(Received for publication, September 8, 1995)
From the
WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We isolated and analyzed a clone of genomic WI-1 to characterize this key adherence mechanism of the yeast. The 9.3-kilobase insert contains an open reading frame of 3438 nucleotides and no introns. The amino acid sequence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned gene is authentic WI-1. The complete coding sequence has 30 highly conserved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersiniae adhesin invasin, the C terminus displays an epidermal growth factor-like domain, and the N terminus has a short hydrophobic sequence that may be a membrane-spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the adhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nucleotide positions 2287-2290), TATA box(2380-2385), and CT motif(2399-2508). Transcription is initiated within the CT motif at nucleotide 2431. A 5.5-kilobase subclone containing the full coding sequence of WI-1 was expressed as a histidine-tagged fusion protein in Escherichia coli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is strongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. This work clarifies a key adherence mechanism of B. dermatitidis and will permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.
Medically important fungi pose a serious health hazard, especially for the growing numbers of immunocompromised patients worldwide(1) . At the same time, there are few antifungal drugs, and little is known about pathogenic mechanisms that might serve as new drug or vaccine targets in the fungi. To establish themselves in a host, pathogens must be able to adhere to tissues. Characterization of adherence promoting molecules, or adhesins, has therefore yielded insights into a key mechanism of microbial invasion of the host. Several adhesins of the opportunistic fungus Candida albicans regulate attachment, invasion, and dissemination of the fungus and help explain the molecular pathogenesis of disseminated candidiasis(2, 3, 4) . However, adhesins of other medically important fungi have not been characterized.
Blastomyces dermatitidis is a dimorphic fungal pathogen that infects the host through inhalation of conidia(5) . Upon transformation into the pathogenic yeast phase, B. dermatitidis multiplies within the lung and disseminates via the blood stream and lymphatics to cause disease in the skin, bone, genitourinary tract, and brain(5, 6) . Inflammatory reactions occur at the initial site of infection and at these metastatic foci. Cellular immunity is the major protective response of the host in preventing progressive disease in blastomycosis (7, 8, 9, 10) . The interaction of B. dermatitidis yeasts with host macrophages is therefore a critical event in the pathological process.
WI-1 is a 120-kDa surface protein on B. dermatitidis yeasts (11) and the major target antigen of cellular (12) and humoral (13) immunity in infected humans. Analysis of a partial cDNA from cloned WI-1 showed 4.5 copies of a 25-amino acid repeat arrayed in tandem near the C terminus. The repeat sequence is similar to invasin, an adhesion-promoting protein on Yersiniae(14) . Binding studies demonstrated WI-1 is an adhesin, which mediates attachment of yeasts to macrophages; the 25-amino acid repeat interacts with both CR3 and CD14 receptors(15) . In view of the importance of adherence in understanding and intervening in the pathogenesis of invasive fungal diseases, and because of the key role of WI-1 in binding yeasts to host tissues, we sought to characterize WI-1 more carefully at the molecular level. We describe here the complete cloning of genomic WI-1 and express and functionally analyze the adhesive properties of mature recombinant WI-1. We show the striking finding that the full-length gene contains 30 copies of the invasin-like repeat within the coding sequence. This information sheds new light on a key adherence mechanism in this pathogenic fungus.
Genomic DNA was resolved
over 1 h at 100 V on a 1% agarose gel and stained with ethidium
bromide. For Southern analysis, genomic DNA digested with restriction
enzyme was resolved overnight at 30 V on a 1% agarose gel, transferred
to nitrocellulose, and probed using previously described
methods(17) . A WI-1 cDNA designated p1.1 and described under
``Results'' was prepared as a purified NotI fragment
and used as a probe. A 100-ng aliquot of the probe was radiolabeled
with [-
P]dCTP to a specific activity of
approximately 10
cpm/µg using random oligonucleotides
as primers (Pharmacia Biotech Inc.). Washed Southern blots were used to
expose Kodak XAR-5 film with intensifying screens at -80 °C.
