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The opportunistic yeast Cryptococcus neoformans is surrounded by a polysaccharide capsule comprised primarily of glucuronoxylomannan (GXM). GXM is a key component of the antigenic character of the capsule. Expression of the epitope that allows for binding of mAbs that require O-acetylation of GXM for mAb recognition was greatly influenced by cell age, growth conditions and serotype. Yeast cells of serotype A grown in vitro under capsule induction conditions showed considerable cell-to-cell variability in binding of two O-acetyl-dependent mAbs, and such mAbs uniformly failed to bind to GXM that covers yeast buds. Expression of the O-acetyl-dependent epitope increased with cell age. In contrast, all serotype A cells harvested from brain tissue bound the same O-acetyl-dependent mAbs. The ability of the cryptococcal capsule to activate the complement cascade and bind C3 occurred uniformly over the surface of all yeast cells, including the bud. Finally, the cell-to-cell variability in binding of O-acetyl-dependent mAbs with strains of serotype A was not found with strains of serotype D; almost all cells of serotype D showed homogeneous binding of O-acetyl-dependent mAbs. These results indicate that variability in expression of antigenic epitopes by GXM should be considered in selection of mAbs used for immunodiagnosis or immunotherapy.
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Cryptococcus neoformans is a fungal pathogen that is surrounded by an antiphagocytic capsule. C. neoformans produces a life-threatening meningitis, particularly in individuals with impaired cell-mediated immunity, including those with AIDS. C. neoformans has multiple virulence factors that contribute to yeast pathogenesis; one such virulence factor is the capsular polysaccharide, glucuronoxylomannan (GXM), a linear polysaccharide with a (1–3)-α-d-mannopyranan backbone with single β-d-xylopyranosyl and β-d-glucopyranosyl-uronic acid substituents (Bhattacharjee et al., 1984; Cherniak et al., 1998; Casadevall et al., 2003). There are four major serotypes of GXM that are distributed among three distinct varieties. C. neoformans var. grubii produces serotype A GXM; C. neoformans var. neoformans produces serotype D, and C. neoformans var. gatii produces serotypes B and C (Bennett et al., 1977; Franzot et al., 1999). GXM serotypes are distinguished primarily by the extent of O-acetylation and xylose substitution on the mannose backbone (Turner and Cherniak, 1991; Brandt et al., 2003). O-acetyl groups are of particular interest because they are immunodominant epitopes recognized by monoclonal and polyclonal antibodies to GXM (Cherniak et al., 1980; Belay and Cherniak, 1995).
Several groups have made monoclonal antibodies that bind to distinct epitopes within the C. neoformans capsule (Dromer et al., 1987; Eckert and Kozel, 1987; Mukherjee et al., 1992). Previously, we examined the interaction between GXM-specific mAbs and the cryptococcal capsular matrix by a variation in the classical quellung reaction (Neufeld, 1902) in which we utilized differential interference contrast (DIC) microscopy to evaluate mAb binding. We found three different reactions. MAbs with different epitope specificities may (i) fail to react with GXM and thus cannot visualize the capsule; (ii) produce an annular or ‘rim’ pattern that is characterized by a sharp increase in the optical gradient at the edge of the capsule followed by a decrease in the optical gradient; or (iii) produce a diffuse pattern termed ‘puffy’ where there is an increase in the optical gradient at the capsular edge marked by the absence of the immediate decrease in the optical gradient that is observed with the rim pattern (MacGill et al., 2000). Importantly, mAbs that produce a rim pattern are also protective in murine models of cryptococcosis, whereas mAbs that give a puffy pattern fail to protect in the same model (MacGill et al., 2000).
