INFECTION AND IMMUNITY, Jan. 1994, p. 79-850019-9567/94/$04.00+0Copyright (C 1994, American Society for Microbiology

Isolation, Characterization, and Cloning of cDNA and theGene for an Elastinolytic Serine Proteinase

from Aspergillus flavusMATUR V. RAMESH, TATIANA SIRAKOVA, AND PAPPACHAN E. KOLATTUKUDY*

Ohio State Biotechnology Center, The Ohio State University, Columbus, Ohio 43210

Received 26 July 1993/Returned for modification 4 October 1993/Accepted 29 October 1993

An elastinolytic serine proteinase produced byAspergiUusflavus 28 that was isolated from a patient who diedof aspergillosis has been purified and characterized. The enzyme was inhibited by the serine proteinaseinhibitors phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. The metal-chelating agents EDTAand EGTA [ethylene glycol-bis(1-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not severely inhibit theenzyme. A cDNA and a 2.95-kb segment of genomic DNA containing the proteinase gene were sequenced. Theopen reading frame that would code for a protein containing 403 amino acids was interrupted by three introns.The mature protein lacks 121 N-terminal amino acids including a putative 21-amino-acid signal peptide. Thepurified mature protein showed a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, whereas that calculated from the deduced protein sequence was 30 kDa. This elastinolyticserine proteinase ofA. flavus has 83 and 82% sequence homology to the similar proteinases from A. fumigatusand A. oryzae. The catalytic properties and the sequence homology around the putative catalytic amino acidssuggest that this enzyme belongs to the serine proteinases of the subtilisin family.

Invasive aspergillosis is a major threat to the long-termsurvival of immunocompromised patients, such as bonemarrow transplant patients (7). Even with the best antifungalagents currently available, the mortality rate is very high (2,5). An approach targeted against the major virulence factorsinvolved in aspergillosis could be effective in combating thisinfection. However, little is known about the factors in-volved in this infection.Use of extracellular enzymes to degrade the barriers in the

host seems to be a common strategy used by pathogenicfungi to invade their hosts (15). Since the fungal conidiaenter the immunocompromised host mainly via the lungs,the main barrier penetrated by the fungus would have elastinas a major structural component (29). Therefore, the invad-ing fungus might use elastase to breach such host barriers. Infact, Aspergillus fumigatus and A. flavus, two prominentagents that cause invasive aspergillosis, have been reportedto produce elastinolytic enzymes when grown on elastin-containing media. Kothary et al. (16) correlated the produc-tion of elastinolytic proteinase activity ofA. fumigatus withits ability to cause invasive pulmonary aspergillosis in im-munocompromised mice. Such elastinolytic enzyme pro-duced by A. fumigatus has been purified and characterizedto be a serine proteinase of the subtilisin family (14, 22, 24).An elastinolytic metalloproteinase from A. flavus was re-

ported (25). It appeared surprising that two closely relatedAspergillus species would use proteinases that have differentmechanisms of action to invade the host tissues. We ob-tained evidence that A. flavus does produce an elastinolyticserine proteinase that is related to the enzyme from A.fumigatus (14). However, such an enzyme has not beenisolated from A. flavus. In this paper, we report purificationand characterization of an elastinolytic serine proteinase

* Corresponding author. Mailing address: Ohio State Biotechnol-ogy Center, 1060 Carmack Rd., The Ohio State University, Colum-bus, OH 43210. Phone: (614) 292-5670. Fax: (614) 292-5379. Elec-tronic mail address: kolattukudy.2@osu.edu.

from A. flavus. Cloning of the cDNA and the gene for thisenzyme and comparison of its structure with the enzymefrom A. fumigatus and other organisms are also presented.These results demonstrate that A. flavus and A. fumigatusproduce highly homologous elastinolytic serine proteinaseswhen grown on elastin.

MATERIALS AND METHODS

Microorganisms. A. flavus isolate 28 was obtained from a

patient who died of aspergillosis. Escherichia coli DH5a wasused for cloning the proteinase gene and plasmid replication.

