Molecular Pathogenesis

Antigenic and Biological Characteristics of Mutant Strains of Cryptococcus neoformans Lacking Capsular O Acetylation or Xylosyl Side Chains

Thomas R. Kozel, Stuart M. Levitz, Françoise Dromer, Marcellene A. Gates, Peter Thorkildson, Guilhem Janbon
DOI: 10.1128/IAI.71.5.2868-2875.2003

ABSTRACT

Cryptococcus neoformans is surrounded by an antiphagocytic polysaccharide capsule whose primary constituent is glucuronoxylomannan (GXM). Three prominent structural features of GXM are single xylosyl and glucuronosyl side chains and O acetylation of the mannose backbone. Isogenic pairs of O-acetyl-positive and O-acetyl-negative strains (cas1Δ) as well as xylose-positive and xylose-negative strains (uxs1Δ) of serotype D have been reported. The cas1Δ strains were hypervirulent, and the uxs1Δ strains were avirulent. The goal of this study was to examine the effects of the cas1Δ and uxs1Δ mutations on the following: (i) binding of anti-GXM monoclonal antibodies (MAbs) in capsular quellung reactions, (ii) activation of the complement system and binding of C3, (iii) phagocytosis by neutrophils, and (iv) clearance of GXM in vivo. The results showed that loss of O acetylation produced dramatic changes in the reactivities of five of seven anti-GXM MAbs. In contrast, loss of xylosylation produced a substantive alteration in the binding behavior of only one MAb. O-acetyl-negative strains showed no alteration in activation and binding of C3 from normal serum. Xylose-negative strains exhibited accelerated kinetics for C3 deposition. Loss of O acetylation or xylosylation had no effect on phagocytosis of serum-opsonized yeast cells by human neutrophils. Finally, loss of O acetylation or xylosylation altered the kinetics for clearance of GXM from serum and accumulation of GXM in the liver and spleen. These results show that O acetylation and/or xylosylation are important for binding of anti-GXM MAbs, for complement activation, and for tissue accumulation of GXM but do not impact phagocytosis by neutrophils.

Cryptococcus neoformans is an opportunistic yeast that may produce a life-threatening form of meningitis, particularly in patients with deficiencies in cellular immunity, such as AIDS. C. neoformans is surrounded by an antiphagocytic capsule that is essential for virulence (6, 16, 32). The primary constituent of the cryptococcal capsule is glucuronoxylomannan (GXM), a polysaccharide with a linear (1→3)-α-d-mannopyranan backbone with substitutions of single β-d-xylopyranosyl and β-d-glucopyranosyl-uronic acid residues (4, 9). The mannose backbone is also variably O acetylated at C-6 (24, 47). GXM occurs in five major serotypes, A, B, C, D and A/D, and eight chemotypes (4, 9, 50).

Cryptococcal polysaccharide and the cryptococcal capsule have numerous biological activities that may contribute to the virulence of the yeast, such as inhibition of phagocytosis (5, 28), induction of immune unresponsiveness (26, 36), binding to phagocyte surface receptors, such as Toll 2, Toll 4, CD14, and CR3 (13, 44, 46), induction of shedding of l-selectin from neutrophils (12), potent activation of the complement system via the alternative pathway (30, 31), contribution to cerebral edema and increased intracranial pressure (11, 20, 21), alterations in cytokine secretion by leukocytes (10, 41, 48), and enhanced infectivity of human immunodeficiency virus (38, 39). The biological and immunological activities of cryptococcal polysaccharide and the cryptococcal capsule are likely due individually or in combination to the following: (i) the large molecular size of the polysaccharide, (ii) the repeating nature of individual units within the polysaccharide, or (iii) the presence or absence of specific substituents in the polysaccharide. For example, studies of complement activation by the cryptococcal capsule found that the capacity of the capsule for accumulation of C3 fragments is increased by de-O acetylation of the cells (51), and the efficiency with which the capsule can act as an acceptor for metastable C3 is related to the extent of xylose substitution (42). In contrast, neither O acetylation nor carboxylation of glucuronic acid is required for inhibition of phagocytosis, since cells that were chemically de-O acetylated or carboxyl reduced showed a resistance to phagocytosis that was identical to that of unmodified yeast cells (25). In another example, the degrees of xylose substitution and O acetylation are major antigenic determinants that distinguish the structures of each serotype (9). Moreover, the O-acetyl substituent is a critical epitope for recognition of GXM by polyclonal antibodies (8, 25) and many monoclonal (2, 14) antibodies (MAbs).

