P R O D U C T I O N OF G L Y C O P R O T E I N BY C A N D I D A ALBICANS IN A SYNTHETIC MEDIUM AND ITS I N F L U E N C E O N T H E G R O W T H OF N E W B O R N MICE*
by Z. THADDEUS MANKOWSKI,
M.D.
Western Institute /or Cancer and Leukemia Research, Santa Monica (Cali/ornia)
(with 1 fig.) (29.V.1967) It has been previously reported (MANKOWSKI, 1962) that the metabolic products of Candida albicans have a pathogenic affect on newborn mice. These metabolic products were used in crude form and were not defined chemically. It appeared useful to investigate more exactly and express in more accurate terms the composition and the pathogenicity of the extraneous products of Candida atbicans. TRIMBLE (1957) in his search for specific antigens from Candida albicans obtained potysaccharide substance using absolute alcohol precipitation in the medium on which Candida albicans was grown. It would be interesting therefore, if by similar procedure one could obtain a biological active substance. MATERIAL AND METHODS
Candida albicans was obtained from a human source and maintained on Sabouraud's glucose agar. In some experiments Candida albicans strain No 1775 was used which was kindly provided by Dr. M. SILVA from Columbia University College of Physicians and Surgeons, New York City. As in previous experiments (MANKOWSKI, t962), organisms of Candida albicans were grown on NICI~ERSON'S (1956) synthetic medium of the following composition: 20 g glucose; 3 g (NH~)2SO4; 3 g KH2PO,; 1 g Na glutamate; 0.25 g MgSQ. 7H20; 20 ~# biotin; 25 ml of 1 N NaOH; 3 ml 10 % CaC12.2H~O; 1000 ml distilled water. The cultures were grown in two liter * This work was supported b y Damon R u n y o n Memorial F u n d G r a n t 720 and J o a n Sloan Memorial Fund.
114
z. TH. MANKOV~rSKI
Erlenmeyer flasks and aerated on Model V rotary action shaker, (New Brunswick Scientific Co., New Brunswick, New Jersey). To measure non-nitrogenous polysaccharides, anthrone procedure was used (MORRIS, 1948). Proteins were measured with a modified Kjeldahl method (HILLER et al., 1948). Hydrolysates of the obtained substance, prepared in 6 normal HC1 for eight hours at 105°C and evaporated under vacuum, were employed in tile analysis of glucosamine b y the Elson Morgan method (1953). For identification and evaluation of monosaccharides by paper chromatography, the method of WILSON (1959) was performed using 1 N hydrolysates of the substance. ]~ESULTS The samples of tile growing cultures, obtained at weekly intervals, were spun down at 4000g for 15 minutes. Supernatant was precipitated with absolute alcohol. At the third week of growth, precipitation was observed which increased with consecutive samplings. After about 6 weeks, the whole contents were centrifuged at 4000g for 15 minutes. The supernatant was lyophilized and afterwards dialised against tap and distilled water for 6 days. After dialisis, the liquid was passed through Gelman GA-8 cellulose triacetate filter, porosity 0.2 #. Absolute alcohol was used for precipitation to obtain the final 50 to 70 ~o concentration of alcohol. On some occasions 3 ~/o sodium acetate has been used to facilitate the precipitation. The precipitate was washed twice with alcohol, twice with absolute acetone, and dried in a desiccator over P~Q. A white substance was obtained which was water soluble. The water solution was ninhydrin negative indicating tile absence of free amino acids. The absence of the characteristic cuprous oxide precipitate with Fehling's solution proved the absence of reducing sugars, For evaluation of polysaccharide, anthrone reaction has been used and it was found that polysaccharide constituted 70.5 ~o of the substance. Proteins were found to constitute 1.8 °/o as measured b y Kjeldahl method. Glucosamine was evaluated by Elson Morgan method, and was found to constitute 0.5 mg ~o. One dimensional paper chromatography showed that the polysaccharide is composed of mannose and glucose in the approximate ratio 3 : 1. Amino acid analysis was performed on Technicon amino acid analyser. For amino acid study 1 mg of substance was hydrolysed with 6 NHC1 for 24 hours in a sealed gless tube. See following table. Analytic centrifugation was performed with this substance using model E Beckman ultracentrifuge at speed 200,000g. Sedimentation coefficient was found to be 6.3. One but rather wide peak was observed. Calculated from tile sedimentation coefficient, approximate molecular weight was 100,000. This substance was found to be toxic to Swiss mice. Its LD 50 when injected intravenously, was found to be 0.75 mg/g of body
