Abstract Extracellular lipolytic activity enabled the hu-man pathogen Candida albicans to grow on lipids as thesole source of carbon. Nine new members of a lipase genefamily (LIP2–LIP10) with high similarities to the recentlycloned lipase gene LIP1 were cloned and characterised.The ORFs of all ten lipase genes are between 1281 and1416 bp long and encode highly similar proteins with upto 80% identical amino acid sequences. Each deduced li-pase sequence has conserved lipase motifs, four con-served cysteine residues, conserved putative N-glycosyla-tion sites and similar hydrophobicity profiles. All LIPgenes, except LIP7, also encode an N-terminal signal se-quence. LIP3–LIP6 were expressed in all media and at alltime points of growth tested as shown by Northern blotand RT-PCR analyses. LIP1, LIP3, LIP4, LIP5, LIP6 andLIP8 were expressed in medium with Tween 40 as a solesource of carbon. However, the same genes were also ex-pressed in media without lipids. Two other genes, LIP2and LIP9, were only expressed in media lacking lipids.Transcripts of most lipase genes were detected during theyeast-to-hyphal transition. Furthermore, LIP5, LIP6, LIP8and LIP9 were found to be expressed during experimentalinfection of mice. These data indicate lipid-independent,highly flexible in vitro and in vivo expression of a largenumber of LIP genes, possibly reflecting broad lipolyticactivity, which may contribute to the persistence and viru-lence of C. albicans in human tissue.

Keywords Lipases · Gene family · Candida albicans ·Human pathogen

Abbreviations Sap Secreted aspartic proteinase · Lip Lipase

Introduction

The number of individuals predisposed to infections withopportunistic microorganisms such as Candida albicanshas increased significantly over the last decade. Patientsat risk include those being treated for cancer, organ trans-plantation or HIV infections (Vartivarian and Smith 1993).While impaired host immune function is the most com-mon cause of severe Candida infections (Casadevall andPirofski 1999), the fungus must possess attributes whichfacilitate the transition from a harmless commensal to anaggressive pathogen. Moreover, these attributes are appar-ently able to adapt to very different environments sinceC. albicans is able to cause several different types of in-fections such as oral, vaginal or systemic candidiasis.

A number of virulence factors have been suggested tobe important during different types of C. albicans infec-tions. These factors include the yeast-to-hyphal transition,adhesion factors or surface hydrophobicity, phenotypicswitching, thigmotropism, molecular mimicry and the se-cretion of hydrolytic enzymes (Cutler 1991; Odds 1994).While secreted aspartic proteinases (Saps) and phospholi-pases B (PLB) are well characterised (Ibrahim et al. 1995;Mirbod et al. 1995; Hube 1996; Hoover et al. 1998; Leidichet al. 1998; Sugiyama et al. 1999), other hydrolytic enzymesof C. albicans such as secreted lipases and esterases havebeen widely neglected.

Esterases and lipases (EC 3.1.1.1 and 3.1.1.3) are char-acterised by their ability to catalyse the hydrolysis of esterbonds of triacylglycerols, although several esterases andlipases display activity towards mono- and diacylglycerolsor even phospholipids (Anthonsen et al. 1995). A funda-mental difference between esterases and lipases is theirability to act on soluble substrates (Derewenda and Sharp

Bernhard Hube · Frank Stehr · Michael Bossenz ·Anna Mazur · Marianne Kretschmar ·Wilhelm Schäfer

Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members

Received: 26 June 2000 / Revised: 6 September 2000 / Accepted: 11 September 2000 / Published online: 18 October 2000

ORIGINAL PAPER

Bernhard Hube and Frank Stehr contributed equally to this work.

B. Hube (✉ )Robert Koch-Institut, NG4, Nordufer 20, 13353 Berlin, Germany e-mail: HubeB@rki.de, Tel.: +49-1-888-7542116, Fax: +49-1-888-7542328

F. Stehr · M. Bossenz · A. Mazur · W. SchäferInstitut für Allgemeine Botanik, AMP III, Universität Hamburg,Ohnhorststrasse 18, 22609 Hamburg, Germany

M. KretschmarInstitut für Medizinische Mikrobiologie und Hygiene, Klinikum Mannheim, Postfach 10 00 23, 68135 Mannheim, Germany

Arch Microbiol (2000) 174 :362–374DOI 10.1007/ s002030000218

© Springer-Verlag 2000

1993). Lipases hydrolyse ester bonds at the interface be-tween the insoluble triacylglycerol phase and the aqueousphase in which the enzyme is dissolved. In contrast, es-terases act on soluble substrates and thus display normalMichaelis-Menten activity (Anthonsen et al. 1995).

Besides their important role in biotechnology, lipasesare discussed as potential virulence factors in pathogenicbacteria and fungi. For example, it has been shown thatPropionibacterium acnes is able to promote colonisationand persistence by the release of fatty acids from triacyl-glycerols on human skin (Gribbon et al. 1993). Anti-lipaseIgG antibodies have been detected in patients suffering fromStaphylococcus aureus and Pseudomonas aeruginosa in-fections, suggesting that lipases from these bacteria are ex-pressed during infection. Also, an S. aureus lipase has beenshown to block phagocytic killing of bacterial cells, prob-ably by damaging granulocyte surface structures (Rollofet al. 1988; Jaeger 1994). In vitro studies revealed that alipase from P. aeruginosa significantly inhibited mono-cyte chemotaxis (Jaeger et al. 1991, 1992). In synergywith a phospholipase C, the same lipase triggered the re-lease of 12-hydroxyeicosatetraenoic acid (12-HETE)from human platelets, which are involved in inflamma-tory processes (König et al. 1994). For Hortea werneckii,the causative agent of tinea nigra, cell-surface hydropho-bicity and lipolytic activity seem to be critical factors, andfor the facultative pathogenic yeast Malassezia furfur, se-creted lipases are essential for viability (Ran et al. 1993;Göttlich et al. 1995). Thus, lipases may play a role in ad-hesion, interactions with the immune system or nutrition,providing a carbon or energy source for the pathogenicmicroorganism.

Extracellular lipase activity of C. albicans was first de-scribed by Werner (1965), and a secreted esterase has sincebeen characterised by Tsuboi et al. (1996). The esterase isinduced by lipids, such as Tween 80, and has an optimalactivity on α-naphthyl palmitate at pH 5.5. The esterase isnot able to hydrolyse triolein, tripalmitin and α-lecithinand is therefore defined as a monoester hydrolase. Sheri-dan et al. (1996) have observed that C. albicans is able togrow in media with triolein as a sole source of carbon,suggesting that other lipolytic enzymes must exist. One ofthese proteins is the gene product of LIP1 (Fu et al. 1997).When the non-lipolytic yeast S. cerevisae was transformedwith LIP1, transformants produced a secreted lipase whichhydrolysed Tween and triacylglycerols. The same lipidswere able to induce LIP1 expression in C. albicans. WhenLIP1 was used as a probe against genomic DNA fromC. albicans in a Southern blot analysis, several bands hy-bridised, suggesting that a number of similar genes existin this fungus and may constitute a lipase gene family (Fuet al. 1997).

We focus here on the identification, characterisationand expression pattern of nine additional lipase genes(LIP2–LIP10) in C. albicans with strong similarities to LIP1.No homologous genes were present in Saccharomycescerevisiae. Members of this new gene family were ex-pressed in several media with and without lipids, possiblyreflecting a broad lipolytic activity thatmay contribute to

the persistence and virulence of C. albicans in human tis-sue.

Material and methods

Strains, culture media and growth conditions

The clinical Candida albicans wild-type strain SC5314 (Gillum etal. 1984) was used to study the expression of lipase genes. Sequencedata for LIP2–LIP4 were obtained from single clones of a fosmidlibrary, which was produced with DNA from strain 1161 (Mageeand Scherer, 1998). The fosmid library was kindly provided by Dr.S. Scherer (University of Minnesota, USA). Escherichia coliXLBlue (Stratagene) or DH5α (GibcoBRL/ Life Technologies) wereused for transformation with LIP gene fragments. Clinical isolatesof the Candida species C. tropicalis 4959, C. parapsilosis 4237,C. glabrata 332 and C. krusei 1900 and of S. cerevisae strainJRYα were used for Southern blot analysis.

