Cytochemical Demonstration of Aminopeptidase in Candida Albicans*

Vol. 51, No. 1

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1968 by The Williams & Wilkins Co.

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CYTOCHEMICAL DEMONSTRATION OF AMINOPEPTIDASE IN CANDID A ALBICANS* L. F. MONTES, M.D.t AND V. S. CONSTANTINE, M.D.t

Several years ago Kim et al. (1) using the medium is as follows: 1% aqueous solution of biochemical techniques demonstrated substantial amounts of aminopeptidase activity in cells of Candida albican.s. The availability of cytochemical staining procedures for aminopeptidase, and the increasing evidence (2—5) that yeasts lend themselves well to cytochemistry,

L-leucyl-p-naphthylamide (1.0 ml); distilled water

led us to undertake a eytochemical staining study of the distribution of this enzymatic activity in C. albican.s. Also this work has been a preliminary to an investigation of the

(9, 10). The incubations were carried out at 370 C

(40 ml) 02 M tris buffer pH 7.1 (10 m1).

Both methods consist of the adaptation for

cytochemistry of the chromogenic substrates used in colorimetric determination of aminopeptidase. They depend upon hydrolysis of a peptide bond adjacent to a terminal a-amino group, which re-

sults in the liberation of a chromogenic moiety

for periods ranging from 15 minutes to 4 hours. After incubation the smears were rinsed in distilled water for 2 to 3 minutes and then placed in a solution of 0.1 M cupric sulfate for 15 to 30 minutes. Following this, the smears were mounted on glass slides using glycerol gelatin as a mounting medium. Occasionally the smears were fixed in glutaraldehyde (7) or in formol calcium (9) either before or after incubation. Since serious

role of aminopeptidase in candidiasis. Results of this study are presented. MATERIAL AND METHODS

Cultures—Three different isolates of C. albi- diffusion artifacts of reaction products occur can.s were cultivated at room temperature on rapidly (7), microphotographs were taken shortly Bacto-Sabouraud dextrose agar for 1—14 days. after incubation. Control incubations were carried Cells were harvested by scraping them from the surface of the colony with the minimum possible amount of culture medium followed by an immediate wash in normal saline solution (0.86%) for

out simultaneously using a substrate free medium.

coverslips (0.085—0.13 mm thick) and after 3—4 minutes of exposure to air they were immersed in Petri dishes containing the incubation medium for

(11).

et al. (6) was used. It employs L-leueyl-4-methoxy-

mixture of peptidases, the more general term

Because the intracellular localization of aminopeptidase was a primary purpose of our investigation, the distribution of cellular organdIes was de5 minutes. termined by electron microscopic examination of Cytochemistry.—Thin smears were prepared on ultrathin sections of cells from the same cultures RESULTS AND DISCUSSION aminopeptidase. Sometimes the incubation was carried out in test tubes containing 5 ml of the In agreement with current views (9) that medium plus the organisms and the smears were the so-called "leucine aminopeptidase" is a prepared after incubation. The method of Monis

aminopeptidase is used in this paper to

-naphthylamide HCl as substrate. The composition of the incubating medium was that suggested by Monis et al. (7): phosphate buffer 0.1 in pH

designate the enzymes which hydrolyze various amino acid naphthylamides.

7.4 (2.5 ml); L-leucyl-4-methoxy-p-naphthylamide

Preliminary incubations were carried out using the procedure of Monis et al. (6) or the method of Burstone and Folk (8). A

HC1 10 mg/ml (0.25 ml); saline 0.86% (2.0 ml); KCN 2 X 1Q° M (0.25 ml); Fast blue B (5 mg). Also the procedure of Burstone and Folk (8) was utilized. This technique employs L-leucyl-pnaphthylamide as substrate. The composition of

comparison of the results obtained revealed yellow diffusion artifacts with the latter that * From the Department of Dermatologyt and were not present with the former. Thus the Department of Pathology, University of Alabama procedure of Monis et a2. (6) was chosen. Medical Center, Birmingham 35233. Supported by TJSPHS Research Grant AI-0763502, by Research Career Development Award 7-K3-

§ L-leucyl-4-methoxy-p-naphthylamide HC1, L-

AI-31, 210-02 (Dr. Montes) and by U. S. Veterans Administration Research Associateship Program (Dr. Constantine).

leucyl--naphthylamide, and glycerol gelatin were purchased from Sigma Chemical Company, St. Louis, Missouri. Fast Blue B was obtained from

Gwen Ramer is gratefully acknowledged. Accepted for publication January 26, 1968.

N. J. Bacto-Sabouraud dextrose agar was obtained from Difco Laboratories, Detroit, Michigan.

Matheson, Coleman and Bell, East Rutherford,

The technical assistance of Betty Henkel and

1

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(Smears were photographed at different levels of focus) FIG. 1. Aminopeptidase activity in cells of C. olbicon.s. Round granules of different sizes are intensely stained. The cytoplasm of many cells shows a diffuse weaker reaction. X 1700

FIG. 2. Intense diffuse activity in two cells of C. olbicons (arrow) which are in the process of budding. Compare the reaction in these two cells with the reaction in other

cells in the photograph. >< 1900 FiG. 3. Other dividing cells of C. elbicons showing intense diffuse activity. X 1700 FIG. 4. albicans showing several round reactive bodies and a vacuole (arrow) with strong peripheral activity. >< 1900

FIG. 5. Aminopeptidase activity is seen diffusely and in granules of a pseudohypha and

blastospores of C. olbicans, >< 1700 FIG. 6. Intense activity in granules of pseudohyphae of C. olbicans. X 1700

3

AMINOPEPTIDASE IN CANDIDA ALE lOANS

The following description of the localization of aminopeptidase activity is based on results

Cells of C. albicans grown at room tempera-

obtained with this technique. As described in animal cells, the end product

ture in Sabouraud's dextrose agar were incubated for cytochemical demonstration of

of the cytochemical reaction marking the sites

aminopeptidase activity utilizing L-leucyl-4-

SUMMARY

of enzymatic activity was visualized as a methoxy--naphthylamide as substrate. Enpurple pigment. Aminopeptidase activity was present in blastospores and in pseudohyphae as well. In most cells the reaction was strongly localized as round cytoplasmic granules, ap-

zymatic activity was mainly localized as round cytoplasmic granules. In many cells there was

of yeast cells. The fact that some of the

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also activity in the vacuoles, particularly at their periphery. Diffuse activity was present proximately 0.5 to 1 jc in size (Figs. 1—6). When in the cytoplasm of some cells, but it was looking at the larger granules it was difficult weaker than in the granules and vacuoles. to be certain if they were not the vacuoles REFERENCES vacuoles also showed activity, particularly at their periphery (Fig. 4), raised the question of a transformation of the large granules into vacuoles. Furthermore, it supports Matile and

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