Zbl. Bakt. Hyg., 1. Abt, Orig. A 255, 537-548 (1983)

Secretion of Acid Proteinases by Different Species of the Genus

Candida!

Sekretion saurer Proteinasen bei verschiedenen Candida-Spezies

R. RUCHEL, K. UHLEMANN, and B. BONING

Abt. Med. Mikrobiologie, Zentrum fur Hygiene und Humangenetik, Universitat Cortingen

With 4 Figures' Received March 16, 1983

Abstract

Strains of several Candida species that are isolated from humans aside of C. albicanshave been screened for secretion of acid proteinases. A third of the strains of C. tropicaliswas strongly proteolytic, other strains of this species were moderately or virtually non­proteolytic; the variety of activities resembled pattern found among strains of C. albicans(32). Strains of C. parapsilosis produced 24% of the proteolytic activity of strongly proteo­lytic strains of C. tropicalis. The proteolytic activity of C. pseudotropicalis, C. krusei, andC. glabrata accounted for 7.5, 2.0, and 0.8% respectively. Four strains of C. guilliermondiideveloped strain-specific proteolysis after prolonged cultivation only. Five proteinases fromstrains of C. tropicalis cross reacted immunologically as did five proteinases from C. albi­cans. Interspecific cross reaction, though, was not observed when certain sera from rabbitswere employed.

Zusammenfassung

Eine Anzahl von Stammen jener Candida-Spezies, die neben C. albicans vom Menschenisoliert werden, wurde auf Sekretion von sauren Proteasen untersucht. Bei C. tropicalisfan den sich etwa ein Drittel stark proteolytischer Stamme sowie maRig- und scheinbarnicht-proteolytische Starnme in einer Vielfalt, die bereits bei C. albicans vorgefunden wor­den war (32). Verglichen mit stark proteolytischen Stammen von C. tropicalis erreichteC. parapsilosis 24% proteolytischer Aktivitat bei einheirlichem Spaltmuster. Die Werte furC. pseudotropicalis, C. krusei und C. glabrata lagen bei 7,5, 2,0 und 0,8%. Vier Starnmevon C. guilliermondii wurden erst bei vcrlangertem Kultivationsintervall proteolytisch. Die

1 This investigation was supported by a grant from the Deutsche Forschungsgemein­schaft.

Abbreviations used: BSA: bovine serum albumin; DAN: diazoacetyl-Dl.snorleucine­methylester; DEAE: diethylaminoethyl ; EPNP: 1,2-epoxy-3-(p-nitrophenoxy)-propane;Tris: tris(hydroxymethyl)aminomethane; YCB: yeast carbon base",

538 R. Riichel, K.Uhlemann, and B.Boning

Bedeutung der sekretorischen Proteinasen hinsichtlich der Persistenz und Virulenz vonHefen wird diskutiert.

Zwischen den Proteinasen von fiinf C. tropicalis-Stemmesi fand sich eine immunologi­sche Kreuzreaktion, obwohl die Enzyme gegeniiber Inhibitoren unterschiedliche Empfind­lichkeit aufwiesen. Eine Kreuzreaktion fand sich auch zwischen fiinf Proteinasen vonC. albicans. Keine Kreuzreaktion trat hingegen zwischen Enzymen beider Spezies ein, wennbestimmte Antiseren von Kaninchen verwendet wurden.

Introduction

A few species only of the genus Candida are found on human mucous membranesand are considered opportunistic pathogens. Experimental evidence has been ac­cumulated in favour of a strain-specific virulence of such yeasts (19, 36, 40, 44).Aside of the fungal dimorphism, germ tube formation, secretion of toxins andhydrolytic enzymes have been discussed with respect to pathogenicity (for reviewsee 6). Among the hydrolases, acid proteinases have drawn most attention (18,29,31).

Previously, we have described the incidence and substrate specificity of suchenzymes among strains of C. albicans (32), and the present communication dealswith the secretory proteinases from other species of the genus Candida that areisolated from humans.

