments used for probes were labeled with [�-32P]dCTP by using theDECA prime II DNA labeling kit (Ambion, Austin, Tex.). PCR amplificationconditions and primers chs51, chs53, chs5F, and chs5R, used for amplifying theWdCHS5 fragment, have been described previously (19, 47). Primers used forreverse transcription-PCR (RT-PCR) were designed as follows: pATG, 5�-ATGGCCACTCGAGGGAACGTC; pIntron1, 5�-GCTTTGGAAGCAGTTGGGTCG; pTGA, 5�-TCACAGTTGCCCAGACAAAAT; pIntron2, 5�-CTTCCCACTGGCTCTGTCTAT. Primers used to amplify 5� upstream sequences were asfollows: psmaI, 5�-TGTCCCGGGGGTGAACTTCAATGGC (the SmaI site isunderlined); p1.2, 5�-TCAGGGCCCATCAGAAGGAGCGGTA; p1.0, 5�-TCAGGGCCCAACTTGACCTCGACTT; p0.88, 5�-TCAGGGCCCAGAGGTAGGTTGGAAT; p0.68, 5�-TCAGGGCCCTATTCTAGAGGGTCTA; and p0.2, 5�-TCAGGGCCCTTGATTACGACTTGA (the ApaI sites are underlined).Methods for the cDNA synthesis and subsequent PCR and RT-PCR have beendescribed previously (47). Methods used for construction of the cosmid libraryand the ZAPII cDNA library have also been described previously (12, 49). Thepartial genomic library was constructed by using genomic DNA completelydigested with EcoRI. Following electrophoresis, the resulting 2.5- to 3.5-kbfragments were excised from the gel and ligated into the pBS-KS(�) vector,which was cut by EcoRI and dephosphorylated with calf intestine alkaline phos-phatase (Promega, Madison, Wis.). The ligation product was then transformedinto XL-l Blue competent cells, and the resulting subgenomic library of about6,000 independent clones was screened by colony hybridization using the PCRproduct generated by primers chs51 and chs53 as a probe.
The WdCHS5 integrative gene disruption plasmids, pHB0320 and pHB0510,were constructed by cloning 1.8-kb SacI and 1.2-kb SalI fragments from the 5�and 3� ends of WdCHS5 into corresponding sites of vectors pCB1636 andpCB1004 (50), respectively. After the resulting vectors were linearized with BclIor BstEII, respectively, to target integration into genomic sites of WdCHS5, theywere used to transform W. dermatitidis competent cells by electroporation asdescribed previously (48, 54). To construct the one-step replacement disruptionvector pHB0280, the 1-kb EcoRI-BamHI fragment of the 3� end was cloned intothe EcoRI and BamHI sites, and the 0.5-kb KpnI-PstI fragment (blunt ended byfilling in the PstI site with Klenow enzyme) of the 5� end of WdCHS5 was clonedinto the KpnI and ApaI sites (blunt ended by filling in the ApaI site with Klenowenzyme), of vector pCB1636, which positioned the hph gene to the middle of theconstruct. The 4.5-kb fragment obtained by digestion of pHB0280 with KpnI andBamHI was then used for transformation. The complementation vectorpHB2080, which contained the full-length WdCHS5 gene, was constructed bycloning a 3-kb EcoRI fragment of the 3� end of WdCHS5 into the EcoRI site ofplasmid pHB0051, which contained the 5� end of WdCHS5. The combinedfull-length WdCHS5 was then released with BssHII digestion, filled in withKlenow enzyme, and subsequently blunt-end ligated into the SmaI site ofpCB1551 (provided by J. Sweigard), which has a sur gene marker for selection.
TABLE 1. Strains used in this study
Strain or mutationa Parent strain Genotype or properties Reference orsource
Wd8656 Wild type ATCC 34100Mc3 Wd8656 wdcdc2; grows isotropically at 37°C ATCC 38716Hf1 Wd8656 Temperature-sensitive mutant; grows as hyphae at 37°C 49wdchs5�11 Wd8656 wdchs5::hph, WdCHS5 replacement This workwdchs5�236 Wd8656 wdchs5::hph, myosin-motor domain �b This workwdchs5�316 Wd8656 wdchs5::hph, chitin synthase domain �b This workwdchs5�11-1 wdchs5�11 wdchs5::hph-WdCHS5-sur This workwdchs�236-1 wdchs5�236 wdchs5::hph-WdCHS5-sur This workwdchs�316-1 wdchs5�316 wdchs5::hph-WdCHS5-sur This work
a All mutants used in this study were derived from the wild-type parental strain 8656.b � indicates site of WdCHS5 disruption.
