Glycolytic Enzymes of Candida albicans Are Nonubiquitous ...

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A cDNA library was made with mRNA from Candida albicans grown under conditions favoring the ... patients with oral candidiasis and five uninfected patients.
Vol. 61, No. 10

INFECTION AND IMMUNITY, OCt. 1993, p. 4263-4271

0019-9567/93/104263-09$02.00/0 Copyright X 1993, American Society for Microbiology

Glycolytic Enzymes of Candida albicans Are Nonubiquitous Immunogens during Candidiasis R. K. SWOBODA,l* G. BERTRAM,1 H. HOLLANDER,2 D. GREENSPAN,2 J. S. GREENSPAN,2 N. A. R. GOW,1 G. W. GOODAY,1 AND A. J. P. BROWN' Molecular & Cell Biology, Marischal College, University of Aberdeen, Aberdeen AB9 IAS, United Kingdom, and Oral AIDS Center, Department of Stomatology, University of California, San Francisco, California 941432 Received 26 April 1993/Returned for modification 22 June 1993/Accepted 16 July 1993

A cDNA library was made with mRNA from Candida albicans grown under conditions favoring the hyphal form. The library was screened for sequences that encode immunogenic proteins by using pooled sera from five patients with oral candidiasis and five uninfected patients. Most of these patients were human immunodeficiency virus positive. From 40,000 cDNA clones screened, 83 positive clones were identified. Of these, 10 clones were chosen at random for further analysis. None of these 10 cDNAs were derived from a multigene family. The 5' and 3' ends of all 10 clones were analyzed by DNA sequencing. Two cDNAs were separate isolates of a sequence with strong homology to pyruvate kinase genes from other fungi (59 to 73%) and humans (60%). A third cDNA had strong sequence homology to the Saccharomyces cerevisiae and Kluyveromyces lactis alcohol dehydrogenase genes (68 to 73%). A fourth cDNA was homologous (81%) to an S. cerevisiae protein of unknown function. The functions of the remaining six C. albicans cDNAs are not known. A more detailed analysis of the clones encoding glycolytic enzymes revealed that sera from few patients recognized them as antigens. Therefore, although glycolytic enzymes constitute a group of C. albicans proteins that are immunogenic during oral and esophageal infections, their detection cannot be exploited as an accurate marker of infection.

attempted to elucidate the immunological relationship between C. albicans and its host by characterizing specific C. albicans antigens (12, 13, 40, 45). Using an alternative approach, we have attempted to identify C. albicans antigens which (i) can be used for diagnosis of candidiasis and (ii) define new proteins which may play a role in infection. We have analyzed, at random, a set of cDNAs that encode C. albicans proteins which elicited an immune response during infections in humans. Our data confirm that numerous antigens are recognized by sera from patients and show that some glycolytic enzymes are nonubiquitous immunogens

The imperfect fungus Candida albicans is an opportunistic pathogen that causes a wide range of infections which frequently occur in the gastrointestinal, respiratory, and genital tracts in many animals, including humans (25). It normally exists as a commensal organism, but it can cause severe infections, particularly in an immunocompromised host. Only a limited number of safe and effective drugs are available to combat C. albicans infections. Genetic approaches to an understanding of the pathogenicity of C. albicans have been hampered because the organism is diploid and has no known sexual cycle (36). In addition, the relative importance of various virulence factors of C. albicans during pathogenesis remains unclear (7). This organism is able to undergo a morphogenetic switch from a yeast form (which undergoes cell division by budding) to a hyphal form (that generates mycelia). The hyphal form is able to penetrate epithelia, but both forms are found in infected tissue, so the relationship between cell morphology and pathogenicity is not yet clear (7, 20, 25, 30). Other potential virulence factors include the production of an extracellular proteinase (4, 15, 19, 27), the synthesis of receptor-ligand molecules that promote adherence to the host (6), and a high degree of genetic flexibility, which allows the conversion to a variety of phenotypic forms at high frequencies (3, 26, 29, 42). Pathogenicity is also dependent on the immunological status of the host. However, mucocutaneous infections rarely become invasive, even in severely compromised hosts (12). Mechanisms other than the cell-mediated immune response appear to be important in protection; for example, circulating and secretory antibodies may have protective effects in the disease situation (21). Some groups have *

during C. albicans infections.

MATERUILS AND METHODS Sera. Serum samples were obtained from 30 different patients, 22 of whom were suffering from acute candidiasis (oral or esophageal) (Table 1). Twenty-seven of these patients were positive for human immunodeficiency virus (HIV) infection. Culture conditions. C. albicans Robin, Berkhout strain 3153, was from the London Mycological Reference Laboratory. The organism was grown in the yeast form to the late exponential growth phase at 25°C with shaking at 200 rpm in YPD (2% glucose, 2% bacteriological peptone, 1% yeast extract). To induce hyphal growth, 10 ml of this culture was then used to inoculate 100 ml of YPD containing 10% bovine calf serum at 37°C (approximately 2 x 107 cells per ml). Growth in the yeast and hyphal forms was monitored by light microscopy. Preparation and analysis of nucleic acids. Genomic DNA was prepared from C. albicans as described previously (18), and total RNA was prepared by the method of Lindquist (17). Southern blotting (34) and Northern (RNA blot) analysis (24) were performed as described previously with radio-

Corresponding author. 4263

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TABLE 1. Reactivities of cDNAs 3 and 4 with individual seraa Patient no.

