The Elastases of Pathogenic Fungi and Actinomycetes*

vol. 50, No. 1

THE JOtrnNAr. op IwvEsnaATIvE DEaMAToLoay Copyright 1868 by The Williams & Wilkins Co.

Printed in U.S.A.

THE ELASTASES OF PATHOGENIC FUNGI AND ACTINOMYCETES* JOHN W. RIPPON, Pu.D. AND DAVID P. VARADI, M.D.t

Many enzymes have been described from fungi pathogenic for plants (ccllulase, pectinase) as well as from bacteria pathogenic for

Waring blender. After centrifugation the extract was dialyzed against distilled water overnight at 4° C and lyophilizcd. The powder was then assayed for elastase.

human beings (coagulase, leeithinase) that Assay of elastase: An clastase may be defined may play a role in their ability to produce as an enzyme that causes rapid dissolution of pardisease. Few such enzymes, however, have ticulate elastin. Elastase activity was determined been found in fungi and actinomycctcs path- by a modification of the clastin-orcein colorimetric technique of Sachar (5). The enzyme powder was dissolved in 02 M Tris HC1 buffer (pH 8.0) to a protein, keratin, is a physiologic property final concentration of 1 mg/ml. The reaction mixcommon to the dermatophytes. This ability ture contained 20 mgs. of orcein-clastin, 1 ml. of buffer (pH 8.0) and 1 ml. of enzyme solution is shared by only a few saprophytic fungi Tris (final concentration 0.5 mg/ml). The mixture was and actinomycetes, and by a few species of incubated at 37° C or 25° C in a Dubnoff shaking insects. Previous work from this laboratory water bath for various time intervals. The reaction described two medically important species, one was stopped by adding 2 mls. of 0.5 M phosphate fungal and the other actinomycotic, each capable buffer pH 6.0. The sides of the flasks were washed

ogenic for man. The utilization of the sclero-

1 ml. of distilled water and the contents of elaborating a collagcnase (3). It appears that with filtered. Absorbency of the colored filtrate was this enzyme plays a significant role in the patho- read at 590 mm against a blank similarly treated genesis of maduramycosis caused by the myce- but containing no enzyme. Activity is expressed as

toma agent, Streptomyces madurae (4). The O.D. at 590 mm relative to a standard curve using present report is concerned with the ability of pancreatic elastase (Worthington). Partial purification and characterization procepathogenic fungi and actinomycetes to utilize dure: The elastase elaborated by Nannizzia fulva the other major human sclcroprotcin, elastin.

2181 (+) was selected for characterization and purification studies. The enzyme was extracted from agar cultures as noted above. The extract was precipitated with ammonium sulfate at 40%

MATERIALS AND METHODS

Screening plates for elastase activity: Elastase

test plates were prepared by adding 1 gram of and 60% concentrations. The resulting precipitates were dialyzed against distilled water and lyopbilized. The pH optimum of the enzyme in the most active fraction was determined. Tris HC1 buffers (0.2 M) at pH 6, 7, 7.5, 8 and 8.5 were used. In one set of experiments the enzyme was incubated Those cultures that showed zones of clearing were in each buffer at its respective pH for 10 minutes grown on agar without elastin and assayed for prior to enzyme assay at pH 8.0. This was done activity to eliminate the possibility of induced to ascertain any irreversible effects of pH on the enzyme. In another set of experimcats the enzyme enzyme formation. Isolation of the enzyme: Selected organisms and substrate were incubated for the entire durashowing positive activity on the screening plates tion in each buffer at its respective pH. Inhibitor were grown as giant cultures on 140 mm. pctri studies included: Etbylenediamine tetraacetic acid dishes. After 14 days the agar was mixed with an 10 M (EDTA); p-chloromercuribenzoic acid equal amount of distilled water and stirred in a (CMB in glycylglycine buffer), 102 M; cysteine, 0.05 M; iodoacetic acid, 10° M; NaCl, 0.14 M; * From the Section of Dermatology, University and Urea, 7 M. The enzyme was incubated in the of Chicago, Department of Medicine, Chicago, presence of inhibitor prior to assay and in addiIllinois. tion, untreated enzyme was assayed in the preselastin powder (Worthington) to 100 ml. Trypti-

case soy agar or Czapek Dox agar. Fungi were planted and the plates incubated at 25° C. Examination for zones of clearing around growing colonies was made at 7, 14 and 21 days (Fig. 1).

t Research trainee in Dermatology. Present ence of inhibitor. address: University of Toronto, Department of Medicine, Ontario, Canada.

