The conserved and divergent roles of carbonic anhydrases in the filamentous fungi Aspergillus...

The conserved and divergent roles of carbonic anhydrasesin the filamentous fungi Aspergillus fumigatus andAspergillus nidulansmmi_7058 1372..1388

Kap-Hoon Han,1 Yoon-Hee Chun,1

Bárbara de Castro Pimentel Figueiredo,2

Frederico Marianetti Soriani,2 Marcela Savoldi,2

Agostinho Almeida,3 Fernando Rodrigues,3

Charlie Timothy Cairns,4 Elaine Bignell,4

Jaqueline Moisés Tobal,2 Maria Helena S. Goldman,5

Jong-Hwan Kim,6 Yong-Sun Bahn,7

Gustavo Henrique Goldman2* andMárcia Eliana da Silva Ferreira2

1Department of Pharmaceutical Engineering, WoosukUniversity, Wanju, Korea.2Laboratório Nacional de Ciência e Tecnologia doBioetanol (CTBE) and Faculdade de CiênciasFarmacêuticas de Ribeirão Preto, Brazil.3Life and Health Sciences Research Institute (ICVS),School of Health Sciences, University of Minho, Braga,Portugal.4Department of Microbiology, Imperial College London,London, United Kingdom.5Faculdade de Filosofia, Ciências e Letras de RibeirãoPreto, Universidade de São Paulo, São Paulo, Brazil.6Department of Bioinformatics, Soongsil University,Seoul, Korea.7Department of Biotechnology, Center for FungalPathogenesis, Yonsei University, Seoul, Korea.

Summary

Carbon dioxide (CO2) and its hydration product bicar-bonate (HCO3

-) are essential molecules in variousphysiological processes of all living organisms. Thereversible interconversion between CO2 and HCO3

- isin equilibrium. This reaction is slow without catalyst,but can be rapidly facilitated by Zn2+-metalloenzymesnamed carbonic anhydrases (CAs). To gain an insightinto the function of multiple clades of fungal CA, wechose to investigate the filamentous fungi Aspergil-lus fumigatus and A. nidulans. We identified four andtwo CAs in A. fumigatus and A. nidulans, respectively,

named cafA-D and canA-B. The cafA and cafB genesare constitutively, strongly expressed whereas cafCand cafD genes are weakly expressed but CO2-inducible. Heterologous expression of the A. fumiga-tus cafB, and A. nidulans canA and canB genescompletely rescued the high CO2-requiring pheno-type of a Saccharomyces cerevisiae Dnce103 mutant.Only the DcafA DcafB and DcanB deletion mutantswere unable to grow at 0.033% CO2, of which growthdefects can be restored by high CO2. Defects in theCAs can affect Aspergilli conidiation. Furthermore, A.fumigatus DcafA, DcafB, DcafC, DcafD and DcafADcafB mutant strains are fully virulent in a low-dosemurine infection.

Introduction

Carbon dioxide (CO2) and its hydration product bicarbon-ate (HCO3

-) are essential molecules in various physiologi-cal processes of all living organisms. CO2 is the end-product of cellular respiration in aerobic microorganismsand mammals while plants, algae and cyanobacteria areable to undergo CO2 fixation during photosynthesis. CO2

represents only 0.033% of the atmospheric gases, but it isfound at concentrations of roughly 5–6% in the humanbloodstream and in tissues, where respiration takes place(Bahn and Mühlschlegel, 2006). CO2 is able to diffusethrough lipid-containing cell membranes because it is anon-polar molecule, but bicarbonate is negatively chargedand not permeable to phospholipid bilayers (Casey, 2006;Missner et al., 2008). The reversible interconversionbetween CO2 and HCO3

- is in equilibrium and it is spon-taneously balanced. This reaction is slow without catalyst,but can be rapidly facilitated by Zn2+-metalloenzymesnamed carbonic anhydrase (CA, carbonate lyase; carbon-ated dehydratase; E.C.4.2.1.1).

Based on their amino acid sequence and protein struc-ture CAs are categorized into five distinct classes, a, b, g,d and z, which share no sequence similarity, and appearto have evolved independently (Hewett-Emmett andTashian, 1996; Tripp et al., 2001; Supuran, 2008a,b).Alpha-CA isoforms, but not beta-CA, have been isolatedin mammals, while plants and fungi encode both a- and

Accepted 13 January, 2010. *For correspondence. [email protected]; Tel. (+55) 16 36024280/81; Fax (+55) 1636331092.

Molecular Microbiology (2010) 75(6), 1372–1388 � doi:10.1111/j.1365-2958.2010.07058.xFirst published online 9 February 2010

© 2010 Blackwell Publishing Ltd

b-class CAs (Tripp et al., 2001; Bahn and Mühlschlegel,2006; Fabre et al., 2007). Archaea and eubacteria haveg-class, but this class has also been identified in plantmitochondria (Smith et al., 1999; Parisi et al., 2004).Finally, both z- (containing a cadmium in the active centreinstead of zinc) and d-classes have been discovered inmarine diatoms (McGinn and Morel, 2008; Xu et al.,2008).

All the CAs that have been functionally characterized infungi belong to the b-class although some fungi appear tocontain a-CA (for reviews, see Bahn and Mühlschlegel,2006; Supuran, 2008a,b; Elleuche and Pöggeler, 2009b;2010). Fungal b-CA was first characterized in the buddingyeast Saccharomyces cerevisiae (Götz et al., 1999;Amoroso et al., 2005). S. cerevisiae CA, named Nce103, isrequired for growth under ambient conditions, but dispens-able in high CO2 conditions (Götz et al., 1999; Amorosoet al., 2005). Another ascomycete fungus, the humanpathogen Candida albicans, contains the NCE103 geneand its deletion generates a mutant that requires elevatedCO2 levels (~5%) for growth (Klengel et al., 2005). In C.albicans, the b-CA works as a CO2 scavenger essential forpathogenicity in niches where the available CO2 is limited,such as epithelial cell surfaces (Klengel et al., 2005). Incontrast, the basidiomycete human pathogen Cryptococ-cus neoformans, causing fungal meningitis, contains twob-CAs, Can1 and Can2, whose functions have recentlybeen elucidated (Bahn et al., 2005; Mogensen et al.,2006).Among these, the CAN2 gene was shown to encodeits major CA and is required for growth of C. neoformansunder ambient air (Bahn et al., 2005). Interestingly, Can2 isdispensable for infection of C. neoformans in the mamma-lian host which provides high CO2 (Bahn et al., 2005).

Besides its role in cellular growth, CA also plays animportant role in signalling pathways that mediate viru-lence and differentiation of fungal pathogens. In C. albi-cans, the activity of adenylyl cyclase producing cAMP andthus activating the cAMP-signalling pathway can beenhanced in the presence of bicarbonate and CA (Klengelet al., 2005; Mogensen et al., 2006). In C. neoformans,adenylyl cyclase activity was also shown to be activated byCA in vitro (Mogensen et al., 2006). Furthermore, themating ability of C. neoformans that is diminished by highCO2 can be partially restored by mutation of the CAN2gene (Bahn et al., 2005). Therefore, fungal CAs are crucialnot only for cell survival and proliferation, but also forvarious CO2-related signalling cascades that are importantfor virulence and differentiation of pathogenic fungi.