Purification and N-terminal sequencing of the enzyme.Elastinolytic serine proteinase was isolated from culturesupematant of a 4.5-day-old culture, grown in elastin me-dium (yeast carbon base, 1.17%; insoluble elastin, 0.2%;calcium carbonate, 0.3%) in Roux bottles at 37°C. Themedium was filtered through Miracloth (Calbiochem), and tothe supernatant ammonium sulfate was added to 80% satu-ration. The recovered protein was dialyzed against 50 mMsodium phosphate buffer (pH 6.5), and the solution was

brought to 10% ammonium sulfate. This preparation wasloaded onto a phenyl-Sepharose column that was equili-brated with phosphate buffer containing 10% ammoniumsulfate. After the column was washed with 6 bed volumes ofthe loading buffer, the enzyme was eluted with a 10 to 0%ammonium sulfate concentration gradient. The fractionscontaining the highest elastinolytic activity were collectedand stored at -80°C in 10% ammonium sulfate. The enzymesample from the phenyl-Sepharose column chromatographywas further purified by gel filtration on a Superose 12(Pharmacia) column by fast protein liquid chromatography(FPLC). Only the fraction containing the highest proteinaseactivity was collected and used for other studies. Through-out the purification procedure and storage, the protein wasmaintained in sodium phosphate buffer containing 10% am-monium sulfate to prevent autodigestion. To prevent auto-digestion during electrophoresis, the purified enzyme was

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80 RAMESH ET AL.

precipitated by 5% trichloroacetic acid and washed withacetone before sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). From the polyacrylamide gel,the protein was electroblotted onto a Pro-Blot membrane(Applied Biosystems Inc.) and stained, and the protein bandwas cut and used for N-terminal amino acid sequencing withan automated pulsed liquid protein sequencer (Applied Bio-systems model 475-A).Enzyme assay. With the synthetic substrate, N-succinyl-

Ala-Ala-Pro-Leu-p-nitroanilide (Sigma Chemical Co.), theactivity was measured at 25°C with 1 mM substrate in 20 mMTris-HCl (pH 8.0) in a total volume of 1 ml. The release ofp-nitroaniline was monitored at 410 nm. Tritiated elastinused for the enzyme assay was prepared as reported previ-ously (1, 14). With labeled elastin, the activity was assayedin a total volume of 400 pl containing 1 mg (0.2 ,Ci) ofsubstrate. The radioactivity released was measured by scin-tillation spectrometry. Usually enzyme assays were carriedout with 1 and 10 jig of enzyme protein for the syntheticsubstrate and labeled elastin, respectively. For activitymeasurement at different pHs, the buffers used were acetate(4.0 to 5.0), sodium phosphate (5.0 to 7.5) and Tris-HCl (7.5to 9.0). To determine the effect of inhibitors, the enzyme wasincubated with the inhibitor for 30 min at room temperatureprior to measuring the residual proteinase activity by addingthe substrate.

Electrophoresis, Western blot, and tests for glycosylation.SDS-PAGE was done by the method of Laemmli (18) with a12.5% separating gel and a 4% stacking gel. The electro-phoresed proteins were transblotted onto a polyvinylidenedifluoride membrane and tested for glycosylation by a peri-odate oxidation and biotin-streptavidin detection system, theGlycotrack kit (Oxford GlycoSystems). Western blotting(immunoblotting) of the purified proteinase of A. flavustransblotted onto nitrocellulose membrane was done withthe antibodies raised against A. fiumigatus elastinolyticserine proteinase, 125I-protein A, and 5% nonfat dry milk asthe blocking agent, as described before (4, 14).

Southern hybridization. A. flavus genomic DNA (4 to 5 p,g)digested by different restriction enzymes was used forSouthern blotting as described by Sambrook et al. (26).cDNA of elastinolytic proteinase labeled by the randomprimer method (Boehringer-Mannheim) was used as theprobe. Hybridization of the probe with DNA was carried outin 30% formamide, at 42°C, for 36 to 48 h. The filters werewashed with 3x SSPE, lx SSPE-0.1% SDS, and 0.1 xSSPE-0.05% SDS at 370C (lx SSPE is 0.18 M NaCl, 10mMNaPO4, and 1 mM EDTA [pH 7.7]).

Construction of cDNA library. Total RNA was isolated asdescribed by Sambrook et al. (26). Poly(A)+ RNA waspurified by using magnetic beads coupled with streptavidin-bound oligo-dT (Dynal Inc.), following the manufacturer'sinstructions. Double-stranded cDNA was synthesized frompoly(A)+ RNA by using a cDNA synthesis kit from Pharma-cia LKB Biotechnology. After the addition of NotI-EcoRIlinkers-adapters, the cDNA was ligated with Agtll arms andpackaged in vitro with Gigapack-plus (Stratagene). The sizeof the resultant library was 4.5 x 106 recombinant plaques.The Xgtll cDNA library were screened by plaque hybridiza-tion using 32P-labeled cDNA of A. fiumigatus elastinolyticproteinase (14).