During mammalian infection, an increase in capsule size occurs, resulting in encapsulated yeast equipped to evade phagocytosis and modulate immune responses through the shedding of capsular material into the host (Bergman, 1965; Casadevall et al., 1998; Feldmesser et al., 2001). Additionally, acapsular strains of C. neoformans are avirulent (Bulmer et al., 1967; Kozel and Cazin, 1971; Kwon-Chung and Rhodes, 1986). Despite the importance of encapsulation to virulence, assembly of the capsular matrix surrounding each cell is a complex process that is still poorly understood. The goal of our study was to examine expression of capsular epitopes by use of a library of mAbs that are reactive with GXM. The experimental design included examination of mAb reactivity with parent versus budding cells, cells of different ages, and cells cultured in different conditions, including the use of cells isolated from infected murine brain tissue. The results showed that expression of epitopes reactive with two mAbs that are dependent on GXM O-acetylation for binding occurs independent of capsule formation and is greatly influenced by growth conditions.
Variability in mAb binding to parent and budding cells
A library of mAbs reactive with GXM has been developed in our laboratory. These mAbs differ in their binding to cells of different serotypes and their requirements for O-acetylation of GXM for binding to cells of serotype D (Kozel et al., 2003) (Table 1). These mAbs exhibited a striking variability in binding to the capsule that surrounded serotype A CN6 cells that had been grown in synthetic medium. mAbs 3C2, 471, 1255 and 339 showed a uniform bright staining of the entire population of cells (Fig. 1A and C). In contrast, there was considerable variability in the binding of mAbs 302 and 1326 to these same cells. Some cells showed a pattern of bright binding to the capsule that surrounded the parent cell; other cells showed a fine punctate binding pattern, but most cells failed to bind the antibodies. The variability in mAb binding patterns by mAbs 302 and 1326 is illustrated in Fig. 1B. An examination of yeast cells that exhibited the bright pattern of binding showed an additional difference between the mAbs. mAbs 3C2, 471 and 1255 bound to capsular material that surrounded both buds and parent cells; mAbs 339, 302 and 1326 did not bind to capsule surrounding buds of cells that exhibited the bright, uniform binding pattern (Fig. 1A).
Table 1. Characteristics of mAbs used for this study.
Cell-to-cell variability in binding of mAbs 302 and 1326 could be due to the presence of a mixed cell population or to phenotypic variability within a population. As a consequence, CN6 was streaked for isolation, and binding of mAbs was assessed for 10 individual colonies. The results showed that each of the clones displayed the same variability in binding of mAbs 302 and 1326 that was observed with the parent isolate (data not shown).
Variability in expression of the epitope recognized by mAbs 302 and 1326 within a population of clonal cells as well as the absence of mAb binding to capsule surrounding buds suggested that cell age might be a factor influencing epitope expression. This question was examined further by evaluation of binding of mAb 1326 to cells of different ages. For this and several subsequent experiments, mAb 1326 was chosen to evaluate the expression of the epitope recognized by mAbs 302 and 1326. We refer to this as the 1326 epitope. In order to study changes that may occur with cell ageing, a system was designed to identify original ‘parent’ cells that would, with increasing time in culture, be surrounded by an increasing number of daughter cells. This was achieved by fluorescently labelling the cell wall of live C. neoformans cells with Alexa Fluor 488. After labelling, parent cells were returned to culture conditions in fresh synthetic medium and allowed to continue growth. Samples were taken daily and examined for mAb 1326 binding patterns to parent and daughter cells (Fig. 2A). As parent cells aged, there was a significant increase in the frequency of cells with bright binding patterns after 4 or 6 days of continued growth (P < 0.001 versus time 0). In contrast, there was no change in the ratio of binding patterns among the growing population of daughter cells. Images captured at days 0 and 6 illustrate the impact of cell age on the pattern of mAb 1326 binding to parent and daughter cells (Fig. 2B and C). At day 0 (Fig. 2B), most parent cells (shown by labelling of the cell wall with the green Alexa Fluor 488) are negative. At day 6 (Fig. 2C), the Alexa Fluor 488-labelled parent cells show a much higher percentage of bright staining with mAb 1326.
