I. Nonencapsulated Mutants

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1967, p Vol. 94, No. 5 Copyright 1967 American Society for Microbiology Printed in U.S.A. Cryptococcus neoformans I. Nonencapsulated Mutants G. S. BULMER, M. D. SANS, AND C. M. GUNN Department of Microbiology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma Received for publication 1 September 1967 Seven nonencapsulated mutants of Cryptococcus neoformans were isolated from an encapsulated strain of human origin. Initially, the mutants were avirulent for mice. After several months of subculturing, six of the seven isolates reverted LO the encapsulated state and possessed varying degrees of virulence. The results of these experiments suggest that a strong correlation exists between the presence of a capsule and the virulence of C. neoformans. Studies on the relationship of the capsule of Cryptococcus neoformans to the virulence of the organism are limited and the results have been conflicting. Drouhet, Segretain, and Aubert (2) stated that the virulence of the fungus is directly related to the size of the capsule. However, Kao and Schwarz (4), Littman and Tsubura (6), and Hasenclever and Mitchell (3) failed to observe such a correlation. A small-capsule variant isolated by Kase and Metzgar (5) induced central nervous system symptoms in mice in 7 to 12 days, whereas the parent large-capsule strain required 14 to 120 days to produce the same symptoms. It was the purpose of these studies to isolate nonencapsulated strains of C. neoformans and contrast the virulence of such strains to the encapsulated parent. MATERIALS AND METHODS An encapsulated strain of C. neoformans (serotype A; J. E. Walter, personal communication; strain CP-5, Veterans Administration Hospital, Oklahoma City, Okla.), hereafter referred to as CIA, was obtained from a human case of cryptococcal meningitis. This organism was subcultured weekly, as were all other strains, on Sabouraud Dextrose Agar (Difco) and maintained under mineral oil. Ncnencapsulated mutants of the parent strain (CIA) were obtained by irradiating an aqueous suspension of cells (106) with an ultraviolet (UV) lamp (Mineralight, model R51, Ultra Violet Products, San Gabriel, Calif.) at a distance of 25 cm. To insure maximal exposure to UV light, the cell suspension was placed in the bottom of a petri dish and kept under constant agitation with a magnetic stirrer. The time required to kill 99% of the cells was determined in preliminary experiments. Samples of cells irradiated to that point were plated onto Sabouraud Dextrose Agar and incubated for S to 7 days at 26 C. India ink preparations of cells from dry-appearing colonies were examined microscopically for the presence of a capsule. When cells from a given colony were observed to be nonencapsulated, subcultures were prepared on Sabouraud Dextrose Agar in an attempt to obtain homogeneously nonencapsulated clones. This process was repeated several times. Assimilation studies were performed in collaboration with Frances Felton, Veterans Administration Hospital, Oklahoma City, Okla., according to the procedures of Wickerham (8). Pathogenicity of all of the strains was examined by injecting 106 viable cells suspended in 0.5 ml of saline intraperitoneally (ip) and 0.05 ml of saline intracerebrally (ic) into waite mice obtained from the mouse colony at the University of Oklahoma Medical School. Four to six animals were used for each test. India ink preparations of brain tissues from animals which died were examined microscopically for yeast cells. Tissue samples were also streaked onto Sabouraud Dextrose Agar. The cultures were incubated for 1 to 2 weeks at 26 C and examined for growth of C. neoformans. Animals surviving 140 days (ic injection) or 250 days (ip injection) were sacrificed, and the organisms with which they were inoculated were considered to be avirulent. Cells from the various strains of C. neoformans were examined for capsular constitutents by paper chromatography. For this purpose, 1.5 ml of a thick aqueous suspension of whole cells was hydrolyzed by adding 1.5 ml of 3 N HCI and heating at 100 C for 4 hr. The hydrolysate was evaporated to dryness in vacuo to remove HCI. The residue was then taken up in a minimal amount of 80% ethyl alcohol or distilled water and assayed for carbohydrates by means of descending paper chromatography with SS filter paper and solvent systems consisting of n-butanolpyridine-0.1 N HCI (5:3:2, v/v, 16 hr) and n-butanolethyl alcohol-water (5:1:4, v/v, 20 hr). Chromatograms were developed with 2-amino biphenyl. Standards consisted of D-glucose, D-xylose, D-galactose, D-mannose, D-ribose, D-glucuronolactone, and D-glucuronic acid. RESULTS In several experiments, the time required for UV light to kill 99% of the cells of the 1475