Partial genomic library. The genomic DNA of A. flavuswas digested with XbaI and separated on 0.8% agarose gels.Part of the gel corresponding to 2.5 to 3.5 kb ofDNA was cutout, and the DNA was electroeluted. The recovered DNAwas ligated to dephosphorylated XbaI-digested phagemid

kDa-200

,!9768.4

_ 4336kDa~

*-29

- 14.3

FIG. 1. SDS-PAGE of elastinolytic serine proteinase purifiedfrom culture filtrate of A. fiavus.

pBlueScript II (Stratagene) and used to transform E. coliDH5at cells. The library was screened by colony hybridiza-tion using labeled elastinolytic proteinase cDNA.

Northern (RNA) hybridization. Total RNA was isolatedfrom 4.5-day-old mycelium grown on elastin by the alkalinelysis and phenol-chloroform extraction method. ClonedcDNA ofA. flavus was labeled and used as the probe. Otherconditions used with the 1.2% formaldehyde agarose gelswere as described by Sambrook et al. (26).DNA sequencing. DNA sequencing was done by the chain

termination method of Sanger et al. (27) with a Sequenasekit, version 2.0 (U.S. Biochemical Co.). The sequences ofboth strands were determined by using synthetic oligonucle-otides as primers.Measurement of protein. Protein was measured by the

Bio-Rad assay kit based on the Bradford (3) method of dyebinding.

Nucleotide sequence accession number. The GenBank ac-cession number for the primary nucleotide sequence of thisenzyme is L08472.

RESULTS

Purification and characterization of elastinolytic proteinase.The proteinase of A. flavus was purified by ammoniumsulfate precipitation and hydrophobic column chromatogra-phy using phenyl-Sepharose. When the resulting enzymepreparation was subjected to FPLC gel filtration on a Super-ose-12 column, the proteinase activity emerged in a singleprotein peak, suggesting that there is only one proteinase inthe enzyme preparation. However, the presence of anotherproteinase of the same molecular size can not be ruled out.The purified enzyme preparation, when analyzed by SDS-PAGE, revealed a major protein band at 36 kDa with twosmall faint bands below (Fig. 1). A Western blot with theantibodies raised against A. fumigatus elastinolytic protein-ase showed cross-reactivity only with the major protein band(Fig. 2). The major protein band from the SDS-PAGE, whensubjected to N-terminal amino acid sequencing, yielded asequence of 14 amino acids, DLTTQSDAPWGLGS. Thepurified proteinase showed optimum activity from neutral toalkaline pH (Fig. 3), indicating that it belongs to the alkalineproteinase family. This proteinase ofA. flavus was stronglyinhibited by the serine proteinase inhibitors, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride (Fig. 4),when tritiated elastin was used as the substrate. WithN-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide as the substrate,no inhibition of the proteinase activity was observed withmetal-chelating agents such as EDTA and EGTA [ethyleneglycol-bis(,B-aminoethyl ether)-N-N-N'-N'-tetraacetic acid]

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A. FLAVUS ELASTINOLYTIC SERINE PROTEINASE 81

kDa A B

97-68.4-

43-.

29

FIG. 2. Western blot of A. flavus (A) and A. fiumigatus (B)elastinolytic alkaline_proteinases with antibodies raised against A.

fiumigatus enzyme. 1"I-protein A was used for detection.

(data not shown). These metal-chelating agents, even at 10mM, did not severely inhibit the proteinase activity onelastin. The elastinolytic proteinase retained 92, 82, and 73%of activity after incubation with 1, 5, and 10 mM of EDTA,respectively. Similarly, it retained 66% of its proteinaseactivity after incubation with 10 mM EGTA.

Cloning and sequencing of cDNA. Screening of the AgtllcDNA library with labeled elastinolytic proteinase cDNA ofA. fiumigatus yielded four hybridizing clones. The largest ofthese, containing a 1.4-kb insert, was selected for sequenc-ing. The nucleotide sequence of this insert showed one openreading frame coding for a protein containing 403 aminoacids. The 5' end of this cloned cDNA starts with ATG withno noncoding segment. The polyadenylation signal wasfound at 150 bp after the stop codon.