The following liquid media were used for C. albicans growthcurves and expression studies: Tween 40 medium: 0.67% (w/v)yeast nitrogen base with (NH4)2SO4 and without glucose andamino acids (YNB) (Difco, Detroit, Mich., USA), 2.5% (v/v)Tween 40 (Sigma, Munich, Germany); olive oil medium: 0.67%(w/v) YNB, 2.5% (v/v) olive oil (Sigma); proline medium: 0.67%(w/v) YNB, 2% (w/v) proline (Sigma); protein medium (Hube etal. 1994); modified Lee’s medium (Morrow et al. 1992). For allcultures, test media were inoculated with 2×105 cells ml–1 of asemi-synchronised preculture of C. albicans grown in YPG(Schaller et al. 1998) for growth curves or 1–5×107 cells ml–1 forexpression studies. Cultures were shaken in an orbital incubator ateither 25°C or 37°C. For hyphal growth, either C. albicans yeastcells were suspended in 5% (v/v) calf serum (final cell density ap-proximately 1–3×107 cells ml–1) (Gow and Gooday 1982) or theregime of pH/temperature-regulated yeast-to-hyphal transition wasused (Buffo et al. 1984). Growth was measured by counting cells.Yeast-to-hyphal transition was checked microscopically.

Plate assay

The release of fatty acids was monitored by a plate test using thedye rhodamine B (Kouker and Jaeger 1987), which produces a flu-orescing complex with free fatty acids. Aliquots of various culturesupernatants were tested for lipolytic activity by incubation inpunched holes in olive oil agar (0.7% (w/v) YNB, 2% (v/v) oliveoil, 0.001% (w/v) rhodamine B, 2% (w/v) agar) for up to 24 h.Fresh culture medium was used as a negative control. A lipasefrom Rhizopus arrhizus (Sigma) was used as a positive control.

Northern and Southern blot analysis

Northern blot analysis was performed as described by Hube et al.(1994). For Southern blot analysis genomic or fosmid DNA wasdigested with restriction endonucleases, size-fractionated by gelelectrophoresis and blotted onto nylon membranes (Sambrook etal. 1989). For hybridisation of Southern blots, PCR fragments ofLIP genes were labelled by using a non-radioactive digoxigenin(DIG) labelling kit (Boehringer Mannheim, Germany). For hy-bridisation of Northern blots, a 545-bp PCR fragment of LIP1(Table 3) and a 700-bp PCR fragment of TEF3 (Colthurst et al.1992; Hube et al. 1994) were purified by using the Prep-a-Gene(Bio-Rad) or NucleoSpin (Macherey-Nagel) extract kits and la-belled with [α-32P]dCTP (~3000 Ci/mmol) (Amersham) using arandom-primed DNA labelling kit (Boehringer Mannheim).

Northern blots were hybridised as described by Hube et al.(1994). For Southern blots, membranes were hybridised with DIG-labelled probes as described by the manufacturer (BoehringerMannheim).

363

Blotting and screening of fosmid library

A C. albicans fosmid library (Magee and Scherer 1998) was usedto isolate LIP2–LIP4. The library was amplified in 20 microtitredishes. E. coli cells from a copy of each set (96 clones) were lysedand fosmid DNA was denatured with 0.5 M NaOH, 1.5 M NaCl.After neutralisation with 1 M Tris-HCl, pH 8.0, 1.5 M NaCl andwashing with 2×SSC, fosmid DNA was blotted onto nylon mem-branes by using a vacuum dot-blot system (Schleicher and Schuell,Dassel, Germany). The DNA was fixed by baking, and membraneswere hybridised as described for Southern blot analysis.

Isolation of fosmid DNA

In order to isolate DNA from positive fosmids, E. coli clones wereincubated overnight at 37°C in LB medium containing 20 µg chlo-ramphenicol/µl. Cell cultures (400 ml) were centrifuged for 20 minat 2,000×g at 4 °C, and the pellet was resuspended in 5 ml solutionI (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA) con-taining 9 mg lysozyme. After 10 min incubation at room tempera-ture, 10 ml of solution II (0.2 N NaOH, 1% SDS) was added. Themixture was then incubated for 10 min on ice and neutralised with7.5 ml of solution III (5 M potassium acetate, pH 4.8). After a further30-min incubation on ice, the mixture was centrifuged at 24,000×gand 4°C for 10 min. The supernatant was then precipitated with0.6 volumes of isopropanol, and the pellet was dissolved in 8 mlTE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). For RNA precipita-tion, 8 ml of 5 M LiCl solution were added and the mixture was in-cubated for 15 min on ice. After further centrifugation for 15 minat 20,000×g at 4 °C, the supernatant was again precipitated withone volume of isopropanol. The DNA pellet was dissolved in 5 mlTE containing 10 µg RNase I ml–1 and incubated for 60 min at 37°C to remove remaining RNA. To remove the RNase, the DNAwas extracted twice with phenol/chloroform (1:1, v/v) and oncewith chloroform/isoamyl alcohol (24:1, v/v). The plasmid DNAwas finally precipitated for 1 h at 4 °C with 0.8 volumes of iso-propanol. The DNA pellet was washed with 70% ethanol and re-suspended in 150–500 µl TE.

Subcloning and sequence analysis

Fosmid DNA of positive clones was digested with restriction en-zymes and analysed by Southern blotting. DNA fragments con-taining hybridising sequences were excised from agarose gels, pu-rified and subcloned into pBluescript KS (+/–) (Stratagene).

DNA subcloned into pBluescript was sequenced by Seqlab Lab-oratories (Göttingen, Germany). In addition to the T7/T3 primer ofpBluescript, gene-specific primers were designed for sequencingboth strands of LIP2, LIP3, and LIP4 genes. The DNA sequence ofLIP1 was determined directly from a purified PCR-amplified frag-ment by Seqlab Laboratories. PCR fragments of LIP2 (3′ end ofthe ORF), LIP5 and LIP8 were cloned into pGEM-T (Promega,Mannheim, Germany) and sequences were obtained from the puri-fied plasmids.

Nucleic acids were analysed using the program DNASIS, V 2.1(Hitachi Software Engineering 1994, 1995). For similarity searches,the program BLAST 2.0.3 of the National Center for Biotechnol-ogy Information server (http://www.ncbi.nlm.nhi.gov; Altschul etal. 1997) along with the SwissProt Data Base of the ExPASyserver (http://www.expasy.ch; Bairoch and Apweiler 1996), Gen-Bank of NCBI, the EMBL database of the European Bioinfor-matics Institute (http://www.ebi.ac.uk) and the DNA DataBank ofJapan (http://www.ddbj.nig.ac.jp) were used. Sequence data forC. albicans were obtained from the Stanford DNA Sequencing andTechnology Center Web site at http://www-sequence.stanford.edu/group/candida and used to prepare PCR primers. Sequencing ofC. albicans by the Stanford DNA Sequencing and Technology Cen-ter was accomplished with the support of the NIDR and the Bur-roughs Wellcome Fund. Multiple alignments of protein sequenceswere done by the program ClustalW (Higgins and Sharp 1988)[http://dot.imgen.bcm.tmc.edu:9331/multi-align/Options/clustalw.

html; ClustalW Multiple Sequence Alignment (@BCM)] andprocessed with GeneDoc 2.1.

RNA isolation

For Northern blot analysis and RT-PCR, total RNA from shock-frozen cell pellets and infected organs was isolated using RNA-Pure (Peqlab Biotechnologie) according to the manufacturer’s in-structions. To obtain infected organs, mice were infected intraperi-tonally as described by Kretschmar et al. (1999). Mice were killedby cervical dislocation. Infected organs were cut out and immedi-ately shock-frozen in liquid nitrogen.

Reverse Transcriptase-PCR

cDNA was synthesised with Superscript II reverse transcriptase(Gibco BRL/Life Technologies) following the manufacturer’s in-structions. For PCR, a single protocol was used with all ten primersets. The samples were subjected to 35 cycles of denaturation for30 s at 94°C, annealing for 30 s at 55°C, and extension for 1.5 minat 72°C.

