Phenotypic switching of Cryptococcus neoformans occurs in vivo and influences the outcome of infection

Phenotypic switching has been linked to the virulence of many pathogens, including fungi. However, it has not been conclusively shown to occur in vivo or to influence the outcome of infection. Cryptococcus neoformans undergoes phenotypic switching in vitro to colony types that differ in their virulence in mice. In this study, we asked whether C. neoformans undergoes phenotypic switching in vivo and whether this phenomenon contributes to virulence. By using a small inoculum to preclude the introduction of variants that had already switched during in vitro propagation, we demonstrated that in vivo switching to a mucoid phenotype occurred in two mice strains and was associated with a lethal outcome. Phenotypic switching resulted in changes of the capsular polysaccharide that inhibited phagocytosis by alveolar macrophages. This promoted a more vigorous inflammatory response and rapid demise. These data document in vivo switching in a fungus and associate this phenomenon with enhanced virulence and a lethal outcome. The importance of this finding is underscored by the increased likelihood of phenotypic switching in chronic cryptococcosis; thus this mechanism may account for the inability to eradicate the organism in immunocompromised hosts.

Cryptococcosis affects 5–30% of patients with AIDS. It is caused by Cryptococcus neoformans, an encapsulated yeast that is acquired by inhalation and ultimately results in a chronic meningoencephalitis with a high tendency to relapse despite effective antifungal therapy (1). Several virulence factors (2–5) including a polysaccharide capsule (6, 7) are described for C. neoformans. Most virulence factors are invariably present (8) in clinical strains. Genetic inactivation of their regulatory proteins, which are part of signaling pathways, renders the yeast avirulent (9, 10). It is, however, unclear whether regulation of virulence factors occurs in vivo during chronic infection in order to enhance the virulence of an infecting C. neoformans strain. Therefore, it is often assumed that failure to eradicate the fungus is a function of a diminished cell-mediated immune response (CMI). Several observations, however, indicate that pathogen-specific factors contribute to the pathogenesis of cryptococcosis. First, prolonged in vitro and in vivo passage of C. neoformans isolates can result in phenotypic changes associated with differences in virulence (11–14). Second, serial isolates from chronically infected patients can exhibit differences in virulence (15). Third, C. neoformans variety gattii strains cause symptomatic infections primarily in immunocompetent hosts even in areas where AIDS is endemic (16).

Reversible phenotypic switching can result in microevolution, defined as rapid changing of an organism to a stable, inherited phenotype (17). Microevolution and phenotypic switching can be achieved by several mechanisms, some of which involve changes of genes and follow a mendelian inheritance pattern whereas others (e.g., silencing) are epigenetic and inherited in a nonmendelian fashion. For certain pathogens, the emergence of phenotype variants is associated with invasion of mucosal barriers and immune evasion (18). For fungi, phenotypic switching is an in vitro phenomenon defined as the spontaneous emergence of colonies with altered colony morphology at rates higher than the somatic mutation rates (19–21). Phenotypic switching was demonstrated for C. neoformans in vitro in three different clinical strains and resulted in enhanced virulence (22, 23). Despite an association with virulence, phenotypic switching in vivo has been demonstrated only in pathogens with complex life cycles that require the switch for survival in the host (24). Most infections require high doses, and because of the expected in vitro switching rates (10^–2 to 10^–5), these inocula are likely to be contaminated with in vitro switch variants. Therefore, it is difficult to demonstrate that the emergence of new phenotypes in the infected host are the result of in vivo switching events and do not represent in vivo selection of variants from an initially heterogeneous pathogen population (25).

Our objective was to investigate whether phenotypic switching of C. neoformans occurred in vivo and, if so, to explore its consequences. Our data show that phenotypic switching occurs in vivo and results in changes of a key virulence factor, the capsular polysaccharide. These findings suggest a unique mechanism of increased virulence, namely, an enhanced inflammatory response with altered cytokine and chemokine expression that ultimately interferes with the host ability to mount an effective immune response.

Characterization of the C. neoformans strain RC-2 as a switching system. Strain RC-2 grows as a SM colony on SDA with mucoid MC colonies arising spontaneously at a frequency of 0.5 in 10^–4 (Figure 1a). Both are common colony phenotypes in clinical isolates. The reversion of MC to SM occurs in vitro at a comparable frequency. Karyotype analysis and RFLP patterns with an α-P³²–labeled probe to repetitive sequences in the C. neoformans genome (CNRE) (27) did not detect DNA rearrangements and confirmed that SM and MC are one strain (data not shown). Comparison of several virulence traits including doubling time and the ability to melanize revealed no major differences between SM and MC (Table 1). Small differences in cell and capsule size were observed, but cell charge and sugar assimilation profile were not affected by phenotypic switching (data not shown).