This study was supported in part by USPHS grant A1-06444 and USPHS Training grant No. T01-AM5263. Accepted for publication Juno 16, 1967.

RESULTS

As shown in Table I elastase activity was found in several dcrmatophyte species and in a

54

ELASTASES OF FUNGI

55

Fie. 1. Elastin agar plate with a colony of Trichophyton verrucosum showing large area of clearing.

few subcutaneous and deep-infecting fungi. A reducing agent cysteine, in a high salt concenspecies was considered an elastase producer tration or from EDTA (Table IV).

only if activity was detectable within three DI5CU55ION days. Of special interest was the association of elastase activity with the plus strain of The ability to utilize the seleroprotein Nannizzia fulva while not with the minus elastin is widespread among the dermatophytes. This property is also shared by a few strain (Table II). Most of the activity of the elastase isolated subcutaneous and deep-infecting fungi. In gen-

from N. fulva was found in the 60% am- eral, the organisms characterized by their

monium sulfate fraction (Table III). Similarly, ability to induce marked inflammatory reac-

most of the activity of the elastases from tions were the ones that produced elastase. Trichophyton schoenleinii and Trichophyton verrucosum, respectively, were found at this ammonium sulfate concentration. Optimal activity of the enzyme was markedly pH-dependent with its peak at pH 8.0 (Table III).

None of the pathogenic actinomyeetes were elastase producers. A late clearing was noted

for Streptomyces madurae. However, late

clearing is probably an induced enzyme phenomenon as no activity was detectable in an Incubation in less than optimal pH did not extract of a culture grown without elastin. In irreversibly alter its activity. The enzyme was some species all strains were able to hydrolyze inhibited by urea, p-CMB and iodoacetic acid. elastin, while in others only an occasional No enzyme inhibition was observed from the strain could do so. This phenomenon is sim-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

56

TABLE I Survey of organisms for elastase activity on elastin particle agar plates Organisms

Organisms

(at one Y)

(at one

Arthroderma cuniculi IRI CMI

—

Trichophyton tonsurans

—

+

Art hroderma multifidum 1S2 Arthroderma quadrifidum 1Q7 (T. terrestre) Epidermophyton floccosurn

strains) Trichophyton tonsurans var snif. (3 strains)

+

(4

(5

+ *

—

Eidernilla diflexa 69-66 Keratinornyces ajelloi (6 strains)

Micros porum canis

(4 strains) Microsporum cookei

(2 strains) Micros porum ferrugineum

(2 strains) Micros porurn nanum X467 Micros porum rivalierii

(2 strains) Micros porurn vanbreuseghernii X322 Nannizzia gypsea 618 A Nannizzia gypsea 1317 a Nannizzia gypsea 1315 A

Nannizzia incurvata 5912 A Nannizzia incurvata 5913 a Trichophyton concentricum. (Marples)

—

—*

(4 strains)

strains)

Trichophyton rnentagrophytes

(8 strains) Trichophyton mentagrophytes 125—65 Trichophyton mentagrophytes 17—65 Trichophytors rubrurn (6 strains)

Trichophyton schoenleinii

strains)

Trichophyton son danense Trichophyton terrestre (Hedgehog) (One

—

+ —*

(3 strains)

Paracoccidioides brasiliensis — — — —* — — — — — —

(3 strains)

Histoplasma capsulaturn (5 strains)

Candida albicans

(7 strains) Candida guilliermondi Candida paropsilosis Candida pseudotropicalis Candida stellatoidea Candida tropicalis Cryptococcus neoforinans (2 strains)

— — —

Fonsecaea

+ +

(4 strains) Nocardia brasiliensis

pedrosoi

Geotrichum candidum

Nocardia astroides

—

Streptornyces madurae

—

,Streptornyces

+ + —

+ + +

+ at 6 weeks.)