To prove the absence of contaminating genomic DNA, primersspecific (Table 3; Fig.6) for the intron-containing gene encodingelongation factor 1 (EFB1) were used (Maneu et al. 1996).

Results

Growth of C. albicans in media with lipids as the sole source of carbon

One possible role for secreted lipases may be to supplycells with fatty acids or glycerol for nutritional purposes.To determine whether C. albicans is able to use lipids assubstrates, cells were grown in minimal media with oliveoil, which contains triacylglycerols, or Tween 40 (poly-oxyethylene sorbitan monopalmitate) as the sole source ofcarbon. In these media the fungus grew more slowly thanin media such as YPG, but cell numbers increased from2×105ml–1 up to 2×108 ml–1 within 6 days. During the first

364

Fig.1A,B Detection of lipolytic activity in Candida albicans cul-ture supernatants with Tween 40 as the sole carbon source. A Onehundred microlitres of a solution containing 1, 10, 100, and 1000µg lipase ml–1 from Rhizopus arrhizius (1–4) were used as positivecontrols. B One hundred microlitres of culture supernatants fromYPG medium (1), Tween 40 medium (2), the YPG preculture (3),and Tween 40 medium inoculated with 5×107 C. albicans cellsml–1 after 30, 60 and 180 min of growth (4–6). Weak lipolytic ac-tivity detected in the YPG preculture indicated that cells alreadyproduced a slight lipolytic activity which released fatty acids

two days of growth, yeast cells tended to form aggregates,and in both media hyphal formation was observed inabout 30% of the cells. Possibly due to the release of fattyacids into the medium, the pH of the culture media de-creased from 5.5 to 2.8 after 6 days of growth. The release

of fatty acids was monitored by a plate test using the dyerhodamine B (Kouker and Jaeger 1987), which produces afluorescent complex with free fatty acids. Lipolytic activ-ity of supernatants clearly increased within the first 3 h ofgrowth in Tween 40 medium (Fig.1). These data suggestthat C. albicans secretes lipolytic enzymes that are able tohydrolyse both the neutral triacylglycerols in olive oil andthe monopalmitate ester Tween 40 and that the fungus iscapable of using the hydrolysed products as carbon sources.

Identification, cloning and sequencing of LIP2, LIP3 and LIP4

The gene product of LIP1 is a secreted lipase which isable to hydrolyse neutral fats and is likely to be involvedin extracellular lipase activity (Fu et al. 1997). To deter-mine whether additional lipase genes exist in the genomeof C. albicans, we screened for genes similar to LIP1 us-ing a major part of the ORF of LIP1 as a probe. In C. al-bicans strain SC5314, at least nine different bands thatstrongly hybridised to EcoRI-digested genomic DNA in aSouthern analysis under low stringency conditions (Fig. 2)were identified. Since LIP1 contains a single EcoRI site(Fu et al. 1997), at least seven bands may belong to se-quences with high similarity to LIP1. When screening afosmid library (Magee and Scherer 1998), 17 fosmids hy-bridised with the same probe. Seven strongly hybridisingpositive fosmids were further analysed by Southern analy-sis. Fosmids 4A9 and 14D10 showed a restriction patternidentical with the LIP1 locus. Fosmids 3A9 and 4D8, and

365

Fig.2 Southern blot analysis using the LIP1 gene as a probe. Ge-nomic DNA of C. albicans (1), Saccharomyces cerevisiae (2),Candida glabrata (3), Candida parapsilosis (4), Candida tropi-calis (5) and Candida krusei (6) was digested with EcoRI and hy-bridised to LIP1 under low stringency conditions

LIP1 LIP2 LIP3 LIP4 LIP5 LIP6 LIP7 LIP8 LIP9 LIP10

ORF (bp) 1407 1401 1416 1380 1392 1392 1281 1380 1362 1398Pre-protein 468 466 471 459 463 463 426 460 453 465

(amino acids)Signal peptide 16 16 16 14 14 16 – 14 14 16

(amino acids)kDa 50.8 50.4 51.4 49.6 50.0 50.5 47.9 49.7 49.2 50.4pI 4.68 4.60 4.94 5.99 4.94 6.28 5.82 4.94 5.52 9.00Cys 112, 285, 112, 285, 8, 112, 110, 281, 110, 281, 112, 285, 13, 108, 75, 110, 110, 281, 112, 285,

364, 409 364, 409 285, 364, 359, 404 359, 404 364, 409 219, 269, 281, 359, 359, 404 364, 409409 274, 341, 404

385GXSXG 196 196 196 194 194 196 190 194 194 196YHG 344 344 344 339 339 344 FHS 321 339 339 344NXS/T 79, 231, 111, 231, 231, 319, 229, 266 229 111, 231, 74, 175, 229, 291, 36, 229, 231, 319

319, 417, 319, 331, 417, 422, 422 179, 223, 417 266, 269, 422, 451 422, 451 451 378, 379, 417

422, 423Chromosomal 1 n.d. 1 6 7 1 R 7 n.d. 1

location Accession number AF AF AF AF AF AF AF AF AF AF

188894 189152 191316 191317 191318 191319 191320 191321 191322 191323

Table 1 Sequence data of all ten C. albicans lipase genes andtheir deduced proteins. Preprotein Size of the deduced protein,signal peptide size of the signal peptide according to Von Heijne(1986), pI calculated isoelectric point of the protein, Cys positionsof the cysteine residues, GXSXG position of the serine residue in

the lipase motif of the catalytic triad, YHG position of the histidineresidue of the catalytic triade (FHS for Lip7), NXS/T positions ofpossible N-glycosylation sites, n.d. not determined. Location onchromosome as determined for known sequences linked to the li-pase genes. No signal sequence was identified for Lip7

18F3 and 5C1 showed identical hybridisation patterns.Fosmid 18D4 displayed a unique restriction pattern. Hy-bridising EcoRI and PstI bands of the selected fosmids3A9, 5C1 and 18D4 were subcloned into pBluescript andsequenced. A 2.4-kb EcoRI fragment of fosmid 18D4contained an ORF with strong similarities to LIP1 and

was therefore called LIP2. Since a small fragment of the3′ end of the ORF was absent on the EcoRI band, wesearched for homologous gene sequences in the Candidadatabases (http://www.ncbi.nlm.nih.gov/BLAST/credits/5476.html) and prepared primers to amplify the missingpart by PCR. This PCR fragment was cloned into pGEM-Tand sequenced. The complete ORF contained 1401 nu-cleotides, including the stop codon. According to pub-lished criteria, the first 16 amino acids of the deduced pro-tein sequence encode a putative signal peptide, suggestingthat Lip2 is translated into the endoplasmatic reticulum(Von Heijne 1986). The complete Lip2 protein consists of466 amino acids, with an apparent molecular mass of 50.4 kDa and a calculated isoelectric point of pH 4.60

366

Fig.3 Alignment of the protein sequences deduced fromLIP1–LIP10. The proteins are arranged as clustered in the dendro-gram (Fig.4). Signal peptides, conserved lipase motifs, and a po-tentially conserved N-glycosylation site are framed. Conservedcysteine residues are framed and marked C. Areas of 100% iden-tity are shown on a black background, 80–90% homologous re-gions are on a grey background. CUG codons were translated intoserine (Santos et al. 1993)

(Table 1). Four cysteine residues, which may form disul-fide bridges, were identified at amino acid positions 112,285, 364 and 409 (Table 1; Fig.3). The serine in theGXSXG motif, characteristic of lipases, was found at aminoacid position 196, and a YHG motif, which may containthe postulated histidine of the active site, was found at po-sition 355 (Fig.3).

A 5.3-kb EcoRI fragment of fosmid 3A9 contained anORF of 1416 bp encoding a protein (Table 1) with strongsimilarities to Lip1 and Lip2 (Fig.3) and was thereforecalled LIP3. Details of the characteristics of Lip3 are sum-marised in Table 1. Since URA1, which was also found onfosmid 3A9, was mapped to chromosome 1 (http://alces.med.umn.edu/Candida.html), LIP3 must be located on thesame chromosome.