2 1476 BULMER, SANS, AND GUNN J. BACTERIOL. encapsulated parent strain, CIA, was found to be approximately 4 min. India ink preparations of 35 isolates revealed that the organisms were, for the most part, devoid of a visible capsule. In many instances, the cells were peculiar in shape, i.e., long and slender, ovoid, twisted, or bananashaped. Many of the isolated strains were composed of cells which were similar microscopically to other Cryptococcus spp. For a period of 5 to 6 weeks, samples of these colonies were subcultured weekly onto Sabouraud Dextrose Agar in attempts to isolate dry-appearing colonies which, microscopically, were composed of cells that were homogeneously devoid of a capsule and had a morphology similar to that of C. neoformans. From the original 35 isolates, 7 were selected which met these criteria. These mutants will be referred to hereafter as MI, M2, M3, M4, M5, M6, and M7. India ink preparations of the encapsulated parent strain, CIA (A), and one of the nonencapsulated mutants (B) are shown in Fig. 1. The mucoid appearance of the colonies of CIA (C), as compared with the dry, wrinkled colonies of the nonencapsulated mutant (D), is also shown. All seven of the nonencapsulated mutants grew at 37 C, were urease-positive, and turned brown on Niger seed medium (7). The results of assimilation studies indicated that, with the exception of M3, all of the mutants had an assimilation pattern similar to the parent strain (Table 1). In a further attempt to determine more precisely whether or not the mutants were truly nonencapsulated, cells from all of the strains, plus the parent CIA, were hydrolyzed and examined for capsular constituents by paper chromatography. The results of these studies (Fig. 2) revealed that Downloaded from on July 7, 2018 by guest FIG. 1. Encapsulated Cryptococcus neoformans CIA shown microscopically in India ink preparation (A) antd in gross appearance (C) of colony. Nonencapsulated strain shown microscopically in India ink preparation (B) and in gross appearance of colony (D).

3 VOL. 94, 1967 NONENCAPSULATED CRYPTOCOCCUS NEOFORMANS MUTANTS 1477 TABLE 1. Results of assimilation tests for Cryptococcus neoformans, parent strain (CIA), and seven nonencapsulated mutants (MJ-M7) 4) 4) 4) ~~~04) 0 0 o u C's 0 Z C OC CIA M M M M M M M the parent encapsulated strain, CIA, contained galactose, mannose, xylose, and glucuronic acid (also shown in the lactone form), all of which, according to Blandamer and Danishefsky (1), are constituents of the capsule of C. neoformans. M7, as well as all of the other mutants, lacked all four of these chemicals. Since, except for the absence of a capsule, the mutants appeared to be similar to the encapsulated parent strain, it was felt that the function of the capsule in the pathogenesis of cryptococcosis might be assessed by determining whether or not these strains were virulent for mice. The results of these studies are recorded in Table 2 (1 month postisolation). The encapsulated strain CIA killed mice in 8 days (ic inoculation) or 24 days (ip inoculation). All seven of the nonencapsulated mutants, regardless of the route of inoculation, failed to kill mice. All seven of the mutants were subcultured weekly, and colonies were observed for gross appearance. The cells were observed microscopically in India ink preparations. Afier 2 months of subculturing, all of the mutant strains were still nonencapsulated (Table 2). The results of virulence studies indicated that MI and M2 killed mice slowly when they were inoculated ic, and the brains of the animals contained encapsulated yeast cells. All of the mutants were avirulent after ip inoculation. After 4 months of weekly subculturing, colonies of the M4 organisms began to appear mucoid; however, in India ink preparations the cells still appeared to lack a capsule. A thick, aqueous suspension