The genomic library was screened for clones of WI-1 by colony hybridization (17) using the WI-1 cDNA p1.1 as a probe. Positive colonies were isolated from original replica plates, replated, and rescreened with the probe until they were purified.
Protein sequencing was performed (Keck Facility,
Yale University, New Haven, CT) on peptides of native WI-1 from ATCC
strain 60636 (20) following treatment of WI-1 in situ in acrylamide gel slices with endoproteinase LysC and separation
by narrow bore, reverse-phase high performance liquid chromatography
(HPLC) ()using previously described techniques(21) .
Peptides were selected for sequencing on Applied Biosystems instruments
by screening HPLC fractions for purity using electrospray ionization
mass spectrometric analysis.
Figure 2: Complete nucleotide and deduced amino acid sequence of genomic WI-1 in B. dermatitidis yeasts. The nucleotide sequence is numbered on the left. The start codon is underlined, the stop codon is designated by an asterisk, and the promoter elements are in boldface and underlined. The transcription initiation site at nucleotide 2431 is in uppercase. The deduced amino acid sequence is displayed below the nucleotide sequence and is numbered on the right. Deduced amino acids that correspond to those derived independently in peptide fragments of native WI-1 appear in boldface type; deduced residues that do not match the residues in the peptide are italicized. Nonmatching residues reflect variation between strains 26199 (genomic WI-1) and 60636 (peptide fragments); see Fig. 5, and (14) .
Figure 5: N-terminal amino acid sequence of internal peptide fragments of native WI-1 protein isolated from ATCC strain 60636. Underlined residues in italicized, boldface type are those that do not match the deduced sequence of genomic WI-1 from ATCC strain 26199 (see corresponding amino acid positions in the complete sequence of genomic WI-1 in Fig. 2). However, the peptide sequence shown matches identically the deduced amino acid sequence of a WI-1 cDNA cloned from ATCC strain 60636 (14) .
For expression of recombinant WI-1, transformed E. coli was grown in LB media containing 100 µg/ml
ampicillin and 25 µg/ml kanamycin in a gyrator shaker at 37 °C.
When the density of the culture reached A at 0.6
(after about 6 h of growth), 5 mM
isopropyl-1-thio-
-D-galactopyranoside was added. Optimal
production of the fusion protein was examined at 1-h intervals after
induction. Bacteria were centrifuged at 5,000
g for 15
min at 4 °C. A lysate was prepared by resuspending the pellet in 1
gel-loading buffer (50 mM Tris, pH 6.8, 100
mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol)
equivalent to 0.1 volume of the original culture. The lysate was heated
to 100 °C for 3 min and centrifuged at 12,000
g for 1 min at room temperature. Lysates were examined by
SDS-polyacrylamide gel as described by Laemmli(24) . Gels were
stained for protein with Coomassie Brilliant Blue.
Recombinant WI-1 containing 6 histidine residues at the N terminus was purified over Ni-NTA resin (23) according to the supplier's specifications. To assess antibody recognition of the fusion protein, Western blots of gels were performed as described(14) . Anti-WI-1 antisera used in these experiments were from immunized rabbits and blastomycosis patients in whom antibody responses to WI-1 had previously been demonstrated by radioimmunoassay(11) .
Human monocytes
were purified from buffy coats by sequential centrifugation on
Ficoll-hypaque and Percoll gradients and cultured in RPMI 1640
containing 12.5% human serum and 10 µg/ml gentamicin. Macrophages
were harvested after 4-7 days, washed, and suspended to
0.5-1.0 10
/ml in Hanks' balanced salt
solution containing 20 mM HEPES, 0.25% bovine serum albumin,
and 0.3 units/ml aprotinin. Five µl of macrophage suspension were
added to each well of a Terasaki tissue culture plate, which had been
previously coated with 1 mg/ml human serum albumin, and the cells were
allowed to adhere for 1 h at 37 °C.