Materials

The Candida strains employed in this investigation were provided by Prof. R. Ansorg,Gottingen (a), Priv.-Doz. Dr. ]. Miiller, Freiburg (b), Deutsche Sammlung fiir Mikroorga­nismen, Gottingen (c), Prof. F.Staib, Berlin (d), and DipI.-BioI. Ortrud Zimmermann, Got­tingen (e).

Species Number ofstrains a b

C. tropicalis 29 22 2C. glabrata 17 12C. krusei 12 9C. parapsilosis 11 3 2C. pseudotropicalis 10 5 3C. guilliermondii 4

Sourcec

2

3

d

211

e

55321

The classification of the strains was confirmed by the API-20C-Auxanogram® (API­BioMerieux, Niirtingen) and, in certain cases, by a serotest (Candida Check", Iatron,Tokyo).

Freund's adjuvants were from Behring, Marburg; azocoll was from Calbiochem, GielSen;Yeast Carbon Base? was from Difco, Detroit; 1,2-epoxy-3(p-nitrophenoxy)propane (EPNP)was from Eastman, Rochester; bovine serum albumin, butanedione, o-phenylenediamineand all other laboratory reagents were from Merck, Darmstadt; Df.Ali-Sephacel", Sepha­cryl-S 200®, and Aminohexyl-Sepharose" were from Pharmacia, Freiburg; all reagents forpolyacrylamide gel electrophoresis were from Serva, Heidelberg; diazoacetyl-Dl.-norleucine

Acid Proteinases 539

methylester (DAN), bovine hemoglobin, pepstatin-A, and horseradish peroxidase wereobtained from Sigma, Taufkirchen.

The IgA2-myeloma serum was a gift of Dr. F.Skuaril, Berne, and purified IgGI (heavychain) was kindly provided by Dr. H. Kratzin, Giittingen. Protovita® was a gift of Roche,Basle. The micro testplates for enzyme immunoassay were from Nunc, Copenhagen(No. 62162).

Methods

All the Candida strains used in this investigation were tested for secretion of totallyhydrolytic proteinases by the BSA-agar criterium of Staib (39) employing YCB-BSA agar(32). The cleavage profile of BSA was analyzed by polyacrylamide gel gradient electro­phoresis (30); concomitantly the decline of pH in YCB-BSA broth was monitored as ameasure of the metabolic activity of the individual strains. Gross proteolytic activity of thestrains was tested by azocoll- and hemoglobin assay at pH 3.5 (17, 24). The secretoryproteinases of all strains used were classified by reaction with the specific inhibitor pep­statin-A (4).

Five strongly proteolytic strains of C. tropicalis (190, 293, 328, 1093, 3780), as well asC. albicans strains HP, GM, and DSM 70010 were grown in 1 liter of YCB-hemoglobinbroth for partial purification of the acid secretory proteinases from the culture supernatantas described previously (32). The enzyme of strain 293, in addition, was submitted to gelfiltration on Sephacryl 5-200 and affinity chromatography on pepstatin aminohexyl Se­ph arose (31). This enzyme was used for subcutaneous immunization of rabbits, employingFreund's adjuvant as usual. Partial purification of the immunoglobulins was accomplishedby the ion exchange procedure of Mollison (23); the linkage of horseradish peroxidase toimmunoglobulins was performed by the two step glutaraldehyde method of Avrameas andTernynck (3).

The optimum pH of proteolytic activity was tested between pH 2 and 6 as describedpreviously (32).

The course of alkaline denaturation was determined between pH 6 and 9.5 employing0.1 M citric acid - NaOH, 0.1 M Tris-HCl, and 50 mM sodium tetraborate-NaOH buf­fers respectively; after preincubation for 60 min at 22 DC, residual proteolytic activity wasdetermined by the hemoglobin assay at pH 3.5. The hydrolysis, by Candida proteinases,of human immunoglobulins A, and G] (heavy chain) was tested as described previously (32).

The inhibition of these enzymes by diazoacetyl-DL-norleucine merhylcster (DAN) and1,2-epoxy-3(p-nitrophenoxy)propane (EPNP) was tried according to Kaehn et al. (14).Butanedione(diacetyl) was employed as described by Gripon and Hofmann (9).