An 8.3-kb PstI fragment of fosmid 5C1 contained anORF of 1380 bp encoding 459 amino acids (Table 1) withstrong homologies to Lip1, Lip2 and Lip3 (Fig.3); it wascalled LIP4 (Table 1; Fig.3). The gene ALS2, which is lo-cated on chromosome 6, was also found on fosmid 5C1(http://alces.med.umn.edu/Candida.html), indicating thatLIP4 is also present on this chromosome.

These data suggested that LIP2–LIP4 encode secretedlipases. The sequences of LIP2–LIP4 are filed in the

EMBL/GenBank database, accession numbers AF189152,AF191316, and AF191317, respectively.

Sequence analysis of LIP1 revealed an ORF of 1407 bp

When the sequences of LIP2, LIP3 and LIP4 were com-pared to the sequence of LIP1, we found strong similari-ties upstream from the ATG start codon published by Fuet al. (1997) at both the DNA and amino acid level. Fur-ther analysis of the 5′ region of LIP1 showed that, accord-ing to the published sequence (Fu et al. 1997), two frame-shift mutations must have occurred at positions –41 and–132 bp from the ATG codon. This fragment of LIP1 wasamplified by PCR and sequenced. The sequence revealedan additional T at position –41 bp and an additional A atposition –132 bp. Therefore, the ORF of LIP1 was foundto be 1407 bp long, encoding a preprotein with 468 aminoacids (Table 1). The first 16 amino acids were identifiedas a possible signal sequence. The molecular mass of thecomplete lipase 1 protein was calculated at 50.8 kDa withan apparent isoelectric point of pH 4.68 (Table 1). Thenew sequence of LIP1 was filed in the EMBL/GenBankdatabase, accession number AF188894.

367

Table 2 Pairwise comparisonof Lip1–Lip10 using the pro-gram CLUSTAL (Higgins andSharp 1988). The proteins areshown in the order of similar-ity from left to right. Totalnumbers and percent of identi-cal amino acids, similar aminoacids and gaps (in the align-ment) are given. For example,Lip5 and Lip8 share 80% iden-tical amino acids (374 aminoacids) and 89% similar aminoacids (416 amino acids). Thealignment between these pro-teins contained three gaps

Lip5 Lip8 Lip4 Lip9 Lip1 Lip3 Lip2 Lip10 Lip6 Lip7

Lip5 463 80% 79% 73% 52% 48% 49% 49% 50% 34%0 89% 92% 85% 68% 68% 65% 65% 66% 55%0 0% 0% 2% 1% 2% 1% 1% 2% 7%

Lip8 374 460 78% 69% 51% 46% 47% 47% 49% 34%416 0 88% 83% 68% 67% 64% 65% 67% 55%3 0 1% 1% 2% 2% 1% 1% 2% 7%

Lip4 370 362 459 74% 53% 47% 48% 48% 50% 35%426 411 0 87% 69% 69% 66% 67% 69% 55%4 5 0 1% 2% 2% 1% 1% 1% 7%

Lip9 342 321 342 453 50% 46% 48% 46% 48% 34%397 385 403 0 69% 66% 65% 66% 66% 57%10 7 6 0 3% 4% 2% 3% 2% 5%

Lip1 244 240 249 239 468 72% 58% 54% 58% 35%321 319 328 324 0 88% 78% 76% 77% 53%7 10 11 17 0 0% 0% 1% 1% 8%

Lip3 230 221 226 221 343 471 57% 55% 62% 34%321 318 327 315 415 0 76% 75% 78% 55%10 13 14 20 3 0 1% 1% 1% 9%

Lip2 231 222 224 227 273 270 466 65% 64% 33%306 302 309 306 366 362 0 82% 82% 53%7 8 7 13 4 7 0 0% 0% 8%

Lip10 231 222 226 219 257 260 304 465 64% 35%307 307 313 308 359 354 385 0 80% 54%8 9 8 14 5 8 1 0 0% 8%

Lip6 234 230 234 226 272 295 300 299 463 35%313 313 323 309 364 370 383 374 0 54%10 11 6 12 5 8 3 4 0 7%

Lip7 162 158 163 156 167 162 158 164 165 426258 254 257 259 252 261 247 253 251 037 34 33 27 42 45 40 41 37 0

Identification and sequence analysis of LIP5–LIP10

Since four closely related lipase genes were identified, wequestioned whether additional genes encoding lipases ex-isted in the genome of C. albicans. Southern blots with re-stricted genomic DNA were prepared and probed withLIP1, LIP2 and LIP4. In addition to the nine EcoRI bandsthat hybridised to the LIP1 probe, four further bands wereidentified using the LIP2 and LIP4 probes. This indicatedthe possible existence of additional lipase genes. Whengene sequences of LIP1–LIP4 were used to identify addi-tional lipase genes in the Candida databases, six moreORFs, LIP5–LIP10, were identified that had strong simi-larities to LIP1–LIP4 (Fig.3). Two of these ORFs, LIP5and LIP8, were up to 90% identical in many regions,while other areas were markedly different, suggesting thepossibility that these differences may be due to sequenc-ing errors or to the addition of sequences from differentgenes into the same contig. To clarify this, LIP5 and LIP8were amplified, the PCR products were cloned, and theORFs and flanking regions were sequenced. LIP5 andLIP8 were found to be clearly different, but closely re-lated genes. The ORFs were 80% identical at the aminoacid level, but the promoter regions were distinct. The se-quences of LIP5, LIP6, LIP8, LIP9 and LIP10 werehighly similar to those of LIP1- LIP4 and contained puta-tive signal peptides, conserved cysteine residues and li-pase motifs (Table 1, Fig.3). Characteristics of all ten LIPgenes are summarised in Table 1. The deduced amino acidsequence of LIP7 also had conserved cysteine residuesand lipase motifs, but had no obvious signal peptide se-quence. Uncertain sequence data of these new lipase geneswere verified or corrected by additional sequencing ofamplified PCR products. The sequences of LIP5–LIP10were filed in the EMBL/GenBank database, accessionnumbers AF 191318, AF 191319, AF 191320, AF 191321,AF 191322 and AF 191323, respectively.

LIP1–LIP10 constitute a lipase gene family in C. albicans

All LIP genes encode lipases with high similarities toeach other and with the same overall structure, suggestingthat these ten lipase genes are members of a gene family.All the lipases, except Lip7, encode an N-terminal signalsequence. Each enzyme had four conserved cysteineresidues, conserved lipase motifs, and a conserved puta-tive N-glycosylation site (Table 1; Fig.3). Hydrophobicityplots of all the deduced proteins showed similar profiles(not shown). Amino acid sequence identities ranged from33% (between Lip2 and Lip7) to 80% (between Lip5 andLip8) (Table 2). When clustered, the Lip isoenzyme fam-ily could be divided into two subgroups (Fig.4). Lip4,Lip5, Lip8 and Lip9, which were more than 73% identicalto each other (Fig.4; Table 2), and Lip1, Lip2, Lip3, Lip6and Lip10, which were at least 54% identical to eachother. Lip7 was the most divergent lipase in this isoen-zyme family.

When the Candida Lip sequences were compared withsequences from databases, high similarities to several li-pases in conserved regions were found. However, theoverall similarities were low when compared to most li-pases, including those from other Candida species, suchas the lipase isoenzyme family from C. rugosa. No lipases

368

Fig.4 Dendrogram of the lipase isoenzyme family of C. albicans(Lip1–Lip10) and lipases from C. rugosa (Canru1–5; Candida ru-gosa protein for lipase precursor 1–5; accession numbers P20261,P32946, P32947, P32948, P32949), C. antarctica (CananB; lipaseB; accession number P41365), Geotrichum candidum (Geoca1–2,protein for triacylglycerol lipase; accession numbers P17573,P22394), Galactomyces geotrichum (Galge; mRNA for lipase; ac-cession number X78032), Trichosporon fermentans WU-C12(Trife; lipase I; accession number BAA19072), Yarrowia lipolyt-ica (Yarli; lipase I; accession number Z50020), Humicola lanugi-nosa (Humla; lipase; Derewenda et al. 1994), Rhizopus delemar(Rhidl; mRNA for triacylglycerol lipase; accession numberP21811), Rhizomucor miehei (Rhimi; protein for triacylglycerol li-pase; accession number P19515), S. cerevisiae (Yb54; accessionnumber P38139; chromosome II ORF YBR204c), S. cerevisiae(Yj77; accession number P47145; chromosome X ORF YJR107w)and a putative lipase from Mycobacterium tuberculosis (unknownprotein; accession number Z95586)

with high similarities were found in S. cerevisiae (Fig.4).In contrast, high similarity to an unknown protein (acces-sion number Z95586) of Mycobacterium tuberculosis wasdiscovered (Fig.4). This protein contained typical lipasemotifs and was therefore likely to be a lipase.