For binding assays, 5 µl
(10) of WI-1- or HSA-coated microspheres were added to
macrophage monolayers and incubated at 37 °C for 30 min. Unattached
microspheres were removed by washing, and the monolayers then were
fixed with 2% glutaraldehyde, 1% sucrose in 0.01 M phosphate
buffer, pH 7.2. The attachment of microspheres to macrophages was
quantified by counting the number of particles bound to 100 macrophages
by phase-contrast microscopy on a Nikon diaphot-inverted microscope.
Peptide inhibition of the binding of microspheres was investigated
by adding 5 µl of the inhibitor to the well just prior to the
addition of the microspheres. The tandem repeat peptide inhibitor used
in these experiments displays 4.5 copies of the repeat and was
synthesized and purified in recombinant form as described
previously(20) . For monoclonal antibody blocking of macrophage
receptors, 5 µl of antibody (50 µg/ml) were added to each well,
and the plates were incubated for 30 min at 4 °C before
microspheres were added. Monoclonal antibodies (1B4, specific for the
common -chain of CD18 receptors(26) ; 904, specific for
the
-chain of CD11b/CD18(27) ; 3C10, specific for
CD14(28) ; and 3G8, specific for the low affinity FcR of
neutrophils, FcRIII, CD16(29) ) were generously provided by Dr.
Simon Newman (University of Cincinnati College of Medicine, Cincinnati,
OH).
The results of binding assays are presented as the percentage of binding, which is the percentage of macrophages binding one or more particles, and the attachment index, which is the total number of microspheres bound per 100 macrophages. All experimental points were performed in duplicate, and experiments were repeated with macrophages from three to five different donors.
Figure 1: Restriction analysis and mapping of genomic WI-1 in B. dermatitidis yeasts. Panel A displays a Southern analysis of genomic DNA from ATCC strain 26199 probed with WI-1 cDNA p1.1. Prior to analysis, DNA was digested with the restriction enzymes shown. Panel B displays a restriction map of the isolated, genomic WI-1 and respective size and orientation of WI-1 cDNAs p1.1 and BK1.
Nearly 20,000 recombinants from an XbaI genomic DNA library of B. dermatitidis strain 26199 were screened by colony hybridization using p1.1 as the cDNA probe. Three independent clones were identified, and each contained the predicted 9.3-kb insert anticipated from the Southern analysis. Comparison of the three clones to WI-1 cDNAs p1.1 and BK1 by further restriction analysis and Southern hybridization confirmed the identity of the three clones as WI-1 genomic DNA.
One clone was studied more thoroughly to investigate whether the 9.3-kb genomic insert contained all of the WI-1 gene. A restriction map of the insert relative to the cDNAs is illustrated below (Fig. 1B). Overlapping WI-1 cDNAs (p1.1 and BK1) and vector sequence were used to probe Southerns of a BglI digest of the clone to define the orientation of the restriction fragments and the location of the WI-1 gene. We predicted that the 3.2-kb BglI fragment was most 5` and contained 2.8 kb of DNA extending beyond the 5` end of p1.1, sufficient to contain the remainder of the WI-1 gene.
In a previous report(14) , we described a 25-amino acid repeat arrayed in tandem and present in 4.5 copies near the 5` end of the 1-kb WI-1 cDNA isolated from ATCC strain 60636. This repetitive sequence also was of interest for its homology over a stretch of 17 residues with the Yersiniae adhesin invasin. Recently, we demonstrated that the tandem repeat promotes WI-1-mediated attachment to macrophage CD18 and CD14 receptors(15) . The tandem repeat from genomic WI-1 of ATCC strain 26199 is 24 amino acids in contrast to the 25-amino acid repeat of the other strain. The sequence of the repeat of strain 26199 is HEKYDW(K/E/D/V)LW(N/D)KWCKD(F/P/S)YNC(D/E)WD(K/S)(F/S). Fig. 2, amino acids 407 to 430, illustrates an example of a copy of the repeat. Fig. 3A shows the alignment and homology of 17 residues of the WI-1 tandem repeat in these two fungal strains with invasin.
