Mycopathologia et Mycologia applicata, voI. 54, 3, pag. 391-404, 1974

ENDOTOXIC SUBSTANCE OF CRYPTOCOCCUS NEOFORMANS

by

Takashi Kobayashi 1, Izumi Nakashima & Nobuo Kato

Abstract

In this study an endotoxic substance was extracted from the cells of Cryptococcus neoformans and the physicochemical and biological properties of this substance (Cr-ET) were investigated. In comparison with endotoxin of gram-negative bacteria, the lethality of Cr-ET for mice and chick embryos was low and such biological activities were weak as the pyrogenic effect on rabbits and effects on the leucocyte count and blood sugar level in rabbits. Skin reactions (both primary and Shwartzman reactions) were elicited in rabbits by relatively large dose of Cr-ET. Unlike bacterial endotoxin, hyperreactivity to Cr-ET was not induced in mice by prior infection with BCG.

Introduction

Cryptococcosis of man is one ofthemost important deep-seated mycosis with high mortality, and about a half of the patients was found to be primarily infected without any basic diseases like leukemia and carcino- ma (13). A knowledge of the factors relating to the virulence of Crypto- coccus neoformans is essential to a understanding of the disease produced and of host responses to this fungal infection. Although many workers studied with the capsular substance of C. neoformans, little studies have been done on the existence of endotoxin of this fungus (1). In this paper, we describe the results of study of the isolation and physiocochemical and biological properties of a cryptococcal endotoxic substance.

Materials and methods

Strains

C. neoJbrmans Duke, N-1 and N-2 atrains were used. The Duke strain was supplied by Dr. H. Kobayashi, Chest Disease Research Institute, Kyoto University. The N-1 and N-2 strains were the stock strains of this laboratory. BCG was supplied by the Japanese Tuberculosis Pre- ventive Society, Tokyo.

i Department of Bacteriology, Nagoya University School of Medicine, 65 Tsurumai- cho, Showa-ku, Nagoya 466, Japan. Accepted for publication: 7. VII. 1973

392

Animals

Male SMA mice, 4 weeks old, and female albino rabbits weighing about 2.5 kg were used.

Chick embryos

Eleven-day-old embryonated eggs were used.

Preparation o fan endotoxie substance of C. neoformans ( Cr-ET)

C. neoJbrmans was cultivated at 37 °C for 4 days in Sabouraud's broth containing 3 ~ of glucose, 200 #g/mt of streptomycin and 200 units/ml of penicillin. The organisms were killed by the addition of formalin to a final concentration of 0.4 ~ followed by further incubation at 37 °C for 48 hours. The organisms were collected by centrifugation at 7500 rpm for 30 minutes and washed 3 times with M/100 phosphate-buffered saline (PBS). The organisms were dried by washing 3 times with acetone. Dried organisms were suspended in PBS to a concentration of 0.1 ~ . The suspension was sonicated (10 kc, Reytheon sonic oscillator) for 20 minutes. The sonicated preparation was used as the starting material (sonic lysate). Cr-ET was extracted according to the. phenol-water procedure (28). The water phase obtained after centrifugation at 3000 rpm for 30 minutes were poured into a dialysis bag and dialyzed against tapped water for 48 hours. To the fluid an equal volume of ethanol was added and the precipitate was removed by centrifugation at 3000 rpm for 30 minutes. The supernatant fluid was concentrated approximately 10-fold in vacuo at 37 °C, and 6 volumes of ethanol and sodium acetate at a final concentration of 5 ~ were added. The precipitate which settled overnight in the refrigerator was collected by centrifugation at 3000 rpm for 30 minutes, the supernatant was descarded, the precipitate was dis- solved and dialyzed in water. Precipitation with ethanol and dissolution of precipitate were further repeated twice. The precipitate was dissolved in distilled water and centrifuged at 100,000 G for 2 hours. The sediment was lyophilized and dissolved in PBS prior to use.