GLYCOPROTEIN INFLUENCE
115
ON THE GROWTH OF MICE
A minoacid composition o[ glycoprotein [rom Candida albicans. *n mg % Aspartic acid Threonin Serine Glutamic acid Proline
4.4 4.7 4.3 1.3 2.0
Glycine Alanine Cystine Valine Methionine
2.0 2.5 0.61 1.9 0.9
Tyrosine Phenylanaline Lysine Histidine Arginille
0,9 1.3 3.4 0.9 1.7
weight. Heating at 60 ° for one hour destroyed the toxicity of the compound. It has been previously reported (MANKOWSKI, 1962) that concentrated filtrates of Candida albicans have an inhibitory effect on growth of newborn mice, and therefore it remained to be determined if the presently isolated substance had a similar effect. Various amounts of isolated glycoprotein were injected in newborn mice five times a week; the results of these experiments m a y be seen in the following table. THE INFLUENCE OF GLYCOPROTEIN FROM CANDIDA ALBICANS ON THE GROWTH OF NEWBORN MICE Time 8 in days Saline mg
Saline
5 mg
2 Saline mg
Saline
0.5 0.1 mg Saline mg
1
1.8
1.8
2.1
2.1
1.8
1.7
1.6
1.6
2.1
2.1
3
2.9
2.9
3.2
3.0
2.1
2.9
2.1
2.0
2.4
2.2
6
3.8
3.1
4.6
3.7
3.3
3.1
2.9
2.8
4.2
3.7
9
7.4
5.8
5.6
4.4
5.4
4.6
4.9
4.4
5.8
5.2
12
8.3
6.6
7.3
6.1
6.4
5.8
5.8
5.1
7.2
7.3
15
8.7
6.9
8.8
6.9
7.0
6.3
6.3
5.4
8.7
8.9 11.3
18
10.8
7.7
11.8
8.0
7.8
7.6
6.8
6.3
11.0
21
12.0
8.5
12.2
8.4
9.2
8.5
8.5
8.3
12. 1 12.2
27
18.8
8.6
20.6
18.5
16.4
4.0
16.0
3.1
17.2 i19.5
(Weight of mice in g) Each litter of newborn mice was divided in half; one half was injected with isolated glycoprotein in saline and the other with the same amount of sterile solution of saline. Injections were performed subcutaneously five times a week. Since each dosage used in this experiment has its own control from the same litter, one has to read the control on the left side of each concentration used. One m a y see that the dosage of 8 mg produces more than 50 % growth inhibition in 27 days. The growth inhibition was still observed,
116
z. TH. MANKO~,VSKI
but to a lesser degree when the dosages of 5 and 2 mg were used. Slight growth inhibition was observed up to the 9th day when the dosage 0.1 mg was used; whereas after the twelfth day the experimental group showed a higher weight than the controls. On the 32nd day the animals injected with 0.1 mg weighed 20 % more than the controls injected with saline. Some of the experimental animals, which can be seen in the following f~gure, also showed areas of depilation. DISCUSSION The production by Candida albicans of toxic substances is of interest because of increased recognition of the significance of the toxic metabolic products of fungi, covered by the name of mycotoxins (FORGACS, 1962). The interest in the production of toxic substances b y Candida albicans is increased by the fact of frequent occurrence of this organism in the human population. WINNER (1964) gives this subject only a small place in his book. SALVIN (1951) in his study of hemolytic substance from fungi, used the supernatants of the broken cells, and therefore endotoxins and not exotoxins were studied. ~[ANKOWSKI, (1957) reported that culture filtrates from the media in which Candida albicans was grown were toxic when injected intravenously to C3H mice, without apparent toxicity to Swiss mice when the same dosage was used. Concentrated filtrates from the synthetic media on which Candida albicans were grown produced a variety of pathological changes following injection to newborn Swiss mice (MANKOWSKI, 1962). Most spectacular of these changes has been inhibition of growth with occasional occurrence of areas of extensive depilation. TRIMBLE (1957) used alcohol precipitation of the media in which Candida albicans was grown in search for specific antigenic substance, which could differentiate Candida albicans from Candida stdlatoidea. On the basis of the positive Molisch reaction, he called it a polysaccharide. Alcohol precipitation has been used also in this experiment, and the substance gave positive Molisch reaction. It is very probable that the same substance has been obtained. Ultracentrifugation showed the presence of a single peak. Since this peak was broad, it may be that the isolated substance is not homogenous.
