PRODUCTION OF SERINE CHYMOTRYPSIN-LlKE ELASTASE BY ASPERGILLUS FUMIGATUS STRAINS

Thirtyfour AspergiUus fumigatus strains isolated fron! air, horse-hai1; agricultural soil and human samples were screened lO evaluale lhe produclion of elastase. MpergilLus fumigatus strains were grown in elastin solid medium, showing a widespread elastin solubilization. H owever, isolares from human and agricultural soil samples were found to be the highest elastase producers. Then, eight out of 34 strains were grOWI! in four different ¡iquid media, on wich we investigated total and specific proleolytic activity. Results from this experiments suggesllhar the elastase production is induced by the presence of elastin as a substrate and that the elastase is a chymonypsin like enzyme. InhibilO/)' pro file slzowed Ihar the A.fumigalus elastase is a serine proteinase.

MpergilLus fumigatus strains were grown in elastin solid medium, showing a widespread elastin solubilization.
H owever, isolares from human and agricultural soil samples were found to be the highest elastase producers.
Then, eight out of 34 strains were grOWI! in four different ¡iquid media, on wich we investigated total and specific proleolytic activity.Results from this experiments suggesllhar the elastase production is induced by the presence of elastin as a substrate and that the elastase is a chymonypsin like enzyme.

INTRODU CTION
Aspergillus fumigatus Fres. is a widespread thermotolerant species.Ir is also a pathogenic fungus able to induce a variety of lung disease in man, Le. alIergic aspergillosis fungus balls, and invasive aspergillosis.The la ter disease is particularly severe in immonocompromised patients.(2,4).It is known that members oF the genus AspergilIus may produce a number of proteolytic enzymes (3,10) and it has been suggested that the production ofsuch enzymes maybe associatedd wi th virulence of the fungal strain.
In this contribution, we examined in vitro elastase production by 34A.fllmigatus strains isolated from different sources (soil, p lant, human and horsehair) and we achieved a pania!characterization ofthis enzyme in me mbers of this species group.
The reasons why A.fitmigatus is more invasive than the other species of the genus remain as unexplained: we wiIl present and discuss sorne of our own experimental data concerning the elastase production from these strains in relation to their ecoIogy and to pathogenic activity.

Elastase prodUlctiolill in solid medium
The strains were cultured (25° C) on Czapekagar (Biolife) until sufficient growth hadoccurred.The cultures were used to provide the ¡Doculum required; spores were collected in sterile 0.1 % Tween 80 (pol-yoxyethyleneSorbitan monooleate) and adro~ ofspore suspension at the concentration of 10 Iml was inoculated, in a central spot, onto agar medium (25 mI in Petri dishes) containing elas tin, according to Kothary et al. (5).
Cultures were incubated at 37° C for 7 days, elas tase activitywas detected visually as a clearing zone around and benea th the colony.
A drop ofthe suspension obtained aS previously described was inoculated into 50 mI of each growth medium in a 250 mI.baffled flask and incubated on a shaker, 100 rpm) at 37°C for 72 h.
A sample of each medium without fungus in-oculation, was processed in the same fashion and used as control.
The reaction was stopped after 15 minutes with 200 ul of acetic acid IN and the absorbance read at 440 nm (PYE Unicam).AH samples were run in duplicate with appropriate blanks.
Values were expressed as mg.azocasein degraded over time.

HydroBysis of synthetic peptide substrates
To this aim 20 ul of each sample were incubated for 1 h.at 37°C in phosphate buffer 0.2M pH 7.4, with the following substrates: I At the end of incubation the reaction was stoppedand theabsorbance read at41Onm.Thevalues were expressed as nMoles ofsubstrate hydrolyzed over time.

Inhibitory study
In order to assign the hydroIytic actlvlty on the synthetic peptide substrates tb the class o f metallo orserine-proteinase, somesamples were preincuba ted with the metalloproteinase inhibitor EDTA 10 mM (Merck) and the serine-proteinase inhibito r phenylmethylsulfonylfluoride 10 mM (PMSF; Sigma) for 30 minutes.Thqe after we proceeded as aboye described.