The immunological cross reactivity of Candida proteinases was tested by double im­munodiffusion (28) and by the direct enzyme immunoassay designed for detection of Can­dida proteinases (35).

Results

After two days of growth on YCB-BSA agar at 3rC and subsequent stainingwith amidoblack, 30% of the strains of C. tropicalis only were found to be stronglyproteolytic; strains of C. parapsilosis were moderately proteolytic (Table 1). Allstrains of the other species tested appeared negative, which does not exclude limitedproteolysis of the nitrogen source (BSA). The proof for this distinction was providedby hemoglobin tests of culture supernatant after growth of the yeasts in YCB-BSAbroth (4 days, 3rC). The calculation of the degree of hydrolysis of hemoglobinwith respect to strongly proteolytic strains of C. tropicalis revealed that all species,

540 R. Riichel, K.Uhlemann, and B.Boning

Table 1. Hydrolysis of BSA by Candida species as judged by the protein agar criterium

Species

C. tropicalis (29)C. glabrata (17)C. krusei (12)C. pseudotropicalis (11)C. parapsilosis (11)C. guilliermondii (4)Rhodotorula spp. (5)Saccharomyces cerevisiae (2)

++

9

+

12

8

(+)

5

3

o

3171211

452

( ): number of strains+ + : strong proteolysis exceeding the fringe of the colony+ : moderate proteolysis limited to the area of the colony(+): slight proteolysis, partial clarification only of the agaro : no visible proteolysisRhodotorula and Saccharomyces were added for comparison.

Table 2. Hydrolysis of hemoglobin and formation of hyphal elements. The degree of hydro­lysis of the substrate by culture supernatants of strongly proteolytic strains of C. tropicaliswas defined as 100% ; strongly proteolytic strains of C. albicans reached the same degree.The growth in mycelial form was tested on Tween rice agar as usual

C. tropicalis (9)C. parapsilosis (11)C. pseudotropicalis (10)C. krusei (12)C. glabrata (17)C. guilliermondii (4)

( ): number of strains

Pseudomyce1ia

++++

Proteolysis

100.0%24.0%

7.5%2.0%0.8%

with the exception of C. guilliermondii, secrete acid proteinases (Table 2). Appar­ently, strains of dimorphic species are better producers of acid proteinases than thestrains of C. glabrata and C. guilliermondii that grew in yeast phase only.

The metabolic activity of Candida strains in YCB-BSA broth at 26°C was moni­tored over a period of ten days by measurement of the pH (Fig. 1). As expected,acidification of the medium among the four groups of C. tropicalis is proportionalto the degree of proteolytic activity. Among the other species, C. parapsilosis withits comparatively high proteolytic activity (compare Table 2) was the fastest toacidify the medium, while C. glabrata and c.guilliermondii, virtual non-producersof secretory acid proteinase, were the slowest; c.guilliermondii, though, in thelong run reached low pH values, while C. glabrata had most difficulties to utilizethe nitrogen source. The pH profile of C. guilliermondii suggests the delayed in­duction of an acid proteinase as was demonstrated by electrophoretic analysis ofBSA-fragments.

Acid Proteinases 541

o

~---+ _ ~

+ V)+

'"V)

0-' -'< V)U

0..0.. <0 0::0:: <.... 0..

U

lI') 0 lI') lI') 0 lI')

::J: vi vi -i vi lI'i ...Q.

--+-'.....<

'" u

-' 0..< 0U 0::....0.. 00 Cl0:: :::>.... w

V)

0..

U

Fig. 1. Acidification of the YCB-BSA broth by various Candida species at 26°C. Top level:Strains of C. tropicalis distinguished by their proteolytic activity, symbols as in Table 1.Lower level: Species other than C. tropicalis. The standard deviation x 2 has been de­picted for every measurement. For comparison the pH of day four has been marked by adashed line.