Northern hybridization. Northern hybridization of themRNA, isolated from the culture actively synthesizing theenzyme in elastin medium, with the labeled cDNA as theprobe showed a single hybridization band at about 1.4 kb

pHFIG. 3. Effect of pH on alkaline proteinase activity using N-Suc-

Ala-Ala-Pro-Leu-p-nitroanilide as substrate. The buffers used wereacetate (pH 4.0 to 5.5), phosphate (pH 5.5 to 7.5) and Tris-HCI (pH7.5 to 9.0).

PMSF (mM)FIG. 4. Inhibition of elastinolytic proteinase activity of purified

proteinase from A. flavus on tritiated elastin by serine proteinaseinhibitors diisopropyl fluorophosphate (DFP) (a) and phenylmethyl-sulfonyl fluoride (PMSF) (b). %Res. Activity, elastinolytic activityremaining after treatment with the inhibitor expressed as a percent-age of the control.

(Fig. 5), a size close to that of the cDNA; thus, the cDNArepresents near full length of the transcript.

Cloning and sequencing of elastinolytic proteinase gene. A.flavus genomic DNA was subjected to restriction enzymedigestion and Southern blot analysis with labeled cDNA ofelastinolytic proteinase as the probe. With XbaI a 2.95-kbfragment showed hybridization with no additional strongbands in the autoradiogram, whereas with other enzymeseither multiple hybridization bands or very large hybridiza-tion bands were observed (Fig. 6). Therefore, the 2.5- to3.5-kb size DNA fragments generated by XbaI digestion ofA. flavus genomic DNA were cloned in phagemid pBlue-Script II. Screening of this partial genomic library by colonyhybridization yielded one positive clone. The miniprep plas-mid DNA from this clone showed a 2.95-kb insert thathybridized with the cDNA probe. The nucleotide sequenceof the cloned insert showed one open reading frame of 1,209bp. Comparison with nucleotide sequence of the cDNA

DFP ( mM)

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82 RAMESH ET AL.

kb

9.49-1

2.35-

9 .34 5

FIG. 5. Northern hybridization of total RNA isolated from elas-

tin-grown A. fiavus with labeled cDNA of elastinolytic serine

proteinase from A. flavus as the probe.

showed that the open reading frame was interrupted by three

introns of 61, 62, and 58 bp (Fig.7), and the coding

sequences were identical.

The calculated molecular mass of the mature protein is 30

kDa, whereas the protein purified from the culture filtrate

migrated on SDS-PAGE at 36 kDa. The amino acid sequence

ofA. flavus elastinolytic proteinase shows only one potential

glycosylation site, Asn-253-Met-Ser, whereas four such

regions are found in A. fiumigatus proteinase. However, the

purified protein tested negative for carbohydrates even with

one of the most sensitive methods available for detecting

glycosylation (data not shown) on polyacrylamide gels.

E EX

kb

-212

-4.2

3.5

-2.0

-1.5

-1.3

FIG. 6. Southern hybridization of restriction enzyme-digestedgenomic DNA from A. flavus with cDNA of A. flavus elastinolyticserine proteinase as the .probe. Control, unlabeled probe DNA.

DISCUSSION

The elastinolytic proteinase purified from A. flavus 28isolated from a patient migrates at 36 kDa on SDS-PAGE,while the calculated molecular mass for the mature enzymefrom the deduced amino acid sequence is 29.1 kDa. Such adifference between the purified protein and the one deducedfrom the DNA sequence was also observed for the A.fumigatus and A. oryzae proteinase (12, 14, 32) and appearsto be a common feature for serine proteinases fromAspergil-lus species, although the reason for this difference is notclear. One of the possible reasons for such a difference inmolecular mass of the proteins could be protein glycosyla-tion. However, we could not find any protein glycosylationeven with a highly sensitive method of detection, despite thepresence of a putative glycosylation site in the proteinsequence. These results are similar to those reported forA.fumigatus and A. oryzae proteinases which contain putativeglycosylation sites but do not seem to be glycosylated (12,32).The purified elastinolytic proteinase showed highest activ-