Figure 3: Homology of WI-1 with other sequences in the GenBank data base. Panel A shows sequence similarity between a 17-amino acid portion of the tandem repeat of WI-1 (total repeat is 24 amino acids in strain 26199 and 25 amino acids in strain 60636) and Yersiniae invasin. Vertical solid lines indicate identity between residues, vertical dashed lines indicate a conservative amino acid substitution. Panel B shows the similarity between a cysteine-rich sequence near the C terminus of WI-1 and the consensus sequence for epidermal growth factor-like domain(45) . Conserved cysteines are marked with asterisks.
The 30 repeats are clustered in two noncontiguous areas of the protein. In the first area, four repeats are arrayed in tandem near the N terminus, corresponding to amino acid positions 211-307. In the second area, 26 repeats are arrayed in tandem corresponding to amino acid positions 407-1031. The amino acid sequence flanking these two regions also displays repeat-like sequence, but with varying amounts of degeneracy. For example, the four highly conserved repeats near the N terminus are flanked on both the 5` side (amino acid positions 182-210) and the 3` side (positions 307-335) by an elongated repeat of 28 amino acids. Even longer, more degenerate repeat-like sequence (positions 144-182) or, in some instances, short 9- or 10-amino acid elements of the tandem repeat (positions 335-406) flank these two elongated repeats of 28 amino acids and also flank the 3` end of the 26 repeats arrayed in tandem.
We previously described sequence homology of a cysteine-rich, nonrepetitive sequence near the C terminus of WI-1 cDNA BK1 with epidermal growth factor (EGF)-like domains(14) . This sequence also is conserved in positions 1058-1093 of the deduced amino acid sequence of genomic WI-1 from ATCC strain 26199 ( Fig. 2and Fig. 3B).
The predicted molecular mass of the WI-1 protein coded by this nucleotide sequence is 146 kDa, which is in close agreement with the SDS-PAGE relative mobility of approximately 120 kDa for the native WI-1 protein. In addition, the deduced amino acid sequence indicates that the protein is rich in tryptophan (146 residues, 18.6 mol %), aspartic acid (161 residues, 12.7 mol %), lysine (126 residues, 11.0 mol %), tyrosine (95 residues, 10.6 mol %), and cysteine (90 residues, 6.4 mol %), as expected from previous published work on the amino acid composition of native WI-1(20) .
A Kyte-Doolittle (30) hydrophilicity plot of the deduced sequence suggests that the protein is predominantly hydrophilic, due mainly to the charge within the tandem repeat, but that the protein has a distinct hydrophobic sequence at the N terminus (spanning amino acids 1-22) indicative of a membrane-spanning domain (Fig. 4, upper panel). Analysis of the amino acid sequence adjacent to the amino-terminal hydrophobic domain failed to identify sequences of a signal peptide cleavage site(31) . Further analysis of the complete amino acid sequence using the Emini surface probability plot (32) indicates that the tandem repeats are predicted to be at the exposed surface of the WI-1 protein (Fig. 4, lower panel). This would explain their ability to mediate attachment of B. dermatitidis yeasts to human macrophage receptors.
Figure 4: Plots of hydrophilicity and surface probability for the deduced amino acid sequence of WI-1. DNAstar was used to access the hydrophilicity plot of Kyte-Doolittle (30) and the surface probability plot of Emini(32) .
We performed primer extension analysis to determine where transcription of WI-1 mRNA initiates in this region. Using three separate primers, we mapped the initiating base to the C at position 2431 within the pyrimidine motif. A representative experiment is shown in Fig. 6. We consistently saw a doublet band at position 2431 and and 2432. The band at position 2432 most likely represents incomplete extension of the band at 2431 due to methylated nucleotides of the mRNA 5` cap(34) . In this experiment, we also saw a smaller band at position 2436. This band may represent incomplete extension or a second initiation site. The band was not always present using either this primer(2711-2728) or the other two primers.