Current Candida infection

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Esophageal Oral Oral Oral Oral Oral Oral Oral

cDNA3 (pyruvate kinase) IgM/IgG

IgA

+ +++ + + +

cDNA4 (alcohol dehydrogenase) IgA IgM/IgG

+++

+ ++

+++

+

-

+

+

-

-

-

-

-

-

-

-

-

-

-

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-

-

-

a The immunoreactivities of C. albicans cDNAs encoding alcohol dehydrogenase and pyruvate kinase were compared with negative controls prepared by using Agtll phage lysates and the whole lambda cDNA library. The signals were scored blindly and independently by four people. Average scores minus the negative-control values are presented.

labelled probes prepared by random priming of purified DNA restriction fragments (10) or by in vitro transcription (14). Unless stated otherwise, other DNA and RNA manipulations were performed by standard procedures (32). Construction of cDNA library. RNA was prepared from C. albicans grown for 105 min following the induction of hyphal growth by the addition of serum combined with an increase in temperature to 37°C. Poly(A)-containing RNA was then isolated by one passage through oligo(dT)-cellulose with 10 mM Tris-HCl-0.2% (wtlvol) sodium dodecyl sulfate (SDS)-5 mM EDTA-0.5 M NaCl (pH 7.5) as the loading buffer and 10 mM Tris-HCl-0.2% (wtNvol) SDS-1 mM EDTA (pH 7.5) as the elution buffer. A cDNA library was then constructed with 10 ,ug of poly(A)-containing RNA and the XZAPII kit following the manufacturer's instructions (Stratagene Ltd., Cambridge, U.K.). Library screening. A portion of the cDNA library was incubated with 100 ,ul of an Escherichia coli XL-1 Blue culture at 37°C for 20 min. This mixture was then plated with 8 ml of NZYM agarose onto NZYM agar plates (NZYM: 1% NZ medium, 0.5% NaCl, 0.5% Bacto extract, 0.2% MgSO4- 7H20 [pH 7.5]) and incubated for 4 h at 42°C. A nitrocellulose filter saturated with 50 mM IPTG (isopropylthiogalactopyranoside) was laid onto each plate, and the plate was incubated for a further 4 h at 37°C. Filters were blocked by incubation with lx TBS (50 mM Tris-HCl, 150 mM NaCl [pH 8.0]) containing 5% (wttvol) nonfat dry milk.

Before use in the cDNA screening procedure, samples of 10 sera were combined and absorbed twice against protein extracts made from E. coli XL-1 Blue and from C. albicans 3153 harvested in the yeast form to enrich for antibodies that react with proteins synthesized in the hyphal form of the organism. These 10 sera were from five patients with candidiasis (Table 1, patients 2, 4, 5, 18, and 23) and five uninfected patients (Table 1, patients 25, 26, 27, 30, and one other patient not included in this experiment). Preabsorbed antibodies were diluted 1:100 (final dilution) in lx TBS containing 5% nonfat dry milk and incubated with the filters overnight at 4°C. Filters were washed twice in lx TBS containing 1% (vol/vol) Tween 20 and then twice in 1 x TBS. Alkaline phosphatase-labelled second antibody against human immunoglobulin M (IgM) and IgA (Sigma, Poole, Dorset, U.K.) were diluted 1:500 in lx TBS containing 5% nonfat dry milk and incubated with the filters for 2 h at room temperature. The filters were washed twice in 1 x TBS containing 1% (vol/vol) Tween 20 and then three times in 1 x TBS. The staining reaction was carried out in AP buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2 [pH 9.5]) containing 132 ,ug of nitroblue tetrazolium and 72 ,ug of 5-bromo-4-chloro-3-indolylphosphate per ml until a clear signal was visible. The reaction was stopped by washing the filters in 20 mM Tris-HCl-5 mM EDTA (pH 8.0). Positive plaques were picked, incubated with 1 ml of SM (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl [pH 7.5], 0.01% [wt/vol] gelatin), and 10 randomly selected clones were screened and purified a second time by the same procedure. Finally, positive phage were converted into Bluescript plasmids by the Stratagene protocol. DNA sequencing. Bluescript clones were sequenced by dideoxy procedures (33) with commercial T3 and T7 oligonucleotide primers (Promega, Southampton, U.K.), and the sequences were extended with 17-base synthetic oligonucleotide primers. Double-stranded DNA templates were sequenced with Sequenase (USB, Cambridge BioScience, Cambridge, U.K.) and 35S-dATP or with Taq polymerase (Promega, Southampton, U.K.) and 32P-end-labelled primers. DNA sequences were analyzed with the Genetics Computer Group programs (9) on the SERC computer at the

Daresbury Laboratory. Immunological dot blots. NZYM agar plates were overlaid with 8 ml of NZYM-agarose containing 100 ,ul of E. coli RY1090 (47). To test the reactions to the proteins encoded by the 10 cDNA clones, 10 ,ul of each phage was spotted onto the plates; 10 ,ul of a Xgtll lysate were used as a negative control. To test reactivity to the glycolytic enzymes, only phage for C albicans PYK1 (cDNA3) and C. albicans ADHI (cDNA4) were used. Xgtll and the total cDNA library were used as negative controls. For this experiment, the phage stocks were diluted to 100 PFU/10 ,lI. All plates were incubated for 3 h at 42°C, overlaid with nitrocellulose filters saturated with 50 mM IPTG, and incubated for 4 to 5 h at 37°C. All further procedures were similar to the library-screening procedure with two exceptions. (i) Twenty individual sera were used to analyze the reactivity of the glycolytic enzymes, whereas a pooled mixture of 10 sera was used to analyze the 10 positive clones (the same sera that were used for the library screening). (ii) The second antibodies were specific for different Ig classes. In each case, the patient sera were preabsorbed against bacterial proteins (twice) before use.

