Microbiol. Immunol. Vol. 36 (10), 1099-1104, 1992

Comparison of Secretory Acid Proteinases from Candida tropicalis, C. parapsilosis and C. albicans

Eiko SONO,1 Tomoko MASUDA,1 Kazutoh TAKESAKO,* 4 Ikunoshin KATO,1 Katsuhisa UCHIDA, 2 Somay Yamagata MURAYAMA,3 and Hideyo YAMAGUCHI2,3 1Biotechnology Research Laboratories,

Takara Shuzo Co., Ltd., Otsu, Shiga 520-21,

Japan,

2ResearchCenterfor Medical Mycology, Teikyo University,Hachioji, Tokyo 192-03, Japan, and 3Departmentof Bacteriology, Teikyo UniversitySchoolof Medicine, Itabashi-ku, Tokyo 173, Japan (Accepted for publication, July 23, 1992)

Abstract Acid proteinases secreted by Candida tropicalis and C. parapsilosis were newly isolated. Their physico-chemical and enzymatic properties of molecular weight, pH stability, isoelectric points, specific activity, and N-terminal amino acid sequences were determined and compared with those of a C. albicans acid proteinase. The two acid proteinases secreted by C. parapsilosis were found to be new enzymes in their molecular weights. The acid proteinases from C. tropicalis and C. parapsilosis showed lower activity at neutral pH, less resistance to neutral and alkaline pH than that from C. albicans, and a half or a third of the specific activity of the C. albicans enzyme. These differences seemed to be associated with the difference of pathogenesis between Candida species. Of the 31 N-terminal amino acids, the enzymes of these three Candida species revealed 12 homologous amino acids.

Candida albicans, C. tropicalis and C. parapsilosis are all major pathogens of systemic candidiasis in humans. All three species secrete an acid proteinase when grown in a medium containing a protein as a sole nitrogen source (9, 10, 12). Particularly, clinical isolates of the former two Candida species frequently secrete acid proteinases (12), and these proteinases are generally considered one of the major virulence factors of these pathogenic yeasts (6, 8). Acid proteinases of C. albicansand C. tropicaliswere isolated and their amino acid sequences and DNA sequences were reported from different laboratories (2, 3, 5, 11). An acid proteinase of C. parapsilosiswas also isolated and characterized (10). Biological and pathogenic aspects of C. albicansacid proteinase have been extensively studied, while much less information is available on the comparable enzymes from C. tropicalis and C. parapsilosis and on enzymatical difference of these three Candida acid proteinases. We here newly isolated one acid proteinase (50 kDa) from C. tropicalisand two (41 and 42 kDa) from C. parapsilosis and first revealed their enzymatical properties such as stability, specific activity and N-terminal sequences in comparison with the C. albicansenzyme (45 kDa) (12). 109 9

110 0

E. SONO

ET AL

The acid proteinase activity was determined by the standard hemoglobin assay (4) at pH 3.5 with 0.5% hemoglobin as a substrate. One unit of enzyme activity was defined as the amount that released trichloroacetic acid-soluble fragments of 1.0 absorption units (275 nm) from hemoglobin in 10 min at 30 C. The amount of protein was determined by Bio-Rad protein assay. For enzyme production, all Candida strains tested were cultured in a liquid medium containing 0.2% bovine serum albumin (BSA) and 1.17% yeast carbon base (Difco) and incubated for 3 days at 30 C. The culture supernatant of C. tropicalis TIMM 0318, C. parapsilosis TIMM 0290, and C. albicans No. 114 contained 4.3, 3.1, and 9.2 units/ml of proteolytic activity, respectively. The culture filtrates of the three Candida strains were separately purified by DEAE-Sephacel (Pharmacia) column. After washing the column with 10 mM Na-citrate buffer (pH 6.3), the enzyme was eluted with 200 nM Na-citrate buffer (pH 6.8). Fractions with proteolytic activity were further purified by Mono-Q column (1 ml, Pharmacia) using a gradient elution with 0 to 1 MNaC1 in Na-phosphate buffer (pH 6.0). Purified acid proteinases were obtained in the yield of 2.3, 22.4, and 29.0% from the culture filtrates of C . tropicalis,C. parapsilosis,and C. albicans,respectively. The yield of C. tropicalisacid proteinase was very low. C. parapsilosis produced two acid proteinases with molecular mass of 42 and 41 kDa, which were separated by Mono-Q column chromatography. Purities of the prepared enzymes were then confirmed by SDS-PAGE (Fig. 1). The other three strains (TIMM 0314, 0326, and 1753) of C. tropicaliswere found to secrete the same acid proteinase with TIMM 0318 by analyses with SDS-PAGE and Mono-Q column chromatography of their culture filtrates purified by DEAE-Sepacel (data not shown). C. parapsilosis TIMM 0288 secreted the 41 kDa acid proteinase. The proteolytic activity of these enzymes was fully inhibited by 10 /IMof pepstatin A, revealing the enzymes to be aspartic proteinases. The molecular mass of the enzyme isolated from C. tropicalisTIMM 0318 was estimated to be 50 kDa, larger than the 40 kDa reported by Togni et al (11) for the corresponding C. tropicalisenzyme, and rather close to the 49 kDa reported by Banerjee et al (1). The molecular mass of each of the two aspartyl proteinases isolated from C. parapsilosis TIMM 0290 was larger than the value reported by Riichel et al (10) for the comparable C. parapsilosis enzyme (33 kDa) and the 36 kDa reported by Banerjee et al (1). Thus, the two C. parapsilosis acid proteinases were found to be new enzymes. There may exist some different secretory acid proteinases within strains of C. tropicalisand those of C. parapsilosis,as is the case of C. albicans (5, 7). Some physico-chemical and enzymatic properties of the four Candida acid proteinases are shown in Table 1. To determine the optimum pH of proteolytic activity, hemoglobin assay of the enzyme samples was performed in the pH range of 1.7 to 7.4 in 0.1 MHC1-citrate buffers, 0.1 MNa-citrate buffers or 0.1 MNa-phosphate buffers. Alkaline denaturation of proteinases was performed with incubation (45 min, 22 C) of the enzymes in 0.1 MTris-HC1 buffers ranging from pH 7 to 9 and 0.1 MNa-phosphate buffers ranging from pH 6 to 8, and subsequent hemoglobin assay was carried out at pH 3.5. To compare substrate specificity among the four acid proteinases, BSA, hemoglobin, and casein were used as substrates, and the reaction products at pH 3.5