LIP1 (Fig.2) and LIP4 (not shown) were used to probegenomic DNA from other Candida species and S. cerevisiaein a Southern analysis under low stringency conditions. Anumber of weak bands were identified in the genomes ofC. tropicalis, C. parapsilosis and C. krusei, while no similarsequences were detected in C. glabrata and S. cerevisiae,confirming the results from Fu et al. (1997). Using LIP2as a probe, two bands from S. cerevisiae and C. glabratahybridised. These data suggest that similar lipase gene se-quences may exist in other related fungal species.

Expression of the LIP gene family in media containing lipids

Since C. albicans contains a family of LIP genes, a num-ber of experiments were designed to determine whetherthese genes are expressed and how they may be regulated.First, we investigated which genes were expressed in me-dia with lipids as the sole source of carbon.

Total RNA from lipolytic C. albicans cells grown inTween 40 medium was isolated at different time points,blotted and probed to LIP1 (Fig.5). Levels of mRNAwere measured relative to ribosomal RNAs by loading ap-proximately equal amounts of total RNA in each lane andby probing the TEF3 mRNA as described by Hube et al.(1994). Strong expression of LIP1 was observed after 1 h.

The mRNA level of LIP1 increased within the next 4 hand then decreased, but was still detectable at a relativelyhigh level after 74 h.

To analyse the expression of all ten LIP genes in Tween40 medium, LIP-specific primers (Table 3) were designed,and RT-PCR was used as a sensitive and specific methodfor studying the expression of closely related genes(Schaller et al. 1998). The specificity of each set of primerswas confirmed using genomic DNA, plasmids and fos-mids containing the lipase genes (not shown). Primer pairswere chosen that yielded PCR bands of different sizesspecific for each gene (Table 3).

The absence of genomic DNA contamination and thesensitivity of the RT-PCR analysis were tested usingprimers that amplified the intron-containing gene EFB1,as described by Schaller et al. (1998). In addition to twointron-flanking EFB1 primers (Schaller et al. 1998), athird intron-specific primer was introduced (Table 3) thatallowed the detection of trace amounts of contaminatingDNA. Furthermore, samples without the addition of re-verse transcriptase were used to show the absence of ge-nomic DNA. When LIP1 specific primers were used forRT-PCR analysis of the same RNA which was used forNorthern analysis, the expression pattern of the Northernanalysis was confirmed (Fig.5).

Using LIP2–LIP10-specific primers, expression of LIP3,LIP4, LIP5, LIP6 and LIP8, but not of LIP2, LIP7, LIP9and LIP10, was observed during growth in Tween 40 me-dium (Fig.6, Table 4). Therefore, some but not all lipasegenes are expressed in medium containing Tween 40 asthe sole source of carbon.

369

P 0.5 1 3 5 7 9 12 30 74 P 1 3 6 9

← TEF3

← LIP1

LIP1

Time [h] Time [h]

Tween 40 Proline

← 18S

← 26S

P 0.5 1 3 5 7 9 12 30 74 P 1 3 6 9 C C

A

B

Fig.5 Expression of LIP1 inTween 40 and proline media asexamined by Northern blotanalysis (A) and RT-PCR (B).Samples were taken after 30min, 1 h, 3 h, 5 h, 7 h, 9 h, 12 h, 30 h, and 74 h of growthin Tween 40 medium, and after1 h, 3 h, 6 h, and 9 h in prolinemedium. Northern blots of theprepared RNA were hybridisedto radiolabelled LIP1. ProbedTEF3 mRNA and ethidium-bromide-stained ribosomalRNAs are shown for all sam-ples (A). Specific LIP1 primerswere used to amplify a 545-bpfragment of LIP1 cDNA byRT-PCR (B). P Preculture,C control with genomic DNA

370

Table 3 Primers used forpreparing probes, RT-PCR andsequencing

Primer Sequence Expected length of PCR fragment

LIP1a 5′-ACAAATTCACTGGGATCAAGAG-3′ 545 bpLIP1b 5′ATAAGTGACATGGACGTTACTG-3′LIP2a 5′-TTTCCGACTTTGCTGTTCCAG-3′ 589 bpLIP2b 5′-ATAATACTGCTTACAAGACCAAG-3′LIP3a 5′-AAATGCCAGGCAAAATGAGC-3′ 694 bpLIP3b 5′-TGTTGTAAAGCTCCTTCATATC-3′LIP4a 5′-TGATCAATTATATTGGTAAGCAC-3′ 455 bpLIP4b 5′-TCCTTTTTGGATGAGTATATTC-3′LIP5a 5′-AATCGTCCCTATTGTCGATACC-3′ 283 bpLIP5b 5′-AAGTCCGAGATGGAGAACAAC-3′LIP6a 5′-TTAAACCTGGTGCCAAAGCTG-3′ 441 bpLIP6b 5′-TCGATGCCCTGGTGGTGAAC-3′LIP7a 5′-TTCCATCATTCGAGACATTTCAG-3′ 878 bpLIP7b 5′-ACGGAAGTACTGACTGAGAAATG-3′LIP8a 5′-AGAGTGATACAGACAAAAAATCAG-3′ 521 bpLIP8b 5′-AAGACCATTCAGCATGGTG-3′LIP9a 5′-TTTATAAAGTATGTGGGAGCTAG-3′ 466 bpLIP9b 5′-TAGGACCAAGCCCTTGTTGTG-3′LIP10a 5′-TTGGGTTTGCAACTGCTAAGC-3′ 733 bpLIP10b 5′-ATAGTAGATCTAGCACTGAGC-3′EFB5′ 5′-ATTGAACGAATTCTTGGCTGAC-3′ 916 bp (with intron),

551 bp (without intron)EFB3′ 5′-CATCTTCTTCAACAGCAGCTTG-3′EFB5′ 5′-ATTGAACGAATTCTTGGCTGAC-3′ 264 bp (with intron)EFBint 5′-TCTTGAGGCCACCTCATAAAC-3′

Fig.6 Expression of the LIPgene family in Tween 40 andproline media by RT-PCR.RNA samples were taken atdifferent time points as indi-cated, translated into cDNAand used to amplify LIP-tran-scripts. Expression of LIP1 isshown in Fig.5. EFB1 primerswere used to demonstrate theabsence of genomic DNA con-taminations. Amplification of a551-bp EFB1 fragment (arrow)indicated the presence ofcDNA; amplification of a 264-bp and a 916-bp fragmentoccurred when DNA was usedas a template. The larger intron-flanking fragment was fre-quently amplified when highamounts of DNA were present(lane C). P Preculture

Most members of the LIP gene family were expressed in minimal media containing proline as a sole source of carbon

When RNA from YPG precultures, used to inoculateTween 40 cultures, was analysed by RT-PCR, transcriptsfor LIP1, LIP3, LIP4, LIP5, LIP6 and LIP8 were detected(Figs. 5, 6). Although conditions for precultures, includingincubation times, were kept constant, this expression wasnot always detected, indicating that only small amounts ofmRNA were present. This also suggested that LIP expres-sion may occur in the absence of lipid substrates. To testthis, minimal medium containing proline as a sole sourceof carbon was used. The expression of LIP genes wasanalysed by Northern analysis for LIP1 and RT-PCR forLIP1–LIP10. LIP1 was strongly expressed after 1 h ofgrowth, but expression decreased within the next 8 h, asshown by Northern analysis (Fig.5). Using RT-PCR to ex-amine the expression of the entire LIP gene family, a sim-ilar expression pattern as found for Tween 40 medium(LIP3, LIP4, LIP5, LIP6 and LIP8) was observed, buttranscripts of LIP2 and LIP9 were also detected (Figs. 5,6). Therefore, lipids in the medium are not essential to in-duce LIP genes, and at least two LIP genes are up-regu-lated in medium containing proline as a sole source of car-bon.