VOL. 61, 1993

RECOGNITION OF C. ALBICANS PROTEINS DURING INFECTIONS TABLE 2. cDNA clones analyzed

A

ii 2

Insert

.4

mRNA

cDNA clone

length'

1 2

0.4 1.7

1.8 1.9

3

1.7

1.9

pyX1b

4 5 6 7 8 9 10

1.2 0.9 0.5 2.2 1.1 1.0 0.9

1.3 1.8 0.6 3.5 1.2 1.0 1.0

ADHlC

lengtha

4265

Identity

_ -, *11._

.-

t

-;

t

49411 12

\

9

0,p )I(

pYKJb 1

4Q
> 11.(.,(>D

Northern

RESULTS Isolation of cDNA clones encoding immunogenic proteins. A cDNA library was made from poly(A)-containing RNA isolated from the hyphal form of C. albicans 3153. At least 99% of the 1.2 x 106 independent clones in the library contained cDNA inserts, as judged from the frequency of insertional inactivation of the lacZ sequence in the vector. The library was constructed in the XZAPII vector, which is designed to clone cDNAs in a directional manner, with the 5' end of the mRNA sequence located close to the T3 primer site and the 3' end close to the T7 primer. This facilitated subsequent analysis of the cDNAs by DNA sequencing. The library was screened with pooled sera from 10 patients, five of whom had oral and/or esophageal candidiasis. Nine of these patients were HIV positive. Before screening, the sera were absorbed with cell extracts to reduce the titer of antibodies that react with E. coli proteins and proteins present in the yeast form of C. albicans. Positive reactions were clearly identifiable for 83 of 40,000 clones screened (data not shown). Ten of these 83 positive clones were chosen at random for further analysis. All 10 clones reacted consistently with the pooled sera during subsequent rounds of plaque purification. The 10 clones were converted from their lambda phage form into their Bluescript plasmid form, and the approximate length of each cDNA insert was measured by restriction analysis (Table 2). Northern analysis of the corresponding mRNAs revealed that 7 of the 10 cDNAs were nearly full length (Table 2). Quantification of these Northern blots showed that the relative abundances of the mRNAs varied considerably (more than 100-fold), with cDNAs 2, 3, and 4 having the most abundant mRNAs (43a). Preliminary characterization of cloned sequences. Since differential expression of specific isoenzymes during morphogenesis in C. albicans has been observed (16), it was important to determine whether any of our cDNAs was derived from a multigene family. None of the cDNAs generated complex hybridization patterns associated with multigene families when they were used as probes against Southern blots of C. albicans genomic DNA. For example, single bands were observed in each lane of the Southern blot probed with cDNA2 when genomic DNA was digested with enzymes that do not cleave within the cDNA insert (Fig. 1). Furthermore, when Northern blots of C. albicans RNA were probed with each cDNA, all gave a single band (for example,

FIG. 1. Southern and Northern analysis of C. albicans PYKI. Southern and Northern blots were prepared with genomic DNA and RNA from C. albicans, respectively, and probed with the purified insert from cDNA2 (PYKI). (A) Southern analysis of genomic DNA digested with EcoRI, EcoRI plus BamHI, EcoRI plus HindIII, EcoRI plus PstI, EcoRI plus XbaI, BamHI, BamHI plus HindIII, BamHI plusXbaI, HindIII, HindIII plusXbaI,XbaI, and PstI (lanes 1 to 12, respectively). (B) Northern analysis of 20 1Lg of RNA isolated from the yeast (Y) and hyphal (H) forms of the organism.

cDNA2, Fig. 1). Therefore, the proteins encoded by the cDNAs were recognized as antigens by sera from infected patients and stimulated an immunological response. Northern analysis of the mRNA corresponding to cDNA2 showed that this mRNA was induced approximately threefold when cells growing in the yeast form at 25°C were compared with those growing in the hyphal form in the presence of serum at 37°C (Fig. 1). A similar result was obtained for cDNA4. We are currently investigating whether changes in the expression of these sequences correlate with the dimorphic switch. Antibody type involved in recognition. Immunological dot blots were performed to confirm the reactivity of the proteins encoded by the 10 clones and to identify the antibody type involved in the immunological response. In particular, we were interested in the reactivity with IgA antibodies, since mucosal tissue is infected in oral candidiasis. Duplicate dot blots were prepared on nitrocellulose membranes for each plaque-purified lambda cDNA clone. The 10 pooled sera were incubated with both membranes, and then individual membranes were incubated with goat antibodies against either human IgM or human IgA. Binding by antibodies was detected by the conjugated alkaline phosphatase reaction

(Fig. 2).