Activation of the alternative complement pathway is an important biological activity of the cryptococcal capsule that leads to deposition of iC3b within the capsular matrix. Given the variability in epitope expression between capsule that surrounds the parent and buds (Fig. 1A), we examined deposition of C3 fragments over parent and buds in relation to the binding sites of the various mAbs. Yeast cells were incubated with normal human serum to deposit C3 in the capsular matrix. Alexa 488-labelled C3 was added to the human serum in trace amounts to allow for recognition of the sites for binding of C3 fragments (Gates and Kozel, 2006). This was followed by incubation with Alexa 555-labelled mAbs. The results (Fig. 3) showed deposition of C3 in the matrix over both the parent and bud. mAbs 3C2, 471 and 1255 bound to the capsule surrounding both parent and bud, whereas mAbs 339, 302 and 1326 only bound to capsule over the parent, confirming results shown in Fig. 1A.
Dependence of mAb binding on GXM acetylation
Our previous studies reported the binding of this set of mAbs to CAS1 (O-acetyl positive [NE167 and NE 169]) and cas1Δ mutants [isogenic O-acetyl-negative strains (NE168 and NE 170)] of a serotype D strain of C. neoformans (JEC156) (Kozel et al., 2003). mAbs 3C2, 471 and 1255 bound to both the parent and mutant strains; mAbs 339, 302 and 1326 bound to the parent cells but not the O-acetyl-negative mutant strain. These prior studies suggested that variability in binding of mAbs 302 and 1326 to cells within the population of CN6 cells could be due to differences in O-acetylation. JEC156 is derived from a serotype D strain, whereas CN6 is a serotype A strain. Since cells of serotype A and D differ in the extent of O-acetylation, mAb binding was assessed for untreated and chemically de-O-acetylated cells of CN6. The results (Fig. 4) showed that the pattern of binding of mAbs 3C2, 471 and 1255 shifted from a rim-type pattern to a puffy pattern with de-O-acetylated cells, but all cells continued to bind mAbs. mAb binding was evident by both the capsule reaction (Fig. 4A) and by use of directly labelled mAbs (Fig. 4B). In contrast, mAbs 339, 302 and 1326 failed to bind to the de-O-acetylated cells. These results indicate that binding of mAbs 339, 302 and 1326 to cells of serotype A strain CN6 is dependent on an intact O-acetyl group. Note the speckled pattern of binding of mAb 1255 to the de-O-acetylated cells, suggesting a partial contribution of O-acetylation to binding by mAb 1255.
Influence of growth conditions on mAb binding
Variable expression of an O-acetyl-dependent epitope within clonal cells grown in synthetic medium prompted additional experiments to explore the influence of growth conditions on presentation of the 1326 epitope. In previous studies, we examined CN6 cells isolated from brain tissue of infected mice (Gates et al., 2004). Yeast cells isolated from brain tissue have an increased concentration of GXM within the capsular matrix compared with the original in vitro cultured cells used to infect the mice. To investigate expression of the 1326 epitope further, yeast cells isolated from brain tissue were incubated with Alexa 555-labelled mAbs (Fig. 5A). In contrast to the cell-to-cell variability in binding of mAbs 302 and 1326 to yeast cells grown in synthetic medium (Fig. 1), the mAbs showed homogenous bright binding to all cells isolated from brain. To confirm the importance of O-acetylation in binding of mAbs 339, 302 and 1326, the yeast cells derived from brain tissue were de-O-acetylated by treatment at pH 11 with NH4OH and mAb binding was evaluated. The results showed bright uniform binding of the O-acetyl-independent mAbs 3C2 and 471, a speckled binding of mAb 1255 and an absence of binding by the O-acetyl-dependent mAbs 339, 302 and 1326 (Fig. 5B).