InsolubBe elastñn degradation
To this aim we used the method described by Shotton (15).Briefly,7 mlofboratebuffer O.02 M,pH8,8 containingelastin Congo red (Sigma) at concentrationof 1 mg/ml, were added to 2 mI ofthe sample (strain N° 13 grown for 72 h on liquid elastin medium).The mixture was incubated at 37°C. on shaker and every 15 minutes an aliquot was drawn, centrifu ged and the absorbance of the superna te read at 495 nm.In separate tube, elastin Congo red was incubated with 40 ug of porcine T able 1. Elastinolytic activity on solid medium oC different A.fumigatus strains.
In sorne cases the growth and elastinolysis had the same degree (fig.lA), whereas in the other Gases the diameter of elastinolysis exceeded the diam-eter€lf gmwth (fig.lB).Strains ofA.fumigatus, isolated mom agricultural soils were the high elastase producers.
As it may be ascertained from the inspection of table 2, none of eight strains, selected on the basis of previ€lus results and grown in Czapek liq\lid medium, o Iievealed a pro teo lytic activity, as detected byazocasein assay.In nve out of the eight strains, this activity was fairly enhanced when the liquid medium was the yeB + peptone.A drama tic increase in the proteolytic activity was determinated by the growth of strains in elastin medium.In most of the cases the addition of aibumin to elastin medium induced only a modera te fun1her inorease of proteo lytic activi ty.
With regard to the hydrolysis ofthe three syn-theJic peptide substrates used, the eight strains did not reaetéitherwith SApNaorMe(O) AAPVpNasubstrates (data notshown).
Concerning the inhibitory profile of the fiydr@lytic activity, we found that the deavage of the SAAPPhepNa substrate was fully abolished by PMSF, whereas it was unaffected by EDTA (data not shown).'Therefmewe may assign tbis activity to theclass ofserine proteinase.
Finally theelastin Congo red degradationassay all0wed us to establish that the elastase produced by A. ftlmigatus in elastin medium has a modera te elastinolytic capacity, when compared to the elasti-Production ofserine chymotrypsin-like elastaseo Mo GlIglielminetti et al.

DISCUSSION
OurdatashowthatthemostoftheA.fumigoLus strains tested are able to produce an elastase when cultured in agar solid medium containing insoluble elastin, with the exception of most ofthe strains which wereisolatedfromairsamples.Thisfindingisconsistent with the putativevirulence role ofthe elastase (5) in the pathogenesis of aspergillosis in which A. fumigatus is the primary agent.However differences in thedegree of elastase production were found within strains isolated from the same solirce.
Several speculations may be drawn from the liquid media results.First of aH, the preference of the elastase for a synthetic peptide substrate containing a Phe residue in PI position suggests a chymotrypsinlike activity (17).Our results are consistent with those previously reported by Reichard and coworkers (12).The lack of activity of AJumigatus elastase with substrates containing Valor Ala residues in PI position are consistant with previously results obtained by someofus (8) onalkaline proteinase (called "seaprose") produced by A.mel/eus Yukawa, a nonpathogenic mould belonging toA.alutaceus (formerly A. ochraceus) group (6).The inhibitory profile shows that the AJumigatus elastase is a serine-proteinase, whereas the ability to degrade insoluble elastin, as suggested by the elastin-oeongo red assay, is rather weak when compared with that showed by porcine pancreatic elastase.Both of these characteristics are shares by seaprose.
Since A.ochraceus has been reported as human pathogenic only occasionally (11), the homology between AJumigatus elastase and seaprose, the A.
Production 01 sel'ine chymotrypsin-üke elastase.M. Guglielminetti et aL me/leus elastase,actually raises concem on the role of A.fumigatus elastase as a virulence factor in human infection, at least as a main factor from this point of view (4)(5).This concem recently found a confirmation in a paper ofTony and coIleagues (16).
In addition to this functional homology between seaprose and A.fumigatus elastase, our resuIts suggest other speculations.As previously reported (4,5) theA.fumigatuselastaseacts as inducibleenzyme, whose production is particularly evident in the presence of elastin as a substrate.The addition of albumin to elastin further enhances the production of the elastase, but the elastin albumin enriched liquid medium seems less favourable for purification purposes, as deduced from the results corrected by the protein amount (table2).
It has been also reported that the enzyme is induced by presence of collagen as a substrate (9).
In our hands, the good agreement between activity of A.fumigatus liquid media on specific syn• thetic peptide substrate and total proteolytic activity, i.e. azocasein assay, would suggest that the serine• chymotrypsinlikeelastaseisthemajorelastaseproduced by A.fumigatus und~r experimental conditions.
Our results in agreement with Rhodes et al. ( 13) demonstrated elastinolytic activity in all strains of A. fumigatus isolated from clínical cases, bUl also a strong elastinolytic activity in saprophytic strains iso• lated from agricultural soils.