All of the cDNAs were found to encode proteins that were recognized by IgA antibodies, although the reactions with cDNA3 were stronger than with the other clones. IgM antibodies reacted with the proteins encoded by all 10 cDNAs, and once again, the reactions were stronger for cDNA3. These observations were reproducible. Therefore, the proteins encoded by these cDNAs, and especially by cDNA3, appear to be potent immunogens during C. albicans infections. Sequence analysis of each cDNA. Preliminary DNA sequencing was performed on each cDNA in an attempt to assign its function. Between 150 and 300 nucleotides of single-stranded DNA sequence was generated from each end of all 10 cDNA clones with the commercial T3 and T7 oligonucleotide primers. These expressed sequence tags (1) were subjected to computer searches in an attempt to

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1

()

FIG. 2. Immunological reactivity of the proteins encoded by each cDNA clone. Plaques for each lambda cDNA clone (1 through 10) and a Agtll control (nos. 11) were prepared, and the phage lysates were transferred to a nitrocellulose membrane following IPTG induction. Filters were blocked and incubated with pooled sera from 10 patients, and detection was done with alkaline phosphatase-labelled second antibody against human IgM. Positive responses were obtained reproducibly for all 10 clones in further experiments.

oligonucleotide primers. These expressed sequence tags (1) were subjected to computer searches in an attempt to identify possible sequence homologies within the data bases. Significant homologies (>50% on overlaps of >100 bp) were identified for four of the cDNAs. The 5' sequences of cDNAs 2 and 4 were then extended to 548 and 510 bases, respectively (Table 3). The 5' sequences of cDNA clones 2 and 3 show a high degree of homology to the 5' regions of pyruvate kinase gene sequences from Saccharomyces cerevisiae, Trichoderma reesei, Yarrowia lipolytica, Aspergillus nidulans, and human muscle (Fig. 3; Table 3). In addition, the 3' ends of cDNAs 2 and 3 are homologous to the 3' region of S. cerevisiae PYKI (59.9% identity over 142 bases; not shown). Furthermore, the significance of these homologies increased when noncoding sequences were excluded and the comparison was limited to the coding region. Therefore, cDNAs 2 and 3 appear to be two separate isolates of the pyruvate kinase mRNA (C. albicans PYK1). The 5' sequence of cDNA4 shows strong homology with the 5' regions of alcohol dehydrogenase genes from S. cerevisiae and Kluyveromyces lactis (Fig. 4; Table 3). Also, the 3' sequence of cDNA4 is homologous to the 3' ends of the ADH1 and ADH3 genes from S. cerevisiae (73.2% over TABLE 3. Significant sequence homologiesa Identity

overlap

Reference(s)

S. cerevisiae PYK1 T. reesei PKI Y lipolytica PYK1 A. nidulans PKIA Human M-type Pyk

73.0 59.3 61.5 69.6 59.8

534 332 423 148 132

5, 23 37 43 8 44

S. cerevisiae ADHI S. cerevisiaeADH2 S. cerevisiae ADH3 K lactisADH2 K lactisADH3 K lactisADH4

70.8 73.2 68.1 72.2 71.4 70.8

383 351 304 331 346 346

2 28 46 38 31 31

cDNA

Homologous sequence

2+3

4

S. cerevisiae ORFb 80.8 313 41 a The alignments for C. albicans PYKI and ADHI are presented in Fig. 3

198 bases and 70.1% over 201 bases, respectively; not shown). Furthermore, the 5' sequence of cDNA4 overlaps the partial sequence of C. albicansADHI published by Shen and coworkers (40). These sequences are identical over a 107-base overlap (Fig. 4). Therefore, cDNA4 probably encodes alcohol dehydrogenase (C. albicans ADH1). cDNA10 is homologous to a 177-amino-acid-coding open reading frame from S. cerevisiae located downstream from the SIR3 locus (41) (Table 3). No functional analysis of this open reading frame has been performed, and therefore no function can be assigned to cDNA10. The functions of the six remaining cDNAs remain obscure (Table 2). Recognition of glycolytic enzymes by individual sera. The phage lysates for cDNAs 2, 3, and 4 gave strong reactions against IgA and IgM antibodies in the pooled sera. These reactions could provide the basis of a diagnostic test for C. albicans infections if a high proportion of infected individuals generate antibodies to the proteins encoded by these cDNAs. Therefore, replicate immunological dot blots were prepared for cDNA3 (PYKJ) and cDNA4 (ADHJ), and the membranes were incubated separately with preabsorbed sera from 30 patients. Twenty-two of these patients had oral and/or esophageal infections when their blood samples were taken, and eight were control patients. Antibody binding was detected by using an alkaline phosphatase-conjugated secondary antibody against human IgA or a mixture of antihuman IgM and IgG antibodies. The reactions were scored on a scale (from - to + + +), blindly and independently, by four people. The average scores (after subtraction of background values from the control values) are summarized in Table 1. Only 6 of the 24 patients with C. albicans infections (oral or esophageal) showed clearly detectable IgA, IgM, or IgG antibodies against C. albicans alcohol dehydrogenase or pyruvate kinase (+ or greater, Table 1). Two of the clear positive samples had detectable levels only of IgA antibodies against either of the glycolytic enzymes, two had only IgM or IgG antibodies against these enzymes, and one gave strong reactions with both IgA and IgM plus IgG (patient 2, Table 1). In three of these cases, a reaction was detected against pyruvate kinase but not against alcohol dehydrogenase. However, none showed a reaction against alcohol dehydrogenase in the absence of a reaction for pyruvate kinase. The strength of these reactions varied considerably. Four of the eight patients in the control group (Table 1, patients 13 through 20) carried IgA, IgM, or IgG antibodies against pyruvate kinase (two reactions were clear, but two were questionable). None of these sera showed a reaction against alcohol dehydrogenase. Therefore, there is no clear correlation between the existence of systemic C. albicans infection and the presence of antibodies against these glycolytic enzymes in the serum of patients. Our attempts to isolate the C. albicans alcohol dehydrogenase and pyruvate kinase fusion proteins from E. coli phage lysates by affinity chromatography with anti-,-galactosidase antibodies were unsuccessful, possibly because of instability of the fusion protein (47). This precluded quantification of the reactions by enzyme immunoassay. DISCUSSION