Lipase genes are expressed during the yeast-to-hyphal transition

One of the predominant virulence factors of C. albicans isthe ability to grow in a yeast or hyphal form. To testwhether lipase genes are expressed during this yeast-to-hyphal transition, yeast cells were grown in modifiedLee’s medium pH 4.5 at 25°C and transferred into thesame medium at pH 6.0 and 37°C (Buffo et al. 1984). Af-ter 1 h, 33% of all cells had formed germ tubes; after 3 h,89% had transformed into hyphal cells; and after 5 h more

than 99% grew as hyphae. Samples were taken after 1, 3,and 5 h and used for Northern and RT-PCR analysis. Noexpression of LIP1 was observed by either of these meth-ods. Furthermore, LIP2, LIP7, LIP9 and LIP10 mRNAwere not detectable. In contrast, LIP3–LIP5, and weaklyLIP6, were expressed at all time points. Expression ofLIP8 was observed 1 and 3 h after hyphal induction, butwas not detected after 5 h. When the yeast-to-hyphal tran-sition was induced by incubation in 5% serum, the sameexpression pattern was observed, except for the additionalexpression of LIP1 and stronger expression of LIP6.When RT-PCR analysis during hyphal induction was re-peated under more sensitive conditions, transcripts for al-most all LIP genes were detected at various time points(Table 4), indicating that at least some cells of the culturepopulation expressed lipase genes constitutively underthese conditions. However, because levels of transcriptsdid not increase during the hyphal formation and sincetranscripts of most lipase genes were also found in othernon-hyphal-inducing media, we concluded that LIP genesare expressed during the dimorphic transition but are notdependent on the morphology of the fungus.

Simultaneous expression of lipase and proteinase genes

Secreted aspartic proteinases (Saps) are known virulencefactors of C. albicans and have been shown to contributeto tissue damage during infection (Schaller et al. 1999).To investigate whether the lipase genes are expressed to-gether with the proteinases, cells were grown in pro-teinase-inducing medium and expression by RT-PCR wasanalysed. Samples were taken after 2, 4, 6 and 8 h incu-bation in medium with protein as the sole source of nitro-gen. Rapid growth indicated that proteinases (mostlySap2) (Hube et al. 1994) were efficiently secreted. RT-

371

Table 4 Summary of the in vitro expression of LIP genes in allmedia tested. Expression of LIP genes in the YPG precultures wasnoted, but was not systematically studied and is, therefore, notshown in the table. Lee’s Yeast-to-hyphal transition under theregime of pH/temperature shift (Buffo et al. 1984), serum yeast-to-hyphal transition with 5% serum, + expression of gene, – nomRNA detectable, +/– weak expression in a few samples. Expres-sion of LIP8 was observed under all conditions, but not at all timepoints of growth

LIP Tween 40 Proline Lee’s Protein Serum

1 + + +/- + +2 – + +/– + +/–3 + + + + +4 + + + + +5 + + + + +6 + + + + +7 – – +/– +/– +/–8 + + + + +9 – + +/– + +/–

10 – – +/– +/– –

EFB1

LIP5

LIP6

LIP8

LIP9

916 551

[bp]1 2 3 4 C

Fig.7 Expression of LIP genes in infected liver 24 h after in-traperitoneal (i. p.) infection with 2×107 C. albicans cells. Lanes1–4 RT-PCR results from four different organs. Transcripts ofLIP5, LIP6, LIP8 and LIP9 were detected in two, one, four or threeinfected organs, respectively. No transcripts of LIP1–LIP4, LIP7and LIP10 were detected in these organs. C Control with genomicDNA

PCR analysis revealed that all LIP genes were expressedduring at least one time point (Table 4), indicating thatproteinases and lipases are simultaneously expressed un-der these conditions.

LIP gene expression in vivo

To investigate whether LIP genes were expressed in vivo,a screening for mRNA transcripts in infected tissue wascarried out. Mice were infected in a model of C. albicansperitonitis (Kretschmar et al. 1999), and infected liver tis-sue containing C. albicans cells was used to isolate totalRNA. mRNA was amplified by RT-PCR using the LIP-specific primers. Transcripts of LIP5, LIP6, LIP8 andLIP9 were detected in infected organs 7, indicating thatLIP genes are expressed during systemic candidiasis.

Discussion

The presence of gene families in microorganisms often al-ludes to their importance in cell biology and viability. Inpathogenic microorganisms obligatorily associated with ahost, such gene families may be involved in persistenceand virulence. For example, gene families encoding cell-surface adhesion glycoproteins (Als), mannosyl transferases(Mnt), transporter proteins (Mdr) or proteinases (Saps)have been shown to be important attributes for adhesion,drug resistance and tissue damage of the opportunisticfungus C. albicans (Hoyer et al. 1998; Vanden Bossche etal. 1998; Buurman et al. 1998; Schaller et al. 1999).

The LIP genes described in this study constitute a largegene family that may have evolved to adapt to the perma-nent association of C. albicans with the human or animalhost and, therefore, may also have important functionsduring persistence and infection processes.

Southern blot analysis and screening of a genomic li-brary with the known lipase gene LIP1 (Fu et al. 1997),sequencing of positive clones, and comparison of all LIPgene sequences with data from the Candida genome pro-ject revealed that the closely similar LIP genes constitutea family with at least ten members. Strong similarities toLip1 and the conserved lipase motifs of all deduced pro-teins indicated that each of the new LIP genes encodes alipase. All deduced amino acid sequences contain fourconserved cysteine residues, which may form disulfidebridges contributing to a similar three-dimensional struc-ture. Analogue hydrophobicity plots and conserved putativeN-glycosylation sites also indicated similar isoenzymestructures. In addition, all Lip proteins, except Lip7, con-tain a putative N-terminal signal sequence. Heterologousexpression of LIP1 in S. cerevisae showed that this geneencodes a secreted lipase (Fu et al. 1997). The strong sim-ilarities and the similar structures of LIP2–LIP10 (exceptLIP7) to LIP1 therefore suggest that the new lipases, likeLip1, are secreted. The secreted lipases may well con-tribute to the observed extracellular hydrolysis of neutrallipids, which enables C. albicans to grow in media con-

taining olive oil or Tween 40 as a sole source of car-bon.

Comparison of specific regions of LIP1–LIP10 withother lipase genes confirmed that these genes code forlipases. However, when comparing the complete aminoacid sequences with lipase sequences from the data bases,the overall similarities to other lipases were found to beweak. In particular, we found minor similarities to thoselipases of the closely related, apathogenic non-lipolyticyeast S. cerevisiae (Abraham et al. 1992) and the apatho-genic lipolytic species C. rugosa (Brocca et al. 1995). Incontrast, Southern blot analysis using LIP probes sug-gested that similar genes may exist in C. tropicalis,C. parapsilosis and C. krusei, indicating that this genefamily may be unique to pathogenic Candida species.

Interestingly, the highest similarity of the LIP geneswas found towards an ORF in the genome of the bacterialpathogen Mycobacterium tuberculosis (Fig.4). Conservedlipase motifs showed that this unknown protein is likely tobe a lipase. Lipolytic enzymes have been discussed as pu-tative virulence factors of this important human pathogen(Camacho et al. 1999; Cole 1999). However, it remains tobe tested whether the high similarities between the lipasesof these two organisms reflect a similar enzyme activity.

The fact that secreted lipases of C. albicans are en-coded by a family of genes may indicate that different li-pase genes are needed during different stages or types ofinfection. This has been demonstrated for another familyof genes in C. albicans encoding the Saps. It has beenshown, for example, that SAP1-3 are important for super-ficial infections (Schaller et al. 1999), while SAP4-6 playa fundamental role during systemic infections (Kretschmaret al. 1999). These patterns of tissue- and infection-spe-cific gene expression strongly suggest that different SAPgenes are differentially regulated in vitro and in vivo, de-pending on the environment and the morphological form(Hube et al. 1994; White and Agabian 1995; Monod et al.1998; Schaller et al. 1998; Naglik et al. 1999).