To date, structure-function studies of GXM have been dependent on differences in structure that can be attributed to GXM of different serotypes, spontaneously occurring variants, and chemical modification of GXM. Two recent reports of capsule structure mutant strains now provide an alternative approach to structure-function studies (24, 35). The first gene studied, named CAS1, encodes a putative glycosyl transferase. Analysis of capsule structure after CAS1 deletion shows that the gene is required for GXM O acetylation. As a consequence, cas1Δ strains are O-acetyl negative (24). The second gene, termed UXS1, encodes UDP-xylose synthase, which catalyzes the transformation of UDP-glucuronic acid into UDP-xylose, which is required for xylosylation of GXM (1). As a consequence, uxs1Δ strains are xylose negative (35). Analysis of the virulence of the capsule mutant strains found that cas1Δ strains are more virulent than the original strains, whereas the uxs1Δ strains are avirulent (24, 35). The availability of isogenic strains of C. neoformans that are (i) CAS1 or cas1Δ or (ii) UXS1 or uxs1Δ allows for an unambiguous assignment of biological or antigenic activity to the function of each gene. The goal of the present study was to assess selected antigenic and biological activities of soluble GXM or encapsulated cells that were attributable to CAS1 or UXS1.

MATERIALS AND METHODS

Yeast strains and GXM.The characteristics of yeast strains used in this study are summarized in Table 1. The isogenic sets of strains were obtained by backcrossing the initial disrupted strain with the original strain one and three times for UXS1 and CAS1, respectively (24, 35). The cas1Δ strains were derived from strain JEC156 (MATaura5 ade2), and the uxs1Δ strains were derived from strain JEC155 (MATα ura5 ade2). The original strains belonged to serotype D. The final progenies were screened for the presence of the disruption cassette, and their mating types were determined. Two MATa and two MATα strains were recovered: two containing the disruption cassette and two containing the wild-type gene. Analysis of GXM from the original (UXS1 and CAS1) and mutant (uxs1Δ and cas1Δ) strains by nuclear magnetic resonance determined the structural modification produced by each mutation (24, 35). Yeast cells used to assess antibody-dependent capsule reactions and C3 binding were grown in the presence of bicarbonate-CO2 for production of large capsules and were killed by treatment with formaldehyde (18, 37). Use of formaldehyde to kill C. neoformans has no discernible effect on either the reactivity of GXM with antibody or the antiphagocytic action of the cryptococcal capsule (T. R. Kozel, unpublished observations). Yeast cells used to assess phagocytosis by polymorphonuclear neutrophils (PMN) were grown as described previously (34). GXM was purified from culture supernatant fluids by differential precipitation with hexadecyltrimethylammonium bromide (7, 24).

View this table:
TABLE 1.

C. neoformans strains used in this study

MAbs and immunoglobulin fragments.The characteristics of MAbs used in this study are summarized in Table 2. All MAbs except 471 were produced by in vitro culturing in a Tecnomouse system (Integra Biosciences, Ijamsville, Md.) and purified by affinity chromatography on protein A. MAb 471 was purified from mouse ascites by differential precipitation with caprylic acid and ammonium sulfate followed by immunoaffinity chromatography with a GXM-Sepharose column (27) and affinity chromatography with protein A. Concentrations of MAbs were determined by UV spectroscopy using an optical density at 280 nm of 1.43 for 1 mg of immunoglobulin G/ml (43).

View this table:
TABLE 2.

MAbs used for this study

DIC microscopy.MAb-induced capsular reactions were done as previously described (33). Capsular reactions were assessed by differential interference contrast (DIC) optics. Capsule reactions were classified as rim or puffy (33). The rim pattern is characterized by a sharp increase in the optical gradient at the capsular edge, followed directly by a decrease in the optical gradient. The hallmark of the puffy pattern is an increase in the optical gradient at the capsular surface and the absence of the immediate decrease that is characteristic of the rim pattern.

Activation and binding of C3 to encapsulated cryptococci.Formalin-killed cryptococci were incubated at a concentration of 4 × 105 cells/ml at 37°C in a medium that consisted of 40% pooled human serum (PHS) in phosphate-buffered saline (PBS). Samples were taken at various time intervals, and EDTA was added to a final concentration of 10 mM to stop the complement cascade. The yeast cells were washed three times with PBS, incubated for 1 h at 4°C with a 1/50 dilution of fluorescein isothiocyanate (FITC)-conjugated antiserum to human C3 (Kent Laboratories, Bellingham, Wash.), washed three times with PBS, resuspended in 25 μl of Vectashield (Vector Laboratories, Inc., Burlingame, Calif.), and mounted under a coverslip. Binding of C3 to the yeast cells was qualitatively assessed with a Nikon Eclipse 800 microscope equipped for epifluorescence. Images were captured using both DIC and epifluorescence microscopy. Fluorescence images were collected in a FITC channel at 0.4-μm intervals through the z axis with a Spot RT Color charge-coupled device camera (Diagnostic Instruments, Inc., Sterling, Mich.) that was run with SimplePCI software (Compix Inc., Cranberry Township, Pa.). Fluorescence images were deconvolved and projected onto a single plane and overlaid with greyscale DIC images using SimplePCI software.