542 R.Riichel, K.Uhlemann, and BiBoning

All the strains of Candida species used in this investigation were grown for fourdays in YCB-BSA broth at 37°C. Subsequently, culture supernatant of every strainwas submitted to anodic gel gradient electrophoresis under non-den aturing condi­tions. As revealed by staining with Coomassie blue, BSA was hydrolyzed to a vary­ing extent by proteinases of C. tropicalis (Fig. 2). Certain strains caused fast hydro­lysis of the substrate into low molecular weight fragm ent s (channels 8, 9); suchpattern are dominated by residu al intact BSA monomer and by fast migratingfragments. Other enzymes of C. tropicalis, at a comparable ratio of undigestedBSA, produced fragments pref erentially of intermediate molecular weight (i. e.channel 7). A third group of strains caused little if any destruction of the substrate(i.e. channel 6). At least in part the se differences are due to enzymic variants ratherth an different amounts of secreted enzyme, as proven by the pattern of specificinhibition (see below).

6 7 8 9

a t

Fig. 2. Hydrolysis of BSA by randomly selected strains of C. tropicalis. Polyacrylamide gelgradient electrophoresis of culture supernatants under non-denaturing conditions at pH 8.4after four days of growth in YCB-BSA broth at 37 DC (for details see methods). A, and A2

are BSA monomer and dimer ; a and t are controls (c. albicans CBS 2730 and C. tropi­calis 35 respectively); the arrow indicates the position of prealbumins that do not neces­sarily represent cleavage products.

Under identical conditions, some hydrolysis of BSA by enzymes of C. parapsilosiswas observed (Fig. 3). In contrast to C. tropicalis,strains of C. parapsilosis producedidentical electrophoresis pattern th at differed from those found among strains ofC. tropicalis. A similarity of the pattern was found also among the strains ofC. pseudotropicalis, C. glabrata, C. hrusei, and C. guilliermondii (Fig. 3). Strainsof th ese species in a four day interval hardly caused degrad ation of BSA, thusconfirming results of the BSA-agar test. Some differentiation among the ses strains,th ough , was observed when cultivation was extended up to 12 days.

Five strains of C. tropicalis th at were stro ngly proteolytic versus BSAwere selectedfor partial purification of th e secretory acid proteinase. The pH-dependent profile

'-.J '.....J '-J l--J 1_' '- '_ '_ '_ '-

Acid Proteinases 543

- ~ - -

,..~~!-,.-~----

•+L-__ po

Fig. 3. Hydrolysis of BSA by randomly selected strains of C. parapsilosis (pa), C. guiltier­mondii (gu), C. pseudotropicalis (ps), C. tropicalis (tr), C. glabrata (gl), and C. albicansCBS 2730 (a). Culture conditions and electrophoresis as in Fig. 2. A, and A2 are BSA mono­mer and dimer. Note the fairly identical cleavage pattern produced by C. parapsilosis; thispattern differs from those produced by C. tropicalis (see Fig. 2).

Table 3. Pattern of inhibition of acid Candida proteinases by the active site directed in­hibitors pepstatin-A, DAN, and EPNP, and the photoreagent butanedione. The data onporcine pepsin have been added for comparison; for details see methods

C. tropicalis293/801093328/793780/81190

Pepsin

Pepstatin DAN EPNP Butanedione

++ ++ ++++ ++ ++++ ++ ++++ ++ +++ + ++

++ ++ ++ ++

C. albicansCBS 2730113HPGMDSM 70010

++ : total inhibition+ : > 50% inhibition

++++++++++

+

(+): < 50% inhibition-: no inhibition

++++++++

(+)

544 R.Riichel, K.Uhlemann, and B.Boning

of these enzymes revealed an optimum of gross proteolytic activity around pH 3.6,proteinase 3780 (pH 3.4) and proteinase 293 (pH 3.8) reaching the lowest andhighest value respectively. The alkaline denaturation of all five enzymes at roomtemperature occurred between pH 7.3 and 7.6. The enzymes at pH 3.5 cleaved bothimmunoglobulin A2 and the heavy chain of IgG}.