ity in the neutral to alkaline pH, like the proteinases from A.fumigatus and A. oryzae (22, 32). A. flavus was reported toproduce an elastinolytic metalloproteinase of 24 kDa (25)that was highly sensitive to metal ion chelators. Data show-ing that the purified enzyme from A. flavus isolate 28 is notseverely inhibited by the metal-chelating agents EDTA andEGTA strongly suggest that it is not a metalloproteinase.The activity of the enzyme on insoluble labeled elastin wasinhibited by the serine proteinase inhibitors phenylmethyl-sulfonyl fluoride and diisopropyl fluorophosphate in a dose-dependent manner and hence, it is a serine proteinase.Comparison with the other serine proteinases suggests thatAsp-41, His-71, and Ser-238 ofA. flavus mature elastinolyticproteinase constitute the catalytic triad (34). The proteinsequence around these three amino acids is highly homolo-gous with those of serine proteinases of the subtilisin family(Fig. 8) (19, 20, 33); this result further confirms that the A.flavus enzyme belongs to the subtilisin family.

A. flavus elastinolytic proteinase gene encodes 403 aminoacids and contains three introns. The elastinolytic serineproteinase genes in A. fumigatus and A. oryzae also containthree introns (12). However, the serine proteinase encodinggene in A. niger contains only one intron at the positioncorresponding to the first of the three introns of the gene inthe above three organisms (8). The sequence of the first 21amino acids in the A. flavus protein shows all the propertiesof a signal sequence of a secreted fungal protein (23). Theamino-terminal sequence determined for the mature proteinmatches with the protein sequence deduced from the DNAsequence; the N terminus of the mature protein started atamino acid 122 of the open reading frame. Thus, the elasti-nolytic proteinase of A. flavus must be synthesized as apreprotein with a putative signal sequence. Moreover, se-quence of the first 121 amino acids is also homologous to thesignal sequence and prepro sequences of the proteinasesfromA. oryzae (32) andA. fumigatus (12), further supportingthatA. flavus proteinase is synthesized as a preprotein whichis processed to form an active enzyme. The proteolyticprucessing of serine proteinase in A. niger is different in thatit was proposed to be processed at either or both termini toform the mature enzyme (8) whereas the serine proteinasesin the other three species of Aspergillus are processed onlyat the N terminus (32). The amino acid sequence of themature proteinase from A. flavus has 83, 82, 61, and 63%identity and 89, 90, 80, and 82% similarity to those of A.

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A. FLAVUS ELASTINOLYTIC SERINE PROTEINASE 83

1 agttctagaaagccccctggtctcccgctggtctctatatccaatcatccagaacgcgacgtgataggcaaggcaaaaga 8081 cgcgggcagcagcggatatccggtgagttgccaagcacctgagaaagattaaggtcacattgatgccaaagacaaggcca 160

161 gcccatccactctgtagggctcgtttgcgtgatgctgggcccccagcaatgacgtcgatttgctctgggagcgacgcatg 240*241 ttcatgacttgcttctgggccaacgcagctgcaagtgtcgatcgcccatgctcctcagggactcgggcttggcaaagcag 320321 cagctccgataaccatgtgtgcatggtctgcggatggcctatgaaatgtcttggaaggcagtcggccttccgtgatgatc 400401 gccgttgacatccacattgtccctttcatttacagcttctccaarargcgccaagtccgacggagcacaatggagtcg 480481 tctgaggatgcaatgtcacctgtgagtttggaacaccctctaggtacaccgagtcttgggcacgcgtgaactgcgacttt 560561 gcatggcggagatggcaaatggacatggtctcgtgaagggtAtAtAgggcggetcgccctcaccttctgtcgtgcttctc 640641 ctcatcaagccactgagatatctcaaqcttaactcttctttgcaaaccactccgggcatctcacc ATG CAG TCC 714

1 M Q S- 3715 ATC AAG CGC ACT CTC TTG CTC CTG GGA GCT GTC CTT CCC GCG GTA CTG GCC GGT CCA ATC 7744I K R T L L L L G A V L P A V L A G P I 23

775 TTT CCA CAC CGT CGC GCG CCT ACC ACT ATT CCT GGC AAG TAC ATT GTC ACC TTC AAG TCG 83424 F P H R R A P T T I P G K Y I V T F K S 43

835 GAT GTC GAC CAG GCC GCG ATT GAC AAG CAC ACC GCG TGG GCG ACC GAT ATC CAC AAG CGC 89444 D V D Q A A I D K H T A W A T D I H K R 63

895 AAC CTG CAG CGG CGC GAC TCG TCC GAG GAG GAC CTC CCC ATC GGC ATT GAG CGG AAC TTC 95464 N L Q R R D S S E E D L P I G I E R N F 83