The comparison of binding by the O-acetyl-dependent mAbs 302 and 1326 to yeast cells grown in synthetic medium and cells isolated from brain tissue suggested that yeast culture conditions influence expression of the 1326 epitope. Previous studies found induction of large capsule synthesis by growth in PBS + 10% fetal bovine serum (FBS) (Zaragoza et al., 2003). An evaluation of binding by mAbs 302 and 1326 to yeast cells grown for 48 h at 37°C in 10% FBS with 5% CO2 found that > 80% of the cells bound the mAbs (not shown). Growth conditions that did not produce a change in the heterogenous binding of mAbs 302 and 1326 included: (i) growth on Sabouraud's dextrose broth; (ii) incubation in PBS; (iii) growth in synthetic medium at different temperatures (21°C versus 30°C versus 37°C); or (iv) growth in synthetic medium in the absence of shaking (not shown).
The shift from limited binding of O-acetyl-dependent mAbs 302 and 1326 to cells grown in synthetic medium to extensive binding of these same mAbs to cells shifted to 10% FBS could be due to new expression of the O-acetyl epitope by GXM in the capsule of existing cells or to the growth of new cells that expressed the epitope. In an effort to better understand the nature of the shift from limited O-acetyl epitope expression to extensive epitope expression, yeast cells were grown for 48 h in synthetic medium, directly labelled with Alexa Fluor 488, which stained the cell wall, washed with PBS to remove excess Alexa 488, inoculated into 10% FBS in PBS and cultured for an additional 18 h. In this manner, the original parent cells carried a green label from the Alexa 488 on the cell wall, whereas the progeny cells lacked a labelled cell wall. Examples of binding of the O-acetyl-dependent mAb 1326 to parent and daughter cells are shown in Fig. 6A.
Examination of the binding of mAb 1326 to parent cells at various times after the shift from synthetic medium to 10% FBS showed only 11% of the cells with bright homogeneous binding at the time of the shift (Fig. 6B). The percentage of cells displaying bright homogeneous binding increased to 44% after 18 h incubation in FBS (P < 0.001 versus time 0). Conversely, the percentage of parent cells that that did not bind mAb 1326 dropped from 60% at the time of the shift to 24% after 18 h in FBS (P < 0.001 versus time 0). These results indicated that the parent cells either synthesized new GXM that bound the O-acetyl-dependent mAb after the shift to growth in FBS or that the 1326 epitope was newly expressed on pre-existing GXM. This result is consistent with results shown in Fig. 2 that the percentage of parent cells showing bright staining increases with cell age. An examination of daughter cells found that the progeny cells were apparent 6 h after the shift to FBS. All of the cells bound mAb 1326 with either the bright or punctate patterns at 6 h, and almost all cells showed bright patterns after 18 h; no daughter cells were negative at any time point. This result contrasts with the high percentage of daughter cells that showed no binding of mAb 1326 after shift to synthetic medium (Fig. 2).
Strain and species variability in expression of the mAb epitope
Studies described above showing variability in expression of the 302/1326 epitope were done with a single strain of serotype A (CN6). As a consequence, an experiment was done with two additional serotype A strains to determine if the observed variability in expression of the O-acetyl-dependent epitope recognized by mAb 1326 was generalizable to other strains. In addition, we examined three isolates of serotype D (C. neoformans var. neoformans). Cells from three strains of serotype A and three strains of serotype D were incubated with Alexa 555-labelled mAb 1326 (O-acetyl-dependent), and the patterns of mAb binding were assessed. The results (Fig. 7) showed that the cell-to-cell variability in expression of the mAb 1326 epitope by strain CN6 was also observed with serotype A strains MU-1 and H99. In contrast, almost 100% of cells of serotype D strains 3501, 24067 and EJ127 produced a bright homogeneous binding pattern.
Variability in epitope expression with soluble GXM
Studies shown in Figs 1–7 describe the binding of O-acetyl-dependent and O-acetyl-independent mAbs to GXM as it is presented in an intact capsule. GXM is also shed into body fluids in large amounts during infection and exerts a number of biological effects that may impact pathogenesis. As a consequence, we also examined variability in binding of mAbs to soluble GXM.