Differences between the five enzymes became apparent upon inhibition by DAN,EPNP, and butanedione (Table 3). While EPNP, a reagent directed against theactive site of aspartic proteinases, caused inhibition to a varying degree, only oneenzyme (3780) was sensitive to another active site directed reagent (DAN). Thisenzyme, on the other hand, was the only one to resist the attack of butanedione,

A

1,0

1,0

J;.-__~

2 4 8 16 32 64

~

128 D

Fig. 4. Direct enzymeimmunoassay of Candida proteinases according to Riichel and Boning(35).Top: Proteinases from C. tropiealis 328 (-0-) and 1093 (-6-) bind to antibodies elicitedagainst proteinase from C. tropiealis 293. The proteinases from C. albieans CBS 2730(-e-) and 113 (-..-) do not bind. Bottom: Proteinases from C. albieans CBS 2730 and 113bind to antibodies elicited against the CBS enzyme; the two proteinases from C. tropiealisdo not react. Corresponding results were obtained with the other fungal enzymes listedin table 3. A: absorption at 490 nm, D: serial dilution of enzymestock solution (0.1 Itg/ml).

Acid Proteinases 545

a photoreagent that preferentially modifies tryptophane and tyrosine residues (9).The pattern of inhibition of five secretory acid proteinases from stra ins of C. albicanswere determined as well (T able 3). Th ese enzymes, with the exception of proteinaseHP differ by their lack of sensitivity versus EPNP.

A clearcut interrelationship between enzymes of the same species became apparentwhen the proteinases were submitted to the enzyme immunoassay specific forCandida proteinases (35). Emp loying anti bodies that were elicited against thesecreto ry proteinase of C. tropicalis 293, a strong cross reaction was observedbetween the enzymes from strains of C. tropicalis only; on the opposi te, a com­parable cross reaction was observed among the proteinases from C. albicans, whenanti bodies against the prot einase of C. albicans CBS 2730 were employed. No inter­specific cross reaction was observed (Fig. 4). The restr icted cross reactivity wasconfirmed by double immunodiffusion,

When another rabbit (of the Chinchilla breed) was immunized against proteinase293, the enzyme immunoassay revealed a strong interspecific cross reaction, thusshedding some light on the vagaries of immunobiology.

Discussion

In a previous communica tion we have dealt with the varie ty of secreto ry acidpr oteinases found in th e culture supern atant of most strains of C. albicans (32).Subject of the pre sent investigat ion was the occur rence of secretory acid proteinasesamong those Candida species that are isolated from hum ans aside of C. albicans.Th e relat ive frequencies of isolation of Candida species from clinical specimen inth is area were : 81.3% C. albicans, 8.3% C. tropicalis , 4.4% C. glabrata, 2.4%C. krusei, 0.8% C. pseudotropicalis, 0.4% C. parapsilosis , and 2.4% unident ifiedspecies (2). Such distribution is in agreement with figures fro m other Europeancount ries and North America (1, 5, 7, 16).

As compared with 62% of highly pro teo lytic strains of C. albicans (32), suchstrai ns amounted to approx. 30% in C. tropicalis. Among the other species nohighly proteolytic strains were detected . T he strains of C. parapsilosis were mode­rately proteolytic versus BSA, as has alrea dy been recognized by Staib et al. (41),wh ile strains of C. glabrata, C. krusei, and C. pseudotropicalis hardly affected thissubstrate ; the proteolytic activity of these strains was detected by the hemoglobintest. A strain-specific hydr olysis of BSA by C. guilliermondii became evident onlyafte r prolonged cultivation. The growth of these strain s in prot einaceous mediummay be due to intracellular degradation of BSA and may involve membrane-boundproteinases like tho se that have been found in C. albicans (33).

The low acid proteolytic act ivity of C. glabrata was surprising since this speciesrates th ird or even second among the clinica l isolates (27). In contrast to this species,which has only recentl y been added to the genus Candida (45), secretion of acidpro teinases by C. albicans and C. tropicalis reflects the sequence of path ogenicityth at was found both in vitro and in vivo (10, 11, 13). T he highest pathogenicityamong strains of C. albicans appea rs to be associated with mycelium-producersth at are stro ngly proteolytic (19, 36, 42), but other facto rs of viru lence may beinvolved, too.