10

and 4, respectively. The percent identity data are approximate because the DNA sequences were determined on one strand. b S. cerevisiae open reading frame (ORF) 3' to SIR3.

In this study, we screened for C. albicans sequences that encode proteins which are immunogenic during infections in humans. Since expression in E. coli was a prerequisite for detection, only sequences encoding proteins which carry

Ca PYKi Sc PYKi Tr PKI

CACGAGCTCACTCATCTTTATCTTGGTTATCAACTTCAA--TGTTGAmAC--TGTTCCAT AATGTCTAGATTAGAAAGATTGACCTCATTAAACGTTGTTGCTG

Ca Sc Tr Y1

PYK1 PYK1 PKI PYKI

CTAAATATTTG G'&GATCCT-CAAAATCGGTACCAT-TGTCCAA&AhCCAACAACGTGG

Ca Sc Tr Y1

PY1(

ACGTTTTGGTTAAATTGAG&&LCTGGTTTG&ACGTTGTCAGAAGATGAATTTCTCCCA

PYKi PKI

P7YKI

AAACCTTGGTTGCTTTGA AAGCTGGTTTGAACATTGTCCGT--ATGAACTTCTCTCA AGGCCCTCAACAAGCTGCGTGATGCCGGCCTCAACGTCGCCCGC--ATGAACTTCTCCCA ATCATGCCGCAATACTAACCACAGCTGGTCTCAACATTGTTCGA--ATGAACTTCTCCCA

Ca Sc Tr Y1

PYK1 P(YK1 PKI P1YKI

CGGTTCTTACG&&TACC&CAAGTCTGTCATTGLCALCGCCAG ALGTCCGAAGAATTGTA CGGCAGCTACGAGTACCACCAGTCCGTCATCGA ATGTGCGCGCCTCTGTCGCCGCCCA CGGCTCGTACGAGTACCACCAGTCCGTCATTGGA&CGCCCGAGAGTCCGAGCAGCGGTT

Ca Sc Tr Y1

P1YK1 P(YK1

CAAAGGTAGACCATTGGCCATTGCCTTGGATACCAAAGGTCCAGAAACTAGAACTGGTCA CCCAGGTAGACCATTGGCCATTGCTTTGG LCACCAAGGGTCCAG AATCAGAACTGGTAC

TACGCCTGC

GTTCTGACTTGAGAAGACCTCCATCATTGGTACCATCGGTCCAAAGACCAACAACCCAG CCGCAACTTT-CGCCGCACCTCCATCATCTGCACCATCGGCCCCAAGACCAACTCGGTCG CGTATGGACCCATGTGTCT

TGGTTCTTATG&ATACCATCAATCTGTCATTGACAATGCTAGAAAATCTG&AGAAGTCTA

PKI

CCCTGGCCGGCCCGTCGCCITTGCCCTCGACACCA&GGGCCCCGAGATTCGAACTGGCAA

P1YKI

CCGAGGCCGACCCCTAGCCATTGCTCTCGACACCAAGGGCCCCGAGATCCGAACCGGTGT

Ca PYK1 SC PY71

TACCATTGGTG&A&AG&TTCACAA-TTCC&CCAkA CG&AATG&TCTTCICCACTGA CACCACCAACGATGTTGACTACCCAATCCCACCAAACCACGAAATGATCTTCACCACCGA CACGGCCGGCGACGTGGACATTCCCATCAGCGCCGGCACCGTCATGAACTTCACCACCGA CACCAAGGACGLC ALG&CTGGGACGTCAAGGCCGGCCATGTCATGCTCTTCTCCACCAA

Tr PKI Y1 P1YKI Hs PKM1

Ca PYK1 SC PYK1 Tr PKI Y1 P1YKI An PKIA Hs PKM1

Ca PYK1 Sc PYKi Y1 PYKI An PKIA Hs PKM1

Ca PYKi Sc PYK1 Y1 PYKI An PKIA Hs PKM1

Ca PYK1 SC PYK1 Y1 PYKI

CTGAAGAAGGGAGCCACTCTCAAAATCACGCTGGA

TGATGCTTACA&AACTAAATGTG&G&A&GGTCATGATCATTG&CTATACG&&CATCAC TGACAAGTACGCTAAGGCTTGTG&CG&CAAGATCATGTACGTTGACT CALGMLCATCAC CGAGAAGTACGCGACGGCCTGCGACACTCAGAACATG-TAAGAGACTCTTTTT CCCCAAGTA AGGACCAGTGCGACGATAAGATQTGTACATTGACTACACCA&ATTGT AGACTCCGCTGACAAGTCGGATTGTAGGTACCTCGACTACAAG ACATCAC