A quantitative assessment of the rate of formation of focal sites of C3 deposition was determined by examination by immunofluorescence microscopy of cells prepared as described above. The number of focal sites of C3 deposition on each cell was determined by visual inspection. Twenty yeast cells were examined for each time point, and the mean number of focal sites of C3 binding was calculated.

Binding of C. neoformans by neutrophils.Blood was obtained by venipuncture from healthy volunteers, and the polymorphonuclear leukocytes (PMN) were isolated as in previous studies (34). Briefly, the blood was heparinized and then subjected to sequential dextran sedimentation, centrifugation over a Ficoll-Hypaque gradient, and hypotonic lysis. PMN were suspended in RPMI 1640 containing 10% heat-inactivated fetal bovine serum at 106 cells/ml, and 100 μl was added per well of 96-well flat-bottom tissue culture plates. Plates were incubated for 1 h at 37°C prior to challenge with C. neoformans, as described below.

The indicated strains of live C. neoformans were suspended in PBS at a concentration of 2 × 107 yeast cells/ml. The organisms were opsonized in 1.5-ml microcentrifuge tubes by incubating 300 μl of the C. neoformans suspension with 200 μl of PHS for 0 (PHS and EDTA were added concomitantly), 4, 8, 16, or 32 min at 37°C. The reaction was stopped by the addition of 5 μl of 1.0 M EDTA. Yeast cells were washed twice and resuspended in 250 μl of RPMI 1640, and 100 μl was added to wells containing the PMN. C. neoformans and PMN were incubated for 1 h at 37°C; unbound yeast cells were gently washed away; and the cells were fixed in 1% PBS-buffered formaldehyde. The binding index, defined as the average number of C. neoformans cells per 100 PMN, was determined by examining at least 100 PMN per well under an inverted microscope for the presence of cell-associated C. neoformans.

Clearance and distribution of intravenously injected GXM.The use of animals in this study was approved by the Boston University School of Medicine Institutional Animal Care and Use Committee and was compliant with relevant federal guidelines. Experiments to measure in vivo clearance and distribution of GXM were performed as in previous studies (19). Briefly, 250 μg of GXM dissolved in 200 μl of PBS was injected into the lateral tail vein of C57BL/6 mice. Mice were sacrificed at 1, 3, and 7 days after injection. Blood was collected by cardiac puncture and allowed to clot, and the serum was collected following centrifugation. The liver and spleen were harvested and homogenized using a tissue grinder. GXM concentrations were measured by enzyme-linked immunosorbent assay as described previously (19) using unlabeled anti-GXM MAb for the coating antibody and horseradish peroxidase-labeled antibody as the indicator antibody. The anti-GXM MAbs utilized were MAb F12D2 for GXM derived from strains JEC43 and NE30 and MAb 339 for GXM derived from strain NE175. These MAbs were used in each situation because they showed identical reactivities with GXM from the parent and mutant strains. The enzyme-linked immunosorbent assays could reliably detect ≤10 ng of GXM/ml from the samples. The GXM content of the liver and spleen was calculated by subtracting the GXM present in the serum of each organ from the total GXM detected in each organ. The serum content of the organs was estimated based on published data (15).

Statistical analysis.All statistical analyses were done by analysis of variance followed by pairwise comparisons with the Tukey test with the assistance of SigmaStat version 2.03 (SPSS Inc., Chicago, Ill.).

RESULTS

Capsule reactions with anti-GXM MAbs.The reactivity of O-acetyl and xylose mutants of serotype D C. neoformans with a panel of anti-GXM MAbs was assessed by use of capsular quellung reactions. Two isogenic pairs were examined for each class of mutants. The results showed that loss of O-acetyl production (CAS1 disruption) dramatically impacted both the ability of antibody to produce a capsule reaction and the type of capsule reaction that was observed (Fig. 1). Some antibodies (MAbs 339, 302, and 1326) lost the ability to produce a visible capsule reaction; one antibody (MAb 1255) that produced a rim pattern with the O-acetyl-positive strains produced a puffy pattern with the mutant strains; one antibody (MAb F12D2) that produced a puffy pattern with the O-acetyl-positive strains produced a rim pattern with the mutant strains; and two antibodies (MAbs 3C2 and 471) produced puffy patterns with both the O-acetyl-positive and mutant strains.

FIG. 1.
FIG. 1.

Capsule reactions produced by CAS1 (NE167 and NE169 [O-acetyl positive]) and cas1Δ (NE168 and NE170 [O-acetyl negative]) strains. Yeast cells were mixed with each MAb (50 μg/ml), and reactions were assessed by DIC microscopy.

In most cases, loss of xylose substitution (UXS1 disruption) had little effect on reactivity of MAbs in the capsule reaction (Fig. 2). A slight decrease in the intensity of the capsule reaction was noted for MAb 3C2, and MAb F12D2 that was generated by immunization of mice with de-O-acetylated serotype A GXM showed no apparent reactivity with the xylose-deficient UXS1 mutants NE176 and NE178. The capsular patterns produced by all other MAbs were similar in the xylose-positive and mutant strains.