VOL. 3, 2004 CHITIN SYNTHASE 5 (CLASS V) IN W. DERMATITIDIS 41
The resulting circular plasmid was then used for complementation of thewdchs5� mutants directly. Putative randomly reconstituted strains were selectedby prescreening for temperature-insensitive revertants, which were subsequentlyconfirmed to contain WdCHS5 by Southern blot analysis. All the plasmids foranalysis of the 5� upstream sequence of WdCHS5 were derived from pYEX303-gal (53). Briefly, the 5� upstream sequences of different lengths before the ATGstart codon were amplified with primer psmalI (which has a SmaI restrictionenzyme site) from one end and primers p1.2, p1.0, p0.88, p0.68, and p0.2 (whichhave ApaI restriction enzyme sites) from the other end, respectively. All the PCRproducts were subjected to SmaI digestion first and then to partial ApaI diges-tion. The corresponding 1.2-, 1.0-, 0.88-, 0.68-, and 0.2-kb fragments, and the0.45-kb fragment obtained from complete digestion of the 1.2-kb PCR productwith SmaI and ApaI, were used to replace the glaA promoter in plasmidpYEX303-gal to generate pHB8040, pHB9000, pHB9010, pHB9020, pHB9030,and pHB8050, respectively. Prior to transformation, the plasmids were linearizedwith FseI. The WdPKS1 fragment incorporated into these plasmids allowedstrains with site-specific integrations among HmB-resistant transformants to beidentified as white colonies (53).
Photomicroscopy. Light photomicroscopy of W. dermatitidis wild-type andmutant cells was performed by using an Olympus BX-60 microscope. The pro-cedures for staining cell wall chitin with Calcofluor (Sigma) or staining nucleiwith DAPI (4�,6�-diamidino-2-phenylindole; Accurate Chemical, Westbury,N.Y.) have been described previously (54). Scanning electron microscopy wascarried out as described previously (48), except that after fixation with cacody-late-buffered 2.5% glutaraldehyde, cells were not attached to coverslips pre-treated with polylysine.
Chitin synthase activity, chitin content, and �-galactosidase and alkalinephosphatase assays. Chitin synthase activities, chitin contents, and �-galactosi-dase activities were measured by methods described previously (48, 49, 56). Therelative activities of alkaline phosphatase were determined by the modifiedmethod of Nombela and coworkers (26). All assays were carried out at leastthree times. Differences among groups were evaluated for statistical significanceby the parametric one-way analysis of variance using the Newman-Keuls methodfor paired data. Statistical analyses were performed with the PRISM softwarepackage (version 2.0; GraphPad Software, Inc., San Diego, Calif.). Probabilityvalues of �0.05 were considered significant.
DNA sequence analysis. WdCHS5 was sequenced by the Institute of Cellularand Molecular Biology of the University of Texas at Austin. The deduced aminoacid sequence of WdCHS5 was determined, and sequences were compared, byusing the BLAST software system from the National Center for BiotechnologyInformation (NCBI) (http://www.ncbi.rlm.nih.gov/BLAST) and the ClustalWprogram from the European Molecular Biology Laboratory (EMBL) (http://www.ebi.ac.uk/clustalw). Promoter sequence analysis was performed by usingMacInspect software and the TRANSFAC program (version 4.0; BCM SearchLauncher).
Virulence tests and tissue burden analysis. Tests for virulence in an immu-nocompetent mouse model system were performed as described previously (12,48). Survival fractions in the virulence tests were calculated by the Kaplan-Meiermethod, and survival curves were tested for significant difference (P � 0.01) bythe Mantel-Haenszel test using GraphPad Prism software (version 3.0 for Win-dows). Probability values of �0.05 were considered significant. Tissue burdenanalysis involved injecting the wdchs5�11 mutant into mice and sacrificing themat days 1, 3, 5, and 10 postinfection to determine the fungal burdens of the brain,kidney, liver, and spleen.
Nucleotide sequence accession number. The nucleotide sequence of WdCHS5was assigned GenBank accession number AF469116.