TAACGCCTACATGGAAAGTGTGACGAGAACATCCTGTGGCTGGACTACAAGAACATCTG CAQAGTCATTGCTCCAGGTAAAATCATATATGTTGATGALTGGTGTCTTGTCATTTGAAGT CAQGGTCATCTCCGCTGGTAGAATCATCTACGTTG&TGATGGTGTTTTGTCTTTCCAAGT CAAGCAGATTGACATTGGCAAGATCATCTTCGTTGACGACGGTGTTCTGTCCTTCAAGGT CAQGGTGATCTCTCCTGGCAAGCTCATCTATGTTGATGACGGTATCCTTTCCTTCGAGGT CAAGGTGGTGGAAGTGGGCAGCAAGATCTACGTGGATGATGGGCTTATTTCTCTCCAGGT CATCTCCGTTGATGATCAACAAACTTTGAAAGTCAGATCTCTTAACGCTGGAATGATCTC TTTGGAAGTCGTTGACGACAAGACTTTG&AGGTCAAGGCTTTGAACGCCGGTAAGATCTG

TCTCGAGAAGATTGACGGCGAGACCCTCAAGGTCGAGACTCTCAICAACGGTAAGATCTG CCTCGAAGTCGTAGATGACAAGACCATCCGCGTCCGGTGCTTGAACAACGGCAACATCTC GAAGCAGAAAGGTACGTATGGGAGCTGGAGTCCAGTTGTCTAAAACAGTCTTTTG-TCTC TTCCCACAAA

TTCCCACAAGGGTGTCAAC TTCTCGAAAGGGTGTCAAC

TTCCCGCAAGG An PKIA TAAACTTCTCGTCTCTGCCT Hs PKM1 FIG. 3. cDNAs 2 and 3 are homologous to pyruvate kinase. The 510-bp 5' sequence of cDNA2 (represented as Ca PYKI) is aligned with the sequences of the pyruvate kinase genes from S. cerevisiae (5, 23), T. reesei (37), Y lipolytica (43),A. nidulans (8), and the human M gene for Ml- and M2-type pyruvate kinases (44). Homologies to the Candida sequence are in boldface. The percent homology and length for each overlap are presented in Table 3. Of the 5' sequence generated from cDNA3, 292 bp were identical to the corresponding region of cDNA2 (not shown). 4267

Ca Sc Sc Kl Kl Kl

ADHi ADH2 ADHI ADH2 ADH3 ADH4

Ca Sc Sc Ki Kl Kl

ADH1

Ca Sc Sc Kl Ki Kl

ADH2 ADHI

ADH2 ADH3 ADH4 ADHI

ADH2 ADHI

ADH2 ADH3 ADH4

Ca Sc Sc Sc Kl Kl Kl

ADH1

Ca Sc Sc Sc Kl Kl Kl

ADH1 ADH2 ADHI

Ca Sc Sc Sc Kl Kl Kl

ADHI ADH2

ADH2 ADHI

ADH3 ADH2 ADH3 ADH4

ADH3 ADH2 ADH3 ADH4

ADHI

ADH3 ADH2 ADH3 ADH4

.

ACAAATACAAAAACA&TTATGTCTGAACAAATCCCA------AAAACTCAAAAAGCCGTT CTATTAACTATATCGTAATACACAATGTCTATTCCA------GAAACTCAAAAAGCCATT CCAAGCATACAATCAACTATCTCATATACAATGTCTATCCCAGAAACTCAAAAAGGTGTT CTAAAAAGAATATCAATATTAATTAAAAAATGTCCATTCCT-GAAACTCAAAAGGGTGTT TTGGTTCCATCAGAACCTTAGCTACCTCTGTGCCA------GAAACCCAAAAGGGTGTT AGCTTTTTGAGATTAAACTCTTCTTTCGCTATCCCA------GAAACTCAAAAGGGTGTT