Recent phylogenetic analysis of fungal CAs indicatedthat fungal b-CAs can be subgrouped into six differentclades (Bahn and Mühlschlegel, 2006). S. cerevisiae andC. albicans Nce103 proteins belong to the clade III ofb-CAs while C. neoformans and Ustilago maydis CAsbelong to the clade VI (Bahn and Mühlschlegel, 2006).

Multiple fungal CAs were found to be present in manyfungi, including most Aspergillus species, Magnaporthegrisea, Neurospora crassa, which mostly belong to cladesI, II, IV and V (Bahn and Mühlschlegel, 2006). However,the functions of these clades of b-CA remain unknown.

To gain an insight into the function of multiple clades offungal b-CA, we chose to investigate the filamentous fungiAspergillus fumigatus and A. nidulans, which have four(clade I, II, III and IV) and two (clade I and II) b-Casrespectively. Here, we demonstrated by mutant analysisthat CanB in A. nidulans and CafA and CafB in A. fumi-gatus are essential CAs controlling normal growth of thefilamentous fungi in ambient air.

Results

A. fumigatus has four CA-encoding genes

Aspergilli have multiple CAs, mostly of the b-class (Fig. 1;for a review, see Bahn and Mühlschlegel, 2006). Interest-ingly, Aspergillus oryzae has two CAs, one that belongs tothe b-class and a single a-CA (GenBank BAE66418)(Fig. 1A; for reviews, see Bahn and Mühlschlegel, 2006;Elleuche and Pöggeler, 2009b). We were able to identifyfour and two CAs in A. fumigatus and A. nidulans, respec-tively, named cafA-D (CA in A. fumigatus) (Afu4g11250,Afu8g06550, Afu4g09420 and Afu8g06554) and canA-B(CA in A. nidulans) (AN5611.3 and AN1805.3; Fig. 1).Originally the cafD gene (Afu8g06554) was annotated andregarded as the 5′ part of the cafB gene (Afu8g06550),because the two genes are adjacently located. However,cDNA analysis as well as in silico analysis showed that thecafB and cafD genes encode separate CAs and are notclustered together. Although the Central AspergillusData Repository (CADRE) (http://www.cadre-genomes.org.uk/Aspergillus_fumigatus/protview?peptide=AFUA_8G06554&db=core) annotated the cafD gene asAfu8g06554 which encodes a 181-amino-acid-longpolypeptide, our cDNA analysis revealed that the putativeCafD protein contains 30 amino acids more at itsN-terminus (for a more detailed analysis of A. fumigatuscafB-D gene junction, see Fig. 1C). We compared andanalysed the phylogenetic relationship among these sixCAs with other Aspergilli CAs, C. neoformans, C. albicans,Neosartorya fischeri, Sordaria macrospora and S. cerevi-siae (Fig. 1A). To gain a better understanding of the cellularlocalization of CAs in A. fumigatus, we performed in silicoanalysis by using TargetP (http://www.cbs.dtu.dk/services/TargetP/) and MitoProt II (http://ihg2.helmholtz-muenchen.de/ihg/mitoprot.html). CafA and CafD werepredicted to be located in the mitochondria, whereas CafBand CafC were predicted to be cytoplasmic. A. nidulansCanA and CanB were predicted to be cytoplasmic. Theconserved b-CA domain is located in the central part ofCafA, CafB and CafD, and in the N terminus of CafC

Aspergillus carbonic anhydrases 1373

© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 75, 1372–1388

(Fig. 1B). According to the recent classification proposedby Elleuche and Pöggeler (2009b), CafA and CanA areplant-like b-CAS1 CAs whereas CafB and CanB are plant-like b-CAS2 CAs; CafC and CafD are Cab-like b-CAS3CAs. For the gene structure of cafA-D and canA-B, seeTable S1.

To discern caf gene regulation in A. fumigatus, we com-pared mRNA levels of each gene by quantitative reversetranscription-PCR (RT-PCR) with mRNAs isolated from

strains growing at either ambient or high CO2 conditions(5%) for 12 and 24 h incubation (Fig. 2). The mRNA levelsof cafA and cafB were at least 10 and 100 times higherthan cafC and cafD, respectively, at both CO2 concentra-tions (Fig. 2). cafA and cafB mRNA levels increased aboutfour to five times between 12 and 24 h of growth in eitherof 0.033 or 5% CO2 respectively (Fig. 2A and B). Althoughno significant difference in expression of either gene wasobserved between the two CO2 concentrations, cafC and

A

Fig. 1. Phylogenetic tree and multiple sequence alignment of the zinc coordinating region from fungal b-class. The followings proteins wereused for the analysis: A. niger (e_gw1-14.637), A. niger (est_GWPlus_C_110710), A. terreus (ATEG_03956), A. flavus (AFL2G_01941), A.oryzae (AO090003001096), A. clavatus (ACLA_050340), N. fischeri (NFIA_105170), A. fumigatus cafA (Afu4g11250), A. nidulans canA(AN5611.3), A. niger (gw1-10.890), S. macrospora cas1 (CAT00780), C. neoformans can1 (CNAG_02805) e C. neoformans can2(CNAG_05144), S. macrospora cas2 (CAT00781), A. nidulans canB (AN1805.3), A. clavatus (ACLA_007930), N. fischeri (NFIA_099180), A.fumigatus cafB (Afu8g06550), S. cerevisiae (SCRG_03173), A. fumigatus cafD (Afu8g06554), A. clavatus (ACLA_007940), N. fischeri(NFIA_106590), A. fumigatus cafC (Afu4g09420), S. macrospora cas3 (CAT00782), C. albicans (CAWG_01616), A. flavus (AFL2G_06317), A.terreus (ATEG_10070) and A. oryzae (AO090010000582 – a-class CA).A. Phylogenetic tree. This tree was constructed by the neighbour-joining method. Topology was also evaluated by bootstrap analysis (MEGA4program; 1000 repeats). The numerical values in the trees represent bootstrap results. The distance between two strains is the sum of thebranch lengths between them.B. Multiple sequence alignment of the zinc coordinating region from fungal b-class CAs was made in CLUSTAL W2(http://www.ebi.ac.uk/Tools/clustalw2/index.html) using default parameters. Conserved amino acids important for Zn2+-coordination are markedby an asterisk. The dashed box marks the region encoding the part of the protein which was used for the alignment at the top.C. Scheme of the junction region between cafB and cafD genes (bp = base pairs).

1374 K.-H. Han et al. �


cafD mRNA levels were greatly induced by high CO2

compared with 0.033% CO2 at both time points (about 20and 50 times; Fig. 2C and D). Taken together, cafA andcafB are constitutively, strongly expressed genes whereascafC and cafD are weakly expressed, but CO2-induciblegenes.