RESULTS
The WdCHS5 gene encodes a class V chitin synthase(WdChs5p) with a myosin motor-like domain. A 362-bp PCRproduct was amplified by using the degenerate primers chs51and chs53 (19). Sequence analysis of the PCR product showedthat it encoded a peptide with high homology to other class Vchitin synthases. To isolate the whole gene, a cosmid libraryand a 3-kb EcoRI subgenomic library were screened by colonyhybridization using the WdCHS5 PCR product as a probe.Clones from each were then isolated, shown to contain theWdCHS5 gene, and subjected to restriction mapping (19) andsequence analysis. The nucleotide sequence of the cloned
WdCHS5 gene contained a single open reading frame of 5,655bp interrupted by two introns of 53 and 57 bp at its 5� and 3�ends, respectively (data not shown). The WdCHS5 gene en-coded a putative protein (WdChs5p) of 1,885 amino acids thathad six putative transmembrane helices, a calculated mass of208.9 kDa, and a pI of 7.76. Both introns had a consensussplice site that began with GT and ended with AG and weresubsequently confirmed by RT-PCR (Fig. 1) and by sequenceanalysis of the RT-PCR products (data not shown). Compar-isons by BLAST and ClustalW analysis (NCBI and EMBL,respectively) of the deduced protein sequence of WdChs5pwith those of other class V chitin synthases having myosinmotor-like domains indicated that it had 67, 66, 65, 65, 63, and41% identity to CsmA of Aspergillus nidulans (13), Chs2 ofBlumeria graminis (55), ChsA of Glomerella graminicola (1),ChsV of Fusarium oxysporum (20), Csm1 of Magnaporthe grisea(34), and PbrChs5p of Paracoccidioides brasiliensis (sequenceanalysis available at http//www.ncbi.nlm.nih.gov), respectively.The affiliation of WdChs5p with the class V family of chitinsynthases was further supported by the identification in itsN-terminal domain (first 800 residues) of a myosin motor-likeregion, which to date is associated only with members of thisisozyme class (28). Further support for a class V affiliation forWdChs5p was provided by the identification of a characteristicATP- or GTP-binding site motif (P-loop; GESGAGKT), lo-cated in the myosin motor-like region from amino acid resi-dues 99 to 106, and the presence of switch I (TASKAG) andswitch II (DFPGF) motifs at residues 148 to 153 and 407 to411, respectively. Two putative unconventional TATA boxeswere also found at bp �605 and �648. Cloning of the 3� endof the cDNA of the WdCHS5 gene by cDNA library screeningand subsequent analysis revealed that the polyadenylation sig-nal sequence began at a position 225 bp downstream of thestop codon.
Stress conditions result in increased cellular WdCHS5mRNA levels. Regulation of WdCHS5 expression was investi-gated by Northern blot analysis using a WdCHS5-specific PCRprobe amplified with primers chs5F and chs5R. Total RNA forthese assays was obtained from cells grown not only underconditions that included culture at elevated temperature(37°C) for the wild type and two temperature-sensitive mor-phological mutants (Mc3 and Hf1), in which WdCHS5 expres-sion had been assayed previously by semiquantitative RT-PCR(47), but also under additional stress conditions that are knownto initiate development of the sclerotic morphology (acidicconditions or Ca2� limitation induced by increasing EGTAconcentrations) or of hyphae (induced by nitrogen limitation)
FIG. 1. RT-PCR confirmation of the introns of WdCHS5. PrimerspATG and pIntron1 were used for amplification of intron 1 (53 bp)from cDNA (lane 4) and genomic DNA (lane 3), whereas primerspTAG and pIntron2 were used for amplification of intron 2 (57 bp)from cDNA (lane 2) and genomic DNA (lane 1). The introns were alsoconfirmed by direct sequencing of the RT-PCR products (data notshown).
in the wild type. In response to all these conditions, WdCHS5transcripts were detected at higher levels in stressed cells thanin control cells (Fig. 2). Because a similar differential expres-sion pattern was found previously for WdCHS3 (49), it was notsurprising to find two identical regulatory elements,REPCAR1 and STUAP, and several similar regulatory ele-
ments, such as STRE, ABAA, and HAP, in the upstreamregions of WdCHS3 (data not shown) and WdCHS5 (Fig. 3A),suggesting that these two genes may have similar mechanismsfor regulating their transcription.
The 5� upstream sequence of WdCHS5 contains importantregulatory regions. A series of 5� deletion fragments in theWdCHS5 upstream regulatory sequence (URS) was fused inframe with the LacZ gene (Fig. 3B) and then used to replacethe original glaA promoter of plasmid pYEX303-gal, whichalso contains a WdPKS1 targeting sequence. The use of theseconstructs ensured that all alleles were integrated into the
FIG. 2. Northern blot analysis of WdCHS5 expression. Samples oftotal RNA were prepared from the wild-type strain (wt) grown in YPDat 37 or 25°C (A), the temperature-sensitive mutants Mc3 and Hf1grown in YPD at 37 or 25°C (B), (C) the wild-type strain grown at 25°Cin modified Czapek dextrose (MCD) broth at pH 2.5 or 6.5 (C), andthe wild-type strain grown at 25°C in synthetic medium (SD) broth (pH6.5) (D) without nitrogen (first lane) or with the addition of 20, 5, 0.5,or 0 mM EGTA (second to fifth lanes, respectively). After beingprobed with WdCHS5, the same membranes for each sample set (up-per panels) were then stripped and probed with WdACT as controls toshow the approximately equal amounts of RNA loading (lower pan-els).
FIG. 3. Analysis of the 5� URS of WdCHS5. (A) Putative bindingsites for transcription factors. (B) Scheme of the upstream region ofWdCHS5 and truncation sites of constructs used for the �-galactosi-dase expression studies. (C) Expression levels of WdCHS5::LacZ re-porter fusions expressed in cells grown at 25 and 37°C.
