INFECTION AND IMMUNITY,
Apr. 1977, p.
99-106
Vol. 16, No. 1 Printed in U.S.A.
Copyright © 1977 American Society for Microbiology
Non-Encapsulated Variant of Cryptococcus neoformans II. Surface Receptors for Cryptococcal Polysaccharide and Their Role in Inhibition of Phagocytosis by Polysaccharide THOMAS R. KOZEL Division of Biomedical Sciences, School of Medical Sciences, University of Nevada-Reno, Nevada 89557
Reno,
Received for publication 16 December 1976
The binding of cryptococcal polysaccharide to a non-encapsulated strain of Cryptococcus neoformans was studied. Binding of purified polysaccharide to the yeast was determined by inhibition of phagocytosis and by indirect immunofluorescence techniques. The ability of cryptococcal polysaccharide to prevent phagocytosis of the non-encapsulated strain appears to be directly related to adherence of polysaccharide to the yeast via specific receptors on the cell surface. Addition of varying doses of cryptococcal polysaccharide to non-encapsulated yeast cells inhibited phagocytosis only at polysaccharide concentrations at which the polysaccharide could be demonstrated on the yeast surface by immunofluorescence. Macrophages treated with cryptococcal polysaccharide had no detectable amounts of cryptococcal polysaccharide adherent to their surface, and they had a normal ability to phagocytize the yeast. Kinetic studies showed that inhibition of phagocytosis is directly related to the presence of cryptococcal polysaccharide at the yeast surface rather than to some indirect effect by the polysaccharide on serum components necessary for phagocytosis. Purified polysaccharide from C. neoformans serotypes A, B, C, and D bound to the yeast, but type m pneumococcal polysaccharide did not inhibit phagocytosis of the nonencapsulated yeast. Cryptococcal polysaccharide did not bind to cells ofCandida albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., or Saccharomyces cerevtsiae. Cryptococcus neoformans is one of the few pathogenic fungi for which a virulence factor has been clearly identified. The capsular polysaccharide is a significant, if not essential, requirement for virulence. One important biological property of the capsular polysaccharide is inhibition of phagocytosis of cryptococcus by leukocytes. Non-encapsulated or weakly encapsulated cryptococci are engulfed readily by phagocytic cells, whereas encapsulated cells are resistant to phagocytosis by polymorphonuclear leukocytes (1, 5), monocytes (5), peritoneal macrophages (12, 14), and alveolar macrophages (3). Furthernore, microgram amounts of cryptococcal polysaccharide render non-encapsulated cryptococci resistant to phagocytosis (1, 5). Previous studies have shown that cryptococcal polysaccharide inhibits phagocytosis by preventing attachment of the phagocyte to the encapsulated yeast cells (12), but neither the site of action of the polysaccharide nor the mechanism by which attachment is prevented is known. Bulmer and Sans (1) demonstrated that cryptococcal cells grown in a non-encapsulated
state could be preincubated with cryptococcal polysaccharide and washed, and still retain some resistance to phagocytosis. Tacker et al. (15) reported further that cryptococcal polysaccharide inhibited phagocytosis of live cryptococci grown in a non-encapsulated state on a low-pH medium. However, the polysaccharide could not inhibit phagocytosis of killed, nonencapsulated yeast cells, regardless of the treatment used to kill the yeast. These results suggest that cryptococcal cells possess a surface receptor for the capsular polysaccharide which was altered or destroyed by the killing process. The present study was undertaken to provide additional inforrnation on the mechanism by which cryptococcal polysaccharide inhibits phagocytosis. The experiments were designed to: (i) obtain direct evidence for the binding of cryptococcal polysaccharide to a non-encapsulated variant of C. neoformans, (ii) correlate the binding of cryptococcal polysaccharide to the yeast surface with the ability ofthe polysaccharide to inhibit phagocytosis, and (iii) determine the specificity of the yeast receptor for
polysaccharide. 99
100
KOZEL
(This work was presented in part at the 76th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 1976.) MATERIALS AND METHODS Yeast isolates and soluble polysaccharide. C. neoformans 602 is a non-encapsulated strain that produces a low-molecular-weight soluble polysaccharide antigenically similar to type D cryptococcal polysaccharide (10). C. neoformans 613 is a virulent, moderately encapsulated strain of cryptococcal serotype D. Cultures of C. neoformans serotypes A, B, C, and D were obtained from John E. Bennett, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Cultures of Candida albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., and Saccharomyces cerevisiae were obtained from the stock culture collection maintained at the University of Nevada-Reno School of Medical Sciences. Organisms used in all assays were grown for 3 days on slants of potato dextrose agar (Difco) and were washed from the slants with 0.15 M sodium chloride containing formaldehyde at a final concentration of 0.33%. The yeast cells were incubated overnight at room temperature in the formalinized saline, washed three times with Hanks balanced salt solution (HBSS; International Scientific Industries) buffered with sodium bicarbonate to pH 7.2, and resuspended in HBSS. Cell counts were determined with a hemocytometer. The procedure for purification of cryptococcal soluble polysaccharide was described previously (10). Polysaccharide was stored in a lyophilized state at room temperature and was prepared for use at the desired concentration as a saline solution. Type III pneumococcal polysaccharide was generously supplied by Benjamin Prescott, National Institute of Allergy and Infectious Diseases. Phagocytosis. Monolayers of peritoneal macrophages were prepared as described previously (12). Monolayers were prepared in four-chamber tissue culture chamber/slides (Lab Tek Products, Division Miles Laboratories, Inc.) and incubated for 48 h at 37°C in 5% CO2 before use. Each monolayer contained approximately 2.5 x 105 macrophages. For phagocytosis assays, the culture medium was poured off the macrophage monolayers, and 1 ml of a test yeast suspension was added to each monolayer. The test yeast suspension consisted of: (i) unless otherwise indicated, cryptococci at a concentration providing four yeast cells per macrophage; (ii) calf serum (Grand Island Biological Co., lot no. C548421 and C951404) at a final concentration of 10%; (iii) when required by an experimental protocol, 0.25 ml of cryptococcal polysaccharide in saline; and (iv) enough HBSS to give a final volume of 1 ml. Unless otherwise indicated, phagocytosis assays were done by incubating cryptococci with macrophages at 37°C for 2 h. After incubation, the medium was aspirated, and the monolayers were washed gently in HBSS. The monolayers were air dried, fixed in methanol, and stained by the Giemsa procedure. At least 200 macrophages were examined per monolayer, and the percentage of macrophages with ingested yeast (percent phagocytosis) or the number
INFECT. IMMUN. of yeast cells engulfed per macrophage (phagocytic index) was determined. Results are presented as mean values of at least four replications. Immunofluorescence. Indirect fluorescent-antibody techniques were used to direct cryptococcal polysaccharide on the surface of yeast cells. Slides of yeast cells were prepared by a modification of the technique of Goren and Warren (7). Washed yeast cells were diluted in phosphate-buffered saline (pH 7.2) to a final concentration of 2.5 x 107/ml. Onetenth milliliter of a 1% bovine serum albumin solution was mixed with 0.2 ml of yeast suspension and 0.7 ml of distilled water. Small drops of the cell suspensions were placed on microscope slides and allowed to air dry. Slides were fixed in acetone for 20 min before use. Cryptococcal antiserum was prepared in rabbits against whole cells of C. neoformans 613 (11) and against purified cryptococcal polysaccharide complexed with methylated bovine serum albumin (11). The fluorescein-conjugated immunoglobulin G fraction of goat anti-rabbit immunoglobulins (A, G, and M) was obtained from Chappel Laboratories, Inc. (lot no. 7500). The fluorescein conjugate contained 1.7 mg of antibody protein per ml. All immune sera were absorbed three times with 109 cells of strain 602 per ml of serum and diluted 1:30 with phosphatebuffered saline before use. Cryptococcal antiserum was applied to slides containing the yeast suspension and incubated for 30 min at 37°C in a humidified atmosphere. The slides were washed for 5 min in each of three changes of phosphate-buffered saline, reincubated with fluorescein-labeled goat anti-rabbit immunoglobulins for 30 min at 37°C, and washed three times in phosphate-buffered saline. Yeast cells examined for surface polysaccharide were assayed with antiserum prepared against whole cells of C. neoformans 613 and antiserum prepared against cryptococcal polysaccharide complexed with methylated bovine serum albumin. Normal rabbit serum was used as a negative control. Results given are means of results from several anticryptococcal sera and are expressed as intensity of fluorescence on a scale of 0 to 4. A value of 4 corresponded to the intensity seen with fully encapsulated cryptococcal cells, and a value of 0 was given to cells exhibiting only background fluorescence. A very weak background fluorescence was observed with all yeast cells, regardless of serum source, species of yeast, or adsorption of serum with cryptococci. All slides were read in a blind fashion.