Only the DcafA DcafB double deletion mutant wasunable to grow at 0.033% CO2

We generated A. fumigatus cafA, cafB, cafC and cafD nullalleles using an in vivo S. cerevisiae fusion-based

approach both in CEA17-80 and Af293.1 backgrounds(see Experimental procedures). Correct allelic replace-ments of cafA-D were verified by either diagnostic PCR orSouthern blot analysis in several transformants. Indepen-dent mutants for each CA gene exhibited identical pheno-types and one of which was used for further phenotypiccharacterization (Fig. S1). First, we examined which cafdeletion mutants exhibit high CO2-requiring (HCR) pheno-types as found in other fungal CA mutants. Surprisingly,none of the caf deletion strains showed striking growthdefects under either ambient or high CO2 conditions,although the DcafA and DcafC mutants had more pro-

B

C

Fig. 1. cont.



nounced reductions in conidiation in MM (Fig. 3A). Inaddition, the DcafA mutant showed a slightly reducedradial growth when grown in CM at 5% CO2 (Fig. 3A). Inorder to determine which CAs are crucial for growth anddevelopment, we constructed a series of double CA dele-tion mutants. First, we selected for pyrG - auxotrophicstrains by point-inoculating DcafA, DcafB and DcafCmutant strains in YUU plates containing 5-fluoroorotic acid(5-FOA) in a sub-inhibitory concentration of 0.65 mg ml-1.These resulted in the recovery of sectors for three strainsthat were auxotrophic to uridine and uracil and resistantup to 0.75 mg ml-1 of FOA (data not shown). These strainswere named CAFA, CAFB and CAFC. The allelic replace-ment of the cafA, cafB and cafC genes in these strainswas again confirmed either by diagnostic PCR or South-ern blot analysis (data not shown). Strikingly, only theDcafA DcafB double deletion mutant, but not DcafA DcafC,DcafB DcafC, DcafB DcafD and DcafC DcafD doublemutants, was unable to grow in both MM and CM at0.033% CO2 and this growth defect was restored by highCO2 (5%) (Fig. 3B), indicating that CafA and CafB playredundant roles in regulating growth of A. fumigatus underambient air. The double mutant DcafA DcafB has a slightlyreduced growth in CM at 5% CO2 (Fig. 3B). All these

mutants were constructed in both CEA17-80 and Af293.1strains and displayed essentially the same phenotypes inboth strains (data not shown). Unexpectedly, repeatedattempts to construct a DcafA DcafD double mutant wereunsuccessful, suggesting a possible synthetic lethal inter-action between these two genes.

Aguilera et al. (2005) have shown that S. cerevisiae CAplays a physiological role in providing bicarbonate sub-strates for carboxylating enzymes, including pyruvatecarboxylase, acetyl-CoA carboxylase and CPSase(carbamoyl-phosphate synthetase). Thus, we tried tocomplement the defect of the DcafA DcafB double mutantby supplementing the MM with a combination of uracil,adenine, arginine, aspartate and methyl palmitate or eachof these compounds alone. However, we were unable tosee any growth restoration or nuclei duplication in conidiaof this double mutant with the supplements (data notshown), indicating that essential enzymes or proteinsother than the carboxylases may be affected by CA in A.fumigatus. We also tested different concentrations ofbicarbonate (5, 10, 25 and 50 mM), which was not effec-tive for growth restoration of the DcafA DcafB doublemutant (data not shown). As bicarbonate is negativelycharged it cannot freely diffuse into the cells unlike

Fig. 2. mRNA accumulation of the A.fumigatus carbonic anhydrase genes.Real-time RT-PCR was the method used toquantify the mRNA. The measured quantity ofthe mRNA in each of the treated samples wasnormalized using the CT values obtained forthe b-tubulin (Afu1g10910) mRNAamplifications run in the same plate. Therelative quantification of cafA (A), cafB (B),cafC (C) and cafD (D) genes and tubulin geneexpression was determined by a standardcurve (i.e. CT values plotted against logarithmof the DNA copy number). The results are themeans � standard deviation of four sets ofexperiments (*P < 0.001).

1376 K.-H. Han et al. �


Fig. 3. Growth phenotypes of A. fumigatuswild-type and mutant strains grown in differentmedia and carbon dioxide concentrations.A. Wild type, DcafA, DcafB, DcafC and DcafDmutant strains.B. DcafA DcafB, DcafB DcafC, DcafA DcafCDcafB DcafD and DcafC DcafD.



non-polar CO2. Neither of these treatments improved theconidiation in the DcafA and DcafC mutants (data notshown).

We next investigated which A. fumigatus CA genescould complement the S. cerevisiae nce103 mutant byheterologously expressing cDNAs of the cafA-cafDgenes under the control of the constitutively activeADH1 promoter. Heterologous expression of the A. fumi-gatus cafB gene completely rescued the HCR pheno-type of a S. cerevisiae Dnce103 mutant that lacksintracellular CA activity (Fig. 4). However, heterologousexpression of the cafA, cafC and cafD genes wasunable to complement the Dnce103 mutant defect(Fig. 4), indicating that CafB is a functional orthologuefor the S. cerevisiae Nce103.

Taken together, these results suggest that CafA andCafB are the two major CAs, playing important roles forgrowth and conidiation of A. fumigatus under ambient CO2

concentrations whereas CafC plays a minor role inconidiation.

The role of CAs in asexual differentiation ofA. fumigatus

We noticed that both A. fumigatus DcafA and DcafCmutant strains had reduced asexual sporulation in both0.033% and 5% CO2 at 37°C (Fig. 3A). We thereforeexamined and compared the conidiation of the wild typeand DcafA-C mutants upon 24 h of post-asexual develop-mental induction (Fig. 5A). Both DcafA and DcafC mutants

Fig. 4. Heterologous expression ofA. fumigatus cafB gene rescues growth of theS. cerevisiae nce103D mutant. HaploidS. cerevisiae nce103D mutant straincontained vector (pTH19) or cafA-D cDNAsunder the control of ADH1 promoter weregrown on Synthetic Dextrose (SD) mediumlacking uracil and incubated in ambient orhigh CO2 conditions at 30°C.

A

B

Fig. 5. A. fumigatus DcafA and DcafCmutant strains have decreased conidiation.A. Twenty-four hours growth synchronizedasexual developmental induction for A.fumigatus wild type, DcafA, DcafB, DcafC andDcafD.B. Number of conidia from A was evaluatedby sampling four 0.5 cm2 agar fragmentsrandomly distributed at about a quarter of thecolony diameter from wild type and DcafA-Dmutant strains. The conidia were collected in0.01% Tween 20 and counted using ahaemocytometer.C. Fold increase in cafA-D mRNA levelsduring the synchronized asexualdevelopmental induction for A. fumigatus wildtype.D. Fold increase in AfbrlA RNA levels duringthe synchronized asexual developmentalinduction for A. fumigatus wild type, DcafA,DcafB, DcafC and DcafD. Real-time PCR wasthe method used to quantify the mRNA inboth C and D. The measured quantity of themRNA in each of the treated samples wasnormalized using the CT values obtained forthe b-tubulin (Afu1g10910) mRNAamplifications run in the same plate. Therelative quantification of all the genes andtubulin gene expression was determined by astandard curve (i.e. CT values plotted againstlogarithm of the DNA copy number). Theresults are the means � standard deviation offour sets of experiments (*P < 0.05).