VOL. 3, 2004 CHITIN SYNTHASE 5 (CLASS V) IN W. DERMATITIDIS 43
same nonessential WdPKS1 genomic locus, which is requiredfor melanin biosynthesis (49, 53). After the constructs werelinearized in the WdPKS1 sequence with FseI and transformedinto yeast cells, transformants were selected for resistance toHmB, and mutants with a site-specific integration were iden-tified by production of albino colonies. LacZ expression in cellsgrown at 25 and 37°C was quantitatively assessed by a �-galac-tosidase activity assay and by use of o-nitrophenyl-�-galactosi-dase as a chromogenic substrate. The resulting data (Fig. 3C)suggested two conclusions. First, all samples from cells grownat 37°C, except that from cells transformed with the plasmidcontrol without any WdCHS5 URS, had significantly higherlevels of �-galactosidase activity than those with correspondingconstructs from cells grown at 25°C, a finding that was consis-tent with the Northern blot analysis results (Fig. 2). Second,the most dramatic changes in �-galactosidase activities wereobserved among samples from cells with constructs havingtruncations between bp �880 and �450, which is the regionthat includes most of the potential cis-acting elements identi-fied by our sequence analysis. This finding indicated that atleast one negative regulator binding sequence exists betweenbp �880 and �680 and that another regulatory binding site(s)is localized between bp �680 and �450.
Disruption of WdCHS5 produces mutants that are hyper-pigmented and die at 37°C but are like the wild type at 25°C.To begin to elucidate the functions of WdChs5p, as well as thepossible function of each of its domains, three different dis-ruption vectors were constructed. Two were then used forsite-specific integrative gene disruptions that targeted the re-gions encoding the chitin synthase domain and the myosinmotor-like domain, respectively (data not shown). The thirdwas used for one-step replacement of the entire WdCHS5 gene(Fig. 4A). Southern blot analysis then identified numerousmutants (wdchs5�) of each type with site-specific gene disrup-tions that showed the expected band shifts for WdCHS5 (Fig.4B; data shown only for the wdchs5�11 disruption mutant). Allthree types of wdchs5� mutants had identical phenotypes,which differed significantly from that of the wild type at 37°Cbut not at 25°C. For example, at 25°C, all wdchs5� disruptionmutant strains grew normally in the manner of the wild-typestrain on YPD agar medium (Fig. 5A). In contrast, at 37°C, thedifferences between the three types of wdchs5� disruptionmutants and the wild type were very apparent on the agarmedium by 72 h (Fig. 5B) and became even more obvious astime passed: the mutant growth became much darker, andisolated colonies were smaller than wild-type colonies (com-pare colony pigmentation in Fig. 5B; compare colony sizes inFig. 5D, sector 1, with those in sectors 2, 4, and 6). Comple-mentation of each type of wdchs5� disruption mutant withWdCHS5 returned each strain to its wild-type phenotype (com-pare colony pigmentation in Fig. 5B; compare colony sizes inFig. 5D, sector 1, with those in sectors 3, 5, and 7). Darkeningof wdchs5� mutants occurred similarly in YPD broth but wasnot apparent with the complemented strains grown identically(data not shown).
Subsequent quantitative growth studies of the wdchs5� mu-tants and the wild-type strain in YPD broth confirmed thatdisruption of WdCHS5 did not affect the kinetics of growth at25°C when growth was measured by spectrophotometric, he-macytometric, and plate viable counting methods (data not
shown). However, each of these methods provided somewhatdifferent growth kinetic patterns when the mutants and thewild type were grown at 37°C (Fig. 6). Although subtle, thediscrepancies detected were easily explained by correlating themicroscopic and culture color change observations previouslynoted with data showing that death and lysis were not uncom-mon among cells grown at the higher temperature (see Fig. 7and 8 below). For example, the optical density of the wdchs5�cultures at 600 nm continued to increase even after 40 h (Fig.6A). However, this apparent increase was due to the mutantcells becoming much darker than the wild-type cells and notbecause there was an increase in the number of cells, as shownby both hemacytometry counting and viable counting proce-dures (Fig. 6B and C). More importantly, and as shown by thecolony count data, the number of viable wdchs5� cells de-creased rapidly after 40 h. Taken together, these results sug-gested that the wdchs5� mutants grew normally at 25°C but at37°C lost viability during late-log or early-stationary phase.This strongly indicated that WdChs5p is important for sus-tained cell growth and cell maturation at 37°C. This hypothesiswas strengthened by the demonstration that reintroduction ofthe wild-type WdCHS5 gene into the wdchs5� mutant back-grounds restored their wild-type growth kinetics (Fig. 6).