FIG. 2.
FIG. 2.

Capsule reactions produced by UXS1 (NE177 and NE179 [xylose-positive]) and uxs1Δ (NE176 and NE178 [xylose-negative]) strains. Yeast cells were mixed with each MAb (50 μg/ml), and reactions were assessed by DIC microscopy.

Activation and deposition of C3 fragments.Previous studies found that chemical de-O acetylation of C. neoformans cells had no effect on the kinetics for accumulation of C3 on encapsulated cryptococci of all four serotypes (51). In contrast, xylose is a particularly efficient site for binding of metastable C3b, and the capture efficiency for metastable C3b by encapsulated cryptococci was directly related to xylose content (42). As a consequence, we evaluated the effects of the CAS1 and UXS1 gene disruptions on the ability of each strain to activate and bind C3. Yeast cells were incubated for various times in 40% PHS, the reaction was stopped by addition of EDTA, and C3 binding was assessed by immunofluorescence. The results (Fig. 3) showed that the xylose-positive (UXS1) strain activated and bound C3 fragments in an asynchronous and focal manner, a result that is consistent with previous studies of serotype A cryptococci (30). At short incubation times, bound C3 appeared as minute sites of focal binding. With increased incubation, the apparent early sites of C3 binding had increased in size, and new small sites had formed, indicating an asynchronous nuclear appearance and expansion of C3 binding. The xylose-negative (uxs1Δ) strain also accumulated C3 in an asynchronous focal manner, but formation of initiation sites appeared to be much more rapid than was observed for the xylose-positive (UXS1) strain.

FIG. 3.
FIG. 3.

Patterns of C3 deposition on UXS1 (NE177) and uxs1Δ (NE176) strains. Yeast cells were incubated with PHS for 2, 4, 8, 16, or 32 min. The reaction was stopped by addition of EDTA, and the sites of C3 deposition were determined by use of FITC-labeled antibody specific for human C3.

A quantitative assessment of the rate of formation of C3 initiation foci by the xylose-positive and xylose-negative strains was done by counting, via immunofluorescence microscopy, the number of focal sites on at least 20 cells at 1-min intervals from 1 to 8 min of incubation. C3 binding could not be determined at time intervals of greater than 8 min because the foci were beginning to coalesce. The results showed significantly more C3 binding foci on the xylose-negative (uxs1Δ) strain than on the xylose-positive (UXS1) strain at 3 to 8 min of incubation (Fig. 4).

FIG. 4.
FIG. 4.

Kinetics for formation of C3 foci in the C. neoformans capsule. Yeast cells were incubated in 40% PHS. Samples were taken at various time intervals; the reaction was stopped by addition of EDTA; sites of C3 binding were identified by use of FITC-labeled antibodies specific for human C3; and the numbers of focal sites of C3 binding were determined by fluorescence microscopy. Results are given as the mean ± standard error for 20 cells that were examined at each time point.

Activation and binding of C3 fragments by the O-acetyl-positive (CAS1) and O-acetyl-negative (cas1Δ) strains were examined in a similar manner. The results showed no differences between the two strains (data not shown).

Interaction with PMN.The xylose-negative (uxs1Δ) strain showed accelerated deposition of C3 into the capsular matrix (Fig. 4), suggesting that PHS-opsonized mutant strains might differ from xylose-positive strains in their interaction with PMN. To assess this possibility, the effects of preopsonization by incubation with 40% PHS for 0, 4, 8, 16, or 32 min on the binding of cells of the CAS1 (NE167), cas1Δ (NE168), UXS1 (NE177), and uxs1Δ (NE176) strains to PMN were determined. The results (Fig. 5) showed no significant (P > 0.05) differences between the CAS1 and cas1Δ strains or the UXS1 and uxs1Δ strains at any time of preopsonization.

FIG. 5.
FIG. 5.

Binding of PHS-opsonized CAS1 (NE167) and cas1Δ (NE168) strains (upper panel) and UXS1 (NE177) and uxs1Δ (NE176) strains (lower panel) to human PMN. Encapsulated cryptococci were preopsonized by incubation with 40% PHS; samples were taken at the indicated times; C3 deposition was stopped by addition of EDTA; the yeast cells were washed; the opsonized yeast cells were incubated with PMN for 1 h; and a binding index (number of attached or ingested yeast cells per 100 PMN) was determined. Results are given as the mean ± standard error for results from four different PMN donors, with each data point from each donor done in duplicate.