RESULTS polysaccharide to nonsoluble Binding of encapsulated cryptococci. The ability of cryptococcal polysaccharide to adhere to the surface of strain 602 was demonstrated by indirect immunofluorescence. Cells of strain 602 (5 x 107/ ml in saline) were incubated for 30 min at 37°C with an equal volume of strain 613 soluble polysaccharide (2 mg/ml), washed three times with saline, and examined for adherent polysaccharide by immunofluorescence. Indirect immuno-
VOL. 16, 1977
PHAGOCYTOSIS OF C. NEOFORMANS
fluorescence with rabbit cryptococcal antiseand fluorescein-labeled goat anti-rabbit immunoglobulins showed a bright, narrow rim of adherent polysaccharide on the surface of the yeast (Fig. 1B). The intensity of fluorescence was similar to that seen with fully encapsulated cryptococci (Fig. 1A), but the width of the adherent polysaccharide was substantially less than the capsule surrounding the encapsulated yeast. Significant fluorescence was not observed with untreated strain 602 cells or when normal rabbit serum was substituted for the anticryptococcal rabbit serum. Correlation of binding of polysaccharide to yeast surface with inhibition of phagocytosis. Several investigators have demonstrated that purified cryptococcal polysaccharide inhibits phagocytosis of small-capsule (5) or non-encapsulated (1, 11) isolates of C. neoformans. Bul-
and Sans (1) showed further that nonencapsulated cryptococci treated with cryptococcal polysaccharide can be washed with saline and remain partially resistant to phagocytosis. An experiment was done to confirm this observation and to determine what effect cryptococcal polysaccharide might have on the phagocytic activity of the macrophages themselves. Yeast cells (3.3 x 107/ml in HBSS) were incubated for 30 min at 37°C with an equal volume of cryptococcal polysaccharide (2 mg/ml in saline) and washed three times with HBSS. Macrophages monolayers were also incubated for 30 min at 37°C with cryptococcal polysaccharide at a final concentration of 1 mg/ml and washed three times with HBSS. Yeast cells and macrophages were then incubated together for 2 h at 37°C and examined to deternine the percent phagocytosis. In the absence of soluble polysaccharide, cells of strain 602 were engulfed readily by macrophages (Table 1). Treatment of macrophages with soluble polysaccharide had no effect on their ability to engulf the yeast. Examination of polysaccharide-treated macrophages by immunofluorescence techniques showed no observable polysaccharide adherent to the surface of the macrophages. Polysaccharide treatment of strain 602 significantly reduced the ability of macrophages to engulf the yeast; however, the percent phagocytosis was not inhibited to the extent seen with the fully encapsulated strain 613. The previous data strongly suggested that inhibition of phagocytosis was directly related to adherence of cryptococcal polysaccharide to the yeast. An experiment was done to provide a direct correlation between the presence of adherent polysaccharide as seen by immunofluorescence techniques and the ability of cryptococcal polysaccharide to inhibit phagocytosis. Cells of strain 602 were incubated with mono-
rum
101
mer
TABLE 1. Effects of cryptococcal polysaccharide treatment of macrophages and cryptococcal cells on phagocytosis of C. neoformans 602 Cryptococcal cells Strain 613 Strain 602 Strain 602
Polysaccharidetreated strain
FIG. 1. Indirect immunofluorescence of a medium capsule strain of C. neoformans (A) and non-encapsulated C. neoformans 602 preincubated with 2,000 Ag of polysaccharide and washed with saline (B). Indirect immunofluorescence was done with strain 602-adsorbed cryptococcal antiserum prepared in rabbits and fluorescein-labeled goat anti-rabbit immunoglobulins.
Macrophages Untreated Untreated
Polysaccharide treated" Untreated
Phagocytosis 3 + 1 84 + 6 86 ± 4 39 ± 9
602c
SD, Standard deviation. bMacrophage monolayer incubated with strain 613 polysaccharide (1 mg/ml) for 30 min at 37'C and washed three times with HBSS. c Strain 602 cells (3.3 x 107/ml) incubated with an equal volume strain 613 polysaccharide (2 mg/ml) for 30 min at 37'C and washed three times with HBSS. a
102
INFECT. IMMUN.
KOZEL
layers of macrophages for 2 h at 370C in the of varying concentrations of cryptococcal polysaccharide and examined to determine the percent phagocytosis. Identical preparations of strain 602 in cryptococcal polysaccharide were incubated for 2 h at 370C, washed two times with saline, and examined for adherent polysaccharide by immunofluorescence techniques (Fig. 2). Polysaccharide concentrations of 100 and 1,000 ,ug/ml inhibited phagocytosis to the extent seen with the fully encapsulated strain 613. These cells also demonstrated the presence of considerable amounts of adherent polysaccharide as seen by immunofluorescence. No difference was noted in the amount of detectable adherent polysaccharide after treatment with either the 100- or 1,000-,ug/ml polysaccharide concentration. At lesser concentrations of cryptococcal polysaccharide (10,. 1.0, and 0.1 ,g/ml), there was good correlation between the increase in percent phagocytosis and the decline in adherent polysaccharide as seen by immunofluorescence. Inhibition of phagocytosis by adherent polysaccharide could be due to binding of polysaccharide to the yeast per se or to an action by the adherent polysaccharide on various serum components necessary for phagocytosis. Gadebusch (6) reported that cryptococcal polysaccharide is a potent inactivator of the properdin system. Since the alternate complement pathway is an active component in the phagocytosis of cryptococci (4), it could be argued that the adherent polysaccharide seen in Fig. 1 was sufficient to inactivate any properdin present in the experimental system. To answer this question, the effects of cryptococcal polysaccharide on phagocytosis of yeasts other than cryptococci were studied. If cryptococcal polysaccharide inhibits phagocytosis by presence
depleting complement, it is likely that the polysaccharide would inhibit phagocytosis of microorganisms other than C. neoformans. The results (Table 2) showed that addition of cryptococcal polysaccharide (100 ,g/ml) to various yeast isolates did not inhibit engulfinent of C. albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., or S. cerevisiae. Examination of polysaccharide-treated yeast cells by immunofluorescence showed no detectable amounts of polysaccharide adherent to yeast cells other than C. neoformans. It could be argued that complement components were not necessary for phagocytosis of these yeast cells or that cryptococcal polysaccharide might alter or destroy some essential serum opsonin other than complement; consequently, the effects of a varying concentration of yeast upon the inhibitory properties of a constant amount of polysaccharide were studied. It was hypothesized that, if cryptococcal polysaccharide inactivates or inhibits some serum component necessary for phagocytosis, some inhibition of phagocytosis should be observed at all concentrations of yeast, much as with a noncompetitive enzyme inhibitor. If inhibition of phagocytosis is a direct result of binding to the yeast, little inhibition should be observed at very high concentrations of yeast due to dilution of polysaccharide on the surface of the yeast, but increasing degrees of inhibition should be observed if the polysaccharide concentration is held constant and the yeast concentration is decreased. TABLE 2. Specificity of cryptococcal polysaccharide for C. neoformans Untreated Yeast
to0 --
-
1-2+
2-3+
4+
4+
ph tosis (% ± SD-)
4-
IMMUNOFLUORESCENCE
80
60
Candida albicans C. pseudotropicalis Torulopsis sp. Rhodotorula sp. Saccharomyces cere-
Immu-
Phgc-nofluores-
cence
33 ± 10 49 ± 19 23 ± 13 9± 7 39 ± 11
0 0 0 0 0
Cryptococcus neofor- 65 ± 9
0
Polysaccharide
treated
Immu
Phagocy- noflu. tosisb nores ores± SD)
cence'
27 43 23 6.4 45
± 6 ± 14 + 11 ± 11 ± 7
0 0 0 0 0
viesae o a
40
\
CRYPTOCOCCAL POLYSACCHARIDE (ug/mi)
FIG. 2. Dose-response correlation of adherence of polysaccharide to strain 602 as shown by indirect immunofluorescence with inhibition of phagocytosis by cryptococcal polysaccharide.