1101

NOTES

Fig.

I.

SUS

polyacrylaniidc

gcl

cicctropliorcsis

tropicalis I' I NI NI 0318: 2, C. parap.silosis 1-11 k Dal I, albicans No.

Tablc 

l『

Properties

of

imirificil

acid

proteinitscs.

Lancs.:

1,で.

I NI NI 0290 (42 kDal ; 3, C. parapsdosis "111NI NI 0290

of the

Candida

acid

proteinases

were analyzed by SDS-PAGE. IsoGelrm plates (pH 3 7, FMC) and a mixture of markers (pl 3.6 5.5, Sigma) were used to determine the isoelectric points of the purified acid proteinases. As shown in Table 1, the lOur enzymes isolated from the three Candida species differed in their properties. The fOur acid proteinases similarly cleaved BSA, hemoglobin, and casein when incubated at 37 C overnight. The isoelectric, point and optimum pH \ \TIT characteristic for each enzyme. The optimal pHs for C. parapsilosis proteinases had lower values, pH 2.7 (42 kDa) and pH 2.1 (41 kDa), while that for C. tropic-airs was pH 2.9. Rtichel et at (10) reported the optimal pH fOr C. parapsilosis was pH 4.3, and these fOr C. tropicalis were pH 3.4 (strain 3780) and pH 3.0 (strain 293) (0). The difference in values of the optimal pH between these previously reported and our present data may be explained as difference of the

E. SONO

110 2

ET AL

enzyme between strains. At neutral pH, the C. tropicalisand C. parapsilosis (41 and 42 kDa) acid proteinases had 20, 10, and 0% of the activity at optimum pH, respectively, whereas C. albicansacid proteinase retained 50% activity (data not shown). C. tropicalisacid proteinase was most unstable and denatured even at pH 7 (Fig. 2), resulting in a low yield of the purified enzyme as described above; this result confirmed the report of Riichel et al (10) that the alkaline denaturation of the C. tropicalis enzyme occurred between pH 7.3 and 7.6. C. albicans acid proteinase was more stable at alkaline and neutral pHs than the other acid proteinases. The specific activity determined at pH 3.5 of C. tropicalisand C. parapsilosisacid proteinases were a half or a third of the C. albicansenzyme. Such a high stability under neutral and alkaline conditions, the high specific activity, and the high activity at neutral condition of C. albicansacid proteinase suggested its involvement in the higher pathogenicity of C. albicans than other Candida species. To determine N-terminal amino acid sequences, each acid proteinase was first

Fig.

2. C.

pH

albicans

dependency No.

(41

kDa)

0.1

M Na-phosphate

Fig.

3. three with

; •¤, •¥

N-terminal different the

sequences

of

the

114; • , •¡ , C. parapsilosis buffers;

amino Candida of

enzymic

, C.

TIMM closed

0290

acid

sequences Dots(•œ)

albicans

acid

of

TIMM

symbols:

species. C.

activity

tropicalis

of in proteinase.

the

acid

proteinases.