Clearance of GXM in vivo.A comparison was made between the in vivo clearance of GXM isolated from the original (JEC43 [O-acetyl positive, xylose positive]), cas1Δ (NE30 [O-acetyl negative]), and uxs1Δ (NE175 [xylose negative]) strains. Mice were injected intravenously with 250 μg of each polysaccharide, and polysaccharide concentrations in serum, the liver, and the spleen were determined 1, 3, and 7 days after treatment. The results (Fig. 6) showed significantly (P < 0.05) lower levels of GXM from the cas1Δ strain in serum at day 1 and significantly (P < 0.001) lower levels of the same polysaccharide than of GXM from the remaining two strains in the liver on days 1, 3, and 7. A different pattern of clearance was observed for the spleen, where GXM from strain NE175 was present in higher levels (P < 0.001) in the spleen on days 1 and 3 than was GXM from strains JEC43 and NE30.

FIG. 6.
FIG. 6.

In vivo clearance of GXM from wild-type (JEC43), O-acetyl negative (NE30 [cas1Δ]), and xylose-negative (NE175 [uxs1Δ]) strains. Mice were injected intravenously with 250 μg of each polysaccharide, and tissue levels were determined at the indicated time intervals. Data are reported as the mean ± standard error from results from three mice. ∗, P < 0.05 (versus results for JEC43 and NE175 at day 1); ∗∗, P < 0.001 (versus results for JEC43 and NE175 at days 1 and 3) and P < 0.001 at day 7. ∗∗∗, P < 0.001 (versus results for JEC43 and NE30 at days 1 and 3).

DISCUSSION

The production of capsule structure mutant strains of C. neoformans has enabled an evaluation of the contribution of capsule synthesis genes to GXM structure and pathogenesis. Deletion of the CAS1 gene that encodes a putative glycosyl transferase needed for O acetylation of GXM produces a mutant strain that is more virulent than the original strain (24). Deletion of the UXS1 gene that encodes UDP-xylose synthase produces a mutant strain that lacks xylosylation and is avirulent in mice (35). The goal of our study was to examine antigenic and biological consequences with the cas1Δ and uxs1Δ deletion strains in an effort to better understand the roles of the two genes in virulence and the contributions of the two genes to the biological activities of GXM.

O acetylation is important for recognition of GXM by polyclonal antibodies and many MAbs (2, 8, 14, 25). We used the capsule reaction as a robust measure of the reactivities of a battery of anti-GXM MAbs with CAS1 and cas1Δ strains. Production of a rim pattern is a consequence of cross-linking of the capsular surface by antibody; the puffy pattern is a product of antibody binding but does not involve or require cross-linking (33). As a consequence, assessment of the capsule reaction provided qualitative information about the manner of interaction between antibody and the cryptococcal capsule that is not available by use of other immunochemical assays. The results showed that the capsule reaction is dramatically impacted by O acetylation of serotype D GXM. In some cases, a conversion of the capsule reaction was noted, e.g., from rim to puffy or vice versa. This result indicates that depending on the epitope specificity of the antibody, O acetylation is influencing the ability of an antibody to cross-link the capsular surface without blocking antibody binding itself. To date, the structural basis for why one antibody or a given GXM structural motif allows for cross-linking by antibody to produce the rim pattern is not known. However, given the close correlation between protection and production of the rim pattern (33), the CAS1-cas1Δ isogenic set is a powerful means to assess the association between capsule reaction and protection.

Xylosylation of serotype D GXM was a less important determinant of the binding of MAbs used in the present study. The capsule reaction was diminished in intensity in the case of the MAb 3C2, and MAb F12D2 lost reactivity. However, in no instance was there a conversion of the capsule reaction as was observed for cas1Δ strains. Despite the limited impact of xylosylation on the capsule reaction, it is clear that xylosylation is an important epitope for some antibodies because a lack of reactivity with several MAbs was the basis for the initial identification of the UXS1 mutation, and uxs1Δ cells are not reactive with polyclonal factor 8 antisera (35).

The contributions of O acetylation and xylosylation of GXM to complement activation by C. neoformans have been examined previously. Studies of the specificity of the thioester-containing reactive site of human C3 found that xylose is a potent acceptor for metastable C3b (42). A comparison of the complement-activating properties of cryptococci of different serotypes found a strong correlation between the xylose content of polysaccharides of different serotypes and C3b attachment efficiency: serotype C, serotype B, serotype A, and serotype D (listed in order from greatest to least attachment efficiency) (42). Notably, this order for C3b attachment efficiency directly parallels the relative xylose content of GXM in polysaccharides of the four serotypes. Our studies of complement activation by xylose-positive and xylose-negative strains of serotype D found quite a different result. Formation of focal sites of C3 binding occurred much faster with xylose-negative cells than with xylose-positive cells.