Summary Growth of Candida albicans on a synthetic medium for a period of 6 weeks produces glycoprotein substance in the approximate amount of 150rag per liter. The polysaccharide component is formed by glucose and mannose in the approximate ratio 3 : 1. Tile protein component is composed of at least 15 different amino acids. Half percent of glucosamine was also found. The glyco-
GLYCOPROTEIN INFLUENCE ON THE GROWTH OF MICE
] ] 7
Fig. i. A. Litter-mate mice 27 days old. Two small mice since birth date received injections of Candida albicans glycoprotein 8 mg, 5 times a week. The largest one received injections of sterile saline solution in the same amount and frequency as experimental animals. B. Litter-mate mice 27 days old. Mouse with an area of depilation received injections of Candida albicans glycoproteins 5 mg five times a week. The other received injections of sterile solution in the same amount and frequency as experimental mouse.
118
z. TH. MANKOWSKI
protein substance is water soluble and toxic to Swiss mice. LD50 is 0.75 mg/g of body weight when injected intravenously. Subcutaneous injection to newborn Swiss mice produced inhibition of growth. The degree of inhibition varied with the dosage. On one occasion when using a small amount of compound, stimulation of growth was also seen. Acknowledgements
Technical assistance of Mr. PABLO V. VILLANUEVA is greatly appreciated. References ELSON, L. A, & MORGAN, W. T. J., 1933. A colorimetric method for determination of glucosamine and chondrosamine. Biochem. J. 27: 1824--1828. FORGACS, ,[. & CARLL,W-. T , 1962. Mycotoxicoses. Advances in veterinary sciences, 7 : 273--382. HILLER, A,. PLAZI~', J. 6; VAN SLYKn, D. D., 1948. A study on conditions for Kjeldahl determination of nitrogens in proteins. J. biol. Chem. 176: 1401. MANKOWSKI, Z. T., 1957. The experimental pathogenicity of various species of Candida in Swiss mice. New York Acad. Sci. 19: 545--570. MANKOWSKI, Z. T,, 1962. The pathological activity of metabolic products of Candida albicans in newborn mice. Mycopath. et Mycol. appl. 17: 165~174, MORRIS, D. L., 1948. Quantitative determination of carbohydrates with Dregwoods anthrone reagent. Science 1 0 7 : 254--255. 1LXTICKERSON, W. J., TABER, W. A. & FALCONE, G., 1956. Physiological bases of morphogenesis in Fungi: Canad. J. Microbiol. 2: 575. SALVIN, B, S,, 1951. Haemolysin fronl the yeastlike phases of some pathogenic fungi. Proc. Soc. exp. Biol. Med. 76: 852--854. TRI?aELE, J. R,, 1957. The use of a precipitin test to differentiate Candida albicans from Candida stellatoidea. J. invest. Derm. 28: 319--358. VCTILSON,G. M., 1959. Quantitative determination of sugars on papers chromatography. Ann. Chem. 31: 1199--1201. WINNER, H, I. & HURLEY, I~., 1964. Candida albicans. Little Brown and Co. Boston. p, 211.