4 + 2
4+
mans 602 a SD, Standard deviation. Percent phagocytosis determined after incubation of yeast cells (106/ml) with macrophages in the presence of crytococcal polysaccharide (100 ,ug/ml). c Adherence of cryptococcal polysaccharide determined by indirect immunofluorescence with rabbit antiserum to strain 613 and fluorescein-labeled anti-rabbit immunoglobulins. Strain 602 cells (5 x 107/ml) incubated with an equal volume strain 613 polysaccharide (2 mg/ml) for 30 min at 37°C and washed three times with HBSS before examination.
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PHAGOCYTOSIS OF C. NEOFORMANS
Uptake of C. neoformans 602 was determined in the absence of cryptococcal polysaccharide and in the presence of polysaccharide at a final concentration of 0.5, 0.7, and 1.0 ,g/ml (Table 3). Varying concentrations of yeast cells in HBSS containing 10% bovine calf serum and saline or polysaccharide were incubated with monolayers of macrophages for 30 min at 37°C. The macrophages were then rinsed, stained, and examined to determine the mean number of yeast cells phagocytized per macrophage (phagocytic index). Analysis of the data by analysis of variance showed that cryptococcal polysaccharide at a final concentration of 0.5 ,ug/ml produced no significant (P < 0.05) inhibition of phagocytosis at yeast-to-macrophage ratios of 16, 8, and 4; however, significant (P < 0.05) inhibition was seen when the number of yeasts was reduced to two yeast cells per macrophage. Addition of cryptococcal polysaccharide at a final concentration of 0.7 ,ug/ml produced no significant inhibition at a yeast-to-macrophage ratio of 16, but significant (P < 0.05) inhibition was observed at eight, four, and two yeasts per macrophage. Cryptococcal polysaccharide at a final concentration of 1.0 ug/ml inhibited phagocytosis at all concentrations of yeast tested. The data shown in Table 3 are also shown (Fig. 3) as a double-reciprocal plot of 1/phagocytic index versus 1/yeast added per macrophage. Data for a final polysaccharide concentration of 1 ,ug/ml and a yeast concentration of 32 yeast cells per macrophage are also plotted to show the behavior of this concentration of inhibitor at very high yeast concentrations. The data show a linear plot for phagocytosis in the absence of inhibitor. In the presence of 1 ,ug of polysaccharide, the curve was nonlinear and approached the uninhibited curve only at very high yeast concentrations. The nonlinearity of the inhibition curve was more apparent in the presence of 0.7 ,ug of polysaccharide, where a substantial inhibition of phagocytosis occurred at low yeast concentrations; however, as the
yeast concentration increased, the amount of polysaccharide bound per yeast cell decreased below an apparent threshold necessary for inhibition of phagocytosis, and significant inhibition of phagocytosis was not observed at these high yeast concentrations. Thus, under these experimental conditions, inhibition of phagocytosis was associated with binding of polysaccharide to the yeast rather than an indirect effect of the polysaccharide on serum factors necessary for phagocytosis. Specificity of C. neoformans 602 surface receptors for cryptococcal polysaccharide. Bulmer and Sans (1) demonstrated that various polysaccharides other than cryptococcal polysaccharide were unable to inhibit phagocytosis of C. neoformans; however, their studies did not investigate the ability of polysaccharides from various cryptococcal isolates or closely related bacterial polysaccharides to bind to C. neoformans. Consequently, an experiment was
103
40
3.0
0 z
0
a-0
.062
.125
.25
.50
1/(NO. YEAST ADDED PER MACROPHAGE) FIG. 3. Effects of a varying concentration of nonencapsulated cryptococci upon the phagocytosis-inhibiting properties of a constant amount of polysaccharide. Phagocytosis of C. neoformans 602 in the absence of polysaccharide (O) and in the presence of cryptococcal polysaccharide at final concentrations of 0.5 pg/ml (A), 0.7 pg/ml (0), and 1.0 pg/ml (0) is shown.
TABLE 3. Effects of a varying concentration of non-encapsulated cryptococci upon the phagocytosis-inhibiting properties of a constant amount of polysaccharide Phagocytic indexa Treatment (gg/ml)
16b
8
4
2
5.5 ± 1.2
3.1 ± 0.2
1.6 ± 0.2
1.1 ± 0.1
3.3 ± 0.3 1.8 ± 0.3 2.2 ± 0.5 1.2 + 0.3 1.5 ± 0.4 1.1 ± 0.2 1.0 a Mean number of phagocytized yeast per macrophage + standard deviation. b Yeast added per macrophage.
0.82 + 0.15 0.42 ± 0.16 0.25 ± 0.07
Saline Polysaccharide 0.5 0.7
5.7 + 1.2 5.4 + 1.4 2.3 ± 0.5
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INFECT. IMMUN.
KOZEL
done to determine whether soluble polysaccharides obtained from the four cryptococcal serotypes and type III pneumococcal polysaccharide were able to bind to strain 602 and inhibit phagocytosis by macrophages. Cells of strain 602 and each polysaccharide at a final concentration of 100 ug/ml were added to monolayers of macrophages to determine their susceptibility to phagocytosis. Cells of strain 602 (5 x 107/ ml) were also incubated for 30 min at 370C with an equal volume of polysaccharide (2 mg/ml) from the various cryptococcal serotypes, washed three times with saline, and examined for adherent polysaccharide by immunofluorescence techniques with antiserum prepared against strain 613 soluble polysaccharide. The results (Table 4) show no observable difference between the ability of polysaccharides from each cryptococcal serotype to inhibit phagocytosis of strain 602. Type III pneumococcal polysaccharide was unable to inhibit phagocytosis of the yeast. Similarly, immunofluorescence demonstrated that each of the cryptococcal polysaccharides was able to bind to the surface of the yeast. Differences in degree of observed fluorescence were probably due to the use of antiserum prepared against a cryptococcal isolate of serotype D.
DISCUSSION Several investigators (1, 5, 10) have demonstrated that addition of purified cryptococcal polysaccharide to small-capsule or non-encapTABLE 4. Specificity of the polysaccharide receptor on C. neoformans 602 for cryptococcal polysaccharide PhagocytoPolysaccharide
sisa (% ± SD) 90 5
Immunoflu-
orescenceb
0 None Strain 613 4 1 4+ Type A 4 1 3+ Type B 2 1 3+ Type C 3 1 3+ Type D 6 3 4+ Type III pneumococcal 90 ± 2 ND a Percent phagacytosis determined after incubation of cryptococci (106/ml) with macrophages in the presence of cryptococcal polysaccharide (100 ,ug/ml). SD, Standard deviation. b Adherence of cryptococcal polysaccharide determined by indirect immunofluorescence with rabbit antiserum to strain 613 and fluorescein-labeled antirabbit immunoglobulins. Strain 602 cells (5 x 107/ ml) were incubated with an equal volume strain 613 polysaccharide (2 mg/ml) for 30 min at 370C and washed three times with HBSS before examination. ND, Not determined.