Efficient accumulation of C3 on xylose-negative cells indicates that residues other than xylose are able to function as acceptors for binding of C3 to encapsulated cyptococci. Moreover, these results indicate that factors other than efficiency in binding of C3b are the dominant force in determining kinetics of complement activation when xylose-positive and xylose-negative cells are compared. One explanation is a differential affinity of factor B or factor H for particle-bound C3b. Particles that favor binding of factor B and discourage binding of factor H are activators (22). For example, a sixfold- to eightfold-increased binding of factor H to C3b on the surface of M protein-carrying strains of Streptococcus pyogenes compared to M protein-negative strains likely accounts in part for the antiphagocytic action of streptococcal M protein (23). It is quite possible that xylose residues of GXM increase the efficiency with which factor H can function as a cofactor for factor I within the environment of the cryptococcal capsule. This is an attractive hypothesis because previous studies have shown that C3b bound to the cryptococcal capsule is rapidly converted to iC3b (29, 40). Finally, a caveat should be noted in considering the potential role of xylose substitution in regulation of complement activation by the cryptococcal capsule; the UXS1 deletion could also affect protein glycosylation as well as the structure of galactoxylomannan that is produced by C. neoformans.

In a previous study, we used chemical modification of the GXM of encapsulated cryptococci to demonstrate that both the O-acetyl group and carboxylation of glucuronic acid were expendable for inhibition of phagocytosis by the cryptococcal capsule (25). To date, a chemical means for removal of the xylose side chain has not been available for evaluation of the role of xylosylation in inhibition of phagocytosis. Results from the present study confirm the previous report that O acetylation is not necessary for inhibition of phagocytosis and expand the list of expendable substituents to include xylose substitution. One interpretation for such negative results is the possibility that the overall size of the cryptococcal capsule is the major determinant of inhibition of phagocytosis by PMN, with individual components of the polysaccharide having little or no effect.

Similar levels of phagocytosis by PMN were noted for xylose-positive and xylose-negative strains despite the notable difference in rates of C3 deposition on the two strains (Fig. 3). The experimental design for opsonization of cryptococci included short incubation times with PHS that would likely determine whether differences in rates of formation of C3-binding foci were sufficient to impact phagocytosis. Clearly, opsonization time was an important variable (Fig. 5); however, differences in rates of complement activation between xylose-positive and xylose-negative cells were not sufficient to significantly influence opsonization for phagocytosis.

O acetylation and xylose substitution had a considerable impact on clearance of GXM in vivo. O-acetyl-negative GXM was cleared from the serum at a higher rate than O-acetyl-positive GXM and displayed dramatically lower levels in the liver. Previous studies of the in vivo fate of GXM have found that a significant amount of GXM accumulates in the liver (17, 19). Indeed, it is possible that the phagocytic cells of the liver act as a reservoir for GXM to prevent clearance from the body by an as yet unknown mechanism. One explanation for the accelerated clearance of O-acetyl-negative GXM is that O acetylation retards such clearance. Similar levels of xylose-positive and xylose-negative GXM were found in both serum and the liver at all time points. In contrast, xylose-negative GXM showed greater accumulation in the spleen than xylose-positive GXM. The presence of splenic cells with a receptor for xylose-negative GXM is one possible explanation for the high concentration of xylose-negative GXM in spleen.

Our results indicate that O acetylation and xylosylation are important determinants of the antigenic structure of serotype D C. neoformans and that these structures contribute to the biological activities of GXM. There was no clear association between the biological activities that were impacted in the present study by these structural mutants and either the increased virulence of the cas1Δ strain or the avirulence of the uxs1Δ strain. This lack of congruence may be due to the involvement of biological activities other than those studied in the present report or to effects of the mutations that are unrelated to GXM structure.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grants AI14209 (T.R.K.), AI37532 (S.M.L.), and AI25780 (S.M.L.), a Burroughs Wellcome Fund Scholar Award in Pathogenic Mycology (S.M.L.), and a grant from SIDACTION (G.J.).

FOOTNOTES

    • Received 20 December 2002.
    • Returned for modification 24 January 2003.
    • Accepted 18 February 2003.