of these cells was dispensed in 3-ml amounts into test tubes (13 X 100 mm) and shaken at 37 C for 1 hr. The cells were removed by centrifugation, and ethyl alcohol was added to the supernatant fluid to give a final concentration of 90%. After storage for 15 hr at 8 C, a white precipitate was observed. This material was removed by centrifugation, hydrolyzed, and chromatographed according to the techniques described previously. All four capsular constituents were present. After 5 months of subculturing, all but one of the mutants produced mucoid-appearing colonies. The exception (M7) continued to grow in the rough form. Microscopic examination of India ink preparations revealed that the cells were nonencapsulated, whereas 60 to 99% of the cells of strains MI to M6 had capsules. The percentages of encapsulated cells and the results of virulence studies on these strains are recorded in Table 2. It can be seen that three of the reverted strains (Ml, M2, and M3) possessed varying degrees of virulence, whereas M4, M5, M6, and M7 were avirulent. After 12 months of subculturing, from 51 to 99% of the cells of strains Ml, M2, M3, M4, M5, and M6 were encapsulated. Cells of M7 remained iml..6l- M.- :.:i: iia>- - FIG. 2. Chromatogram of hydrolyzed Cryptococcus neoformans. (I) Encapsulated parent strain, CIA. (2) A nonencapsulated mutant. (S) Standards (Ga = galactose; GI = glucose; Ma = mannose; Xy = xylose; Ri = ribose; Gu. La. = glucuronolactone; GI. A. = glucuronic acid).

4 1478 BULMER, SANS, AND GUNN J. BACTERIOL. TABLE 2. Virulence and per cent encapsulation (PE) in vitro of Cryptococcus neoformans, parent straint (CIA) and seven mutants (MJ-M7), during 12 months of subculturing Time postisolation" 1 month 2 months 5 months 12 months Strain Mortality Survival PE Mortality Survival PE Mortality Survival PE Mortality tsurival PE tmb time time time ip ic ip ic ip ic ip ic ip ic ip ic ip ic ip ic % % days % % days days % % days days % % days days CAI Ml Oc M M M4 O - 0O M O M M a After isolation of mutants. b Survival time-mean c days survival of animals that died of cryptococcosis. c Avirulent 140 and 250 days post ic and ip inoculations, respectively. nonencapsulated. Cells of MI through M6 were virulent to varying degrees, whereas M7 was avirulent (Table 2). The results of assimilation studies on the mutants after 12 months of subculturing indicated that all of the mutants, except M3, had patterns identical to that of the parent strain CIA. The assimilation pattern for M3 was the same as is shown in Table 1. DIscussIoN In the present studies, UV irradiation of a heavily encapsulated strain of C. neoformans induced the development of mutants that were devoid of a capsule. Microscopic observations of India ink preparations of the mutants suggested that a capsule was lacking. More definitive proof was obtained by chromatographing hydrolyzed whole cells. By this method, no capsular constituents could be detected. The results of the assimilation tests, growth of the organisms at 37 C, production of urease, and development of a brown pigment on Niger seed medium indicated that six of the seven mutants were similar to the parent strain. One mutant, M3, had a different assimilation pattern. Thus, according to tests used, six of the seven mutants were similar to the parent strain except that they lacked a capsule, formed rough colonies, and were avirulent for mice. Mutant M4, like MI through M6, reverted to the encapsulated state after 5 months of weekly subculturing. After 4 months of subculturing, the colonies of this strain were mucoid. Microscopically, however, the cells still appeared to be nonencapsulated. This suggests that, as a preliminary step to the encapsulated state, the organism produced a soluble capsular material. Thus, this mutant differed from the others, and it seems possible that at least two distinct genetic loci are involved in the development of the typical encapsulated state. A third locus might also be implicated if one interprets the instabilities of strains MI through M6 and the stability of M7 as representing different loci. The results of the experiments comparing the virulence of encapsulated and nonencapsulated strains indicate that virulence for mice is closely associated with the presence of a capsule. After 1 month of subculturing, the mutants lacked