sulated isolates of C. neoformans renders the yeast resistant to phagocytosis. Evidence that this inhibition of phagocytosis might be due to adherence of polysaccharide to the cryptococcal cell was first presented by Bulmer and Sans (1). They showed that cryptococcal cells grown in a non-encapsulated state could be preincubated with cryptococcal polysaccharide and washed, and still be partially resistant to phagocytosis. Further evidence for a cryptococcal surface receptor for soluble polysaccharide was presented by Tacker et al. (15), who reported that capsular material would not inhibit phagocytosis of killed, non-encapsulated cryptococci. This suggested that either the non-encapsulated cells must be viable for the added polysaccharide to inhibit phagocytosis, or the treatment used to kill the cells may have altered sites required for attachment of soluble polysaccharide. The present studies confirm earlier observations that preincubation of cryptococcal cells with polysaccharide render them resistant to phagocytosis, even after a wash with saline. Since this increased resistance to phagocytosis could be explained by either an alteration in the yeast surface by polysaccharide or adherence of polysaccharide to the cell surface, a direct confirmation of the binding of cryptococcal polysaccharide to the non-encapsulated yeast was obtained by immunofluorescence techniques. Non-encapsulated cryptococci that were treated with cryptococcal polysaccharide showed a rim of capsular material that was narrower but equal in intensity to the capsule seen on a virulent, encapsulated strain. A correlation between inhibition of phagocytosis and the binding of cryptococcal polysaccharide to non-encapsulated cells as seen by immunofluorescence was obtained by comparing the concentrations of cryptococcal polysaccharide needed to inhibit phagocytosis with the concentration needed to produce an effect visible by immunofluorescence. As shown in Fig. 2, the concentrations necessary to achieve both effects were similar. No difference was noted between cells treated with polysaccharide concentrations of either 100 or 1,000 ,ug/ml with regard to resistance of the cells to phagocytosis or to the approximate amount of polysaccharide bound to the cell surface. Thus, it appears that the amount of capsular material present influences phagocytosis up to a minimal threshold, but additional polysaccharide has no further effect on resistance to phagocytosis. This may provide an explanation for the observation that there is no clear relationship between in vitro capsule size and virulence (8, 9, 13), since in the present study even a minimal amount of polysaccharide produced inhibition of phagocytosis
VOL. 16, 1977
PHAGOCYTOSIS OF C. NEOFORMANS
comparable to that observed with fully encapsulated cryptococci. At least two mechanisms may be proposed to explain inhibition of phagocytosis by cryptococcal polysaccharide. The capsular material may, by its physical presence around the yeast, prevent an effective interaction between the cryptococcus and the phagocytic cell, or the capsular material may deplete the incubation medium of serum components necessary for phagocytosis. The latter possibility must be considered because cryptococcal polysaccharide has been shown to inactivate properdin (6) and to consume the late components of complement (4). The failure of cryptococcal polysaccharide to inhibit phagocytosis of cells of several yeast genera other than Cryptococcus suggests that cryptococcal polysaccharide does not inhibit or inactivate some essential serum opsonin. Since it could be argued that complement components were not essential for phagocytosis of these yeasts or that cryptococcal polysaccharide affects some serum opsonin specific for C. neoformans, a kinetic analysis of phagocytosis of strain 602 was done to distinguish between the two possibilities. When the effects of a varying concentration of yeast upon the inhibitory properties of a constant amount of polysaccharide were studied, cryptococcal polysaccharide was found to inhibit phagocytosis at low yeast concentrations, but the inhibition decreased progressively as the yeast concentration increased. If cryptococcal polysaccharide inactivated or inhibited some serum component necessary for phagocytosis, some inhibition of phagocytosis should have been observed at all concentrations of yeast. The experimental data suggest that a critical amount of cryptococcal polysaccharide must be bound to the yeast cell for inhibition of phagocytosis. At cell surface concentrations below this threshold, there is no inhibition of phagocytosis. These data also suggest that, under these experimental conditions, inhibition of phagocytosis is an intrinsic result of presence of the capsular material on the yeast rather than an indirect effect on macrophages or essential serum factors necessary for phagocytosis. The role of complement consumption in the antiphagocytic properties of cryptococcal polysaccharide cannot be totally excluded, however, because the ability to consume complement has been reported only with concentrations of polysaccharide considerably higher than concentrations shown in Fig. 3 (4). Thus, it is possible that some additional inhibitory property of cryptococcal polysaccharide might be expressed at very high polysaccharide concentrations. Adherence of cryptococcal polysaccharide to
nomenon mediated by receptors specific for cryptococcal polysaccharide rather than nonspecific adsorption to a yeast cell wall. Type III pneumococcal polysaccharide (Table 4) and a variety of plant polysaccharides are unable to inhibit phagocytosis of cryptococcus (1). Furthermore, cryptococcal polysaccharide does not adhere to a variety of yeasts other than C. neoformans (Table 2). Previous studies (1, 5) have shown that cryptococcal polysaccharide does not inhibit phagocytosis of two yeast genera other than Cryptococcus and two genera of bacteria; however, those reports did not indicate whether cryptococcal polysaccharide was able to bind to those organisms. Tacker et al. (15) studied phagocytosis of cryptococcal cells grown in a non-encapsulated state on low-pH medium. They reported that cryptococcal polysaccharide did not inhibit phagocytosis of killed, non-encapsulated cells regardless of the method used to kill the yeast. The authors suggest that either the non-encapsulated cells must be viable for attachment to occur, or the treatment used on the cells altered the receptors responsible for attachment of the antiphagocytic polysaccharide. The resistance of the receptors on strain 602 to formaldehyde, as reported in the present study, as well as the resistance of these receptors to a variety of physical and chemical agents including methanol, autoclaving, and mild acid and alkali treatment (T. R. Kozel, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, F13, p. 89), suggests an alternate explanation. It is possible that growth of C. neoformans at low pH suppresses formation of the polysaccharide receptor, and the receptor is synthesized upon release of the organism from the low pH. Thus, killing of the cells immediately after harvesting from the low-pH medium would yield cells to which cryptococcal polysaccharide would not adhere.
non-encapsulated cryptococci is a specific phe-
105
ACKNOWLEDGMENTS This research was supported by grants from the Luke B. Hancock Foundation and the University of Nevada Research Advisory Board. LITERATURE CITED 1. Bulmer, G. S., and M. D. Sans. 1968. Cryptococcus neoformans. III. Inhibition of phagocytosis. J. Bacteriol. 95:5-8. 2. Bulmer, G. S., M. D. Sans, and C. M. Gunn. 1967. Cryptococcus neoformans. I. Nonencapsulated mutants. J. Bacteriol. 94:1475-1479. 3. Bulmer, G. S., and J. R. Tacker. 1975. Phagocytosis of Cryptococcus neoformans by alveolar macrophages. Infect. Immun. 11:73-79. 4. Diamond, R. D., J. E. May, M. A. Kane, M. M. Frank, and J. E. Bennett. 1974. The role of the classical and alternate complement pathways in host defenses against Cryptococcus neoformans infection. J. Immunol. 112:2260-2270. 5. Diamond, R. D., R. K. Root, and J. E. Bennett. 1972.
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7.
8. 9.
10.
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Factors influencing killing of Cryptococcus neoformans by human leukocytes in vitro. J. Infect. Dis. 125:367-376. Gadebusch, H. H. 1961. Natural host resistance to infection with Cryptococcus neoformans. I. The effect of the properdin system on the experimental disease. J. Infect. Dis. 109:148-153. Goren, M. B., and J. Warren. 1968. Immunofluorescence studies of reactions at the cryptococcal capsule. J. Infect. Dis. 118:215-229. Hasenclever, H. F., and W. 0. Mitchell. 1960. Virulence and growth rates ofCryptococcus neoformans in mice. Ann. N.Y. Acad. Sci. 89:156-162. Kao, C. J., and J. Schwartz. 1957. The isolation of Cryptococcus neoformans from pigeon nests. Am. J. Clin. Pathol. 27:652-663. 0 Kozel, T. R., and J. Cazin, Jr. 1971. Nonencapsulated variant of Cryptococcus neoformans. I. Virulence studies and characterization of soluble polysaccha-
INFECT. IMMUN. ride. Infect. Immun. 3:287-294. 11. Kozel, T. R., and J. Cazin, Jr. 1974. Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans. Mycopathol. Mycol. Appl. 54:21-30. 12. Kozel, T. R., and R. P. Mastroianni. 1976. Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. Infect. Immun. 14:62-67. 13. Littman, M. L., and E. Tsubura. 1959. Effect of degree of encapsulation upon virulence of Cryptococcus neoformans. Proc. Soc. Exp. Biol. Med. 101:773-777. 14. Mitchell, T. G., and L. Friedman. 1972. In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans. Infect. Immun. 5:491-498. 15. Tacker, J. R., F. Farhi, and G. S. Bulmer. 1972. Intracellular fate of Cryptococcus neoformans. Infect. Immun. 6:162-167.