An antigen capture ELISA was constructed using mAb F12D2 in the solid phase to capture GXM from the samples (Brandt et al., 2003). mAb F12D2 was raised from mice immunized with de-O-acetylated serotype A GXM. As a consequence, it shows uniform binding to cells of all serotypes and to de-O-acetylated GXM. HRPO-labelled mAb 1326 (O-acetyl-dependent) was used in the fluid phase to assess expression of the 1326 epitope by the GXM preparations. Serotype A GXM (strain CN6) was isolated from supernatant fluids of (i) yeast cells grown in synthetic medium, (ii) cells grown in 10% FBS, and (iii) homogenized brain tissue from C. neoformans-infected mice. Although several purification steps were performed on the supernatant from infected brain tissue, the possibility of non-GXM polysaccharide contamination still existed. To ensure accuracy of comparisons made between GXM preparations, F12D2, an O-acetyl-independent GXM mAb, was used to normalize GXM concentrations. The results (Fig. 8) showed identical binding of the O-acetyl-dependent mAb 1326 to GXM captured by ELISA from brain tissue or growth on 10% FBS (ED50 of 0.0049 μg ml−1 and 0.0056 μg ml−1 respectively). In contrast, there was a significant reduction in binding of the mAb to GXM from cultures grown in synthetic medium (ED50 of 0.066 μg ml−1).
Unicellular microbes may exhibit marked variation in their surface components, a property that facilitates adaptation to a changing environment, the sequential colonization of various tissues during infection, and the avoidance of immune surveillance (Beale and Wilkinson, 1961; Saunders, 1989). In an early review, Beale and Wilkinson (1961) noted that the environment plays an important role in determining which out of an array of alternative antigens is formed by a given cell. Given the fact that C. neoformans is a microbe that has an environmental niche and that humans are likely accidental hosts, expression of O-acetyl-dependent epitopes may be a response to the need to adapt to a changing nutritional environment or the need to adapt to changing hosts or predators such as amoebae.
Variable expression of O-acetyl-dependent epitopes might influence the pathogenesis of cryptococcosis. Strains that have a CAS1 deletion are O-acetyl negative and are hypervirulent in mice (Janbon et al., 2001). However, the cas1Δ parent strain is serotype D; serotype D does not show the variability in expression of the O-acetyl-dependent epitope that we found with serotype A (Fig. 7). The O-acetyl group is an immunodominant epitope for GXM (Brandt et al., 2003), and many protective antibodies are O-acetyl dependent (Casadevall et al., 1994). Variable expression of this critical antibody target as cells age or in response to the host environment may influence the ability of an antibody to protect or tissue-specific adaption during infection. Notably, cryptococcal cells of advanced generational age have increased resistance to phagocytosis and killing by antifungals (Jain et al., 2009). These adaptive properties, accompanied by variability in epitope expression on GXM likely contribute to pathogenesis during chronic infection.
Sumner and Avery define phenotypic heterogeneity as ‘non-genetic variation that exists between individual cells within isogenic populations’ (Sumner and Avery, 2002). The authors further note that ‘some of the key parameters that drive non-genetic heterogeneity include cell cycle progression, cell ageing, mitochondrial activity, epigenetic regulation and potentially also stochastic variation.’ Our results clearly implicate cell ageing as an operative variable in cell-to-cell variability in expression of the epitope recognized by the O-acetyl-dependent 1326 mAb.