The wdchs5� mutants have abnormal yeast morphology at37°C. Because the different wdchs5� mutants all had the sameapparent phenotype, the wdchs5�11 mutant produced by the
FIG. 4. Disruption of WdCHS5 by one-step gene replacement.(A) Strategy for construction of the replacement vector. Abbrevia-tions: K, KpnI; P, PstI; A, ApaI; E, EcoRI; B, BamHI; Bg, BglII.Broken arrow indicates the probe used for Southern blot analysis.(B) Southern blot analysis of the wild type (lane 1), a representativedisruption mutant (wdchs5�11) (lane 2), and a representative comple-mentation strain (wdchs5�11-1) (lane 3).
replacement disruption strategy was used for more-extensivemicroscopic investigations. Observations of this mutantshowed that its phenotype could be distinguished from that ofthe wild type as early as 48 h of growth at 37°C in YPD liquidmedium (Fig. 7A and D) and that by 72 h the aberrant cellularmorphologies of the wdchs5�11 mutant had become consider-ably more obvious (Fig. 7G and J). By this time, the mutantcells had clearly begun to clump and form cell aggregations,whereas the wild-type cells did not. Also, individual cells of themutant often had swelled, with some swelling to about doublethe size of normal cells as time increased. Eventually, most ofthese cells lost their smooth surfaces to the extent that theybecame obviously crinkled. Many of these cells also becameflat or irregular and lost their cell wall integrity, and some evenlysed. Calcofluor staining showed that the mutant cells oftentended to have more cell wall chitin by 48 h (Fig. 7E), whichwas not as uniformly localized to septal regions as in the wildtype (Fig. 7B). However, by 72 h the differences between theCalcofluor staining patterns of the two strains had become lessapparent (Fig. 7H and K). Furthermore, DAPI stainingshowed that some of the mutant cells at 72 h had lost nuclei,indicating that they had probably leaked from the cytoplasmdue to the cell wall damage (Fig. 7L). Interestingly, the aber-rant phenotypes of the wdchs5�11 mutant could be reversed by
adding osmotic stabilizers, such as 1 M sucrose (data notshown) or 1.2 M sorbitol, to either YPD broth (Fig. 7M, N, andO) or YPD agar medium (data not shown). Under these con-ditions the mutants did not swell or lyse, which suggested thatthe lysis and death of the mutant at 37°C was, in fact, due tocell wall damage and loss of cell wall integrity.
Scanning electron microscopy confirmed that wdchs5�11cells had severe cellular defects when grown at 37°C for 72 h(Fig. 8A, B, and C) compared to wild-type cells (Fig. 8D, E,and F), with most mutant cells showing various irregularshapes; these cells often were enlarged and usually had morecrinkled surfaces. In addition, adhering materials of unknownorigin were associated with many wdchs5�11 cells; these pos-sibly represented cellular products that had leaked through thepore-like structures seen on the surfaces of some cells. Supportfor our hypothesis that the adhering material was of cytoplas-mic origin was provided by results from assays of alkalinephosphatase activity. These assays showed that by 72 h therelative alkaline phosphatase activity associated with the cul-ture medium of wdchs5�11 cells was about six times higher(5.67 0.626 [mean standard deviation for three indepen-dent measurements]) than that of wild-type cells (arbitrarily setat 1) and that this difference was eliminated by the addition of1 M sorbitol (0.95 0.545 versus 0.72 0.545, respectively).
FIG. 5. Colony characteristics of the wild-type strain, representative wdchs5� disruption mutants, and corresponding wdchs5� disruptionmutants complemented with WdCHS5 after growth on YPD agar medium for 3 days. (A) Strains incubated at 25°C. (B) Strains incubated at 37°C.(C) Key to sectors: 1, wild type; 2, wdchs5�11; 3, wdchs5�11-1; 4, wdchs5�236; 5, wdchs5�236-1; 6, wdchs5�316; 7, wdchs5�316-1. (D) Enlargedimages of the isolated colonies shown in each sector of panel B.
VOL. 3, 2004 CHITIN SYNTHASE 5 (CLASS V) IN W. DERMATITIDIS 45
Disruption of WdCHS5 does not reduce overall chitin syn-thase activity but does induce an increase in chitin content at37°C. No significant differences were detected between thetotal chitin synthase activities of the wild-type strain and thoseof wdchs5� mutant strains at either 25 or 37°C when activitieswere evaluated under zymogenic or nonzymogenic assay con-ditions (data not shown). Interestingly, significantly more (P �0.05) chitin (about 50%) was detected in the wdchs5� mutantsthan in the wild type, but only in cells cultured for at least 48 hat 37°C (data not shown). This paradox indicated that the cell
wall damage caused by the disruption of WdCHS5 induced acompensatory pathway, which caused the other chitin syn-thases to be activated or stimulated to produce more chitin, assuggested previously for mutants with WdCHS4 disruptions(48). Nonetheless, this newly synthesized chitin was not able torescue the damage caused by the loss of WdChs5p, becausethat damage was not repaired by the chitin produced by any ofthe other four WdChsp isozymes. As expected, complementa-tion of the wdchs5�11 mutant with WdCHS5 (the wdchs5�11-1strain) lowered the chitin content to that of the wild-type strainin cells cultured at 37°C for 48 h (data not shown).