Chemical analyses

Proteins were determined by the method of Lowry et al. (23), nucleic acids by UV absorption (34), phosphorus by Bloor's method (8), lipids (bound) by the method of Westphal et al. (35). The quantitative deter- mination of sugars was performed by Dubois's method with galactose as the standard (10). For the analysis of sugars by paper chromatography, Cr-ET (0.5 mg) was hydrolyzed in 1 ml of 1 N HC1 at 100 °C for 1 hour in a sealed tube, the hydrolysate which was freed from HC1 was dissolved in water (0.1 ml) and applied for ascending paper chromatography on Whatman No. 1 paper at 25 °C using the solvent with the composition of

393

n-butanol-pyridine-water (6 :4 :3 ) (18). Sugars were coloured with di- phenylamine-aniline reagent (4). Infrared spectrophotometry was per- formed by KBr method. Gas chromatography for sugar analysis of Cr-ET was kindly performed by Dr. H. Nakayoshi, Taisho Pharma- ceutical Co., Ltd., Tokyo. Sample was hydrolyzed in 5 % methanolic HC1 at 10ff°C for 3 hours. Trimethylchlorosylane derivatives of the methanolysate were analyzed on columns. The column temperature was

180 °C (36).

Test for pyrogenicity

RabNts used for pyrogen test were immobilized in their natural position in holding cages open on top. Sample to be tested was dissolved in pyrogen-free saline and was injected into the marginal auricular vein of rabbits. The rectal temperature of rabbits were measured by a thermis- tor thermometer. Throughout the period of time from 30 minutes before to 8 hours after injection, rabbits were immobilized in the holding cages and temperature measurements were made at 30 minutes or 1 hour intervals. After then, rabbits were set free and immobilized again 30 minutes before each temperature measurement.

Leucocyte counting

Blood was taken from the marginal auricular vein of rabbits. Blood was diluted appropriately with Ttirk's solution. Leucocyte number was counted in a hemocytometer.

Determination of blood sugar

Blood sugar level was estimated by the Glucostat method (25).

Results

Chemical analyses

Chemical compositions of Cr-ET are shown in table 1. The main

Table 1. Chemical compositions of sonic lysate and Cr-ET

Sonic lysate Cr-ET

Proteins 4.02 % 1.46 Nucleic acids 0.16 0.11 P 0.13 0.03 Lipid n.d.* 1.35 Carbohydrates 82.0 67.0 (as galactose)

* not done

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were contained in Cr-ET was in contrast to that Ibund with the capsular polysaccharide of C. neoformans (11, 12, 24).

Paper chromatography with acid hydrolysate of Cr-ET showed the spots corresponding xylose, galactose, mannose, glucmonic acid, glu- cosamine and glucuronolactone. It was reported that glucosamine or hexosaminidic linkage was not contained in the capsular polysaccharide of C. neoformans (7). Gas chromatogram showed that the ratios of sugar contents in Cr-ET were ribose or rhamnose 7.0, xylose 38.0, mannose I 53.8, mannose II 7.2, galactose 9.0, glucose 6.1, and heptose 5.6 (fig. 1). Infrared spectrogram (fig. 2) showed that the polysaccharide portion of Cr-ET was structurally very similar to the type A polysaccharide of C. neoformans (14, 22). At 1725 cm- and 1250 cm-1 regions, prominent absorption bands were recognized, which corresponded to the absorp- tion of ester bond. Absorption at 1620 cm- 1 and 1420 cm- ~ regions showed the presence of carbohydrates. The ratio of absorption at 1725 cm- 1 to that at 1620 cm- 1 was 1.04, indicating the relatively high content of acetyl group.

- - [ W A V E N U M S E R ( ~ m - ' )

Fig. 2. Infrared spectrogram of the sonic lysate (A) and Cr-ET (B) of Duke strain.