The ability of most acid Candida proteinases to cleave hum an immunoglobulin·G1 that is compa ratively resistant to pepsin (38), and the cleavage of immuno-

546 R.Riichel, K.Uhlemann, and B.Boning

globulin A2 that is resistant to bacterial IgA-proteinases (for review see 15) pointsto a role of the fungal proteinases in the persistence of yeasts on mucous membranesthat provide for the acidic milieu necessary for enzymic activity (39). Bound anti­body fragments (of the F(ab)2 type) could allow for a mimicry of the fungal cellsprohibiting the secondary immuno reactions. Asteroid bodies consisting of fungalblastospores surrounded by a thick layer of antibodies (22) may represent themorphological correlate for such aggregates.

The importance of acid Candida proteinase for the virulence of the yeast wassuggested by the detection of the enzyme in the vicinity of invading yeast cells (18).Lytic halos around fungal elements have been observed repeatedly by transmissionelectron microscopy (20, 25, 37). Such defects, appearing as round holes uponscanning electron microscopy, are not related to the action of phospholipase (12),they may rather illustrate the action of acid fungal proteinases in the environmentof the yeast cell that is acidified by metabolites of the fungus (43). A correlationof proteolytic activity and secretion of acids is suggested by our own experiments;it was proven for various fungi of other genera (21).

Specific targets of proteolytic attack by Candida proteinases in the host arezymogens of the blood coagulation and angiotensinogen. Both systems were effectedby acid proteinases from C. albicans and C. tropicalis (33), as was suggested byfindings in vivo (25,26,34).

Surprisingly, even polyclonal antibodies that were elicited in rabbits againstpurified secretory proteinases from C. albicans CBS 2730 and C. tropicalis 293 crossreacted only with enzymes isolated from strains of the same fungal species; theinterspecific cross reaction was negligible. Human antibodies so far were found tocross react with enzymes of both Candida species. The discriminatory power ofrabbit antibodies may allow for a differential diagnosis of candidoses, employingthe antigen directed enzyme immunoassay (35) that proved a powerful tool for thedetection of antigenemia in mice (Ruchel, unpublished).

Acknowledgement

We are indebted to Dipl. BioI. Ortrud Zimmermann, Professor R. Ansorg, Dr. ]. Miiller,and Professor F.Staib for access to their collections of strains. Thanks go to Mrs. I. Teute­berg for excellent secretarial work.

The results of this investigation have been presented at the DGHM-meeting, Mainz,Sept. 24, 1982.

Recently we learned of comparable investigations performed by F. Macdonald, Leicester,England (Sabouraudia, in press).

References

1. Ahearn, D. G.: Medically important yeasts. Ann. Rev. Microbiol. 32 (1978) 59-682. Ansorg, R., R. Bickenbach und I. Miehlmann: Differenzierung hefeahnlicher Pilze aus

klinischem Untersuchungsmaterial mit dem API20 C-System®. Arztl. Lab. 22 (1976)243-248

3. Avrameas, S. and T. Ternynck: Peroxidase labelled antibody and Fab conjugates withenhanced intracellular penetration. Immunochemistry 8 (1971) 1175-1179

Acid Proteinases 547

4. Barrett , A.].: The many fo rms and fun ct ion s of cellular p rote inases. Fed. Proc.39(1980) 9-14

5. Bernhardt , H. und M.Knok e : Zum Vor ko mmen vo n H efep ilzen im Du odenalsaft undin der Galle. M ykosen 20 (1977) 327-338

6. Chattaway , F. W., F.C. Odds, and A. j. E. Barlow : An examina t ion of the productionof hydr olyt ic enz ymes and to xin s by pathogenic strains of Candi da albicans . ]. gen.M icrobiol. 67 (1971) 255-263

7. Derm oumi, H.: Anrimykorika-Empfindlichkeir bei klinisch bedeutsamen Hefen undSchimmelp ilzen im H emmhoftest. M ykosen 25 (1982) 109-117