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Vol. 16, No. 1 Printed in U.S.A.
Copyright © 1977 American Society for Microbiology
Non-Encapsulated Variant of Cryptococcus neoformans II. Surface Receptors for Cryptococcal Polysaccharide and Their Role in Inhibition of Phagocytosis by Polysaccharide THOMAS R. KOZEL Division of Biomedical Sciences, School of Medical Sciences, University of Nevada-Reno, Nevada 89557
Reno,
Received for publication 16 December 1976
The binding of cryptococcal polysaccharide to a non-encapsulated strain of Cryptococcus neoformans was studied. Binding of purified polysaccharide to the yeast was determined by inhibition of phagocytosis and by indirect immunofluorescence techniques. The ability of cryptococcal polysaccharide to prevent phagocytosis of the non-encapsulated strain appears to be directly related to adherence of polysaccharide to the yeast via specific receptors on the cell surface. Addition of varying doses of cryptococcal polysaccharide to non-encapsulated yeast cells inhibited phagocytosis only at polysaccharide concentrations at which the polysaccharide could be demonstrated on the yeast surface by immunofluorescence. Macrophages treated with cryptococcal polysaccharide had no detectable amounts of cryptococcal polysaccharide adherent to their surface, and they had a normal ability to phagocytize the yeast. Kinetic studies showed that inhibition of phagocytosis is directly related to the presence of cryptococcal polysaccharide at the yeast surface rather than to some indirect effect by the polysaccharide on serum components necessary for phagocytosis. Purified polysaccharide from C. neoformans serotypes A, B, C, and D bound to the yeast, but type m pneumococcal polysaccharide did not inhibit phagocytosis of the nonencapsulated yeast. Cryptococcal polysaccharide did not bind to cells ofCandida albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., or Saccharomyces cerevtsiae. Cryptococcus neoformans is one of the few pathogenic fungi for which a virulence factor has been clearly identified. The capsular polysaccharide is a significant, if not essential, requirement for virulence. One important biological property of the capsular polysaccharide is inhibition of phagocytosis of cryptococcus by leukocytes. Non-encapsulated or weakly encapsulated cryptococci are engulfed readily by phagocytic cells, whereas encapsulated cells are resistant to phagocytosis by polymorphonuclear leukocytes (1, 5), monocytes (5), peritoneal macrophages (12, 14), and alveolar macrophages (3). Furthernore, microgram amounts of cryptococcal polysaccharide render non-encapsulated cryptococci resistant to phagocytosis (1, 5). Previous studies have shown that cryptococcal polysaccharide inhibits phagocytosis by preventing attachment of the phagocyte to the encapsulated yeast cells (12), but neither the site of action of the polysaccharide nor the mechanism by which attachment is prevented is known. Bulmer and Sans (1) demonstrated that cryptococcal cells grown in a non-encapsulated
state could be preincubated with cryptococcal polysaccharide and washed, and still retain some resistance to phagocytosis. Tacker et al. (15) reported further that cryptococcal polysaccharide inhibited phagocytosis of live cryptococci grown in a non-encapsulated state on a low-pH medium. However, the polysaccharide could not inhibit phagocytosis of killed, nonencapsulated yeast cells, regardless of the treatment used to kill the yeast. These results suggest that cryptococcal cells possess a surface receptor for the capsular polysaccharide which was altered or destroyed by the killing process. The present study was undertaken to provide additional inforrnation on the mechanism by which cryptococcal polysaccharide inhibits phagocytosis. The experiments were designed to: (i) obtain direct evidence for the binding of cryptococcal polysaccharide to a non-encapsulated variant of C. neoformans, (ii) correlate the binding of cryptococcal polysaccharide to the yeast surface with the ability ofthe polysaccharide to inhibit phagocytosis, and (iii) determine the specificity of the yeast receptor for
polysaccharide. 99
100
KOZEL
(This work was presented in part at the 76th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 1976.) MATERIALS AND METHODS Yeast isolates and soluble polysaccharide. C. neoformans 602 is a non-encapsulated strain that produces a low-molecular-weight soluble polysaccharide antigenically similar to type D cryptococcal polysaccharide (10). C. neoformans 613 is a virulent, moderately encapsulated strain of cryptococcal serotype D. Cultures of C. neoformans serotypes A, B, C, and D were obtained from John E. Bennett, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Cultures of Candida albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., and Saccharomyces cerevisiae were obtained from the stock culture collection maintained at the University of Nevada-Reno School of Medical Sciences. Organisms used in all assays were grown for 3 days on slants of potato dextrose agar (Difco) and were washed from the slants with 0.15 M sodium chloride containing formaldehyde at a final concentration of 0.33%. The yeast cells were incubated overnight at room temperature in the formalinized saline, washed three times with Hanks balanced salt solution (HBSS; International Scientific Industries) buffered with sodium bicarbonate to pH 7.2, and resuspended in HBSS. Cell counts were determined with a hemocytometer. The procedure for purification of cryptococcal soluble polysaccharide was described previously (10). Polysaccharide was stored in a lyophilized state at room temperature and was prepared for use at the desired concentration as a saline solution. Type III pneumococcal polysaccharide was generously supplied by Benjamin Prescott, National Institute of Allergy and Infectious Diseases. Phagocytosis. Monolayers of peritoneal macrophages were prepared as described previously (12). Monolayers were prepared in four-chamber tissue culture chamber/slides (Lab Tek Products, Division Miles Laboratories, Inc.) and incubated for 48 h at 37°C in 5% CO2 before use. Each monolayer contained approximately 2.5 x 105 macrophages. For phagocytosis assays, the culture medium was poured off the macrophage monolayers, and 1 ml of a test yeast suspension was added to each monolayer. The test yeast suspension consisted of: (i) unless otherwise indicated, cryptococci at a concentration providing four yeast cells per macrophage; (ii) calf serum (Grand Island Biological Co., lot no. C548421 and C951404) at a final concentration of 10%; (iii) when required by an experimental protocol, 0.25 ml of cryptococcal polysaccharide in saline; and (iv) enough HBSS to give a final volume of 1 ml. Unless otherwise indicated, phagocytosis assays were done by incubating cryptococci with macrophages at 37°C for 2 h. After incubation, the medium was aspirated, and the monolayers were washed gently in HBSS. The monolayers were air dried, fixed in methanol, and stained by the Giemsa procedure. At least 200 macrophages were examined per monolayer, and the percentage of macrophages with ingested yeast (percent phagocytosis) or the number
INFECT. IMMUN. of yeast cells engulfed per macrophage (phagocytic index) was determined. Results are presented as mean values of at least four replications. Immunofluorescence. Indirect fluorescent-antibody techniques were used to direct cryptococcal polysaccharide on the surface of yeast cells. Slides of yeast cells were prepared by a modification of the technique of Goren and Warren (7). Washed yeast cells were diluted in phosphate-buffered saline (pH 7.2) to a final concentration of 2.5 x 107/ml. Onetenth milliliter of a 1% bovine serum albumin solution was mixed with 0.2 ml of yeast suspension and 0.7 ml of distilled water. Small drops of the cell suspensions were placed on microscope slides and allowed to air dry. Slides were fixed in acetone for 20 min before use. Cryptococcal antiserum was prepared in rabbits against whole cells of C. neoformans 613 (11) and against purified cryptococcal polysaccharide complexed with methylated bovine serum albumin (11). The fluorescein-conjugated immunoglobulin G fraction of goat anti-rabbit immunoglobulins (A, G, and M) was obtained from Chappel Laboratories, Inc. (lot no. 7500). The fluorescein conjugate contained 1.7 mg of antibody protein per ml. All immune sera were absorbed three times with 109 cells of strain 602 per ml of serum and diluted 1:30 with phosphatebuffered saline before use. Cryptococcal antiserum was applied to slides containing the yeast suspension and incubated for 30 min at 37°C in a humidified atmosphere. The slides were washed for 5 min in each of three changes of phosphate-buffered saline, reincubated with fluorescein-labeled goat anti-rabbit immunoglobulins for 30 min at 37°C, and washed three times in phosphate-buffered saline. Yeast cells examined for surface polysaccharide were assayed with antiserum prepared against whole cells of C. neoformans 613 and antiserum prepared against cryptococcal polysaccharide complexed with methylated bovine serum albumin. Normal rabbit serum was used as a negative control. Results given are means of results from several anticryptococcal sera and are expressed as intensity of fluorescence on a scale of 0 to 4. A value of 4 corresponded to the intensity seen with fully encapsulated cryptococcal cells, and a value of 0 was given to cells exhibiting only background fluorescence. A very weak background fluorescence was observed with all yeast cells, regardless of serum source, species of yeast, or adsorption of serum with cryptococci. All slides were read in a blind fashion.