1378 K.-H. Han et al. �


were impaired in conidiation compared with the wild-typestrain and DcafB and DcafD mutants (notice the absenceof green colour in the mycelia from the two DcafA andDcafC mutants versus the wild type and DcafB and DcafDmutants; Fig. 5A). The levels of conidiation in the DcafAand DcafC mutants were dramatically reduced (about30% of the wild-type strain and DcafB and DcafDmutants), indicating that CafA and CafC play important

roles in conidiation of A. fumigatus (Fig. 5B). Surprisingly,the DcafA DcafC double mutant showed conidiation levelscomparable to the wild-type strain (compare Fig. 3A with3B), indicating that CafA and CafC may play opposingroles in controlling conidiation of A. fumigatus. Otherwise,it is possible there could be some increased expression ofeither cafB and/or cafD in this strain that could compen-sate for the conidiation defect of the DcafA DcafC double

C

D

Fig. 5. cont.



mutant; however, this was not investigated in this currentwork.

Accordingly, we observed an increase in cafC mRNAaccumulation during synchronized asexual developmentalinduction (4 and 13 times and 9–280 times at 12–24 hgrowth in the presence of 0.033% and 5% CO2 respec-tively; Fig. 5C). However, cafA has not shown any increaseand cafB showed only a modest increase in their mRNAaccumulation during asexual developmental induction (forcafB, about three times at 6 h growth in the presence of 5%CO2; Fig. 5C). In contrast, cafD showed an increase of 2.5and 2.0 times in its mRNA levels at 12 and 24 h, respec-tively, in the presence of 5% CO2 (Fig. 5C).

In Aspergilli two developmental regulators, AfbrlA andAfwetA are specifically expressed during conidiation (Mahand Yu, 2006). Thus, as a first step to understand thereduced levels of conidiation in the DcafA and DcafCmutants, we measured mRNA abundance for the AfbrlAgene during synchronized asexual developmental induc-tion using real-time RT-PCR (Fig. 5D). In the wild-typestrain, the AfbrlA mRNA levels increased dramaticallyduring the synchronous asexual development (about80–120 times and 200–500 times in the wild type andDcafB mutant strains respectively; Fig. 5D, upper andlower left graphs). AfbrlA mRNA accumulation decreasedin the DcafC (about 20–50 times, except at 12 h growth inthe presence of 0.033% CO2 when reached 180 times;Fig. 5D, lower right graph). Interestingly, the DcafA mutantdisplayed comparable levels of AfbrlA mRNA accumula-tion to the wild type and DcafB mutant strains (Fig. 5D,upper right graph). These results suggest that the conidi-ation defect in DcafA mutant strain does not directly affectbrlA transcription.

Taken together, these results suggest that the CAs playa key role in A. fumigatus conidiation.

A. fumigatus DcafA, DcafB, DcafC, DcafD and DcafADcafB mutant strains are fully virulent in a low-dosemurine infection

Recently, it was shown that CAs are not required for invivo growth or virulence of C. neoformans because themammalian host provides sufficient levels of CO2 tosupport the growth of the CA mutant (Bahn et al., 2005).However, C. albicans Nce103 is a CA that is essential forpathological growth of the pathogen in niches where suf-ficient CO2 is not supplied by the host (Klengel et al.,2005). Therefore, virulence of A. fumigatus DcafA, DcafB,DcafC, DcafD and DcafA DcafB mutants, in comparisonwith the wild-type strain, were determined in a neutro-penic murine model of invasive aspergillosis. Similar tothe C. neoformans Dcan2 mutant, none of DcafA, DcafB,DcafC, DcafD or DcafA DcafB mutants showed differencesin virulence, neither in terms of overall mortality or median

survival times (Fig. 6A–C). Mortality rates among thetested strains were indistinguishable, being above 90%after 14 days post inoculation of the conidia. These resultsindicate that the CAs are not required for virulence.However, we cannot completely discard a possible role of

A

B

C

Fig. 6. A. fumigatus carbonic anhydrases do not contribute tovirulence in neutropenic mice.A. Comparative analysis of DcafA (n = 16), DcafB (n = 15), and wildtype (n = 15).B. Comparative analysis of DcafA DcafB (n = 15) and wild type(n = 15).C. Comparative analysis of DcafC (n = 7), DcafD (n = 7), and wildtype (n = 15) in a neutropenic murine model of pulmonaryaspergillosis. Mice were inoculated with a 40 ml suspension ofconidiospores at a dose of 2.5–5.0 ¥ 105. Mice were sacrificedaccording to an end-point of 20% reduction in body weightmeasured from the day of infection.

1380 K.-H. Han et al. �


CAs in A. fumigatus virulence because we have not con-structed triple or quadruple deletion mutants for the CAs.

A. nidulans CAs

We also verified the mRNA levels for both canA(AN5611.3, the homologue of cafA, Afu4g11250) andcanB (AN1805.3; the homologue of cafB, Afu8g06550)genes under 0.033% and 5% CO2 concentrations(Fig. 7A). The mRNA levels of canA were higher thanthose of canB: at 0.033% CO2, canA showed 2.0–3.8

times higher mRNA levels (after 12 and 24 h of growthrespectively) than canB, while at 5% CO2, canA exhibited4.8–23 times higher mRNA levels (after 12 and 24 h ofgrowth respectively) than canB (Fig. 7A). Interestingly, themRNA levels for both genes at 0.033% CO2 after 24 h ofgrowth were equivalent to those after 12 h of growth at 5%CO2 (Fig. 7A). However, there was 32.4 and 16.5-foldinduction of canA and canB, respectively, from 12 to 24 hat 0.033% CO2 (Fig. 7A). In contrast, there was 2.7 and13.0-fold reduction of canA and canB, respectively, from12 to 24 h at 5% CO2 (Fig. 7A).

As a first step to understand the role of CAs in A.nidulans, we deleted the two CA homologues (see Fig.S1). Both the wild-type strain and DcanA mutant grewwell in complete or minimal medium at 5% CO2 and0.033% CO2 (Fig. 7B, first and second rows). However,the DcanB mutant did not grow at 0.033% CO2 in minimalor complete medium (Fig. 7B, third row). Interestingly,heterologous expression of either A. nidulans canA orcanB genes under the control of the constitutively activeADH1 promoter completely rescued the HCR phenotypeof a S. cerevisiae Dnce103 mutant that lacks intracellularCA activity (Fig. 7C), indicating that both CanA and CanBhave functional CA activity. Both wild-type and DcanAmutant strains conidiate less in complete or minimalmedium at 5% CO2 than at 0.033% CO2 (Fig. 8A). Inter-estingly, the DcanB mutant demonstrates at least atwofold increase in conidiation in MM, relative to the wild-type strain and DcanA mutants (Fig. 8A).