396

Toxicity

Mouse lethality and body-weight reducing effect on mice were tested with both the sonic lysate of C. neoformans and Cr-ET. Among the sonic lysates of three strains of C. neoformans, the preparation of Duke strain was most toxic for mice when injected intraperitoneally (i.p.) (table 2). Even when 5 mg of Cr-ET isolated from Duke strain were injected i.p. to mice, no mice died in a week (table 2). It was found, how- ever, that the mice injected i.p. With Cr-ET were markedly emaciated a few days after injection. Accordingly, changes in the body weight of mice injected i.p. with the sonic lysate or Cr-ET of Duke strain were in- vestigated. As shown in fig. 3, body-weight reducing effect on mice of the sonic lysate and Cr-ET was clearly demonstrated. The effect of the sonic lysate was found to be approximately dose-responsive. It should be noticed that a relatively low sublethal dose of Cr-ET showed an marked body-weight reducing effect on mice.

Chick embryo lethality of the sonic lysate and Cr-ET of Duke strain was tested by the procedures described by Smith et al. (31) (table 3). Chick embryo LDso (CELDs0) was 774.2~#g for CR-ET and 3034.4 #g for the sonic lysate. It was found that the CELDso of CR-ET was about 10 times larger than that of E. coli endotoxin.

Table 2. Toxicity for mice of sonic lysate and Cr-ET*

Dose Numbers of mice Material (rag) Route dead/total

Sonic lysate (Duke) 5 i.p. 0/6 10 i.p. 0/6 15 i.p. 3/6 20 i.p. 6/6

Sonic lysate (N-l) 5 i.p. 0/6 10 i.p. 0/6 15 i.p. 0/6 20 i,p. 0/6

Sonic lysate (N-2) 5 i,p. 0/6 10 i.p, 0/6 15 i.p. 0/6 20 i.p, 0/6

Cr-ET (Duke) 0.05 i.v. 0/15 0.5 i.v. 0/12 5 i.p. 0/8

Cr-ET (N-l) 0.5 i.v. 0/6

LPS of E. coli 0-111 0.5 i.p. 5/5**

* Mice inoculated with indicated doses were observed for week after injection. ** All mice were dead within 24 hours after injection.

397

-I-3 I ~ ' ~ e----, sonic lysate lOmg &r------~ sonic lysate 15rag 4- 2 C 0 Cr-ET O~Smg

0 ~ saline

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Fig. 3. Body-weight reducing effect on mice of the sonic lysate and Cr-ET of Duke strain. Material to be tested (0.5 ml) was injected i.p.

Table 3. Chick embryo lethality of sonic lysate and Cr-ET*

Numbers of chick embryo LDso Material Dose (#g) alcoA/total (yg)

Sonic lysate (Duke) 400 0/8 2000 2/10 3034 4000 7/9

Cr-ET (Duke) 100 2/12 500 4/12 774

1000 7/11

Cr-ET (N-I) 1000 0/4 > 1000

Cr-ET (N-2) 800 1/4 > 800

LPS of E. coli 0--111 50 2/5 100 3/5 75 200 4/4

* Material to be tested (0.05 ml) was inoculated onto the chorioallantoic membrane in an artificial air cavity of 1 l-day old eggs. Chick embryos were examined for their viability

o after 24 hours incubation at 38.5 C.

Pyrogenicity In t ravenous (i.v.) injection o f Cr -ET (75 or 150 #g per kg o f body

weight) induced febrile response in rabbits (fig. 4). Febrile response to Cr -ET was character ized by a short or undetectable lag period, a peak temperature occurr ing 3 to 4 hours post-injection, and markedly pro- longed fever, forming a second peak w h e n administered with 150/~g/l~g of Cr-ET. This fever pat tern by Cr -ET is similar to tha t p roduced by i.v. inoculat ion o f 109 viable cells o f C. neoformans (19).

398

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Fig. 4. Fever curve elicited in rabbits by Cr-ET of Duke strain. The curve shows the mean of 3 rabbits injected i.v. with 75 or 150 #g per kg of body weight of Cr-ET.

Leucopenia and leucocytosis

When 75 pg/kg of Cr-ET of Duke strain was injected i.v. to rabbits, leucopenia occurred rapidly in the peripheral blood and it continued for 6 hours, and it was followed by leucocytosis (fig. 5). Leucocytosis con- tinued at least for 36 hours. The occurrence of leucocytosis by Cr-ET seemed to be significantly late in contrast to that found with bacterial endotoxin (5). The time and duration o f leucopenia after administration of Cr-ET seemed to correspond with those of the initial elevation of fever (fig. 4).