RESULTS polysaccharide to nonsoluble Binding of encapsulated cryptococci. The ability of cryptococcal polysaccharide to adhere to the surface of strain 602 was demonstrated by indirect immunofluorescence. Cells of strain 602 (5 x 107/ ml in saline) were incubated for 30 min at 37°C with an equal volume of strain 613 soluble polysaccharide (2 mg/ml), washed three times with saline, and examined for adherent polysaccharide by immunofluorescence. Indirect immuno-
VOL. 16, 1977
PHAGOCYTOSIS OF C. NEOFORMANS
fluorescence with rabbit cryptococcal antiseand fluorescein-labeled goat anti-rabbit immunoglobulins showed a bright, narrow rim of adherent polysaccharide on the surface of the yeast (Fig. 1B). The intensity of fluorescence was similar to that seen with fully encapsulated cryptococci (Fig. 1A), but the width of the adherent polysaccharide was substantially less than the capsule surrounding the encapsulated yeast. Significant fluorescence was not observed with untreated strain 602 cells or when normal rabbit serum was substituted for the anticryptococcal rabbit serum. Correlation of binding of polysaccharide to yeast surface with inhibition of phagocytosis. Several investigators have demonstrated that purified cryptococcal polysaccharide inhibits phagocytosis of small-capsule (5) or non-encapsulated (1, 11) isolates of C. neoformans. Bul-
and Sans (1) showed further that nonencapsulated cryptococci treated with cryptococcal polysaccharide can be washed with saline and remain partially resistant to phagocytosis. An experiment was done to confirm this observation and to determine what effect cryptococcal polysaccharide might have on the phagocytic activity of the macrophages themselves. Yeast cells (3.3 x 107/ml in HBSS) were incubated for 30 min at 37°C with an equal volume of cryptococcal polysaccharide (2 mg/ml in saline) and washed three times with HBSS. Macrophages monolayers were also incubated for 30 min at 37°C with cryptococcal polysaccharide at a final concentration of 1 mg/ml and washed three times with HBSS. Yeast cells and macrophages were then incubated together for 2 h at 37°C and examined to deternine the percent phagocytosis. In the absence of soluble polysaccharide, cells of strain 602 were engulfed readily by macrophages (Table 1). Treatment of macrophages with soluble polysaccharide had no effect on their ability to engulf the yeast. Examination of polysaccharide-treated macrophages by immunofluorescence techniques showed no observable polysaccharide adherent to the surface of the macrophages. Polysaccharide treatment of strain 602 significantly reduced the ability of macrophages to engulf the yeast; however, the percent phagocytosis was not inhibited to the extent seen with the fully encapsulated strain 613. The previous data strongly suggested that inhibition of phagocytosis was directly related to adherence of cryptococcal polysaccharide to the yeast. An experiment was done to provide a direct correlation between the presence of adherent polysaccharide as seen by immunofluorescence techniques and the ability of cryptococcal polysaccharide to inhibit phagocytosis. Cells of strain 602 were incubated with mono-
rum
101
mer
TABLE 1. Effects of cryptococcal polysaccharide treatment of macrophages and cryptococcal cells on phagocytosis of C. neoformans 602 Cryptococcal cells Strain 613 Strain 602 Strain 602
Polysaccharidetreated strain
FIG. 1. Indirect immunofluorescence of a medium capsule strain of C. neoformans (A) and non-encapsulated C. neoformans 602 preincubated with 2,000 Ag of polysaccharide and washed with saline (B). Indirect immunofluorescence was done with strain 602-adsorbed cryptococcal antiserum prepared in rabbits and fluorescein-labeled goat anti-rabbit immunoglobulins.
Macrophages Untreated Untreated
Polysaccharide treated" Untreated
Phagocytosis 3 + 1 84 + 6 86 ± 4 39 ± 9
602c
SD, Standard deviation. bMacrophage monolayer incubated with strain 613 polysaccharide (1 mg/ml) for 30 min at 37'C and washed three times with HBSS. c Strain 602 cells (3.3 x 107/ml) incubated with an equal volume strain 613 polysaccharide (2 mg/ml) for 30 min at 37'C and washed three times with HBSS. a
102
INFECT. IMMUN.
KOZEL
layers of macrophages for 2 h at 370C in the of varying concentrations of cryptococcal polysaccharide and examined to determine the percent phagocytosis. Identical preparations of strain 602 in cryptococcal polysaccharide were incubated for 2 h at 370C, washed two times with saline, and examined for adherent polysaccharide by immunofluorescence techniques (Fig. 2). Polysaccharide concentrations of 100 and 1,000 ,ug/ml inhibited phagocytosis to the extent seen with the fully encapsulated strain 613. These cells also demonstrated the presence of considerable amounts of adherent polysaccharide as seen by immunofluorescence. No difference was noted in the amount of detectable adherent polysaccharide after treatment with either the 100- or 1,000-,ug/ml polysaccharide concentration. At lesser concentrations of cryptococcal polysaccharide (10,. 1.0, and 0.1 ,g/ml), there was good correlation between the increase in percent phagocytosis and the decline in adherent polysaccharide as seen by immunofluorescence. Inhibition of phagocytosis by adherent polysaccharide could be due to binding of polysaccharide to the yeast per se or to an action by the adherent polysaccharide on various serum components necessary for phagocytosis. Gadebusch (6) reported that cryptococcal polysaccharide is a potent inactivator of the properdin system. Since the alternate complement pathway is an active component in the phagocytosis of cryptococci (4), it could be argued that the adherent polysaccharide seen in Fig. 1 was sufficient to inactivate any properdin present in the experimental system. To answer this question, the effects of cryptococcal polysaccharide on phagocytosis of yeasts other than cryptococci were studied. If cryptococcal polysaccharide inhibits phagocytosis by presence
depleting complement, it is likely that the polysaccharide would inhibit phagocytosis of microorganisms other than C. neoformans. The results (Table 2) showed that addition of cryptococcal polysaccharide (100 ,g/ml) to various yeast isolates did not inhibit engulfinent of C. albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., or S. cerevisiae. Examination of polysaccharide-treated yeast cells by immunofluorescence showed no detectable amounts of polysaccharide adherent to yeast cells other than C. neoformans. It could be argued that complement components were not necessary for phagocytosis of these yeast cells or that cryptococcal polysaccharide might alter or destroy some essential serum opsonin other than complement; consequently, the effects of a varying concentration of yeast upon the inhibitory properties of a constant amount of polysaccharide were studied. It was hypothesized that, if cryptococcal polysaccharide inactivates or inhibits some serum component necessary for phagocytosis, some inhibition of phagocytosis should be observed at all concentrations of yeast, much as with a noncompetitive enzyme inhibitor. If inhibition of phagocytosis is a direct result of binding to the yeast, little inhibition should be observed at very high concentrations of yeast due to dilution of polysaccharide on the surface of the yeast, but increasing degrees of inhibition should be observed if the polysaccharide concentration is held constant and the yeast concentration is decreased. TABLE 2. Specificity of cryptococcal polysaccharide for C. neoformans Untreated Yeast
to0 --
-
1-2+
2-3+
4+
4+
ph tosis (% ± SD-)
4-
IMMUNOFLUORESCENCE
80
60
Candida albicans C. pseudotropicalis Torulopsis sp. Rhodotorula sp. Saccharomyces cere-
Immu-
Phgc-nofluores-
cence
33 ± 10 49 ± 19 23 ± 13 9± 7 39 ± 11
0 0 0 0 0
Cryptococcus neofor- 65 ± 9
0
Polysaccharide
treated
Immu
Phagocy- noflu. tosisb nores ores± SD)
cence'
27 43 23 6.4 45
± 6 ± 14 + 11 ± 11 ± 7
0 0 0 0 0
viesae o a
40
\
CRYPTOCOCCAL POLYSACCHARIDE (ug/mi)
FIG. 2. Dose-response correlation of adherence of polysaccharide to strain 602 as shown by indirect immunofluorescence with inhibition of phagocytosis by cryptococcal polysaccharide.