a capsule and were avirulent. After 2 months of subculturing, all of the mutants still lacked a capsule. Two of them killed mice slowly after intracerebral inoculation; however, encapsulated cells were observed in the brains of the animals. After 12 months of subculturing, six of the seven mutants had a capsule and, for the most part, were virulent. The one mutant which remained consistently nonencapsulated throughout the 12 months of subculturing failed to kill mice. Thus, from the 1, 2, and 12 months data it can be stated that without a capsule C. neoformans CIA is avirulent and with a capsule it is virulent. It should be pointed out, however, that this conclusion is not supported by the virulence studies conducted with mutants that were subcultured for 5 months. In this case, six of the seven mutants had a degree of encapsulation similar to that seen at the 12-month period; yet three of these strains were avirulent and three had some

5 VOL. 94, 1967 NONENCAPSULATED CRYPTOCOCCUS NEOFORMANS MUTANTS 1479 degree of virulence. These results suggest that the capsule, as it existed at that time, did not play a key role in the pathogenesis of cryptococcosis. It is possible that the capsule at the 5-month period differed somehow from that seen at the 12-month period. The only criterion used to determine the nature of the capsular material at the 5-month period was microscopic examination of India ink preparations. Such observations offer no indication as to the chemical structure or antigenicity of the material. The observation that one mutant (M4) progressed through at least two stages of development before an intact capsule could be seen microscopically indicates that reversion from a nonencapsulated to encapsulated state may require several steps. The capsules observed after 5 months of subculturing may, then, have been in an immature state as far as being a functional virulence factor. If the capsule is not a virulence factor, it becomes difficult to explain its apparent close association with virulence at the 1-, 2-, and 12- month periods. Furthermore, M7 never reverted to an encapsulated form, and was consistently avirulent. If the results of further investigations support the contention that the capsule of C. neoformans is a virulence factor, it will be the first such entity described in fungi which are pathogenic for animals. The fact that Cryptococcus spp. possess a capsule and are avirulent does not detract from this suggestion, since virulence need not necessarily be linked solely to a single factor, but rather to the overall integrity of the organism. It is interesting that the results of earlier studies on the significance of the capsule of C. neoformans in virulence are conflicting. It is possible that the strains used previously were not entirely nonencapsulated. Thus, the degree of encapsulation may not be important, but rather the presence or absence of a capsule, and also the possibility that capsular material may be present in a diffusible state in vivo. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Research Career Program Award (G. S. B.) 5-K03-AI from the National Institute of Allergy and Infectious Diseases and Public Health Service grant Al LITERATURE CITED 1. BLANDAMER, A., AND I. DANISHELSKY Investigations on the structure of capsular polysaccharide from Cryptococcus neoformans, type B. Biochim. Biophys. Acta 117: DROUHET, E., G. SEGRETAIN, AND J. P. AUBERT Polyoside capsulaire d'un champignon pathogene Torulopsis neoformans relation avec la virulence. Ann. Inst. Pasteur 79: HASENCLEVER, H. F., AND W. 0. MITCHELL Virulence and growth rates of Cryptococcus neoformans in mice. Ann. N.Y. Acad. Sci. 89: KAO, C. J., AND J. SCHWARZ Isolation of Cryptococcus neoformans from pigeon nests, with remarks on the identification of virulent cryptococci. Am. J. Clin. Pathol. 27: KASE, A., AND J. F. METZGAR Isolation of a dry variant from a mucoid strain of Cryptococcus neoformans and preliminary comparative studies. J. Bacteriol. 83: LrITMAN, M. L., AND E. TSUBURA Effect of degree of encapsulation upon virulence of Cryptococcus neoformans. Proc. Soc. Exptl. Biol. Med. 101: STAIB, F Cryptococcus neoformans and Guizotia abyssinica. Z. Hyg. Infektionskrankh. 148: WICKERHAM, L. J The taxonomy of yeasts. I. Techniques of classification. U.S. Dept. Agr. Tech. Bull