We also verified the mRNA accumulation of both canAand canB genes during synchronized asexual develop-mental induction using real-time RT-PCR (Fig. 8B). Asexpected, in the wild-type strain, the brlA mRNA levelsincreased dramatically during synchronous asexualdevelopment (about 5000 times and 30 000 times after 12and 24 h respectively). canA mRNA accumulationincreased about 20 and 60 times after 12 and 24 h,

A

B

C

Fig. 7. The A. nidulans canB is essential for growth at 0.033%CO2.A. mRNA accumulation of the A. nidulans carbonic anhydrasegenes. Real-time RT-PCR was the method used to quantify themRNA. The measured quantity of the mRNA in each of the treatedsamples was normalized using the CT values obtained for theb-tubulin (tubC) mRNA amplifications run in the same plate. Therelative quantification of canA and canB genes and tubulin geneexpression was determined by a standard curve (i.e. CT valuesplotted against logarithm of the DNA copy number). The results arethe means � standard deviation of four sets of experiments.B. Growth phenotypes of A. nidulans wild type and mutant strainsgrown in different media and carbon dioxide concentrations.C. Heterologous expression of A. nidulans canA and canB genesrescue growth of the S. cerevisiae nce103D mutant. Haploid S.cerevisiae nce103D mutant strain contained vector (pTH19) orcanA-B cDNAs under the control of ADH1 promoter were grown onSynthetic Dextrose (SD) medium lacking uracil and incubated inambient or high CO2 conditions at 30°C.



A

B

C D

1382 K.-H. Han et al. �


respectively, at 0.033% CO2, while it increased about 20and 180 times after 12 and 24 h, respectively, at 5% CO2.canB mRNA accumulation increased about 30 and 180times after 12 and 24 h, respectively, at 0.033% CO2,while it increased about 10 and 150 after 12 and 24 h,respectively, at 5% CO2.

Neither canA nor canB deletion mutants were sterile(data not shown). Interestingly, high CO2 induced moresexual development in both the wild-type strain andDcanA-B mutants, which showed an increased number ofcleistothecia at 5% CO2 than at 0.033% CO2, especially inveA + background (Fig. 8C). This indicates that the CO2-mediated enhancement of sexual development of A. nidu-lans appears to be independent of CA. To addresswhether the canA and canB mRNA levels are related toinduction of sexual differentiation processes, real-timeRT-PCR was performed using total RNA samples isolatedfrom sexual developmental induction stages and sexuallyinduced conditions (Fig. 8D). canA mRNA accumulationonly slightly increased (~3-folds) after 96 h while canBmRNA accumulation significantly increased (~6–10-folds)after 96 and 120 h.

We constructed a DcanA DcanB double mutant bycrossing. As predicted, the double mutant grew as well asthe wild-type strain at 5% CO2 but not at 0.033% CO2

(data not shown). Again, we were unable to complementthe defect of the DcanB or DcanA DcanB double mutant byadding a combination of uracil, adenine, arginine, aspar-tate and methyl palmitate or each of these compoundsalone, or bicarbonate (data not shown).

Complementation of the A. fumigatus and A. nidulansCA deletion mutants

We also investigated if the different A. fumigatus and A.nidulans CA mutants could be homologously or heterolo-gously complemented by different Aspergillus CAs. Wechecked whether growth at 0.033% CO2 and conidiationdefects of the DcafA DcafB and DcanB mutants could becomplemented by different Aspergillus CAs. The growthdefects of the DcafA DcafB and DcanB mutant at 0.033%CO2 can be complemented only by cafA or cafB, andcanB respectively. Interestingly, the complementation ofthe DcafA DcafB mutant by cafB was only partial. The

DcafA DcafB + cafB complemented strain was not able toconidiate, providing an additional evidence that CafAplays an independent role in conidiation (Table S2). Inter-estingly, similar partial complementation was observedwhen canB was used to complement the DcafA DcafBmutant. The canB gene was able to complement thegrowth defects, but not the conidiation defect, of the DcafADcafB mutant at 0.033% CO2 (Table S2). Taken together,multiple CAs play conserved and divergent roles in A.fumigatus and A. nidulans.

Discussion

In this study we aimed to characterize the function ofCAs in two ascomycete filamentous fungi, A. fumigatusand A. nidulans. We found that two CAs, CafA andCafB, play a major role in regulating growth underambient air conditions in A. fumigatus whereas in A.nidulans, a single CA, CanB, plays a predominant role.The growth defects of the Aspergillus CA mutants canbe rescued by high CO2 concentration, which is a typicalHCR phenotype observed in most microbes lacking CAactivity (Bahn and Mühlschlegel, 2006). Thus, CA activ-ity seems to be dispensable for growth and proliferationinside the mammalian host where high CO2 concentra-tions are available. Although these results suggest thatCafA and CafB play redundant roles in regulating growthunder ambient air, some of our data also suggest thatthe two CAs could have different functions. The S. cer-evisiae mutation was only complemented by A. fumiga-tus cafB, but not by cafA, cafC, or cafD. Interestingly, thecafA-B homologues, canA-B, in A. nidulans, can comple-ment the S. cerevisiae nce103 mutation. Second, cafAand cafB are highly expressed under 0.033% and 5%CO2, but cafA appears slightly upregulated after 24 hincubation under high CO2, whereas cafB is slightlydownregulated after 24 h incubation under high CO2.The mRNA accumulation of these two genes is muchhigher than those of the cafC-D genes. CO2-responsivetranscriptional regulation of cafC-D is also notable.Although expression levels of cafC-D are considerablylower in both ambient and high CO2 conditions thanthose of cafA and cafB, cafC and cafD are clearlyupregulated by high CO2. Transcriptional regulation of

Fig. 8. Influence of canA and canB genes on the A. nidulans asexual and sexual development.A. Number of conidia was evaluated by sampling four 0.5 cm2 agar fragments randomly distributed at about a quarter of the colony diameterfrom wild-type and DcanA-B mutant strains. The conidia were collected in 0.01% Tween 20 and counted using a haemocytometer.B. Fold increase in brlA and canA-B mRNA levels during the synchronized asexual developmental induction for A. nidulans wild type.C. High CO2 induces sexual development; bar, 1 mm.D. Fold increase in canA-B mRNA levels during the sexual developmental induction for A. nidulans wild type. Real-time PCR was the methodused to quantify the mRNA in both B and D. The measured quantity of the mRNA in each of the treated samples was normalized using the CT

values obtained for the b-tubulin (tubC) mRNA amplifications run in the same plate. The relative quantification of all the genes and tubulingene expression was determined by a standard curve (i.e. CT values plotted against logarithm of the DNA copy number). The results are themeans � standard deviation of four sets of experiments (*P < 0.05).