The wdchs5� mutants have reduced virulence in mice. Asmight be expected of mutants with a temperature-lethal phe-notype, all three types of wdchs5� mutants showed significantreductions in virulence compared to that of the wild-type strainwhen tested in an acute murine infection model (Fig. 9). Re-introduction of the WdCHS5 gene into each type of wdchs5�mutant background reconstituted virulence. To determine tis-sue burdens of the brain, kidney, and liver or spleen, miceinjected with the wdchs5�11 mutant were sacrificed at days 1,3, 5, and 10 postinfection. Starting on day 3, the viable countsbegan to decrease in all the organs examined, suggesting thatthe cells were being cleared from the different tissues (data notshown). Furthermore, the infection was totally cleared fromthe liver or spleen by day 5 and was 98% cleared from the brainand kidney by day 10. These results indicated that the mutantcells were incapable of sustained growth and survival under theelevated temperature associated with the mice.
DISCUSSION
This report documents that WdChs5p has relevance to thevirulence of W. dermatitidis, because it is essential for sustainedgrowth and viability at 37°C but not at 25°C. Cloning of theWdCHS5 gene and analysis of its deduced amino acid sequenceshowed that a myosin motor-like domain was fused to a chitinsynthase domain, a condition only found among class V chitinsynthases. Although the significance of this type of putativefusion gene remains unknown, it is becoming clear that genesencoding class V isozymes are probably not uncommon infilamentous fungi (28, 37). So far at least 10 similar chitinsynthases with this unique structure have been identified. Fur-thermore, the class V chitin synthases are apparently presentonly in filamentous fungi and some dimorphic and polymor-phic fungi that produce true hyphae (28, 38; this study). How-ever, prior to the present investigation, only CsmA of A. nidu-lans and, to lesser extents, ChsA of G. graminicola and ChsV ofF. oxysporum had received extensive study (1, 13, 16, 20, 46).Particularly, the studies of CsmA established for the first timethe important role of this type of chitin synthase in the main-tenance of hyphal wall integrity and polarized hyphal wallsynthesis, and especially its importance in hyphal growth andmorphogenesis under low osmotic conditions (16, 46). With G.graminicola it was found that ChsA is essential for conidial wallstrength in media with high water potential and contributes tothe strength of hyphal tips, whereas F. oxysporum mutants withdefective ChsV have hyphae with abnormal swellings, whichare eliminated by culture with osmotic stabilizers (1, 20). Theresults of our study similarly demonstrated that WdChs5p
FIG. 6. Comparisons of growth rate and viability of the wild-typestrain, the wdchs5�11, wdchs5�236, and wdchs5�316 disruption mu-tants, and the wdchs5�11-1, wdchs5�236-1, and wdchs5�316-1 comple-mentation strains grown in YPD liquid medium at 37°C and measuredby optical density (A), hemacytometer counts (B), and viable counts(C). The initial inoculation level was 106 cells/ml.
functions in the manner of CsmA, ChsA, and ChsV in main-taining cell wall integrity in W. dermatitidis, but only at 37°C.
Our Northern blot analysis provides support to the priorsuggestion that WdCHS5 is a stress response gene (47).WdCHS5 mRNA was found at significantly higher levels incells grown at 37°C (47; this study) and under a number ofadditional stress conditions known to promote morphologychanges in W. dermatitidis, such as Ca2� limitation, nitrogen
starvation, and low pH. The importance of this gene to viabilityand virulence, and the interesting expression pattern of itstranscription, prompted us to further characterize its promoterregion. Our analyses suggested that a negative regulatory ele-ment(s) exists in the 5� URS of WdCHS5. Studies have previ-ously demonstrated that the CsmA gene of A. nidulans alsodisplays different temporal patterns of expression and that itspromoter region has several cis-acting elements similar to
FIG. 7. Cellular morphologies and staining properties of the wild-type strain and the wdchs5�11 disruption mutant. Wild-type (A, B, C, G, H,and I) and wdchs5�11 disruption mutant (D, E, F, J, K, L, M, N, and O) cells were grown at 37°C in YPD medium for 48 h (A, B, C, D, E, andF) or for 72 h without (G, H, I, J, K, and L) or with (M, N, and O) 1.2 M sorbitol, then fixed with 5% formaldehyde, stained with Calcofluor orDAPI, and viewed by Nomarski phase-contrast or fluorescence microscopy. All cells are shown at the same magnification. Arrows point to mutantcells that were swelled or enlarged and occasionally lost their nuclei.