Figure 6: Primer extension analysis of transcription initiation sites within genomic WI-1. The primer(2711-2728) was annealed to 20 µg of total RNA from strain ATCC 26199. The extended products representing initiation products are represented by arrows. Sequencing reactions using the same end-labeled primer are run alongside (in the order GATC). These results are representative of results observed when two other primers (positions 2513-2529 and 2519-2540) were also used to analyze transcription initiation sites within WI-1.
Direct evidence for promoter activity was accomplished
in functional studies to be reported elsewhere. ()We
confirmed that a 375-base pair piece of DNA spanning nucleotides
2223-2598 in the 5`-flanking region shown in Fig. 2has
promoter activity. This sequence allowed expression of a hygromycin B
resistance gene for transformation of B. dermatitidis yeasts.
Figure 7: Expression and characterization of recombinant WI-1. Panel A shows a 7.5% SDS-PAGE gel of native WI-1 in crude, freeze-thaw extract of B. dermatitidis yeasts (ATCC strain 26199) in lane 1, compared with recombinant WI-1 in a crude lysate of E. coli in lane 2. A molecular size standard of 120 kDa is shown in the left margin. The gel is stained with Coomassie Blue. Panel B shows a 7.5% SDS-PAGE gel of native WI-1 in crude, freeze-thaw extract in lane 3, and purified, recombinant WI-1 following its purification in lane 4. The gel is stained with Coomassie Blue. Panel C shows a Western blot of native WI-1 in lane 5 and purified recombinant WI-1 in lane 6, which were probed with anti-WI-1 antiserum from an immunized rabbit.
Figure 8: Flow cytometry analysis of WI-1 on B. dermatitidis yeasts and WI-1-coated microspheres. Binding of WI-1 specific monoclonal antibody AD3-BD6 was used to quantify the amount of bound native WI-1 (nWI-1) or recombinant WI-1 (rWI-1) adhered to latex microspheres. Staining of B. dermatitidis strain 26199 (positive control) and human serum albumin (HSA)-coated beads (negative control) with monoclonal AD3-BD6 is shown for comparison. Staining of yeasts and beads with the irrelevant IgG2a isotype control monoclonal Leu 5b also is shown. Results are representative of four separate preparations.
To investigate whether recombinant WI-1 retains the adhesive properties of the native protein, we tested binding of the protein-coated microspheres to human macrophages. Both native and recombinant WI-1-coated microspheres bound avidly to the macrophages, yielding percentages of binding between 73 ± 3% and 80 ± 4% and attachment indices between 597 ± 81 and 629 ± 76 (Table 1). In addition, a peptide displaying 4.5 copies of the tandem repeat effectively competed with native WI-1-coated microspheres for binding to the cells. The soluble peptide inhibited binding of these beads in a concentration-dependent manner, yielding a maximum of 81% inhibition at 1 mg/ml of peptide (Fig. 9A). In contrast, 1 mg/ml soluble human serum albumin as a control inhibitor had little effect on binding of the beads, yielding 5% inhibition.
Figure 9: Inhibition of the binding of WI-1-coated microspheres to human macrophages. Panel A shows binding of native WI-1-coated beads inhibited by the soluble ligand HSA, native WI-1, and a recombinant peptide with 4.5 copies of the tandem repeat. Macrophages adhered in the wells of a Terasaki culture dish were incubated with 5 µl of the various soluble ligands and 5 µl of native WI-1-coated beads for 60 min at 37 °C. Nonadherent beads were removed by washing, and the attachment index was quantified by phase-contrast microscopy. The data are the means ± S.E. from two experiments with different donors. Panel B shows inhibition of binding by monoclonal antibodies to CD18 and CD14. Macrophages were adhered in the wells of a Terasaki culture dish and incubated for 30 min at 4 °C with 5 µl of the various monoclonal antibodies at 50 µg/ml. Five microliters of native WI-1 (nWI-1)- or recombinant WI-1 (rWI-1)-coated beads was then added and incubated with macrophages for 30 min at 37 °C. Nonadherent beads were removed, and the attachment index was quantified as above. The data are the means ± S.E. from three experiments with different donors.