955 AAG ATC AAC AAG TTT GCC GCG TAC TCG GGC TCC TTT GAT GAG GAT ACC ATC GCG CAG ATT 101484 K I N K F A A Y S G S F D E D T I A 0 I 103

1015 CGC CAG AGC GAC GAG gttggtgacattgcttgcggtgatcaacaagacgaatgaaaatcgaatctacggatcata 1089104 R Q S D E 108

1090 g GTC GCC GCG GTT GAG GAA GAC CAG GTC TGG CAC CTG TTC GAC CTG ACC ACC CAG TCG 1147109 V A A

1148 GAC GCC CCC128 D A P

1208 TAT GAC ACC148 Y D T

1268 GAC CAC GAG168 D H E

1328 GTG GAC GGT188 V D G

1388 GTG GCC AAG208 V A K

1448 TCC ATC ATC228 S I I

1508 GGA AAG GCA248 G K A

V E E D QTGG GGA TTG GGA AGCW G, T. a S

AAC GGC GGC GAG GGCN G G E GGAA TTT GAG GGC CGTE F E G RGTC GGC CAT GGC ACCV G H G TAAG GCC AAC CTG CTGK A N L LCTG GAC GGC TTC AACL D G F NGCG ATC AAC ATG AGCA I N M S

A'IA(TG(ACiHT

S

Ti

w

T'L

V W

TC TCCS

CC TACy

CG AGTS

AC GTTV

CG GTCV

GG GCGA

H L F D L T T 0 S

GGT CAGG QGTC GTAV V

TAC CACY HACC ATTT ITTC GTCF VGAT ATTD I

CCC AGC ACGP S TGAC ATT GGCD I GGCT GCC GGTA A GGGT GGT AAGG G KGGG GAA TCGG E SGTT TCC AAGV S K

CAC AAGH K

GCT TACA YCTC GCGL ATCT GGTS GAAG GTCK VGCC AATA N

GAC TAC ATCD Y IATC AAC GTGI N VGGC CAG CATG Q HACA TAC GGCT Y GAGC AGC ACAS S TAAG CGC ACTK R T

127120714712671671327187138720714472271507247

ggtatgttatctgccatctatgtgaatgatatcattggagtcac 1578256

1579 acagctgacgcaagcata GGC GGT GGA TAC TCC AAG GCC TTC AAT GAT GCC GTC GAG AAC GCT257 G G G Y S K A F N D A V E N A

1 641271

1642 TTT AAC GAG GGA GTC CTG TCC ATC GTC GCC GCC GGC AAT GAG AAT gtgcgtgccacgttcccccc 1706272 F N E G V L S I V A A G N E N 286

1707 tgtgaatggagtgcggactccgctgactgattcgacag ACC GAC GCC TCG CGC ACC AGC CCG GCT TCT 1774287 T D A S R T S P A S 296

1775 GCT CCT GAT GCC TTT ACC GTC GCT GCG ATC AAC GTG AAC AAC ACC CGT GCC TAT TTC TCC 1834297 A P D A F T V A A I N V N N T R A Y F S 316

1835 AAC TAC GGC TCC GTG GTG GAT ATC TTC GCC CCG GGC CAG AAC ATC CTC TCT GCC TGG ATC 1894317 N Y G S V V D I F A P G 0 N I L S A W I 336

1895 GGC TCC AAC ACG GCC ACC AAC ACC ATT TCG GGC ACT TCC ATG GCC ACC CCC CAC ATT GTC 1954337 G S N T A T N T I S G T S M A T P H I V 356

1955 GGC CTG TCC ATC TAT CTG ATG TCG CTG GAG GTC CTC AGC AGC CCC AAG GCC GTC AGT GAC 2014357 G L S I Y L M S L E V L S S P K A V S D 376

2015 CGC ATC AAG GAG CTG GCG ACT CGA GGC GTC GTC AGC AAC GTC GCC GGT AGC CCC AAC CTG 2074377 R I K E L A T R G V V S N V A G S P N L 396