VOL. 3, 2004 CHITIN SYNTHASE 5 (CLASS V) IN W. DERMATITIDIS 47
those found in the URS of WdCHS5, such as STRE, ABAA,and HAP (46). Similar findings have been made with WdCHS3(37, 49; J.-H. Oh and P. J. Szaniszlo, unpublished data), indi-cating that W. dermatitidis has at least two chitin synthase genesthat are differentially expressed in response to a variety ofconditions. However, unlike the situation with disruption ofWdCHS5, disruption of WdCHS3 does not produce a temper-ature-sensitive phenotype. Nonetheless, we speculate thatthese two genes share a similar regulatory mechanism or globalregulation, although the exact mechanisms responsible for theincreased mRNA levels of both WdCHS5 and WdCHS3 instressed cells have not been determined. While it is still pos-sible that posttranscriptional regulation is also contributing tothe increased mRNA levels of both genes detected, we favorthe hypothesis that the increased mRNA levels of WdCHS5 inthe stressed cells resulted mainly from interactions betweentrans-acting factors and the cis-acting elements identified in itsURS. Support for this possibility is provided by previous datafrom a semiquantitative RT-PCR study that detected compa-rable amounts of WdCHS5 mRNA in cells grown continuouslyat 37°C for 24 h and in cells grown at 25°C for 21 h and thenshifted to 37°C and grown for an additional 3 h (47). Resultsfrom that study, at least for the response to the temperatureshift, which are confirmed by the Northern blot analysis re-ported here, strongly argue that the increased level ofWdCHS5 mRNA detected at 37°C was not due to the increasedmRNA half-life, because the semiquantitative RT-PCR didnot detect a larger amount of WdCHS5 mRNA in cells growncontinuously at 37°C than in cells subjected to the temperatureshift for just 3 h.
Studies of CsmA also suggest that posttranslational process-
ing is necessary for activating the chitin synthase domain in A.nidulans, and they further gave rise to the notion that themyosin motor-like domain might contribute to CsmA localiza-tion. However, no evidence supports the latter hypothesis orsuggests that the putative myosin motor associates with actin.Nonetheless, different myosins with different functions havebeen identified in cells, and some myosin tail domains containa structural motif that may be used to direct the interaction ofa given myosin with its cargo (25, 43). Thus, it remains tempt-ing to suggest that the N-terminal domain of WdChs5p is, infact, a myosin motor that is necessary at 37°C for the properlocalization of the C-terminal chitin synthase domain and con-sequently the newly synthesized chitin by interaction with ac-tin. Support for this idea is provided by results with S. cerevi-siae, where Myo2p, a class V myosin, acts as a transport motorrequired for delivery of chitin synthase 3 to the growing buds(39). The recent finding that ChsZp of Aspergillus oryzae en-codes a second chitin synthase with a myosin motor-like do-main in that fungus, which is different enough from that of itsChsYp isozyme and those of other class V chitin synthases tosuggest its inclusion in a new class (class VI), further indicatesthat the ultimate elucidation of the myosin motor-like do-main’s function is extremely important (7). Studies aimed atdetermining the mechanisms of WdChs5p localization and thepossible interactions of its myosin motor-like domain with ac-tin are in progress. Among the results of these studies was thepreliminary finding that the wdchs5�11 mutant could not becomplemented with just the chitin synthase domain ofWdChs5p, or with only the lysine residue in the conserved Ploop of Wdchs5p mutated to alanine (H. Liu and P. J. Szanis-zlo, unpublished data).
FIG. 8. Scanning electron micrographs of the wdchs5�11 mutant (A, B, and C) and the wild type (wt) (D, E, and F) grown in YPD broth at37°C for 72 h. Arrows in panels A through C point to enlarged or irregular mutant cells; arrowhead in panel C identifies a putative pore. Notealso adhering materials in panels A through C. Bars, 5 �m.
Three different methods were used to disrupt WdCHS5. Ineach case, disruption of WdCHS5 resulted in mutants withtemperature-sensitive phenotypes. At 25°C, the mutant yeastcells grew normally both in agar and in liquid medium, but at37°C, dramatic changes in cell morphology were observed withprolonged incubation. This resulted in mutant cells that be-came hyperpigmented, were often swollen, and died in largenumbers, frequently by lysis. These observations suggest thatthe loss of WdChs5p function results in yeast cell wall weak-ening at the elevated temperature, which in turn brings aboutthe loss of cell viability. This scenario is consistent with thehypothesis that WdChs5p has an essential function at the tem-perature of infection. Reintroduction of the WdCHS5 genesuccessfully complemented the temperature-sensitive pheno-type, confirming that that phenotype resulted solely from theloss of WdCHS5 itself. The fact that wdchs5� mutant cells areidentical to wild-type cells at 25°C further suggests that eitherWdChs5p does not function at this temperature or one ormore of the other chitin synthases compensate for the loss ofWdChs5p at lower temperatures. In either case, it is obviousthat none of the other four WdChsp isozymes can compensatefor the loss of WdChs5p at the higher temperature. Under thescenario that WdChs5p does not have a function at 25°C,together with conclusions from our previous studies that noneof the other four chitin synthases is essential for cell viability at25°C, we hypothesized that the disruption of each other singleWdCHS gene in the wdchs5� background would be withouteffect at 25°C. On the other hand, if one of the other WdChsp’scompensated for the loss of function of WdChs5p at 25°C, then
disruption of WdCHS5 in certain other wdchs� backgroundsshould produce strains with major defects or incapable ofgrowth at both 25 and 37°C. Investigations in progress, aimedat eliminating the latter possibility, have resulted in the pro-duction of all four types of double disruption mutants witheither WdCHS1, WdCHS2, WdCHS3, or WdCHS4 disruptedtogether with WdCHS5 (unpublished data). Preliminary anal-yses of these double mutants indicate that when WdCHS5 isdisrupted in a wdchs� background, the resulting yeast cells aresimilar to those of the wdchs5� single mutant at 37°C andeither have a wild-type phenotype at 25°C or exhibit the veryminor chaining abnormality characteristic of wdchs1� mutantsor an even darker pigmentation than wdchs4� mutants, respec-tively, as reported previously for those single mutants (44, 48).