CA in response to CO2 levels has been also reported inother fungi. In S. cerevisiae, the NCE103 gene is tran-scriptionally upregulated by low CO2 (Amoroso et al.,2005). In S. macrospora, the cas1 and cas3 genes aredifferentially regulated by CO2 levels during development(Elleuche and Pöggeler, 2009a). In C. albicans and C.neoformans, however, CA genes are not regulated inresponse to change of CO2 levels. Interestingly, we werenot able to construct the DcafA DcafD double mutant,suggesting a possible interaction between cafA andcafD. It remains to be determined the contribution ofthese genes at protein levels and activities. Our resultsprovide the molecular basis for a deeper investigation ofthe function of the CA genes in Aspergilli. Takentogether, CA is important for growth and differentiation ofsome filamentous fungi, including A. nidulans and A.fumigatus, under ambient air conditions.

The most important finding in this study is the pres-ence of two major CAs in A. fumigatus, which is in starkcontrast to other pathogenic or non-pathogenicfungi including A. nidulans. In both S. cerevisiae and C.albicans, only a single CA, named Nce103, is respon-sible for CO2 sensing and metabolism essential forgrowth under ambient air (for a review, see Bahn andMühlschlegel, 2006). In contrast, C. neoformans con-tains two CAs, Can1 and Can2 (although CAN2 isexpressed at higher levels than CAN1) and can2 muta-tion, but not can1 mutation, results in a HCR phenotype(Bahn et al., 2005). Similar to C. neoformans, A. nidu-lans also contains two CAs, CanA and CanB, betweenwhich the latter appears to be a dominant CA becauseits deletion causes a HCR phenotype. Interestingly,however, A. fumigatus contains four CAs, CafA, CafB,CafC and CafD, none of which generate HCR pheno-types when mutated. Only DcafA DcafB double muta-tions, but not DcafA DcafC, DcafB DcafC, DcafB DcafDor DcafC DcafD, generate HCR phenotypes in A. fumi-gatus, indicating that the pathogen contains two domi-nant CAs for sensing low levels of CO2 in ambient air.Similar to A. fumigatus, another filamentous ascomycetefungus, S. macrospora, has been also reported tocontain multiple functional CAs, two of which, Cas1 andCas2, are required for growth under ambient air(Elleuche and Pöggeler, 2009a). The presence of mul-tiple, functional CAs for vegetative growth under ambientair conditions is therefore commonly found in filamen-tous ascomycetes.

We also investigated a possible role of the cafA-Dgenes in virulence of A. fumigatus. None of the single CAmutants lost its virulence and surprisingly the DcafA DcafBdouble mutant was also virulent. Similarly, CAN2 is dis-pensable for infection of C. neoformans in the mammalianhost that provides high CO2 (Bahn et al., 2005). In C.albicans, the NCE103 gene is essential for pathogenicity

in niches where the available CO2 is limited, such asepithelial cell surface (Klengel et al., 2005). However, theimportance of CAs in virulence of A. fumigatus cannot becompletely discarded because we have not investigatedthe virulence of other double mutants, or even triplemutants.

Carbonic anhydrase rapidly catalyses the reversibleinterconversion between HCO3

- and CO2, which are thedominant inorganic carbon species at physiological pHvalues. Mutations of the CA genes might disturb the physi-ological balance between CO2/H2O and HCO3

-/H+,decreasing bicarbonate, but not CO2 that is generated bycellular respiration, and thus affect physiological pH regu-lation. CA is known to play a central role in intracellularbuffering and CO2 transport (Nelson and Cox, 2008), and inthe regulation of intracellular (Roos and Boron, 1981) andextracellular pH (Chen and Chesler, 1992) in red bloodcells. Thus, we decided to investigate if there was a con-nection between CA and pH regulation in Aspergilli. As firststep, we checked the sensitivity to different pHs of thewild-type strain and pacC and palB mutants of A. nidulansand A. fumigatus at different CO2 concentrations. PacCbelongs to the conserved family of PacC/Rim101 transcrip-tion factors that control the expression of a subset of pHregulated genes (for a review, see Peñalva et al., 2008).PacC controls acid- and alkaline structural gene expres-sion according to extracellular ambient pH (Caddick et al.,1986; Tilburn et al., 1995). Alkaline pH generates signalsthrough the protein products of six pal genes, including twopotential plasma membrane pH sensors, PalH and PalI(Arst et al., 1994; Denison et al., 1998; Negrete-urtasunet al., 1999), and a signalling protease, probably PalB(Denison et al., 1995), which catalyses the pH-dependentfirst proteolytic step of PacC processing (Diéz et al., 2002).We evaluated if the A. nidulans pH mutant strains weremore sensitive to different pHs at either 0.033% or 5% CO2

than the wild-type strain. As shown in the Table S3, nodifferences were observed in terms of growth rate andconidiation between the wild strain and the caf mutants.Neither are there any differences in growth rate betweenthe A. fumigatus wild-type strain and pacC mutants indifferent pH and CO2 conditions (data not shown).

The A. fumigatus CAs influenced asexual develop-ment because both DcafA and DcafC mutants exhibitedreduced asexual sporulation in both 0.033% and 5%CO2. However, the mechanisms that connect CafA andCafC to conidiation seem to be different. Only cafCmRNA accumulation increases during synchronizedasexual developmental induction, suggesting that othermechanisms exist for translational and/or post-translational modification of CafA. The master transcrip-tional activator of conidiation, brlA, also has decreasedmRNA accumulation in the DcafC mutant but it has nosignificant differences in terms of mRNA accumulation

1384 K.-H. Han et al. �


with the wild-type strain in the DcafA mutant strain. Incontrast, there appears no direct influence of A. nidulansCAs on conidiation. Another key finding made by thisstudy is that high CO2 levels strongly induce sexual dif-ferentiation of A. nidulans. Accordingly, both canA andcanB genes showed increased mRNA accumulationduring the early stages of sexual development. Weobserved that sexual reproduction of A. nidulans isgreatly enhanced by high CO2, which is in stark contrastto the case in C. neoformans (Bahn et al., 2005).However, neither canA nor canB is involved in the CO2-mediated induction of sexual reproduction of A. nidulans.In particular, the DcanB mutant, which is defective invegetative growth in ambient air, shows wild type-likesexual induction upon exposure to high CO2, indicatingthat the CO2-mediated induction of sexual reproductionis independent of CA activity. Involvement of CO2 insexual differentiation has been reported in other fungi.For example, high levels of CO2 repress sexual differen-tiation of C. neoformans by blocking pheromone produc-tion during initial stages of mating (Bahn et al., 2005),which could be one of the reasons why C. neoformanscannot undergo mating in the host. This mating defectcan be partially rescued by mutation of CAN2, indicatingthat increased intracellular bicarbonate by high CO2

inhibits the initial stage of mating (Bahn et al., 2005).However, the Dcan2 mutant is still defective in basid-iospore formation under high CO2 conditions, indicatingthat CA activity is required for the terminal stages ofsexual differentiation in C. neoformans. Similar to C.neoformans, S. macrospora CAS1 and CAS2 are alsoinvolved in sexual reproduction (Elleuche and Pöggeler,2009a). The cas2 mutation causes dramatic reductionsin ascospore germination, whose defects cannot be fullyrestored by 5% CO2 (Elleuche and Pöggeler, 2009b).However, CO2 itself does not appear to affect sexualreproduction of S. macrospora, which is in contrast to C.neoformans. In order to mate, C. albicans must undergohomozygosis at the mating type-like locus MTL, thenswitch from the white to opaque phenotype (for a review,see Soll, 2009). Recently, Huang et al. (2009) haveshown that CO2 stabilizes the opaque phenotype, andfound that physiological levels of CO2 induce white-to-opaque switching and stabilize the opaque phenotype at37°C. These results suggest that the high levels of CO2

in the host induce and stabilize the opaque phenotype,thus facilitating mating. The relationships among CAs,sexual development and high concentrations of CO2

deserve further investigation.In summary, our study shows that CAs are important for

the growth of Aspergilli in ambient CO2 concentrationsand also for asexual conidiation, but not for virulence orpH-responsiveness of the filamentous fungi. Our workwith CAs opens exciting new avenues for research into

environmental sensing and nutrient acquisition in thisimportant fungal genus.