4 + 2
4+
mans 602 a SD, Standard deviation. Percent phagocytosis determined after incubation of yeast cells (106/ml) with macrophages in the presence of crytococcal polysaccharide (100 ,ug/ml). c Adherence of cryptococcal polysaccharide determined by indirect immunofluorescence with rabbit antiserum to strain 613 and fluorescein-labeled anti-rabbit immunoglobulins. Strain 602 cells (5 x 107/ml) incubated with an equal volume strain 613 polysaccharide (2 mg/ml) for 30 min at 37°C and washed three times with HBSS before examination.
VOL. 16, 1977
PHAGOCYTOSIS OF C. NEOFORMANS
Uptake of C. neoformans 602 was determined in the absence of cryptococcal polysaccharide and in the presence of polysaccharide at a final concentration of 0.5, 0.7, and 1.0 ,g/ml (Table 3). Varying concentrations of yeast cells in HBSS containing 10% bovine calf serum and saline or polysaccharide were incubated with monolayers of macrophages for 30 min at 37°C. The macrophages were then rinsed, stained, and examined to determine the mean number of yeast cells phagocytized per macrophage (phagocytic index). Analysis of the data by analysis of variance showed that cryptococcal polysaccharide at a final concentration of 0.5 ,ug/ml produced no significant (P < 0.05) inhibition of phagocytosis at yeast-to-macrophage ratios of 16, 8, and 4; however, significant (P < 0.05) inhibition was seen when the number of yeasts was reduced to two yeast cells per macrophage. Addition of cryptococcal polysaccharide at a final concentration of 0.7 ,ug/ml produced no significant inhibition at a yeast-to-macrophage ratio of 16, but significant (P < 0.05) inhibition was observed at eight, four, and two yeasts per macrophage. Cryptococcal polysaccharide at a final concentration of 1.0 ug/ml inhibited phagocytosis at all concentrations of yeast tested. The data shown in Table 3 are also shown (Fig. 3) as a double-reciprocal plot of 1/phagocytic index versus 1/yeast added per macrophage. Data for a final polysaccharide concentration of 1 ,ug/ml and a yeast concentration of 32 yeast cells per macrophage are also plotted to show the behavior of this concentration of inhibitor at very high yeast concentrations. The data show a linear plot for phagocytosis in the absence of inhibitor. In the presence of 1 ,ug of polysaccharide, the curve was nonlinear and approached the uninhibited curve only at very high yeast concentrations. The nonlinearity of the inhibition curve was more apparent in the presence of 0.7 ,ug of polysaccharide, where a substantial inhibition of phagocytosis occurred at low yeast concentrations; however, as the
yeast concentration increased, the amount of polysaccharide bound per yeast cell decreased below an apparent threshold necessary for inhibition of phagocytosis, and significant inhibition of phagocytosis was not observed at these high yeast concentrations. Thus, under these experimental conditions, inhibition of phagocytosis was associated with binding of polysaccharide to the yeast rather than an indirect effect of the polysaccharide on serum factors necessary for phagocytosis. Specificity of C. neoformans 602 surface receptors for cryptococcal polysaccharide. Bulmer and Sans (1) demonstrated that various polysaccharides other than cryptococcal polysaccharide were unable to inhibit phagocytosis of C. neoformans; however, their studies did not investigate the ability of polysaccharides from various cryptococcal isolates or closely related bacterial polysaccharides to bind to C. neoformans. Consequently, an experiment was
103
40
3.0
0 z
0
a-0
.062
.125
.25
.50
1/(NO. YEAST ADDED PER MACROPHAGE) FIG. 3. Effects of a varying concentration of nonencapsulated cryptococci upon the phagocytosis-inhibiting properties of a constant amount of polysaccharide. Phagocytosis of C. neoformans 602 in the absence of polysaccharide (O) and in the presence of cryptococcal polysaccharide at final concentrations of 0.5 pg/ml (A), 0.7 pg/ml (0), and 1.0 pg/ml (0) is shown.
TABLE 3. Effects of a varying concentration of non-encapsulated cryptococci upon the phagocytosis-inhibiting properties of a constant amount of polysaccharide Phagocytic indexa Treatment (gg/ml)
16b
8
4
2
5.5 ± 1.2
3.1 ± 0.2
1.6 ± 0.2
1.1 ± 0.1
3.3 ± 0.3 1.8 ± 0.3 2.2 ± 0.5 1.2 + 0.3 1.5 ± 0.4 1.1 ± 0.2 1.0 a Mean number of phagocytized yeast per macrophage + standard deviation. b Yeast added per macrophage.
0.82 + 0.15 0.42 ± 0.16 0.25 ± 0.07
Saline Polysaccharide 0.5 0.7
5.7 + 1.2 5.4 + 1.4 2.3 ± 0.5
104
INFECT. IMMUN.
KOZEL
done to determine whether soluble polysaccharides obtained from the four cryptococcal serotypes and type III pneumococcal polysaccharide were able to bind to strain 602 and inhibit phagocytosis by macrophages. Cells of strain 602 and each polysaccharide at a final concentration of 100 ug/ml were added to monolayers of macrophages to determine their susceptibility to phagocytosis. Cells of strain 602 (5 x 107/ ml) were also incubated for 30 min at 370C with an equal volume of polysaccharide (2 mg/ml) from the various cryptococcal serotypes, washed three times with saline, and examined for adherent polysaccharide by immunofluorescence techniques with antiserum prepared against strain 613 soluble polysaccharide. The results (Table 4) show no observable difference between the ability of polysaccharides from each cryptococcal serotype to inhibit phagocytosis of strain 602. Type III pneumococcal polysaccharide was unable to inhibit phagocytosis of the yeast. Similarly, immunofluorescence demonstrated that each of the cryptococcal polysaccharides was able to bind to the surface of the yeast. Differences in degree of observed fluorescence were probably due to the use of antiserum prepared against a cryptococcal isolate of serotype D.
DISCUSSION Several investigators (1, 5, 10) have demonstrated that addition of purified cryptococcal polysaccharide to small-capsule or non-encapTABLE 4. Specificity of the polysaccharide receptor on C. neoformans 602 for cryptococcal polysaccharide PhagocytoPolysaccharide
sisa (% ± SD) 90 5
Immunoflu-
orescenceb
0 None Strain 613 4 1 4+ Type A 4 1 3+ Type B 2 1 3+ Type C 3 1 3+ Type D 6 3 4+ Type III pneumococcal 90 ± 2 ND a Percent phagacytosis determined after incubation of cryptococci (106/ml) with macrophages in the presence of cryptococcal polysaccharide (100 ,ug/ml). SD, Standard deviation. b Adherence of cryptococcal polysaccharide determined by indirect immunofluorescence with rabbit antiserum to strain 613 and fluorescein-labeled antirabbit immunoglobulins. Strain 602 cells (5 x 107/ ml) were incubated with an equal volume strain 613 polysaccharide (2 mg/ml) for 30 min at 370C and washed three times with HBSS before examination. ND, Not determined.