In a final set of experiments, we
investigated whether recombinant WI-1-coated microspheres bound to
macrophage CD18 and CD14 receptors as described previously for the
native WI-1 adhesin(15) . Adherent macrophages were
preincubated with monoclonal antibodies for 30 min at 4 °C and then
were incubated with native and recombinant WI-1-coated microspheres for
30 min at 37 °C. Binding of both native and recombinant WI-1-coated
microspheres to macrophages was markedly inhibited by monoclonal
antibodies specific for the CD18 -chain, CD14, and the LPS binding
site on CR3, but not by a monoclonal antibody specific for macrophage
Fc receptors (Fig. 9B).
Despite the growing number of fungal infections in immunocompromised and normal hosts, little is known about the pathogenesis of fungal infections, especially adherence mechanisms. This is especially true for primary fungal pathogens, i.e. dimorphic fungi Histoplasma capsulatum, Coccidioides immitis, and B. dermatitidis, which are the most common. These pathogens can establish themselves and invade tissues of normal hosts and often cause large and widespread outbreaks of human infection, severe manifestations of disease, or both(35, 36, 37) . A better understanding of pathogenic mechanisms including adherence and adhesins in these fungi will ultimately improve treatment and control strategies. Adhesins could serve as vaccine candidates, similar to the situation in bacteria(38) .
We previously showed that the 120-kDa surface protein WI-1 on B. dermatitidis mediates attachment of the yeast to human macrophages (15) . In that study, WI-1-mediated binding to cells was governed, in part, by the 25-amino acid invasin-like, tandem repeat. Of interest is that the host's humoral immune response is directed against the tandem repeat. Anti-WI-1 antibodies in human serum(14) , and a panel of murine monoclonal antibodies (20) react almost exclusively with the repeat. Although cellular immunity is believed to play the key role in resistance to dimorphic fungi and other fungal pathogens, antibodies directed against the WI-1 tandem repeat may defend the host against B. dermatitidis; we speculate that interfering with attachment to host tissues may neutralize the pathogenicity of the fungus. Monoclonal antibodies directed against the capsular polysaccharide of Cryptococcus neoformans protect mice against experimental, cryptococcal infection(39) , and antibodies against a mannan adhesin of C. albicans protect mice against experimental, invasive candidiasis(40) . Thus, studies with B. dermatitidis, together with these on C. neoformans and C. albicans, suggest that immunity to surface components including adhesins may confer protection, and by inference, that adhesins of pathogenic fungi may be candidates for vaccine development.
In this study, we sought to clone the complete gene encoding WI-1 to characterize its binding domains at the molecular level and permit detailed analyses of WI-1-mediated adherence. Our efforts at cloning a full-length cDNA were unsuccessful but did yield a larger WI-1 cDNA that was useful for restriction analysis and screening of a genomic library to isolate full-length WI. In retrospect, a full-length cDNA was probably difficult to isolate because of the repetitive WI-1 sequence and inability to reverse transcribe it.
We isolated the full-length gene on a 9.3-kb fragment of XbaI-cut genomic DNA. Several lines of evidence indicate we have cloned WI-1 rather than some other WI-1-like gene sequence. First, positive clones were isolated by colony hybridization using a WI-cDNA probe, and the sequence of genomic WI-1 matches that of the probe. Second, rabbit anti-WI-1 antiserum reacts strongly with recombinant WI-1 expressed as a histidine-tagged fusion protein. Finally, and most importantly, amino acid sequence from three internal peptide fragments of native WI-1 match the sequence of the genomic gene, confirming its identity as WI-1.