GTCTTT---GTAACCAATGGTGGTCAATT-AGTCTACAGGATTACCCAGTTCCAACTCCA ATCTTCTACGAATCCAACGG---CAAGTTGGAGCATAAGGATATCCCAGTTCCAAAGCCA ATCTTCTACGAATCCCACGG---TAAATTGGAACACAAGGATATTCCAGTTCCAAAGCCA ATCTTTTACGAA---AACGGTGGTGAATTGCAATACAAGGACATTCCAGTTCCAAAGCCA ATTTTCTATGAG---AATGGTGGTAAATTGGAATACAAGGACATTCCAGTTCCAAAGCCA ATCTTCTACGAA---AATGGTGGTAAGTTGGAATACAAGGATTTGCCAGTTCCAAAGCCA AAGCCAAATAGAATTGTTGATTCACGTCAAATAC-CTGGTGTCTGTCACACTGATTTACA AAGCCCAA-CGAATTGTTAATCAACGTCAAGTACTCTGGTGTCTGCCACACCGATTTGCA AAGGCCAA-CGAATTGTTGATCAACGTTAAGTACTCTGGTGTCTGTCACACTGACTTGCA AAGGCCAA-CGAACTTTTGATCAACGTCAAGTACTCCGGTGTCTGTCACACCGATTTGCA AAGCCAAAT-GAAATCTTGATCAACGTCAAGTACTCCGGTGTGTGTCATACCGATTTGCA AAGGCTAA-CGAAATTTTGATTAACGTTAAGTACTCCGGTGTTTGTCACACCGATTTGCA TGCTTGGAAAGGTGACTGGCCATTGGCTACTAAATTG-CCATTAGTTGGTGGTCACGAAG CGCTTGGCATGGTGACTGGCCATTGCCAACTAAGTTA-CCATTAGTTGGTGGTCACGAAG CGCTTGGCACGGTGACTGGCCATTGCCAGTTAAGCTA-CCATTAGTCGGTGGTCACGAAG TTTACATGCTTGGCACGGCG--ATTGGCCATTACCTGTTAAACTACCAT CGCATGGAAGGGTGACTGGCCTTTGCCAACCAAATTG-CCATTAGTTGGTGGTCACGAAG CGCATGGAAGGGTGACTGGCCATTGCCAACCAAGTTG-CCATTGGTCGGTGGTCACGAAG CGCCTGGAAGGGTGACTGGCCATTGCCAGTTAAATTG-CCATTAGTCGGTGGTCACGAAG

GTGCCGGTGTCGTTGTCGGTATGGGTG-AAACGTCAAGG--TGGAA-ATCGGTGCTTTTN GTGCCGGTGTCGTTGTCGGCATGGGTGAAAACGTTAAGGGCTGGAAGATCGGTGACTACG GTGCCGGTGTCGTTGTCGGCATGGGTGAAAACGTTAAGGGCTGGAAGATCGGTGACTACG TAGTAGGTGGTCATGAAGGTGCTGGTGTAGTTGTCAAAC--TAGGT-TCCAATGTCAAGG GTGCTGGTGTCGTTGTTGCTATGGGTGAAAATGTCAAGGGCTGGATCATTGGTGACTTTG GTGCTGGTGTCGTTGTTGCTATGGGTGAAAACGTCAAGGGCTGGAACATTGGTGACTTTG GTGCTGGTATCGTTGTTGCCAAGGGTGAAAACGTTAAGAACTTCGAAATTGGTGATTACG NNGGATGG AAATCGGTG-ACTTTGCCGGTATCAAATGGTTGAATGGTTCTTGTATGAGT

CCGG-TATCAAATGGTTGAACGGTTCTTGTATGGCCTG--TGAATACT-GTGAATTGGGT CCGG-TATCAAATGGTTGAACGGTTCTTGTATGGCCTG--TGAATACT-GTGAATTGGGT GC---TGSAAGTCGGTG-ATTTAGCAGGTATCAAATGGCTGAACGGTTCTTGTATGACA CTGG-TATCAAATGGTTGAACGGTTCTTGTATGTCCTG--TGAATACT-GTGAATTGTCC CGGG-TATCAAATGGTTGAACGGTTCTTGTATGTCCTG--TGAATACT-GTGAATTGTCC CTGG-TATCAAGTGGTTGAACGGTTCTTGTATGTCTTG--TGAATTGT-GTGAACAAGGT

Ca ADHI

TGTGAAT---TCTGTCAACAAGGTGCTGAACCAAACTGTGGTGAAGCTGACTTGTCTGGT

Sc ADH2 Sc ADHI

AACGAATCCAACTGTCCTCACGCTGACTTGTCAGGTTACACCCA-CGACGGTTCTTTCCA AACGAATCCAACTGTCCTCACGCTGACTTGTCTGGTTACACCCA-CGACGGTTCTTTCCA

Sc Kl Kl Kl

ADH3 ADH2 ADH3

TGCGAAT---TCTGTGAATCAGGTCATGAATCAAATTGTCCAGATGCTGATTTATCTGGT AATGAATCCAACTGTCCCGATGCTGACTTGTCTG AATGAATCCAACTGTCCAGATGCTGACTTGTCTGGTTACACCCA-CGATGGTTCTTTCCA TACGA&TCTAACTGTTTGCAAGCTGACTTGTCTGGTTACACCCA-TGACGGTTCTTTCCA

Ca Sc Sc Kl Kl

ADH1 ADHI

ADH4

.......

ADH3 ADH3 ADH4

...-.............---.--......................