Cell-to-cell variability in expression of capsular epitopes within a clonal population of cryptococcal cells has been described previously. For example, Janbon reported phenotypic variation in expression of an epitope recognized by mAb E1 (Garcia-Hermoso et al., 2004; Janbon, 2004). Variability in structure of GXM over buds versus parental capsule has also been reported (Pierini and Doering, 2001). Our results suggest a link between the two reports. If expression of the 1326 epitope increases with cell age, beginning with no expression of the epitope over budding cells (Fig. 1) to the bright staining pattern observed with aged yeast cells (Fig. 2), this result suggests that an examination of an unsynchronized cell population would show the variability that we and others have observed. The absence of expression of the 1326 epitope in GXM during bud formation also supports the report by Pierini and Doering that GXM produced over the bud is newly synthesized polysaccharide (Pierini and Doering, 2001). Finally, a change in capsule structure as a function of cell age was reported by Pierini and Doering who found an increase in matrix density as a function of culture time (Pierini and Doering, 2001).
Interpretation of results from our study should be limited to what is known about the epitopes that are recognized by the available mAbs. Reactivity of mAbs 339, 302 and 1326 is lost if GXM is chemically de-O-acetylated or if there is a mutation on the pathway to O-acetylation (Kozel et al., 2003). Detailed genetic studies of O-acetylation have been reported for serotype D (Janbon et al., 2001). Our studies with chemically de-O-acetylated encapsulated cryptococci suggest a similar result with cells of serotype A. These studies do not identify a specific structure that is phenotypically varied. It is possible that the O-acetyl group itself is the relevant structure. Alternatively, variability in O-acetylation could mask or unmask structures other than the O-acetyl group, or the presence or absence of an O-acetyl group could have a secondary effect on epitopes that are not by themselves O-acetylated.
A shift in expression of GXM epitopes has been reported previously. For example, Cherniak et al. noted variation in the structure and antigenic make-up of GXM in isolates from patients with recurrent cryptococcal meningitis, despite the fact that the isolates were indistinguishable by DNA RFLP analysis (Cherniak et al., 1995). Garcia-Hermoso et al. (2004) reported structural evolution of GXM epitope expression during mouse passage and prolonged in vitro culture. Such changes in epitope structure were relatively stable traits that were evident on yeast subculture. Our report of phenotypic variability in epitope expression in response to environmental cues most closely resembles the report by Charlier et al. (2005) that the cryptococcal capsule undergoes a rapid change in epitope expression on passage across the blood brain barrier. Invasion of the brain after intravenous infection was associated with the acquisition of reactivity with mAb CRND-8 and loss of reactivity with mAb E1. CRND-8 is an O-acetyl-dependent mAb raised against serotype D that does not react with cells of serotype A that have been grown in vitro (Ikeda et al., 1996); mAb E1 produces serotype-specific binding patterns to cells of serotypes A, B or D and no binding to cells of serotype C (Dromer et al., 1993). Notably, Charlier et al. used a serotype A strain (H99), the serotype that our studies found to undergo phenotypic variability.
In contrast to expression of the O-acetyl-dependent epitope that is dependent on cell age, the capsular structure that is responsible for activation of the complement cascade and deposition of C3 into the capsular matrix is expressed on all cells in a non-synchronized cell culture. C3 was also deposited in the capsular matrix that surrounded yeast buds. This independence of complement activation from expression of an O-acetyl-dependent epitope is in agreement with our previous reports that de-O-acetylated cryptococci are fully able to activate the complement system and bind C3 fragments (Young and Kozel, 1993; Kozel et al., 2003).
Cryptococcus neoformans serotypes A and D were previously designated as C. neoformans var. neoformans, whereas serotypes B and C were designated as C. neoformans var. gattii (Vanbreuseghem and Takashio, 1970; Kwon-Chung et al., 1982). More recently, designation as C. neoformans var. neoformans has been limited to isolates of serotype D, and isolates of serotype A are designated C. neoformans var. grubii (Franzot et al., 1999). Despite considerable genetic differences, C. neoformans var. grubii and var. neoformans are distinguished by only a few phenotypic characteristics (Franzot et al., 1999). Our results suggest an additional difference; isolates of serotype A show considerable phenotypic variability in expression of the 1326 epitope, whereas isolates of serotype D show high constituitive expression of this same epitope. It is likely that the cryptococcal capsule evolved as a means to survive in a hostile environment such as soil amoebae (Ruiz et al., 1982) and desiccation (Ophir and Gutnick, 1994). If this is the case, differences in variability of epitope expression by strains of serotypes A and D raise the possibility of different environmental niches for the two varieties.