2075 TTG GCG TAC AAC GGC AAC G C TAA cctcggcaagacttggcatagcgcacgtgaccgcgtcgacttgggata 2146397 L A Y N G N A * 404

21472227230723872467254726272707278728672947

tggaagccagcaggtgatgtgatct'agcttcaatgtacatgtactatactagcatcgcttctcgaggatcaacgtggaagttatttgacttgtccgatacaacctcctattacatctcccgttctctttaaactaggcacatggccccgtatatcccgcataacctgtttggcgccgagcacctgcagaggcctagtatgcgccatatctcgagctatcatcacgtttctccgccggat

caggtaaggaagggatagcaggtc-gggatcttcacggcggcggcgaccattaggcgaatgagttgtccgtcgcgcggaatgtggaccggggacattgcgtcgagagcacgatatagccaccaccgggaaggacggcagacttgccagacacgggggtctccagtcgcagctcatacgtacttagagctgtgaaatgcatcacgagctccttggaggtcttggagatccgccatgacgacgaccagcctagagggtcgagcccgtagacctgctggggggaggatctgcacgtcgaccgggcccctttacattctgatgcaaagtttgccggctggggagttatcgttgcgcaatccgctcgtcgttgtgcaggtctgggttaaagccgatgtcatacgctgcgtcggatgtgatatcaacgctggggagttcattctgggcgttctcctcgtgcaggtttcttgttcttctgtctatagccgtctaga

22262306238624662546262627062786286629462955

FIG. 7. Nucleotide sequence of the cloned DNA fragment showing the open reading frame coding for elastinolytic serine proteinase.N-terminal amino acid sequence determined from the purified mature enzyme and the upstream sequences resembling the TATA boxes, areunderlined.

fumigatus (12), A. oryzae (32), Acremonium chtysogenum(11), and Trichoderma harzianum (9) proteinases, respec-tively. On the basis of the high sequence homology of serineproteinase gene approaching 90% among all the three As-pergillus spp. (Fig. 9), it is highly likely thatA. fumigatus,A.

oryzae, and A. flavus acquired this gene from a commonancestor. All the three serine proteinases share about 40%homology with that ofA. niger serine proteinase (8), thoughall of them belong to the same genus.

It was apparent that A. fumigatus contains only one copy

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84 RAMESH ET AL.

A.flaV 34GTYAYVVDIGIN45Prot B GVTSYVIDTGVNSubtilisin NVKVAVIDSGIDThermitase GAKIAIVDTGVQ

A.flav 67DGVGHGTHV75Prot B DGNGHGTHCSubtilisin DNNSHGTHVThermitase NGNGHGTHC

A.flav 234ISGTSMATPH243Prot B LSGTSMASPHSubtilisin YNGTSMASPHThermitase LSGTSMATPH

FIG. 8. Comparison of conserved regions and putative catalyticamino acids ofA. flavus elastinolytic serine proteinase with those ofserine proteinases of subtilisin family, reported from other organ-isms. (Prot B, proteinase B of Saccharomyces cerevisiae; Subtilisin,Bacillus amyloliquifaciens subtilisin; Thermitase, Thermoactinomy-ces vulganrs alkaline proteinase). The highlighted amino acids rep-resent the putative catalytic triad.

of the elastinolytic serine proteinase gene whereas Southernblots of A. flavus genomic DNA with A. fumigatus probeindicated multiple copies of the gene (30). Our Southernhybridization results using elastinolytic proteinase cDNA ofA. flavus or A. fumigatus as the probes also indicated thepossible presence of multiple or related genes of elastinolyticproteinase in the A. flavus genome. The copy number ofserine proteinase gene may vary among different strains ofA. flavus. In Fusanium solani pisi such differences in copynumbers of the cutinase gene were found (13).

Correlation of extracellular elastase level with virulence(16, 28) was consistent with the hypothesis that elastinolyticenzyme is used to penetrate structural barriers in the lung.A. fumigatus produced the same elastinolytic proteinasewhen grown either on insoluble structural material frommurine and bovine lungs or elastin (14). Similarly, A. flavusproduced an elastinolytic proteinase when grown on lungstructural material (27a). Immunological localization showedthat the elastinolytic proteinase was secreted by A. fumiga-tus spores germinating and penetrating the lungs of neutro-penic mice (14). Furthermore, A. fumigatus mutants defi-cient in elastinolytic activity were found to be less virulentthan the wild type in causing mortality. Such evidencestrongly suggested that extracellular elastinolytic activity isa significant virulence factor. On the other hand, disruptionof elastinolytic proteinase gene did not render the disruptedmutants avirulent in murine models (21, 31). However, inboth of these studies the gene-disrupted mutant producedproteinases and elastinolysis was observed in the host lungsinoculated with the gene-disrupted mutant. Obviously, otherproteinases are produced to degrade elastin. Although one ofthe transformants showed no measurable elastinolytic activ-ity, collagenolytic activity was found. The gene-disruptedtransformant produced metalloproteinase (21). Even thoughit was reported that metalloproteinase(s) from A. fumigatusdid not cleave elastin (21), we have isolated an elastinolyticmetalloproteinase from A. fumigatus (18a). From all avail-able evidence it is clear that A. fumigatus produces multiple