Several features of the WI-1 coding and upstream sequence deserve comment. To our knowledge, WI-1 is the first adhesin cloned from a pathogenic fungus and one of the few genomic genes cloned from a pathogenic yeast. This offers insight into the structure and organization of the coding and upstream sequence of genomic genes in these eukaryotic pathogens. Small introns have frequently been observed in filamentous fungi and nonpathogenic yeasts(33) ; none were observed in the coding sequence of WI-1. However, common features in sequence flanking the start codon and characterizing promoter elements in nonpathogenic fungi were evident in the upstream sequence of WI-1.
In a study of 211 higher eukaryotic genes(41) , 205 had a purine three base pairs upstream from the ATG codon. In 79% of the instances, it was an A, and this is the case for WI-1. Elements of a fungal promoter include a CAAT box, TATA box, and a CT motif; all are present in the upstream sequence of WI-1. Moreover, the spatial relationships between these elements are preserved in WI-1. In many filamentous fungal genes, the transcription start site appears in or immediately downstream from a pyrimidine-rich sequence(33) . This has also been seen in yeasts, especially when genes are highly expressed or lack the usual TATA and CAAT motifs. An extreme example of a CT motif is the oliC gene of Aspergillus nidulans in which 96 bases of a 110-base pair stretch are pyrimidines(42) . In the 110-base pair stretch of the CT motif of WI-1, 84 bases are pyrimidines. Primer extension analysis demonstrates that transcription of WI-1 initiates at nucleotide position 2431, within the pyrimidine stretch.
The consensus sequence for the TATA box in fungi is TAT(A/T)A(A/T) (33) . TATAAA is most common and was observed in WI-1. The TATA box is usually found 20-40 base pairs upstream of the transcriptional start site, although it has been found as far away as -112 and -127 in Neurospora crassa (qa-IS gene) and A. nidulans (gdhA gene), respectively(33) . In WI-1, the TATA box is 52 base pairs upstream of the major transcription start site. In addition, the distance between the TATA box and CAAT box in WI-1 (90 base pairs) is similar to that in other fungal promoters.
In filamentous fungi, the CAAT box is usually found 60-120 base pairs upstream of the transcription start site, although longer distances have been found, including ones of -213 and -306 for two genes in A. nidulans(33) . In WI-1, the CAAT box is 145 base pairs upstream of the major transcription start site.
Perhaps the most
intriguing and unanticipated observation here concerns the tandem
repeat. We observed a total of 30 highly conserved copies of a 24-amino
acid repeat arrayed in tandem and a small number of additional,
degenerate repeats, which together comprised much of the WI-1 protein.
Despite the repeat's slight variation in size and sequence
between strains 60636 and 26199, both repeats show homology with
sequence in Yersiniae invasin's adhesive domain, a
76-amino acid disulfide loop at the C terminus of the
molecule(43) . Although invasin binds Yersiniae to
mammalian cells through attachment to 1 chain integrins such as
the fibronectin receptor (44) , WI-1 binds B. dermatitidis to cells through attachment to
2 chain integrins such as
CD11/CD18 and to CD14(15) . The precise sequence(s) in the
tandem repeat that bind these different receptors, the number of
repeats needed for optimal binding, and the role of nonrepetitive
sequence in promoting attachment are questions that need further study.
A nonrepetitive sequence at the C terminus of WI-1 is rich in cysteine and similar to the consensus sequence for EGF-like domains(45) . These domains have been described in molecules involved in cell-cell or cell-matrix interactions. Extracellular matrix components laminin and nidogen interact with one another and with cellular or microbial targets in part by EGF-like domains(46) . We speculate that the EGF-like domain of WI-1 contributes to adhesive functions of the molecule by interacting with extracellular matrix components. Deletion derivatives of the recombinant WI-1 will allow us to investigate whether and how the EGF-like domain binds B. dermatitidis to extracellular matrix components of the alveolar basement membrane and may help establish this pathogen in lung tissues of the host.