REFERENCES

  1. 1.↵
    Bar-Peled, M., C. L. Griffith, and T. L. Doering. 2001. Functional cloning and characterization of a UDP-glucuronic acid decarboxylase: the pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis. Proc. Natl. Acad. Sci. USA98:12003-12008.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    Belay, T., and R. Cherniak. 1995. Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay. Infect. Immun.63:1810-1819.
    OpenUrlAbstract/FREE Full Text
  3. 3.
    Belay, T., R. Cherniak, T. R. Kozel, and A. Casadevall. 1997. Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay. Infect. Immun.65:718-728.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Bhattacharjee, A. K., J. E. Bennett, and C. P. J. Glaudemans. 1984. Capsular polysaccharides of Cryptococcus neoformans. Rev. Infect. Dis.6:619-624.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    Bulmer, G. S., and M. D. Sans. 1968. Cryptococcus neoformans. III. Inhibition of phagocytosis. J. Bacteriol.95:5-8.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Bulmer, G. S., M. D. Sans, and C. M. Gunn. 1967. Cryptococcus neoformans. I. Nonencapsulated mutants. J. Bacteriol.94:1475-1479.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Cherniak, R., L. C. Morris, B. C. Anderson, and S. A. Meyer. 1991. Facilitated isolation, purification, and analysis of glucuronoxylomannan of Cryptococcus neoformans. Infect. Immun.59:59-64.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Cherniak, R., E. Reiss, M. E. Slodki, R. D. Plattner, and S. O. Blumer. 1980. Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans. Mol. Immunol.17:1025-1032.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Cherniak, R., H. Valafar, L. C. Morris, and F. Valafar. 1998. Cryptococcus neoformans chemotyping by quantitative analysis of 1H nuclear magnetic resonance spectra of glucuronoxylomannans with a computer-simulated artificial neural network. Clin. Diagn. Lab. Immunol.5:146-159.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    Delfino, D., L. Cianci, E. Lupis, A. Celeste, M. L. Petrelli, F. Curro, V. Cusumano, and G. Teti. 1997. Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components. Infect. Immun.65:2454-2456.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Denning, D. W., R. W. Armstrong, B. H. Lewis, and D. A. Stevens. 1991. Elevated cerebrospinal fluid pressures in patients with cryptococcal meningitis and acquired immunodeficiency syndrome. Am. J. Med.91:267-272.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Dong, Z. M., and J. W. Murphy. 1996. Cryptococcal polysaccharides induce L-selectin shedding and tumor necrosis factor receptor loss from the surface of human neutrophils. J. Clin. Investig.97:689-698.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    Dong, Z. M., and J. W. Murphy. 1997. Cryptococcal polysaccharides bind to CD18 on human neutrophils. Infect. Immun.65:557-563.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    Eckert, T. F., and T. R. Kozel. 1987. Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide. Infect. Immun.55:1895-1899.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Friedman, J. J. 1955. Organ plasma volume of normal unanesthetized mice. Proc. Soc. Exp. Biol. Med.88:323-325.
    OpenUrl
  16. 16.↵
    Fromtling, R. A., H. J. Shadomy, and E. S. Jacobson. 1982. Decreased virulence in stable, acapsular mutants of Cryptococcus neoformans. Mycopathologia79:23-29.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    Goldman, D. L., S. C. Lee, and A. Casadevall. 1995. Tissue localization of Cryptococcus neoformans glucuronoxylomannan in the presence and absence of specific antibody. Infect. Immun.63:3448-3453.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    Granger, D. L., J. R. Perfect, and D. T. Durack. 1985. Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide. J. Clin. Investig.76:508-516.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    Grinsell, M., L. C. Weinhold, J. E. Cutler, Y. Han, and T. R. Kozel. 2001. In vivo clearance of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans: a critical role for tissue macrophages. J. Infect. Dis.184:479-487.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Hirano, A., H. M. Zimmerman, and S. Levine. 1964. The fine structure of cerebral fluid accumulation. III. Extracellular spread of cryptococcal polysaccharides in the acute stage. Am. J. Pathol.45:1-19.
    OpenUrlPubMedWeb of Science
  21. 21.↵
    Hirano, A., H. M. Zimmerman, and S. Levine. 1964. The fine structure of cerebral fluid accumulation. IV. On the nature and origin of extracellular fluids following cryptococcal polysaccharide implantation. Am. J. Pathol.45:195-207.
    OpenUrlPubMedWeb of Science
  22. 22.↵
    Horstmann, R. D., M. K. Pangburn, and H. J. Muller-Eberhard. 1985. Species specificity of recognition by the alternative pathway of complement. J. Immunol.134:1101-1104.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Horstmann, R. D., H. J. Sievertsen, J. Knobloch, and V. A. Fischetti. 1988. Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H. Proc. Natl. Acad. Sci. USA85:1657-1661.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    Janbon, G., U. Himmelreich, F. Moyrand, L. Improvisi, and F. Dromer. 2001. Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide. Mol. Microbiol.42:453-467.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    Kozel, T. R., and E. C. Gotschlich. 1982. The capsule of Cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages. J. Immunol.129:1675-1680.
    OpenUrlAbstract
  26. 26.↵
    Kozel, T. R., W. F. Gulley, and J. Cazin, Jr. 1977. Immune response to Cryptococcus neoformans soluble polysaccharide: immunological unresponsiveness. Infect. Immun.18:701-707.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    Kozel, T. R., and C. A. Hermerath. 1988. Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column. J. Immunol. Methods107:53-58.