Cell wall weakening and abnormal cell shapes resulting fromthe disruption of a single chitin synthase gene have been doc-umented in a number of different fungi, including Candidaalbicans (CaCHS1), A. nidulans (chsC, chsA, and CsmA), As-pergillus fumigatus (ChsE), Neurospora crassa (Chs1), G. gra-minicola (ChsA), and F. oxysporum (FoCHSV) (1, 14, 16, 20,29, 42, 46, 52). Interestingly, the growth defects associated withthese mutants resulted from the inactivation of chitin synthasesof a number of classes. In addition, the defects were observedpredominantly in older hyphal regions, and those defects couldoften be suppressed with an osmotic stabilizer. In W. dermati-tidis, similarly, the weakening of the yeast cell wall and the lossof cell viability at 37°C caused by the disruption of WdCHS5were observed only when mutant cells entered mature stages ofgrowth. Thus, it appears that this temperature-sensitive phe-
FIG. 9. Mouse survival analyses after injection with the wild type (wt 8656), wdchs5� disruption mutants, and wdchs5� mutants complementedwith WdCHS5. One or more groups of 10 mice received injections of log-phase yeast cells of each strain. The injections contained 9 109 cellsper mouse, and the mice were monitored for 14 days to determine the survival rate. When two or more groups were involved, the data presentedare averages at each time point. Survival fractions were calculated by the Kaplan-Meier method, and survival curves were tested for significantdifference (P � 0.01) by the Mantel-Haenszel test using GraphPad Prism software (version 3.00 for Windows).
VOL. 3, 2004 CHITIN SYNTHASE 5 (CLASS V) IN W. DERMATITIDIS 49
notype is different from that of a wdchs1�wdchs2� doublemutant, which is not able to grow at all at 37°C (44, 48). Thisfinding strongly suggests that WdChs5p, or the chitin synthe-sized by WdChs5p, is essential only for sustained or progres-sive yeast cell growth at elevated temperatures. We speculatethat either WdChs1p or WdChs2p, or residual WdChs5p, isresponsible for the initial cell growth at 37°C but thatWdChs5p is then required for maintaining that growth at hightemperatures. Possibly, mutant cells devoid of WdChs5p cangrow for several generations at 37°C, but when cells becomemature and older, especially while intracellular contents areincreasing, their cell walls require extra strength to resist therise in internal pressures. The observation that the tempera-ture-sensitive phenotype can be rescued by supplementing cul-tures with osmotic stabilizers, such as sorbitol and sucrose,supported this idea and confirmed that the resulting cell lysisand death at 37°C are largely due to the loss of cell wallintegrity. Under this scenario, the loss of virulence of wdchs5�mutants in our mouse model of acute infection would similarlybe due to their inability to maintain cell wall integrity at tem-peratures of infection. This hypothesis is supported by nec-ropsy data showing that the number of viable wdchs5� cells inmice decreased significantly with time: most or all of the in-fection was cleared from all the organs assayed by day 5 (datanot shown). We suspect that the abnormal temperature-sensi-tive phenotype of the wdchs5� single mutants is a major factorcontributing to this clearance and thus that the chitin contrib-uted by WdChs5p is essential for the virulence of the wild-typestrain.
To our knowledge, our data provide the first direct evidencethat a chitin synthase can be a specific virulence factor in aconidiogenous fungal pathogen of humans. Previously, theonly single chitin synthase disruption mutants unequivocallyknown to be less virulent were Chs3 (class IV) mutants of thedimorphic pathogenic yeast C. albicans, although Chs1 in thatfungus can arguably also be considered essential for virulencebecause it is required for viability (28). Interestingly, inactiva-tion of UmChs5p (class IV) in Usilago maydis and of ChsV inF. oxysporum produces mutants with reduced plant virulence(20, 51). Thus, among the filamentous fungi, drugs that targetthe production or function of chitin synthases of class IV orclass V may be more efficacious than those that target mem-bers of the other chitin synthase classes.
ACKNOWLEDGMENTS
We thank J. M. Mendenhall (Institute of Cellular and MolecularBiology, The University of Texas at Austin) for help with scanningelectron microscopy.
This research was supported by a grant to P.J.S. from the NationalInstitute of Allergy and Infectious Diseases (AI 33049).
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