1 50Afum *L .........FG A.VQET. *A QK ...PGT*T*T-Aory .......... ...I....G A-VQET--A EKL-....... *PGI-E-K-Aflav MQSIKRTLLL LGAVLPAVLA GPIFPHRRAP TTIPGKYIVT FKSDVDQAAI

51 100Afum ES--L .- L.*.--E--T TSGEP-V-.. KSY--KD--- -A---DA--Aory QE.-T'. *N* *Q-S-E- GA TGG--...V.. .Y....... A. DA--

Aflav DKHTAWATDI HKRNLQRRDS SEEDLPIGIE RNFKINKFAA YSGSFDEDTI

101 150Afum EE-*KRGD* H...IY DA-...KG........... A.......Aory EE *KNED, Y....IYY- DG-... KS.. .......... . ..Aflav AQIRQSDEVA AVEEDQVWHL FDLTTQSDAP WGLGSISHKG QPSTDYIYDT

151Afum SA'A......Aory SA-.......

Aflav NGGEGTYAYV

201Afum ..........Aory -A.....I--

Aflav IGGKTYGVAK

200--....N-V...S.... N...S...S I-.----A--.V.*--S. . K- N........S I-........

VDIGINVDHE EFEGRASLAY HAAGGQHVDG VGHGTHVSGT

250.T.......Q......... .............-GK--.SI......Q....V.............S..

KANLLSVKVF VGESSSTSII LDGFNWAAND IVSKKRTGKA

251 300Afumn.H. Y..N...D...... S... N....-Aory .................... .EQ ......S-G Q......--

Aflav AINMSLGGGY SKAFNDAVEN AFNEGVLSIV AAGNENTDAS RTSPASAPDA

301 350Afum L......KS- A.-S.-.--.......D ........T...... -

Aory I..--QKS- N-oS... -FK ..V.....D....SS ........Aflav FTVAAINVNN TRAYFSNYGS VVDIFAPGQN ILSAWIGSNT ATNTISGTSM

351 400Afum *.-------V ...-G*-N-oG *A-'TA ..N . 'T' * K"---o-**Aory.o----L * AA-.N-DG 'A--TK... .*KD--KD- K........

Aflav ATPHIVGLSI YLMSLEVLSS PKAVSDRIKE LATR *,SNV AGSPNLLAYN

401Afum ..*Aory ...Aflav GNA

FIG. 9. Comparison of amino acid sequences of the three As-pegillus alkaline proteinases. , no difference in amino acid residue.Afum, A. fumigatus; Aory, A. oryzae; Aflav, A. flavus.

proteinases. Similarly, A. flavus also produces an elasti-nolytic metalloproteinase that has been characterized (25).Thus, it is not surprising that disruption of a gene coding forone proteinase did not show drastically lowered virulence.

Pathogenic organisms cause damage to the host in manydifferent ways rather than using a single virulence factor.Obviously, their ability to colonize the host using multiplemechanisms provided them with an evolutionary advantage.Extracellular enzymes that degrade the host structural bar-riers obviously assist the pathogen to infect the host. Genedisruption studies conducted during the recent years havedemonstrated that multiple enzymes coded by gene familiesare often used by pathogens to break these barriers. Forexample, pectin degradation by bacteria (6) and fungi (10)involves pectate lyases coded by families of genes. Inaspergillosis, proteinases that use different catalytic mecha-nisms and different pH optima are used presumably to retainmaximum flexibility for the pathogen to survive under dif-ferent environmental conditions. Proteinases are likely toplay an important role also in the systemic spread of thefungus to other organs, often a significant factor in mortality(17). In view of the multiple proteinases that are produced bythe fungi, elucidation of the nature of the different types ofproteinases involved is needed before we can assess whethersuch enzymes can be used as targets to protect immunocom-promised patients against invasive aspergillosis.

ACKNOWLEDGMENTThis work was supported by a grant from the National Institutes

of Health (RO1-A130629).

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A. FLAVUS ELASTINOLYTIC SERINE PROTEINASE 85

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