8. Germaine, G. R., L. M . Te llejson, and G. L. j ohnson: Proteol ytic activ ity of Candidaalbicans : Act ion on hu man sa livar y proteins. Infect. Immun. 22 (1978) 861-866

9. Gripo n, j.-c. and T. Hofmann : Inactivation of aspartyl proteinases by butane-2,3­d ione. Biochem. ]. 193 (1981) 55-65

10. Holzscbu, D.L., F. W.Chandler, L. Ajello, and D. G. Ahearn : Evalu ation of industrialyeasts for pathogenicity. Sabouraudia 17 (1979) 71-78

11. Hopfer, R. L., A. Oren go, S. Chesnut, and M. Wenglar: Radiom etr ic detect ion of yeastsin blood cultures of cancer pat ient s. ]. Clin. Microbiol. 12 (1980) 329-33 1

12. Howlett, }. A. and C. A.Squier: Candida albicans ultrastructure : Co lonizat ion and in­vasion of ora l epithelium. Inf ect . Immun. 29 (1980) 252- 260

13. Hu rley, R. and ].De Louvois: Ecological aspects of yeas t- like fun gi of medi cal im­po rta nce: Pathogenic potenti al in the genus Candida . In : Medi cal M ycology, pp. 67-72,H.].Preusser (ed .). Fischer, Stuttgart (1980)

14. Kaehn , K., M. MOTT, and M .-R . Kula : Inh ibition of the acid prote inase fro m Neuro sporacrassa by d iazoacetyl-DL-norleucine methyl ester , 1,2-epoxy-3- (4-nitrophenox y)pro­pane and pepstati n, Hoppe Seyler'S Z . Physiol. Ch em. 360 (1979) 791-794

15. Kornfeld, S.]. and A. G. Plaut : Secreto ry immunity and th e bacterial IgA proteases. Rev.inf ect . Dis. 3 (1981) 521-534

16. Kozinn , P.}., C. L. Ta schdjian, B. E. Kodsi, G.]. W ise, M. SiSeelig, and P. K. Goldberg:Diagnosis of systemic and viscera l candidosis. Canad. Med. Ass. ] . 126 (1982) 1386­1390

17. Lanoe, }. and ] . Dunnigan : Impro vements of the Anson assa y for measuring proteolyt icactivit ies in acidic pH ran ge. Analyt . Bioch em . 89 (1978) 461-471

18. Macdo nald, F. and F. C. Odds: Inducibl e proteinase of Candida albicans in d iagnost icsero logy and in the pathogenesis of systemic candidos is. ] . M ed. M icrob iol. 13 (1980)423-435

19. Mac do nald, F. and F. C. Odds: Virulence for mice of a prot ein ase-secreting strain ofCandia albicans and a pr ot ein ase-de ficient mutant. ]. gen . Mi crobiol., 129 (1983) 431­438

20. MaTTie, T. j. and}. W. Costerto n: T he ultrastructure of Cand ida albi cans infections.Ca na d . ]. Microbiol. 27 (1981) 1156-1164

21. Ma tsushima, K.-j., M. Hayakawa, M . Ito, and K. Shim ada : Fea tures of th e proteolyticenzy me system of hyper-acid-producti ve and non-acid-producti ve fungi. ]. gen. appl.M icro bio l. 27 (1981) 423-426

22. Melcbinger, H. , ]. Mii ller, H. Takamiya und B. No ld : Immunelektron enmikroskopischeUntersuchungen an Asteroid Bod ies in Vagi nalmaterial von Candida-Kolpitis-Patien­t inn en . M ykosen 23 (1980) 161-182

23. Mo llison, P. L.: Blood tr ansfusion in cl inical med icine. Black well Scien tific, Oxford(1975)

4. Moore, G. L.: Use of azo -dye-bo und co llag en to measure react ion veloci t ies of proteo­lyti c en zyme s. An alyt. Biochem. 32 (1969) 122-127

25. Myerotoit z, R. L. : Ultrast ruc tura l observa t ions in disseminated candidias is. Arch. Path.Lab. Med. 102 (1978) 506-511