TACACTCACGATGGTTCATTCGAACAATACGCTACTGCTGATGCTGTCCAAGC -ACAAT-----ACGCTACCGCTGACGCTGTTCAAGCCGC TACACTCATGATGGTTCTTTCCAACAATTTGCGACCGCTGATGCTATTCA -ACAAT----ACCGTA -ACAAT----ACCGT 4268

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FIG. 4. cDNA4 is homologous to alcohol dehydrogenase. The 5' sequence of cDNA4 (Ca ADH1) is aligned with those of the S. cerevisiae ADH1 (2), ADH2 (28), and ADH3 (46) genes and the K lactis ADH2 (38), ADH3 (31), and ADH4 (31) genes. Homologies to the Candida sequence are in boldface. The percent homology and length of these overlaps are presented in Table 3. The dots above the C. albicansADHI sequence indicate the region of overlap with the partial sequence for C. albicansADH1 published by Shen and coworkers (40). The sequences are identical.

polypeptide epitopes would be identified in this study. Therefore, proteins whose epitopes are based on posttranslational modifications (for example, some mannoproteins [45]) would not be detectable with our approach. Five sera from uninfected people were included in the pool of 10 sera used in the screening procedure for two reasons. First, most people are exposed to this pathogen during their lifetime (25), and so we assumed that people without an active Candida infection may also carry antibodies against C. albicans proteins. Second, if certain antibodies confer a protective effect against C. albicans infections, one might expect these antibodies to be present in uninfected subjects. About 0.2% of the cDNAs in the library gave clearly detectable signals in the immunological screening procedure. The proportion of cDNA clones that react positively in such a screen is dependent not only on the proportion of polypeptides that are immunogenic during C. albicans infections but also on the relative abundance of the mRNAs encoding these sequences. We attempted to enrich for those proteins whose expression was increased during hyphal growth in the screening procedure. However, sequences that are expressed during the growth of the yeast form were isolated (for example, those encoding glycolytic enzymes). Presumably, our depletion of antibodies with proteins present in the yeast form was incomplete, because these immunogenic enzymes were abundant. Three of the 10 C. albicans cDNAs that we analyzed appear to encode pyruvate kinase or alcohol dehydrogenase (Fig. 3 and 4). Biases in the cloning and screening procedures may have influenced the number of glycolytic enzymes identified, since glycolytic enzymes can constitute up to 30% of total soluble proteins in S. cerevisiae (11). Nevertheless, the results of the immunological dot blots suggest that these glycolytic enzymes can be major immunogens during C. albicans infections (Fig. 2). This is consistent with previous studies which have analyzed C. albicans proteins as allergens (12, 13, 40). The isolation and DNA sequencing of C. albicans cDNA clones encoding 40- and 48-kDa antigens revealed that these proteins have strong homology to S. cerevisiae alcohol dehydrogenases and enolases (12, 41). In addition, the purification and partial amino acid sequencing of allergens of 37, 43, and 46 kDa indicated that these C. albicans proteins correspond to aldolase, phosphoglycerate kinase, and enolase, respectively (13). Our data extend the above list of immunogenic glycolytic enzymes to pyruvate kinase, and in addition, the results with IgA antibodies demonstrate that glycolytic proteins function not only as allergens but also as antigens during infections (Table 1). Alcohol dehydrogenase and pyruvate kinase sequences were recognized as being among the strongest antigens analyzed in the pool of 10 sera (Fig. 2). However, pyruvate kinase and alcohol dehydrogenase are not general immunogens, since the majority of the sera tested failed to react with them (Table 1). Only one serum was strongly reactive when individual sera were tested (Table 1, patient 2). Interestingly, this serum showed the strongest reactions to both pyruvate kinase and alcohol dehydrogenase. A similar linkage effect (in which sera from some individuals show strong reactions

to more than one glycolytic enzyme) has been observed previously for allergic reactions against C. albicans (13). The basis for the variation in the strength of these reactions among individuals is not known. There was no clear correlation between the severity of infection (esophageal versus oral) and the recognition of the antigens by the sera studied. The coding regions in cDNAs 3 and 4 were full length, as judged from the length of the cDNA inserts and by comparison with the analogous S. cerevisiae sequences (Tables 2 and 3). Therefore, weak or negative reactions to alcohol dehydrogenase or pyruvate kinase were not due to the lack of key polypeptide epitopes. (This may not hold true for cDNAs 1, 5, and 7, which contain only a portion of their corresponding mRNAs [Table 1]). Instead, the variation is probably related to the immunological susceptibility of individual patients and their ability to recognize these proteins as foreign. This notion is supported by the fact that a number of C. albicans proteins can be allergens in some patients but not in others (22, 35, 39, 48). Seven of the patients tested were known to be allergic to antibiotics, but there was no correlation between this property and their response to the glycolytic enzymes. In conclusion, one of our goals was to identify C. albicans antigens that could provide the basis for a diagnostic test. The approach was to isolate cDNAs that encode proteins which are recognized by most individuals during infections. Initially, C. albicans PYKI and ADH1 seemed to be good candidates, as phage lysates made from these cDNAs reacted strongly with the 10 pooled sera (Fig. 2). However, these proteins are unlikely to provide the basis for successful diagnostic tests or vaccines because few sera recognized alcohol dehydrogenase or pyruvate kinase from C. albicans (Table 1). A second goal was to identify new proteins which may play a role in infection. This has been successful, because an additional six C. albicans cDNAs which encode immunogenic proteins have been isolated in this study (Table 2). However, their functions remain obscure, as no significant homologs have been identified in the data bases. Studies are in progress to determine the function and regulation of these sequences and their recognition patterns during infection. ACKNOWLEDGMENTS We thank J. Koehler for her help in obtaining control sera, S. Miyasaki and Y. De Souza for supplying sera from the UCSF AIDS Specimen Bank, and J. Price for his contribution to the DNA sequencing. This work was supported by grants from the Wellcome Trust and

the Oral and Dental Research Trust. This work benefitted from the use of the Sequenet facility at the Daresbury Laboratory, United

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