Experimental procedures

Strains and media methods

A. fumigatus strains used are CEA17-80, DcafA, DcafB,DcafC, DcafD, DcafA + cafA (mutant strain complementedwith cafA), DcafB + cafB, DcafA DcafB, DcafA DCafC, DCafBDCafC, DcafB DcafD and DcafC DcafD. Also, Af293.1 wasalso used for knock-out of the cafA-D genes. Media were oftwo basic types. A complete medium with three variants:YAG (2% glucose, 0.5% yeast extract, 2% agar, trace ele-ments), YUU (YAG supplemented with 1.2 g l-1 each ofuracil and uridine) and liquid YG or YG + UU medium of thesame compositions (but without agar). Trace elements aredescribed by Kafer (1977). A modified minimal medium withthree different pHs was also used: MM pH 6.5, MM pH 5.0(1% glucose, 20 ml l-1 Aspergillus salt solution, 1% OxoidAgar, pH 6.5 or pH 5.0 with 4 M NaOH) and MM pH 8.0(1% glucose, 20 ml l-1 salt solution for pH 8.0 media,2.7 mM NaH2PO4, 50 mM Na2HPO4, 1% Oxoid Agar, pH 8.0with 4 M NaOH). The media was buffered with 200 mM gly-colic acid pH 5.0, 200 mM MES (2-morpholinoethanesul-phonic acid) for pH 6.5 or 200 mM Tris-HCl for MM forpH 8.0.

Aspergillus salt solution: 26 g l-1 KCl, 26 g l-1 MgSO4,76 g l-1 KH2PO4, 50 ml l-1 trace element solution. Salt solutionpH 8.0: 26 g l-1 KCl, 26 g l-1 MgSO4, 50 ml l-1 trace elementsolution. Trace elements: 40 mg l-1 Na2B4O7.10H2O,400 mg l-1 CuCl2.5H2O, 800 mg l-1 FeCl3.H2O, 800 mg l-1

MnSO4.4H2O, 800 mg l-1 Na2MoO4.2H2O. All the growthexperiments for A. fumigatus and A. nidulans were performedat 37°C.

The number of conidia was determined by sampling four0.5 cm2 agar fragments randomly distributed at about aquarter of the colony diameter from wild-type and mutantstrains previously point inoculated and grown on MM for 4days at 37°C in atmospheric air (0.033% CO2) and 5% CO2.The conidia were collected in 0.01% Tween 20 and countedusing a haemocytometer.

For synchronized asexual developmental induction, 5 ¥ 107

conidia of the wild-type and mutant strains were inoculated in50 ml liquid MM with 0.1% yeast extract and incubated at37°C and 150 r.p.m. for 16 h (0 h for developmentalinduction). Then, mycelia were harvested by filtration throughMiracloth (CalBiochem, California), transferred to solid MMwith 0.1% yeast extract, and further incubated at 37°C.Samples for RNA isolation were collected (16 h liquid cultureand 6, 12 and 24 h post-asexual developmental induction),squeeze dried, stored at –80°C and subjected to total RNAisolation.

Sexually developing cultures were propagated as vegeta-tive cultures in liquid MM + CA medium (Han et al., 1990)containing 1% glucose as carbon source, and 0.1% sodiumnitrate and 0.1% casein hydrolysate as sources of nitrogen,and were transferred to solid MM + CA after 18 h of growth.Plates were sealed with parafilm and wrapped with alu-minium foil in order to induce fruiting body formationspecifically.



Several cellular metabolites were used to try comple-menting the absence of growth of double mutant strainDCafA DCafB in 0.033% CO2. The MM was enrichedwith the following combination of supplements: (i) aspartate(100 mg ml-1); (ii) palmitate (2 mM); (iii) uracil (1.2 mg ml-1) +adenine (50 mg ml-1) + arginine (100 mg ml-1); (iv) aspartate(100 mg ml-1) + palmitate (2 mM) + uracil (1.2 mg ml-1) +adenine (50 mg ml-1) + arginine (100 mg ml-1); or (v) bicar-bonate (5, 10, 25 and 50 mM). These experiments weredone with both MM and buffered MM. The plates were incu-bated at 37°C for 4 days in two different atmospheric con-ditions, 0.033% and 5% CO2.

The growth of the mutants and wild-type strains was veri-fied in buffered MM pH 5.0, 6.5 and 8.0. The plates wereincubated at 37°C for 4 days in two different atmosphericconditions, 0.033% and 5% CO2. After this period, the diam-eter of the growth was measured.

Statistical analyses were performed in GraphPad Prismprogram v.5, using two-way analysis of variance followed bythe Bonferroni test. The means were considered significantlydifferent with a P-value below 0.05.

DNA manipulations and construction of the A. fumigatusand A. nidulans mutants

Standard genetic techniques for A. nidulans and A. fumigatuswere used for all strain constructions and transformations(Kafer, 1977). DNA manipulations were according to Sam-brook and Russell (2001). DNA fragment probes for Southernblots were labelled with 32P-a-dCTP using the RTS RadPrime DNA labelling System kit (Invitrogen). PCR primerswere designed for amplifying each DNA fragment by usingPrimer Express Version 1.0 (Applied Biosystems) designsoftware.