mAbs 339, 302 and 1326 all required an intact O-acetyl group for binding. However, the epitope recognized by mAb 339 differed from that recognized by mAb 302 and 1326. The O-acetyl-dependent epitope bound by mAb 339 was found on all parent cells within the population, whereas expression of the epitope recognized by mAbs 302 and 1326 was found only after cells aged. This result suggests two distinct modes by which O-acetyl-dependent epitopes can be regulated and/or expressed. Despite the differences in patterns of expression of epitopes recognized by mAb 339 versus mAbs 302 and 1326 on parent cells, all three failed to bind to capsule overlying buds.
Our results provide further information as to GXM production and modification. GXM is synthesized intracellularly and is secreted from the cell via exocytosis (Yoneda and Doering, 2006). The impact of cell age on binding of mAb 1326 suggests that expression of the 1326 epitope is a late event in capsule formation. These results support the argument that GXM acetylation is the last step in GXM biosynthesis (Janbon et al., 2001). Capsular polysaccharide is clearly evident over the surface of yeast buds (Fig. 1A). Moreover, capsule surrounding yeast buds bind GXM mAbs 3C2, 471 and 1255, activates the complement cascade, and binds C3 in a manner similar to capsule surrounding the parent cells. However, these same buds fail to bind mAbs that require O-acetylation for recognition.
Finally, our results have implications for efforts using mAbs to target GXM for immunodiagnosis of infection or treatment. It is clear from our studies that expression of capsular epitopes recognized by GXM mAbs are influenced by both cell age and growth conditions. These results suggest that efforts to target the capsule for immunodiagnosis or immunotherapy must consider the influence of in vivo growth conditions on expression of epitopes that might be recognized by such mAbs.
C. neoformans cells and GXM
Cryptococcus neoformans serotype A strains CN6 (ATCC 62066) and H99 (ATCC 208821) were obtained from the American Type Culture Collection. Serotype A strain MU-1 and serotype D strains 3501, 24067 and EJ127 were originally provided by Dr Robert Cherniak. Unless indicated otherwise, cells were induced for large capsule production by incubation for 4 days at 37°C with 5% CO2 in synthetic medium (Cherniak et al., 1982) supplemented with 24 mM sodium bicarbonate and 25 mM Hepes (Granger et al., 1985) and killed by overnight treatment with formaldehyde. Some experiments used yeast cells harvested from infected tissue. In this instance, BALB/c mice were injected by the intravenous route with 5000 cells of strain CN6. Once hydrocephalus was observed, mice were sacrificed and yeast cells were isolated from brain tissue as previously described (Gates et al., 2004).
GXM was isolated from culture supernatant fluids and infected brain tissue by differential precipitation with ethanol and hexadecyltrimethylammonium bromide (Cherniak et al., 1991). GXM shed in vivo was isolated from supernatant fluid after brain tissue from CN6-infected mice was minced, passed through cheesecloth to remove cellular debris and then centrifuged to remove yeast cells. De-O-acetylation of cells and soluble GXM was done by alkaline hydrolysis. The pH was adjusted to 11 with ammonium hydroxide followed by an overnight incubation at room temperature. De-O-acetylated cells were washed and resuspended in phosphate-buffered saline (PBS), whereas soluble GXM was dialysed overnight in PBS.