—

—

(6 strains) pelletierii (2 strains) Phialophora verrucosa Piedraea hortai Torulopsis glabrata Hansenula capsulata Saccharornyces cerevisiae Coccidioides irnrnitis Micros porurn gypseum (old stock)

(mated as N. fulva +)

* Late clearing in one strain, no elastase activity by orcein method.

f

+

Fonsecaea corn pactum.

Trichophyton concentricurn (Haldane) Trichophyton equinum (Beneke) Trichophyton gallinae Trichophyton gourvillii CDC X484 Trichophyton gonrvillii IJI Trichophyton megnini

(4

(3 strains)

Trichophyton yaoundi 1L5 (Van.) Trichosporon cutaneum Allescheria boydii Blastomyces dermatitidis

—

1M4

(3

+

Trichophyton violaceum —

(2 strains) Micros porum audouinii

Trichophyton verrucosum

(3 strains)

strains)

— — — —

+ +

57

ELASTASES OF FUNGI

ilar to that found among strains of Pseudo-

TABLE III

menUs aeruqinosa, only a few of which produce

Partial purification and effect of pH on Nannizzia fulva elastase

elastase (2). The two strains of T. mentagrophytes, which were found to produce elastase, were each isolated from severe inflammatory, animal-contracted infections. The infections

resulted in permanent alopecia and pitted scarring in the patients. Of particular interest was the association of elastase with mating type in Nannizia fulva. Only the + mating types produce the enzyme in the strains tested. This may be a convenient method for screening mating types. The dermatophyte T. schoenleinii elaborates enzymes

OD 590 ms

Whole filtrate (pH 8.0) 40% Am. sulfate 60% Am. sulfate

600 360 840

pH 6.0 pH 7.0 pH 7.5 pH 8.0

600 670 700 840 680

pH8.5

capable of solubilizing all three scleroproteins: keratin, elastin and collagen (3).

The enzyme from N. fulva 2181 (+)

was

found to be somewhat different from the three most studied elastases: Pseudomonas elastase (2), hog pancreas elastase (1) and Flavobacterium elastase (1). All enzymes seem to have

optimal activity in alkaline pH (8 or above).

The lack of effect on activity by EDTA is

similar to the Flavobacterium enzyme in contrast to Pseudomonas or pancreatic elastases. Unique to the N. fulva elastase was inhibition by PCMB and iodoacetate suggesting that this TABLE II Elastase activity of Nannizzia fulva (Microsporum fulvum) mating types* Plus strains

2181, 3J2, X642, X198, 86179, 99941, 99944

Minus strains

Negative mating

100718, 98601, X644, 3J3, 0936, 98602, X642, 73E.1., X199, 73, OAP1,

86180, 82774, AZ68, 82775, 99943

100867

All plus strains were positive for elastase production. All minus strains were negative for elastase production.

All negative mating (unable to mate with either plus or minus strains) were negative for elastase except 98607 and 100718. *

Mating types were supplied by Dr. Irene

Weitzman of Columbia University, Dr. Libero Ajello of Communicable Disease Center and Dr. Phyllis Stockdale of the Commonwealth Mycolog-

ical Institute.

TABLE IV Effect of inhibitors on Nannizzia fulva elastase % of Activity

Urea 7 MI pCMB io— MI

NaCl 0.15 M

EDTA 10 M Cysteine 0.05 M lodoacetate 102 MI

13

<10 100 100 95

<10

is an —SH enzyme. All the elastases were in-

hibited by 7 M urea probably indicating a dissociation of tertiary structure by breaking hydrogen bonds. SUMMARY

The first report of elastase from pathogenic fungi is described. Several dermatophytes and a few subcutaneous and deep-invading fungi elaborate this enzyme. In general, the enzyme was produced by organisms associated with the

more inflammatory types of infections. Tnchophyton schoenleinii was notable for producing enzymes capable of lysing the three major acleroproteins: keratin, elastin and collagen. Elastase is associated with the plus mating type of Nannizzia fulva but is absent in the minus type. The enzyme from N. fulva

(+) was partially purified and characterized. REFERENCES

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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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