Effect on blood sugar

As shown in fig. 6, gradual elevation in blood sugar level was induced

399

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Time (hour) Fig. 5. Leucopenia and leucocytosis in the peripheral blood induced in rabbits by Cr-ET of Duke strain. The curve shows the mean of 3 rabbits injected i.v. with 75/~g per kg of

body weight of Cr-ET.

by i.v. ifijection of 75 #g/kg of Cr-ET of Duke strain. A peak of hyper- glycemia was attained 9 hours after injection.

lmmunogenicity

Mice were injected twice i.p. with 0.5 mg of Cr-ET of Duke strain at an interval of 5 days. Two weeks after the last injection, mice were bled from retroorbital plexus. To determine the antibody response to Cr-ET, the sera obtained were examined by both ring test and gel diffusion precipitation test. No precipitins were detected in all of the immunized.

Skin test (primary reaction and Shwartzman reaction)

By an intradermal injection of lO0#g or more of Cr-ET of Duke strain to rabbits, erythema was produced around the skin site of in-

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T i m e (hour) Fig. 6. Effect of Cr-ET of Duke strain on blood sugar level of rabbits. The curve shows

the mean of 3 rabbits injected i.v. with 75 #g per kg of body weight of Cr-ET.

jection within 24 hours after injection (primary reaction) (table 4). After the reacting injection (i.v.) of 500#g of Cr-ET to rabbits primarily injected intradermally with 150/~g of Cr-ET, the local Shwartzman reaction was elicited at the skin site of the primary injection (table 4).

Failure of enhancement of reactivity to Cr-ET by infection with BCG

It has been found that mice infected with BCG become much more susceptible to bacterial endotoxin (26, 33). Experiment was conducted to clarify whether this phenomenon would occur with Cr-ET too, Mice were inoculated i.v. with 1 mg (wet weight) of BCG suspended in 0.2 ml of PBS. Ten days later, toxicity of Cr-ET (Duke) by the i.v. route

401

Table 4. Skin reactions elicited by Cr-ET (primary reaction and local Shwartzman reac- tion)

Local Shwartzman reaction***

Primary reaction** Diameter Diameter Diameter of of of

Dose* erythema erythema hemorrhage Material (/~g) (ram) (mm) (mm)

Cr-ET 50 0 x 0 0 × 0 o x 0 (Duke) 100 25 x 25 0 x 0 0 x 0

150 40 x 30 10 x 10 5 x 15

* Dose for the primary intradermal injection. ** Skin reaction 24 hours after the primary injection.

*** Skin reaction 24 hours after the intravenous provocative injection of 500 #g of Cr-ET.

was tested by using the mice infected with BCG and uninfected normal mice. No enhancement of susceptibility to Cr-ET was observed in this experiment.

Discussion

The purpose of the present study was to extract an endotoxic substance from the cells of C. neoformans. The term 'endotoxin' is generally characterized by its chemical composition and its host-reactive prop- erties, and it is found principally at or near the cell surface of gram- negative bacteria (26). Chemical analyses revealed that Cr-ET was mainly composed of polysaccharide but it contained small amounts of lipids (bound), glucosamine and phosphorus. Cr-ET was found to exhibit the biological activities characteristic of bacterial endotoxin, such as the production of fever, local skin reaction, leucopenia followed by leucocytosis, and transient hyperglycemia, although its effects ap- peared to be generally weaker than those of bacterial endotoxin. The finding that no enhancement of susceptibility to the toxicity of Cr-ET was induced in mice by prior infection with BCG may suggest that the host-reactive properties of Cr-ET somehow differ from those of bac- terial endotoxin. In addition, toxicity of Cr-ET for mice and chick embryos were much weaker than that of bacterial endotoxin. Despite such low toxicity and weak biological activities and although the phys- icochemical properties of Cr-ET was not yet completely elucidated~ it is tempting for us to provisionally consider-this substance extracted from C. neoformans as an endotoxic substance and to serve it for further in- vestigation.