26. Nosa loua, V., T. T rnouec, M . Greguskoua, and R. No sal : The effect of po lysaccha ride-

548 R. Riichel, K.Uhlemann, and B.Boning

protein complex isolated from Candida albicans on the regional blood flow in rats.Experientia 35 (1979) 341-342

27. Odds, F.C. and E. G. V.Evans: Distribution of pathogenic yeasts and humoral anti­bodies to Candida among hospital inpatients. J. Clin. Path. 33 (1980) 750-756

28. Ouchterlony, 0.: Gel diffusion techniques. In: Immunchemie, pp. 13-35, 15. Collo­quium Ges. Physiol. Chern. Springer, Berlin (1965)

29. Remold, H., H.Fasold, and F.Staib: Purification and characterization of a proteolyticenzyme from Candida albicans. Biochim. Biophys, Acta 167 (1968) 399-406

30. Ruebel, R. and ]. Gross: Preparations of continuous gradient gel slabs: A simple techni­que. Analyt. Biochem. 92 (1979) 91-98

31. Riichel, R.: Properties of a purified proteinase from the yeast Candida albicans. Bio­chim. Biophys. Acta 659 (1981) 99-113

32. Ruchel, R., R. Tegeler, and M. Trost: A comparison of secretory proteinases from dif­ferent strains of Candida albicans. Sabouraudia 20 (1982) 233-244

33. Ruebel, R.: On the renin-like activity of Candida proteinases and activation of bloodcoagulation in vitro. Zbl. Bakt. Hyg., 1.Abt. Orig. A 255 (1983a) 368-379

34. Ruebel, R.: On the role of proteinases from Candida albicans in the pathogenesis ofacronecrosis. Zbl. Bakt. Hyg., 1. Abt. Orig. A 255 (1983 b) 524-536

35. Ruebel, R. and B.Boning: Detection of Candida proteinase by enzyme immunoassayand interaction of the enzyme with alpha-2 macroglobulin. J. Immunol. Meth, 61 (1983)107-116

36. Saltarelli, C. G., K. A. Gentile, and S.C.Mancuso: Lethality of Candida strains as in­fluenced by the host. Canad. J.Microbiol. 21 (1975) 648-654

37. Schertoitz, C.: Ultrastructure of human cutaneous candidosis. J. Invest. Dermatol. 78(1982) 200-205

38. Skuaril, F. and L. Theilkaes: IgG subclasses in pepsin resistant normal human IgG.Prot. BioI. Fluids 26 (1979) 701-703

39. Staib, F.: Serum-proteins as nitrogen source for yeastlike fungi. Sabouraudia 4 (1965)187-193

40. Staib, F.: Proteolysis and pathogenicity of Candida albicans strains. Mycopathologia 37(1969) 345-348

41. Staib, F., S.K.Mishra, B.Tompak, G.Grosse, Th.Abel, A. Blisse, V.Folkens, andB.Frohlich: Pathogenic yeast-like fungi in meat products. Zbl. Bakt. Hyg., 1.Abt, Orig.A 248 (1980) 422-429

42. Szarmacb, H., E.Malyszko, A. Wronski und W.Niczyporuk: Versuch zur Bewertungder Virulenz hefeartiger Pilze durch Bestimmung enzymatischer Eigenschaften und miteinem Haurinfektionsrnodell an der weifSen Maus. Mykosen 25 (1982) 210-220

43. Turian, G.: Low pH in fungal bud initials. Experientia 37 (1981) 1278-127944. Wingard,]. R., ]. D. Dick, W. G Merz, G.R. Sandford, R. Saral, and W.H. Burns: Dif­

ferences in virulence of clinical isolates of Candida tropicalis and Candida albicans inmice. Infect. Immun. 37 (1982) 833-836

45. Yarrow, D. and S.A.Meyer: Proposal for amendment of the diagnosis of the genusCandida Berkhout nom. cons. Int. J. system. Bact. 28 (1978) 611-615

Dr. R.Ruchel, Hygiene-Institut, Kreuzbergring 57, D-3400 Gortingen