    OpenUrlCrossRefPubMed
  28. 28.↵
    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.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Kozel, T. R., and G. S. T. Pfrommer. 1986. Activation of the complement system by Cryptococcus neoformans leads to binding of iC3b to the yeast. Infect. Immun.52:1-5.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    Kozel, T. R., M. A. Wilson, and J. W. Murphy. 1991. Early events in initiation of alternative complement pathway activation by the capsule of Cryptococcus neoformans. Infect. Immun.59:3101-3110.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    Kozel, T. R., M. A. Wilson, G. S. T. Pfrommer, and A. M. Schlageter. 1989. Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins. Infect. Immun.57:1922-1927.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    Kwon-Chung, K. J., and J. C. Rhodes. 1986. Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans. Infect. Immun.51:218-223.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    MacGill, T. C., R. S. MacGill, A. Casadevall, and T. R. Kozel. 2000. Biological correlates of capsular (quellung) reactions of Cryptococcus neoformans. J. Immunol.164:4835-4842.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    Mambula, S. S., E. R. Simons, R. Hastey, M. E. Selsted, and S. M. Levitz. 2000. Human neutrophil-mediated nonoxidative antifungal activity against Cryptococcus neoformans. Infect. Immun.68:6257-6264.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    Moyrand, F., B. Klaproth, U. Himmelreich, F. Dromer, and G. Janbon. 2002. Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans. Mol. Microbiol.45:837-849.
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    Murphy, J. W., and G. C. Cozad. 1972. Immunological unresponsiveness induced by cryptococcal capsular polysaccharide assayed by the hemolytic plaque technique. Infect. Immun.5:896-901.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    Netski, D., and T. R. Kozel. 2002. Fc-dependent and Fc-independent opsonization of Cryptococcus neoformans by anticapsular monoclonal antibodies: importance of epitope specificity. Infect. Immun.70:2812-2819.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    Pettoello-Mantovani, M., A. Casadevall, T. R. Kollmann, A. Rubinstein, and H. Goldstein. 1992. Enhancement of HIV-1 infection by the capsular polysaccharide of Cryptococcus neoformans. Lancet339:21-23.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Pettoello-Mantovani, M., A. Casadevall, P. Smarnworawong, and H. Goldstein. 1994. Enhancement of HIV type 1 infectivity in vitro by capsular polysaccharide of Cryptococcus neoformans and Haemophilus influenzae. AIDS Res. Hum. Retrovir.10:1079-1087.
    OpenUrlCrossRefPubMed
  40. 40.↵
    Pfrommer, G. S. T., S. M. Dickens, M. A. Wilson, B. J. Young, and T. R. Kozel. 1993. Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule. Infect. Immun.61:4360-4366.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    Retini, C., A. Vecchiarelli, C. Monari, C. Tascini, F. Bistoni, and T. R. Kozel. 1996. Capsular polysaccharide of Cryptococcus neoformans induces proinflammatory cytokine release by human neutrophils. Infect. Immun.64:2897-2903.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    Sahu, A., T. R. Kozel, and M. K. Pangburn. 1994. Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation. Biochem. J.302:429-436.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    Segal, D. M. 1995. Antibody detection and preparation, p. 2.0.1-2.13.16. In J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current protocols in immunology. John Wiley & Sons, Inc., New York, N.Y.
  44. 44.↵
    Shoham, S., C. Huang, J.-M. Chen, D. T. Golenbock, and S. M. Levitz. 2001. Toll-like receptor 4 mediates intracellular signaling without TNF-α release in response to Cryptococcus neoformans polysaccharide capsule. J. Immunol.166:4620-4626.
    OpenUrlAbstract/FREE Full Text
  45. 45.
    Spiropulu, C., R. A. Eppard, E. Otteson, and T. R. Kozel. 1989. Antigenic variation within serotypes of Cryptococcus neoformans detected by monoclonal antibodies specific for the capsular polysaccharide. Infect. Immun.57:3240-3242.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    Taborda, C. P., and A. Casadevall. 2002. CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are involved in complement-independent antibody-mediated phagocytosis of Cryptococcus neoformans. Immunity16:791-802.
    OpenUrlCrossRefPubMedWeb of Science
  47. 47.↵
    Turner, S. H., and R. Cherniak. 1991. Multiplicity in the structure of the glucuronoxylomannan of Cryptococcus neoformans, p. 123-142. In J. P. Latgé and D. Boucias (ed.), Fungal cell wall and immune response. Springer-Verlag, Berlin, Germany.
  48. 48.↵
    Vecchiarelli, A., C. Retini, D. Pietrella, C. Monari, C. Tascini, T. Beccari, and T. R. Kozel. 1995. Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes. Infect. Immun.63:2919-2923.
    OpenUrlAbstract/FREE Full Text
  49. 49.
    Wickes, B. L., and J. C. Edman. 1995. The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter. Mol. Microbiol.16:1099-1109.
    OpenUrlCrossRefPubMedWeb of Science
  50. 50.↵
    Wilson, D. E., J. E. Bennett, and J. W. Bailey. 1968. Serologic grouping of Cryptococcus neoformans. Proc. Soc. Exp. Biol. Med.127:820-823.
    OpenUrlCrossRefPubMed
  51. 51.↵
    Young, B. J., and T. R. Kozel. 1993. Effects of strain variation, serotype and structural modification on the kinetics for activation and binding of C3 to Cryptococcus neoformans. Infect. Immun.61:2966-2972.
    OpenUrlAbstract/FREE Full Text
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Cryptococcus neoformans
polysaccharides

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