0318; •¢, •£,

(42

kDa).

Open

pH

range

of 7 to

the

four

the

upper

Candida three

C.

Symbols: •œ,

0,

TIMM

0290

parapsilosis

symbols:

pH

9 in

0.1

acid

proteinases

sequences

range

M Tris-HC1

of 6 to

isolated indicate

8 in

buffers.

from

homology

NOTES

110 3

separated on SDS-PAGE and then transferred onto PVDF-Immobilon membrane (Millipore). After staining with Coomassie brilliant blue, the protein band was cut out and analyzed by a gas phase sequenator 470A (Applied Biosystems) with online PTH-derivative analyzer 120A. The sequencing of N-terminal 31 amino acids of the C. tropicalisacid proteinase was completely the same as that reported by Togni et al (11) except for difference in the molecular mass. The first 15 amino acids of the acid proteinase from C. albicansNo. 114 were the same as that for the comparable enzyme reported by Ganesan et al (2) but different from that of Hube et al (3). Of the N-terminal 31 amino acids, 12 (39%) were shared in common by all of the four acid proteinases obtained in this study (Fig. 3). Each of the two proteinases from C. parapsilosis has only two different amino acids (positions 24 and 30) in the Nterminal 31 amino acids, although they markedly differ in optimum pH and isoelectric point. There are 18 (58%) homologous amino acids between the enzymes of C. albicansand C. tropicalis,and 19 (61%) or 20 (65%) between those of C. tropicalis and C. parapsilosis. Such a high homology suggests that substantial differences in stability and optimum pH among the enzymes from different Candida species are not due to the amino acid sequences of N-terminal region. When comparing all amino acid sequences determined by nucleotide sequences between C. albicans (2, 3, 5) and C. tropicalis(11), there are a few low homology regions which are present mainly around the second active site Asp-218 (5). These low homology regions may cause some differences in their enzymatic properties. Determination of cDNA encoding the acid proteinases of C. tropicalisand C. parapsilosisis currently being done. REFERENCES

1) Banerjee, A., Ganesan, K., and Datta, A. 1991. Induction of secretory acid proteinase in Candida albicans. J. Gen. Microbiol. 137: 2455-2461. 2) Ganesan, K., Banerjee, A., and Datta, A. 1991. Molecular cloning of the secretory acid proteinase gene from Candida albicans and its use as a species-specific probe. Infect. Immun. 59: 2972-2977. 3) Hube, B., Turver, C. J., Odds, F.C., Eifert, H., Boulnois, G. J., Kochel, H., and Rachel, R. 1991. Sequence of the Candida albicans gene encoding the secretory asparate proteinase. J. Med. Vet. Mycol. 29: 129-132. 4) Lanoe, J., and Dunnigan, J. 1978. Improvements of the Anson assay for measuring proteolytic activities in acidic pH-range. Anal. Biochem. 89: 461-471. 5) Mukai, H., Takeda, O., Asada, K., Kato, I., Murayama, S.Y., and Yamaguchi, H. 1992. cDNA cloning of an aspartic proteinase secreted by Candida albicans. Microbiol. Immunol. (in press) 6) Ross, I.K., Bernardis, F.D., Emerson, G.W., Cassone, A., and Sullivan, P.A. 1990. The secreted . aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinasedeficient mutant. J. Gen. Microbiol. 136: 687-694. 7) Rtichei, R., T egeler , R., and Trost, M. 1982. A comparison of secretory proteinases from different strains of Candida albicans. Sabouraudia 20: 233-244. 8) Rachel, R. 1983. On the role of proteinases from Candida albicans in pathogenesis of acronecrosis. Zentralbl. Bakteriol. Hyg. Abt. I. Orig. A255: 524-537. 9) Riichel, R., Uhlemann, K., and Boning, B. 1983. Secretion of acid proteinase by different species of the genus Candida. Zentralbl. Bakteriol. Hyg., Abt. I. Orig. A255: 537-548. 10) Riichel, R., Boning, B., and Borg, M. 1986. Characterization of a secretory proteinase of Candida parapsilosisand evidence for the absence of the enzyme during infection in vitro. Infect. Immun. 53: 411-419.

110 4

E. SONO

ET AL

11)

Togni, G., Sanglard, D., Falchetto, R., and Monod, M. 1991. Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis. FEBSL Lett. 286: 181-485. 12) Yamamoto, T., Nohara, K., Uchida, K., and Yamaguchi, H. 1992. Purification and characterization of secretory proteinase of Candida albicans. Microbiol. Immunol. 36: 637-641. (Received for publication,

May 20, 1992; in revised form, June 24, 1992)