Reagents, sera and mAbs
mAbs 3C2, 471, 1255, 339, 1326 and 302 are IgG1 antibodies that are reactive with GXM, the major capsular polysaccharide of C. neoformans. These mAbs are described in further detail in Table 1. mAb F12D2 was raised from mice immunized with de-O-acetylated serotype A GXM (Brandt et al., 2003). Human complement protein C3 was purified from pooled human plasma by differential precipitation with polyethylene glycol, ion exchange chromatography on Mono Q and Mono S and molecular sieve chromatography on Superdex 200 (Tack and Prahl, 1976; Hammer et al., 1981; Alsenz et al., 1992). Purified human C3 and various mAbs were conjugated to Alexa Fluor dyes (488 or 555) according to manufacturer's instructions (Molecular Probes). Complement deposition studies were done with pooled human serum obtained from Innovative Research (Novi, MI).
mAb and C3 binding to the capsule
Cryptococcus neoformans cells (1.2 × 105) were incubated with mAbs (50 μg ml−1) in a 50 μl reaction volume for 10 min at 37°C to assess mAb binding to the capsular matrix by DIC microscopy. Direct immunofluorescence examining antibody binding was done similarly, except the concentration of mAbs conjugated to Alexa Fluor 555 was 10 μg ml−1. Statistical comparisons of antibody binding patterns in populations of cells were done by chi-squared analysis. Direct labelling of the cell wall was accomplished by incubating live yeast at 0°C with Alexa Fluor 488. To remove unbound fluorophore, cells were washed 3 times with cold PBS before transferring to fresh synthetic medium or 10% FBS in PBS. In control experiments, the Alexa Fluor dye was found to colocalize with calcofluor white, a dye that binds to chitin in fungal cell walls (Pringle, 1991). Additional controls found that cells labelled with Alexa Fluor retained the dye for several days without loss of fluorescence and that labelling with the fluorophore had no effect on yeast viability as shown by quantitative culture of labelled and sham labelled cells (not shown). Similar results for labelling of the cryptococcal cell wall were reported by Pierini and Doering (2001) with the exception that labelling was done with amino-fluorescein via a carbodiimide reaction.
Binding of C3 to the capsular matrix was determined by incubating C. neoformans cells (1.2 × 105) in 40% pooled normal human serum supplemented with 50 μg ml−1 of Alexa Fluor 555-conjugated human C3 for 32 min at 37°C. Cells were treated with 10 mM EDTA in PBS to stop complement deposition, then washed with PBS and incubated with fluorescent GXM mAbs as described above. All cells were immediately transferred to poly-l-lysine-coated slides and examined with a Nikon C1 confocal equipped with differential interference contrast (DIC) optics attached to a Nikon Eclipse E800 microscope (100× objective). Confocal images were processed using Nikon EZC1 software, version 1.70. Images were merged and cropped using Simple PCI software (Compix, Cranberry Township, PA, USA).
Assays for antibody activity
An ELISA was used to assess expression of the O-acetyl-dependent epitopes recognized by mAb 1326 in GXM isolated from culture supernatant fluids or from homogenates of brain tissue. In this immunoassay, microtiter plates were coated overnight with the O-acetyl-independent mAb F12D2 (1 μg ml−1 in PBS) (Brandt et al., 2003). Plates were washed with PBS-Tween (PBS containing 0.05% Tween 20, pH 7.4) and incubated for 2 h with a blocking solution (PBS containing 0.5% Tween 20 and 5% skim milk). GXM in blocking solution was added in serial twofold dilutions and incubated for 90 min. After washing plates 3 times with blocking solution, horseradish peroxidase conjugated GXM mAb 1326 (O-acetyl-dependent; 16 μg ml−1) was added and incubated for 90 min. Finally, plates were washed 3 times with PBS-Tween and incubated for 30 min with TMB substrate (KPL, Gaithersburg, MD, USA). Stop solution (H3PO4) was added and the absorbance was read at 450 nm. Immunoassay reactivity with mAb F12D2 in the indicator phase was used to normalize the GXM content in the various samples. The concentration of GXM that produced half maximal OD450 (ED50) was calculated with the assistance of SigmaPlot (Systat Software, San Jose, CA, USA).
This study was supported by Public Health Service grant AI014209 from the National Institute for Allergy and Infectious Diseases.