The expression pattern of the LIP gene family was in-vestigated under a number of different environmentalconditions by Northern analysis and RT-PCR. RT-PCRwas chosen because this sensitive method can distinguishbetween closely related members of a gene family.

All ten LIP genes were expressed in one or other con-dition. Six LIP genes (LIP1, LIP3–LIP6 and LIP8) wereexpressed in media containing lipids as the sole source ofcarbon, suggesting that a number of genes may be in-volved in the observed lipolytic activity of C. albicans.However, the same genes and two more (LIP2 and LIP9)were expressed in media that did not contain any lipids.This would indicate that transcription of these lipase genesis lipid-independent. Furthermore, these lipase genes areexpressed in media that also induced SAP genes. SeveralSAP and all LIP genes were shown to be expressed in me-dia containing protein as the sole source of nitrogen andduring the yeast-to-hyphal transition (Hube et al. 1994;White and Agabian 1995; Smolenski et al. 1997; Monodet al. 1998). Therefore, C. albicans is able to express pro-teinase and lipase genes under the same conditions. How-

372

ever, in contrast to the controlled regulation of distinctSAP genes, expression of some LIP genes seems to be lessspecific or even constitutive. For example, LIP3–LIP6 andLIP8 were expressed under all conditions investigated.

Several functions are possible for the different mem-bers of the LIP gene family. The most obvious function oflipases is the hydrolysis of lipids in order to use fatty acidsand/or glycerol as substrates. C. albicans is able to growon media containing lipids as the sole source of carbon.Although this growth is slow compared to growth in othermedia, the fungus is able to hydrolyse the lipids and totransport the hydrolytic products into the cell. Possiblelipid substrates may be found on human tissue such as theskin or the intestinal tract. The high number of LIP genesmay provide an adaptive advantage to persist on these sur-faces, even in the absence of carbohydrates, and may as-sist C. albicans in competing with the normal microbialflora.

The release of fatty acids may also have additional ad-vantages for the fungus. For example, release of fattyacids may change the pH in the microenvironment of thecells. The pH values decreased from 5.5 to 2.8 duringgrowth in lipid media, and such an acidification may opti-mise the natural microenvironment for other fungal pro-teins such as the secreted proteinases. Similarly, an activealteration of the pH in the microenvironment to allow theproduction and activity of extracellular proteinases has re-cently been shown for the entomopathogenic fungusMetarhizium anisopliae (St. Leger et al. 1999). In addi-tion, release of fatty acids may enhance the adherence ofC. albicans to host surfaces, as shown for P. acnes (Grib-bon et al. 1993).

One surprising observation was that most LIP geneswere expressed in media without lipids. This suggests thatLIP genes may have functions other than providing nutri-ents for the cells. For example, lipases may also developlipolytic activities towards phospholipids. Such non-spe-cific hydrolysis of phospholipids may damage host cellmembranes, as previously shown for phospholipase B ac-tivities (Ibrahim et al. 1995). In addition, C. albicans li-pases, with or without the synergistic action of other phos-pholipases, may affect the host immune system by in-hibiting cell-mediated chemotaxis or by damaging phago-cytotic cells directly. Moreover, lipolytic activities on hostlipid mediators may trigger local inflammatory disorders,which in turn may cause host tissue damage (Heller et al.1998). If LIP genes play such roles during the infectionprocess, they must be expressed in vivo. Several LIPgenes were shown to be expressed during intraperitonealinfections of mice, making it likely that these genes arenot only relicts of a former saprophytic life, but haveadapted to infection processes on or in host tissue.

Acknowledgements The authors thank M. Ghannoum (Centerfor Medical Mycology, University Hospitals of Cleveland, andCase Western Reserve University, Cleveland, Ohio, USA) for pro-viding the LIP1 gene as a probe; M. Strathman and S. Scherer (De-partment of Genetics and Cell Biology, University of Minnesota,USA) for the fosmid library; J. Nörskau, D. Heß and A. Felk (In-stitut für Allgemeine Botanik, Universität Hamburg, Germany) for

RNA samples; and J. Naglik (Guy’s Hospital London, UK) forcritical reading of the manuscript. Sequence data for LIP5–LIP10from Candida albicans were obtained from the Stanford DNASequencing and Technology Center Web site at http://www-sequence.stanford.edu/group/candida and used to prepare PCRprimers. Sequencing of Candida albicans at the Stanford DNA Se-quencing and Technology Center was accomplished with the sup-port of the NIDR and the Burroughs Wellcome Fund. This workwas supported by a grant from the Boehringer Ingelheim Fondsawarded to F. Stehr.

References

Abraham PR, Mulder A, Van’t Riet J, Planta RJ, Raue HA (1992)Molecular cloning and physical analysis of an 8.2 kb segmentof chromosome XI of Saccharomyces cerevisiae reveals fivetightly linked genes. Yeast 8:227–238

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, MillerW, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs. Nucleic AcidsRes 25:3389–3402

Anthonsen HW, Baptista A, Drablos F, Martel P, Petersen SB, Se-bastiao M, Vaz L (1995) Lipases and esterases: a review oftheir sequences, structure and evolution. Biotechnol Annu Rev1:315–371

Bairoch A, Apweiler R (1996) The Swiss-Prot protein sequencedata bank and its new supplement TREMBL. Nucl Acid Res24:21-25

Brocca S, Grandori R, Breviario D, Lotti M (1995) Localization oflipase genes on Candida rugosa chromosomes. Curr Genet 28:454–457

Buffo J, Herman MA, Soll DR (1984) A characterization of pH-regulated dimorphism in Candida albicans. Mycopathology85:21–30

Buurman ET, Westwater C, Hube B, Brown AJ, Odds FC, Gow NA(1998) Molecular analysis of CaMnt1p, a mannosyl transferaseimportant for adhesion and virulence of Candida albicans.Proc Natl Acad Sci USA 95:7670–7675

Camacho LR, Ensergueix D, Perez E, Gicquel B, Guilhot C (1999)Identification of a virulence gene cluster of Mycobacteriumtuberculosis by signature-tagged transposon mutagenesis. MolMicrobiol 34:257–267

Casadevall A, Pirofski LA (1999) Host-pathogen interactions: re-defining the basic concepts of virulence and pathogenicity. In-fect Immun 67:3703–3713

Cole ST (1999) Learning from the genome sequence of Mycobac-terium tuberculosis H37Rv. FEBS Lett 452:7–10

Colthurst DR, Schauder BS, Hayes MV, Tuite MF (1992) Elonga-tion factor 3 (EF-3) from Candida albicans shows both struc-tural and functional similarity to EF-3 from Saccharomycescerevisiae. Mol Microbiol 6:1025–33

Cutler JE (1991) Putative virulence factors of Candida albicans.Annu Rev Microbiol 45:187–218

Derewenda ZS, Sharp AM (1993) News from the interface: themolecular structures of triacylglyceride lipases. Trends BiochemSci 18:20–25

Derewenda U, Swenson L, Wei Y, Green R, Kobos PM, Joerger R,Haas MJ, Derewenda ZS (1994) Conformational lability oflipases observed in the absence of an oil-water interface: crys-tallographic studies of enzymes from the fungi Humicolalanuginosa and Rhizopus delemar. J Lipid Res 35:524–534

Fu Y, Ibrahim AS, Fonzi W, Zhou X, Ramos CF, Ghannoum M(1997) Cloning and characterisation of a gene (LIP1) which en-codes a lipase from the pathogenic yeast Candida albicans. Mi-crobiology 143:331–340

Gillum AM, Tsay EYH, Kirsch DR (1984) Isolation of theCandida albicans gene for orotidine-5‘-phosphate decarboxy-lase by complementation of S. cerevisiae ura3 and E. coli pyrFmutations. Mol Gen Genet 198:179–182