The construction of the deletion cassette was performedaccording to Colot et al. (2006). Briefly, 2 kb regions on eitherside of the open reading frames (ORFs) were selected forprimer design named 5′Fw and 5′Rev for amplification of the5′-UTR flanking region of the CA ORF and 3′Fw and 3′Rev forthe 3′-UTR flanking region. Both fragments were PCR ampli-fied from genomic DNA and used in yeast transformation. Asimilar method was applied to construct the deletion cas-settes for cafA and cafB for Af293.1 transformation (Yu et al.,2004). The pyrG gene was used as a selective marker for thedeletions of four A. fumigatus CAs and for A. nidulans DcanA(AN5611.3). It was amplified from pCDA21 plasmid (Chav-eroche et al., 2000) using the primers 5′Zeo and 3′Pyr. Thepyro gene was used as a selective marker for auxotrophy inthe case of A. nidulans DcanB (AN1805.3). It was amplifiedfrom genomic DNA of A. fumigatus using the primers Pyro Fwand Pyro Rev. The primers 5′Fw and 3′Rev presented cohe-sive ends with the vector pRS426 (bold letters) used for invivo recombination in yeast. This vector was double digestedwith EcoRI and BamHI for linearization and transformed withthese three fragments (5′ and 3′-UTR and pyrG) in S. cerevi-siae strain FY834 (Winston et al., 1995) by the lithium acetatemethod (Schiestl and Gietz, 1989). The DNA of the yeasttransformants was extracted by the method described byGoldman et al. (2003), dialysed and transformed by elec-troporation in Escherichia coli strain DH10B. The deletioncassette was PCR amplified from these plasmids using high-

fidelity Taq-polymerase (Invitrogen) and primers 5′Fw and3′Rev, and used for A. fumigatus and A. nidulans transfor-mation (Osmani et al., 1987). The 50 ml amplification mixtureincluded 1¥ Platinum Taq DNA Polymerase High Fidelitybuffer (Invitrogen), 3 mM of MgSO4, 20 pmol of each primer,0.4 mM deoxynucleotide triphosphate (dNTP) mix, 1.0 U ofPlatinum Taq DNA Hi-Fi polymerase (Invitrogen), and 500 ngof genomic DNA or 100 ng plasmid. PCR amplification of thedeletion cassette was carried out in a PTC100 96-wellthermal cycler (MJ Research), at 94°C for 2 min, and 35times 94°C for 1 min, 58–60°C (depending on the fragment)for 1 min, and 68°C for 6 min, followed by an extension stepat 68°C for 10 min. After the reaction, the PCR products werepurified with a Quiaquick PCR cleanup kit (Qiagen) accordingto the manufacturer’s instructions.

The pyrG + strains DcafA, DcafB and DcafC were pointinoculated in YUU plates containing 5-FOA in a sub-inhibitoryconcentration of 0.65 mg ml-1, resulting in the recovery of asector that was auxotrophic to uridine and uracil and resistantup to 0.75 mg ml-1 of FOA. These strains were comple-mented by co-transformation of a linear fragment amplifiedfrom genomic DNA by using primers 5′Fw and 3′Rev, andplasmid pRG3 that contains the N. crassa pyr4 gene. Trans-formants were scored for their ability to grow on YAGmedium. The complemented strains show essentially thesame behaviour as the wild-type strains.

Transformation of A. fumigatus and A. nidulans strains wasaccording to the procedure of Osmani et al. (1987) using 5 mgof linear DNA fragments. A. fumigatus transformants werescored for their ability to grow on YAG medium in the absenceof UU. A. nidulans transformants were scored for their abilityto grow on MM + UU (AN1805.3) or MM + Pyro (AN5611.3).Southern analysis demonstrated that the deletion cassettehad integrated at the correct locus. All the primer sequencesare described in Table S4.

Real-time RT-PCR reactions

All the A. fumigatus reactions were performed using an ABIPrism 7500 Sequence Detection System (Applied Biosystem,USA). Taq-ManTM Universal PCR Master Mix kit was used forPCR reactions. The thermal cycling conditions comprised aninitial step at 95°C for 10 min, followed by 40 cycles at 95°Cfor 15 s and 60°C for 30 s. The reactions and calculationswere performed according to Semighini et al. (2002). Theprimers and LuxTM fluorescent probes (Invitrogen) used in thiswork are described in Table S4. For the analysis of the A.nidulans canA-B, a real-time PCR was carried out to quantifyand analyse the expression of candidate genes. Total RNAsamples were extracted from the harvested mycelia fromsexual induction stages and sexually induced stages. Sexualdevelopment was induced by sealing culture plates, whichcontain vegetative hyphae obtained from submerged culturefor 16 h, with parafilm for 24 h. The extracted total RNA wasreverse-transcribed into cDNA using SuperScript (Invitrogen)according to the manufacturer’s instructions. The reactionwas primed by 15 oligo (dT) primer.

RNA isolation

Mycelium was disrupted by grinding in liquid nitrogen andimmediately mixed with Trizol (Invitrogen) for RNA extraction

1386 K.-H. Han et al. �


following the supplier’s recommendations. To verify the RNAintegrity, 20 mg of RNA from each treatment was fractionatedin 2.2 M formaldehyde, placed on a 1.2% agarose gel,stained with ethidium bromide, and visualized with UV light.The presence of intact 28S and 18S rRNA bands was usedas a criterion to verify that the RNA was intact.

Heterologous expression of the A. fumigatus andA. nidulans CA genes

To heterologously express the Aspergilli CA genes in the S.cerevisiae nce103D mutant strain, the following plasmidswere constructed. The cDNA clones for Aspergilli cafA-D andcanA-B genes were amplified by RT-PCR using A. fumigatus(Af293) and A. nidulans (A4) total RNA for first strand syn-thesis and then cloned into the pCR2.1-TOPO vector, andsequenced. The cafA-cafD and canA-B cDNA fragmentswere inserted downstream of an ADH1 promoter of plasmidpTH19 (Bahn et al., 2005), and these plasmids were trans-formed into the S. cerevisiae haploid nce103D strain.

Murine infections

Murine infections were performed under UK Home officeproject Licence PPL/70/6487 in dedicated facilities at Impe-rial College London. Outbred male mice (strain CD1,18–22 g, Harlan Ortech) were housed in individually ventedcages. Mice were immunosuppressed as previouslydescribed (Schrettl et al., 2004). A. fumigatus spores forinoculation were grown on Aspergillus complete medium,containing 1% (v/v) glucose and 5 mM ammonium (+)-tartratefor 5 days prior to infection. Conidia were freshly harvestedusing sterile saline (Baxter Healthcare) and filtered throughMiracloth (Calbiochem). Conidial suspensions were spun for10 min at 3000 g, washed twice with sterile saline, countedusing a haemocytometer, and resuspended at a concentra-tion of 1.25 ¥ 107 cfu ml-1. Viable counts from administeredinocula were determined following serial dilution by plating onAspergillus complete medium containing 1% (v/v) glucoseand 5 mM ammonium (+)-tartrate and growth at 37°C. Micewere anaesthetized by halothane inhalation and infected byintranasal instillation of 2.5 ¥ 105–5.0 ¥ 105 conidia in 40 ml ofsaline. Mice were weighed every 24 h from day of infectionand visual inspection made twice daily. In the majority ofcases the end point for survival experimentation was a 20%reduction in body weight measured from day of infection, atwhich point mice were sacrificed. Significance of comparativesurvival was calculated using Log Rank analysis in the Prismstatistical analysis package.

Acknowledgements

This research was supported by the Fundação de Amparo àPesquisa do Estado de São Paulo (FAPESP), ConselhoNacional de Desenvolvimento Científico e Tecnológico(CNPq), Brazil, John Simon Guggenheim Memorial Founda-tion, USA (to M.E.S.F. and G.H.G.), and Medical ResearchCouncil (to E.B.), the University of London Central ResearchFund (OL) (to E.B.), and also supported by the Korea Science

and Engineering Foundation (KOSEF) grant funded by theKorea government (MEST) (R01-2006-000-11204-0).

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