Endotoxins or endotoxin-like substances of fungi were found bY earlier workers in Aspergillus.fumigatus (3, 16, 17, 32), Candida albicans

402

(2, 9, 30) and Rizopus nigricans (15). These fungal endotoxins showed also low toxicity and weak biological activities in comparison with bacterial endotoxin. Azuma et al. (3) reported that the lipopolysaccharide of A. fumigatus showed no lethality for mice even by i.v. injection of 5 mg and no body-weight reducing effect on mice. Formalinized cells of C. albicans showed the mortality rate of only about 10 to 30 % by i.v. inoculation of 25 to 50 mg (30). Recently, Cutler et al. (9) reported that the extracts of C. albieans prepared with hot phenol-water or potassium hydroxide killed mice but were nonpyrogenic for rabbits, the LDso for mice of the extracts was 4.6 mg,. and the CELDso was larger than 5 rag. Endotoxins of A. fumigatus and C. albicans were found to be pyrogenic at the doses of approximately 1 mg per kg of body weight (3, 19).

As to the pyrogenicity, Cr-ET is more potent than these fungal en- dotoxins, namely, 75 #g per kg of body weight could produce a marked febrile response. The fever produced by Cr-ET seemed to continue longer than that produced by the other fungal endotoxins or bacterial endotoxin. The prolonged duration of the biological activities was also found and blood sugar level. This phenomenon is probably due to the retention of Cr-ET in the blood stream (6). Based on its weak host- responsive properties, Cr-ET may resemble to the cell wall components of gram-positive bacteria (29) rather than to endotoxin of gram-negative bacteria.

As to the relation of chemical composition of bacterial endotoxin to its biological activities, it was found that the main component of the lethal fraction was glycolipid and the pyrogenic fraction was sugar fraction (20). Iwata et al. (17) reported that an endotoxin-like substance from A. fumigatus contained almost no lipid by the analysis of infrared spectrophotometry. A toxic substance from C. albicans was of very low content of fatty acids (9). Low toxicity of Cr-ET may be also closely related to low content of lipids.

The effect of this endotoxic substance from C. neoformans on the host resistance to infection with this fungus will be reported in the succeeding paper.

Summary An endotoxic substance (Cr-ET) was isolated from Cr)~Ttococcus neOjbrmans. Cr-ET

was found to consist of carbohydrates (67%), proteins (1.46%), lipids.. (1.35 %), phos- phorus (0.03 %), and nucleic acids (0.11 ~). From the analyses by paper chromatography and gas-liquid chromatography, sugar compositions of Cr-ET were mannose, xylose, galactose, glucose, ribose (or rhamnose), glucuronolactone, glucosamjn~ and glucuronic acid. The lethality of Cr-ET for mice and Chick embryos were low in comparison with bacterial endotoxin, but it showed a marked body-weight reducing effect on mice by in- traperitoneal injection of sublethal doses. In rabbits, Cr-ET exhibited the pyrogenicity, leucopenia followed by leucocytosis and transient hyperglycemia by intravenous in- jection, of relatively large doses. Cr-ET produced local skin reaction in rabbits by intra- dermal injection and Shwartzman reaction at the skin site of intradermal injection fol- lowing intravenous reacting injection of Cr-ET. With respect to the toxicity and host- reactive properties of Cr-ET, it may resemble the toxic components derived form cell walls of gram-positive bacteria rather than endotoxin of gram-negative bacteria.

403

Acknowledgements The authors are grateful to Prof. S. Ata, Institute of Medical Mycology, Nagoya Uni-

versity School of Medicine, for his helpful guidance, to Dr. F. Ohta of this laboratory and Dr. H. Kobayashi, Chest Disease Research Institute, Kyoto University, for supplying the strains, to Pr0f. J'. Okuda, Faculty of Pharmacy, Meijo University~ foi" performing infra- red spectrophometry, 'and to Dr. H. Nakayoshi, Taisho Pharmaceutical Co., Ltd., for performing gas chromatography.

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Key word: Cryptococcal endotoxic substance.