373

Göttlich E, de Hoog GS, Yoshida S, Takeo K Nishimura K, MiyajiM (1995) Cell-surface hydrophobicity and lipolysis as essentialfactors in human tinea nigra. Mycosis 38:489–494

Gow NA, Gooday GW (1982) Vacuolation, branch production andlinear growth of germ tubes in Candida albicans. J Gen Micro-biol 128:2195–2198

Gribbon EM, Cunliffe WJ, Holland KT (1993) Interaction of Pro-pionibacterium acnes with skin lipids in vitro. J Gen Microbiol139:1745–1751

Heller A, Koch T, Schmeck J, van Ackern K (1998) Lipid media-tors in inflammatory disorders. Drugs 55:487–496

Higgins DG, Sharp PM (1988) CLUSTAL: a package for perform-ing multiple sequence alignments on microcomputer. Gene 73:237–244

Hoover CI, Jantapour MJ, Newport G,Agabian N, Fisher SJ (1998)Cloning and regulated expression of the Candida albicansphospholipase B (PLB1) gene. FEMS Microbiol Lett 167:163–169

Hoyer LL, Payne TL, Bell M, Myers AM, Scherer S (1998)Candida albicans ALS3 and insights into the nature of the ALSgene family. Curr Genet 33:451–459

Hube B (1996) Candida albicans secreted aspartyl proteinases.Curr Top Med Mycol 7:55–69

Hube B, Monod M, Schofield DA, Brown AJP, Gow NAR (1994)Expression of seven members of the gene family encoding se-cretory aspartyl proteinase in Candida albicans. Mol Microbiol14:87–99

Ibrahim AS, Mirbod F, Filler SG, Banno Y, Cole GT, Kitajima Y,Edwards JE Jr, Nozawa Y, Ghannoum MA (1995) Evidenceimplicating phospholipase as a virulence factor of Candidaalbicans. Infect Immun 63:1993–1998

Jaeger K-E (1994) Extracellular enzymes of Pseudomonas aerugi-nosa as virulence factors. Immun Infekt 22:177–180

Jaeger K-E, Kharazmi A, Hoiby N (1991) Extracellular lipase ofPseudomonas aeruginosa: biochemical characterization and ef-fect on human neutrophil and monocyte function in vitro. Mi-crob Pathog 10:173–182

Jaeger K-E, Kinscher DA, König B, König W (1992) Extracellularlipase of Pseudomonas aeruginosa: biochemistry and potentialrole as virulence factor. In: Hoiby N, Pedersen SS (eds) Cysticfibrosis, basis and clinical research, Elsevier, Amsterdam, pp 113–119

König B, Jaeger K-E, König W (1994) Induction of inflammatorymediator release (12-hydroxyeicosatetraenoic acid) from hu-man platelets by Pseudomonas aeruginosa. Int Arch AllergyImmunol 104:33–41

Kouker G, Jaeger K-E (1987) Specific and sensitive plate assay forbacterial lipases. Environ Microbiol 53:211–213

Kretschmar M, Hube B, Bertsch T, Sanglard D, Merker R,Schroder M, Hof H, Nichterlein T (1999) Germ tubes and pro-teinase activity contribute to virulence of Candida albicans inmurine peritonitis. Infect Immun 67:6637–6642

Leidich SD, Ibrahim AS, Fu Y, Koul A, Jessup C, Vitullo J, FonziW, Mirbod F, Nakashima S, Nozawa Y, Ghannoum, MA (1998)Cloning and disruption of caPLB1, a phospholipase B gene in-volved in the pathogenicity of Candida albicans. J Biol Chem273:26078–26086

Magee P, Scherer S (1998) Genome mapping and gene discoveryin Candida albicans. ASM News 64:505–511

Maneu V, Cervera AM, Martinez JP, Gozalbo D (1996) Molecularcloning and characterization of a Candida albicans gene (EFB1)coding for the elongation factor EF-1ß. FEMS Microbiol Lett145:157–162

Mirbod F, Banno Y, Ghannoum MA, Ibrahim AS, Nakashima S,Kitajima Y, Cole GT, Nozawa Y (1995) Purification and char-acterization of lysophospholipase-transacylase (h-LPTA) froma highly virulent strain of Candida albicans. Biochim BiophysActa 13:181–188

Monod M, Hube B, Hess D, Sanglard D (1998) Differential regu-lation of SAP8 and SAP9, which encode two new members ofsecreted aspartic proteinase family in Candida albicans. Mi-crobiology 144:2731–2737

Morrow B, Srikantha T, Soll DR (1992) Transcription of the genefor a pepsinogen, PEP1, is regulated by white-opaque switch-ing in Candida albicans. Mol Cell Biol 12:2997–3005

Naglik JR, Newport G, White TC, Fernandes-Naglik LL, GreenspanJS, Greenspan D, Sweet SP, Challacombe SJ, Agabian N (1999)In vivo analysis of secreted aspartyl proteinase expression inhuman oral candidiasis. Infect Immun 67:2482–2490

Odds FC (1994) Candida species and virulence. ASM News 60:313–318

Ran Y, Yoshiike T, Ogawa H (1993) Lipase of Malassezia furfur:some properties and their relationship to cell growth. J MedVet Mycol 31:77–85

Rollof J, Braconier JH, Soderstrom C, Nilsson-Ehle P (1988) In-terference of Staphylococcus aureus lipase with human granu-locyte function. Eur J Clin Microbiol Infect Dis 7:505–510

Sambrook J, Frisch ER, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY

Santos MA, Keith G, Tuite MF (1993) Non-standard translationalevents in Candida albicans mediated by an unusual seryl-tRNA with a 5′-CAG-3′ (leucine) anticodon. EMBO J 2:607–616

Schaller M, Schäfer W, Korting HC, Hube B (1998) Differentialexpression of secreted aspartyl proteinases in a model of hu-man oral candidosis and in patient samples from the oral cav-ity. Mol Microbiol 29:605–615

Schaller M, Korting HC, Schafer W, Bastert J, Chen W, Hube B(1999) Secreted aspartic proteinase (Sap) activity contributes totissue damage in a model of human oral candidosis. Mol Mi-crobiol 34:169–180

Sheridan R, Ratledge C (1996) Changes in cell morphology andcarnitine acetyltransferase activity in Candida albicans follow-ing growth on lipids and serum and after in vivo incubation inmice. Microbiology 142:3171–3180

Smolenski G, Sullivan PA, Cutfield SM, Cutfield JF (1997)Analysis of secreted aspartic proteinases from Candidaalbicans: purification and characterization of individual Sap1,Sap2 and Sap3 isoenzymes. Microbiology 143:349–356

St. Leger RJ, Nelson JO, Screen SE (1999) The entomopathogenicfungus Metarhizium anisopliae alters ambient pH, allowing ex-tracellular protease production and activity. Microbiology 145:2691–2699

Sugiyama Y, Nakashima S, Mirbod F, Kanoh H, Kitajima Y,Ghannoum MA, Nozawa Y (1999) Molecular cloning of a sec-ond phospholipase B gene, caPLB2 from Candida albicans.Med Mycol 37:61–67

Tsuboi R, Komatsuzaki H, Ogawa H (1996) Induction of an extra-cellular esterase from Candida albicans and some of its prop-erties. Infect Immun 64:2936–2940

Vanden Bossche H, Dromer F, Improvisi I, Lozano-Chiu M, RexJH, Sanglard D (1998) Antifungal drug resistance in patho-genic fungi. Med Mycol 36:119–128

Vartivarian S, Smith CB (1993) Pathogenesis, host resistance, andpredisposing factors. In: Bodey GP (ed) Candidiasis: pathogen-esis, diagnosis, and treatment, 2nd edn. Raven, New York, pp 59–84

Von Heijne G (1986) A new method for predicting signal sequencecleavage sites. Nucleic Acid Res 14:4683–4690

Werner H (1965) Untersuchungen über die Lipaseaktivität beiHefen und hefeähnlichen Pilzen. Zbl Bakt Hyg I Abt A 22:113–124

White TC, Agabian N (1995) Candida albicans secreted aspartylproteinases: isoenzyme pattern is determined by cell type, andlevels are determined by environmental factors. J Bacteriol177:5215–5221

374