sulated isolates of C. neoformans renders the yeast resistant to phagocytosis. Evidence that this inhibition of phagocytosis might be due to adherence of polysaccharide to the cryptococcal cell was first presented by Bulmer and Sans (1). They showed that cryptococcal cells grown in a non-encapsulated state could be preincubated with cryptococcal polysaccharide and washed, and still be partially resistant to phagocytosis. Further evidence for a cryptococcal surface receptor for soluble polysaccharide was presented by Tacker et al. (15), who reported that capsular material would not inhibit phagocytosis of killed, non-encapsulated cryptococci. This suggested that either the non-encapsulated cells must be viable for the added polysaccharide to inhibit phagocytosis, or the treatment used to kill the cells may have altered sites required for attachment of soluble polysaccharide. The present studies confirm earlier observations that preincubation of cryptococcal cells with polysaccharide render them resistant to phagocytosis, even after a wash with saline. Since this increased resistance to phagocytosis could be explained by either an alteration in the yeast surface by polysaccharide or adherence of polysaccharide to the cell surface, a direct confirmation of the binding of cryptococcal polysaccharide to the non-encapsulated yeast was obtained by immunofluorescence techniques. Non-encapsulated cryptococci that were treated with cryptococcal polysaccharide showed a rim of capsular material that was narrower but equal in intensity to the capsule seen on a virulent, encapsulated strain. A correlation between inhibition of phagocytosis and the binding of cryptococcal polysaccharide to non-encapsulated cells as seen by immunofluorescence was obtained by comparing the concentrations of cryptococcal polysaccharide needed to inhibit phagocytosis with the concentration needed to produce an effect visible by immunofluorescence. As shown in Fig. 2, the concentrations necessary to achieve both effects were similar. No difference was noted between cells treated with polysaccharide concentrations of either 100 or 1,000 ,ug/ml with regard to resistance of the cells to phagocytosis or to the approximate amount of polysaccharide bound to the cell surface. Thus, it appears that the amount of capsular material present influences phagocytosis up to a minimal threshold, but additional polysaccharide has no further effect on resistance to phagocytosis. This may provide an explanation for the observation that there is no clear relationship between in vitro capsule size and virulence (8, 9, 13), since in the present study even a minimal amount of polysaccharide produced inhibition of phagocytosis
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PHAGOCYTOSIS OF C. NEOFORMANS
comparable to that observed with fully encapsulated cryptococci. At least two mechanisms may be proposed to explain inhibition of phagocytosis by cryptococcal polysaccharide. The capsular material may, by its physical presence around the yeast, prevent an effective interaction between the cryptococcus and the phagocytic cell, or the capsular material may deplete the incubation medium of serum components necessary for phagocytosis. The latter possibility must be considered because cryptococcal polysaccharide has been shown to inactivate properdin (6) and to consume the late components of complement (4). The failure of cryptococcal polysaccharide to inhibit phagocytosis of cells of several yeast genera other than Cryptococcus suggests that cryptococcal polysaccharide does not inhibit or inactivate some essential serum opsonin. Since it could be argued that complement components were not essential for phagocytosis of these yeasts or that cryptococcal polysaccharide affects some serum opsonin specific for C. neoformans, a kinetic analysis of phagocytosis of strain 602 was done to distinguish between the two possibilities. When the effects of a varying concentration of yeast upon the inhibitory properties of a constant amount of polysaccharide were studied, cryptococcal polysaccharide was found to inhibit phagocytosis at low yeast concentrations, but the inhibition decreased progressively as the yeast concentration increased. If cryptococcal polysaccharide inactivated or inhibited some serum component necessary for phagocytosis, some inhibition of phagocytosis should have been observed at all concentrations of yeast. The experimental data suggest that a critical amount of cryptococcal polysaccharide must be bound to the yeast cell for inhibition of phagocytosis. At cell surface concentrations below this threshold, there is no inhibition of phagocytosis. These data also suggest that, under these experimental conditions, inhibition of phagocytosis is an intrinsic result of presence of the capsular material on the yeast rather than an indirect effect on macrophages or essential serum factors necessary for phagocytosis. The role of complement consumption in the antiphagocytic properties of cryptococcal polysaccharide cannot be totally excluded, however, because the ability to consume complement has been reported only with concentrations of polysaccharide considerably higher than concentrations shown in Fig. 3 (4). Thus, it is possible that some additional inhibitory property of cryptococcal polysaccharide might be expressed at very high polysaccharide concentrations. Adherence of cryptococcal polysaccharide to
nomenon mediated by receptors specific for cryptococcal polysaccharide rather than nonspecific adsorption to a yeast cell wall. Type III pneumococcal polysaccharide (Table 4) and a variety of plant polysaccharides are unable to inhibit phagocytosis of cryptococcus (1). Furthermore, cryptococcal polysaccharide does not adhere to a variety of yeasts other than C. neoformans (Table 2). Previous studies (1, 5) have shown that cryptococcal polysaccharide does not inhibit phagocytosis of two yeast genera other than Cryptococcus and two genera of bacteria; however, those reports did not indicate whether cryptococcal polysaccharide was able to bind to those organisms. Tacker et al. (15) studied phagocytosis of cryptococcal cells grown in a non-encapsulated state on low-pH medium. They reported that cryptococcal polysaccharide did not inhibit phagocytosis of killed, non-encapsulated cells regardless of the method used to kill the yeast. The authors suggest that either the non-encapsulated cells must be viable for attachment to occur, or the treatment used on the cells altered the receptors responsible for attachment of the antiphagocytic polysaccharide. The resistance of the receptors on strain 602 to formaldehyde, as reported in the present study, as well as the resistance of these receptors to a variety of physical and chemical agents including methanol, autoclaving, and mild acid and alkali treatment (T. R. Kozel, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, F13, p. 89), suggests an alternate explanation. It is possible that growth of C. neoformans at low pH suppresses formation of the polysaccharide receptor, and the receptor is synthesized upon release of the organism from the low pH. Thus, killing of the cells immediately after harvesting from the low-pH medium would yield cells to which cryptococcal polysaccharide would not adhere.
non-encapsulated cryptococci is a specific phe-
105
ACKNOWLEDGMENTS This research was supported by grants from the Luke B. Hancock Foundation and the University of Nevada Research Advisory Board. LITERATURE CITED 1. Bulmer, G. S., and M. D. Sans. 1968. Cryptococcus neoformans. III. Inhibition of phagocytosis. J. Bacteriol. 95:5-8. 2. Bulmer, G. S., M. D. Sans, and C. M. Gunn. 1967. Cryptococcus neoformans. I. Nonencapsulated mutants. J. Bacteriol. 94:1475-1479. 3. Bulmer, G. S., and J. R. Tacker. 1975. Phagocytosis of Cryptococcus neoformans by alveolar macrophages. Infect. Immun. 11:73-79. 4. Diamond, R. D., J. E. May, M. A. Kane, M. M. Frank, and J. E. Bennett. 1974. The role of the classical and alternate complement pathways in host defenses against Cryptococcus neoformans infection. J. Immunol. 112:2260-2270. 5. Diamond, R. D., R. K. Root, and J. E. Bennett. 1972.
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6.
7.
8. 9.
10.
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Factors influencing killing of Cryptococcus neoformans by human leukocytes in vitro. J. Infect. Dis. 125:367-376. Gadebusch, H. H. 1961. Natural host resistance to infection with Cryptococcus neoformans. I. The effect of the properdin system on the experimental disease. J. Infect. Dis. 109:148-153. Goren, M. B., and J. Warren. 1968. Immunofluorescence studies of reactions at the cryptococcal capsule. J. Infect. Dis. 118:215-229. Hasenclever, H. F., and W. 0. Mitchell. 1960. Virulence and growth rates ofCryptococcus neoformans in mice. Ann. N.Y. Acad. Sci. 89:156-162. Kao, C. J., and J. Schwartz. 1957. The isolation of Cryptococcus neoformans from pigeon nests. Am. J. Clin. Pathol. 27:652-663. 0 Kozel, T. R., and J. Cazin, Jr. 1971. Nonencapsulated variant of Cryptococcus neoformans. I. Virulence studies and characterization of soluble polysaccha-
INFECT. IMMUN. ride. Infect. Immun. 3:287-294. 11. Kozel, T. R., and J. Cazin, Jr. 1974. Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans. Mycopathol. Mycol. Appl. 54:21-30. 12. Kozel, T. R., and R. P. Mastroianni. 1976. Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. Infect. Immun. 14:62-67. 13. Littman, M. L., and E. Tsubura. 1959. Effect of degree of encapsulation upon virulence of Cryptococcus neoformans. Proc. Soc. Exp. Biol. Med. 101:773-777. 14. Mitchell, T. G., and L. Friedman. 1972. In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans. Infect. Immun. 5:491-498. 15. Tacker, J. R., F. Farhi, and G. S. Bulmer. 1972. Intracellular fate of Cryptococcus neoformans. Infect. Immun. 6:162-167.