Figure 1

Characterization of SM and MC. (a) Mucoid colony surface of MC colony compared with the SM colony after growth on SDA for 72 hours. (b) Size exclusion chromatography shows earlier elution of MC GXM compared with SM GXM. (c) Zonal fractionation confirms larger size of MC GXM, which is detected in the earlier fractions after ultracentrifugation over a 5−20% sucrose gradient. mDa, million dalton.

Table 1

Characteristics of SM and MC phenotypes

Analysis of the SM and MC GXM repeat structure by NMR revealed that both GXMs were composed of the M1 structural reporter group, as expected for serotype D (13). However, MC GXM was more viscous than SM GXM, suggesting a difference in molecular weight (Table 1). Analysis of the molecular weight of SM and MC GXM by two independent methods, size exclusion chromatography (Figure 1b) and zonal fractionation by ultracentrifugation (Figure 1c), determined that MC GXM was larger than SM GXM.

Increased virulence of the MC phenotype in mice. MC was more virulent than SM in BALB/c and A/JCr mice (Figure 2a and Table 2). Mice infected with MC (MCmice) died 24 to more than 52 days earlier than SMmice. MC colonies derived from lung and CNS after in vivo switching or from in vitro switching events exhibited similar virulence. Mixed infection experiments using a 1:1 mixture of SM and MC revealed survival comparable to that of mice infected with MC alone, suggesting that cross-protection could not be achieved. These results demonstrate that phenotypic switching from SM to MC results in a more virulent phenotype.

Figure 2

Association of phenotypic switching with virulence. (a) Kaplan Meyer survival curve shows decreased survival of mice infected intratracheally with 10⁴ MC compared with SM. (b) Mice that are infected for a prolonged time have a higher percentage of MC colonies (P value, Spearman coefficient). (c) Mice that died of cryptococcosis have a higher percentage of MC colonies than do long-term survivors (P = 0.0001, t test) and mice prior to onset of disease (P < 0.0001, t test). Comparison of all groups by ANOVA analysis is P < 0.001.

Table 2

Survival difference of A/JCr and BALB/c mice infected with SM and MC^A

Pilot experiments showing that both SM and MC colonies could be recovered from SMmice suggested either that phenotypic switching from SM to MC occurred in vivo, or that rare switching events had occurred in vitro prior to infection and were selected during the infection process. To discriminate between these possibilities, the inoculum was reduced to 10³ to decrease the probability of injecting a cocktail containing at least one MC cell to 5%. Groups of 10 A/JCr and 11 BALB/c mice were injected with 10³ SM cells, and their organs were assayed for MC colonies at day 45, day 73 (after infection), and the time of death. MC colonies were recovered from 9 of 10 A/JCr and 7 of 11 BALB/c mice (range 0.1–30% MC colonies per mouse) after infection. At day 45 no MC colonies were recovered from four BALB/c mice and one A/JCr mouse. Application of the Poisson formula revealed that the probability of recovering MC colonies because of inadvertent inoculation of a MC cell was exceedingly low (P = 1.1 × 10^–12 for A/JCr and P = 4.4 × 10^–8 for BALB/c). Hence, the data imply that the occurrence of MC colonies in SMmice is the result of in vivo phenotypic switching.

To ascertain whether an association existed between phenotypic switching, duration of infection, and/or fungal organ burden (CFU), BALB/c and A/JCr mice (n = 101) were infected with SM (10³ to 10⁶) and analyzed for the percentage of MC colonies and CFU at different times after infection. Pearson correlation analysis revealed a time-dependent increase in the percentage of MC colonies recovered from mouse organs (P < 0.001) (Figure 2b), but no correlation with CFU (data not shown). The percentage of MC colonies ranged from 0.1% to 100% in mice infected with the same dose, and analyzed at the same time. In some cases MC colonies were recovered only from lung or brain, indicating that phenotypic switching occurred after dissemination from lung to brain.

We then investigated the impact of phenotypic switching on the outcome of infection. BALB/c mice (n = 108) were infected with 10⁴ SM cells, an inoculum that is generally cleared within 3 months by BALB/c mice. Mice autopsied before the onset of disease (before day 36) and long-term survivors had significantly (P < 0.001, ANOVA) fewer MC colonies (1.0% ± 2.2% and 3.6% ± 10.2%) than those (41% ± 35%) autopsied at the time of death between day 42 and day 140 (Figure 2c). These data demonstrate that the occurrence of phenotypic switching in vivo increases the likelihood of death following C. neoformans infection.

Mechanisms of enhanced virulence of the MC phenotype: MC cells resist phagocytosis. Given the difference in GXM molecular weight and virulence, we examined the ability of different Mφ types to phagocytose and support intracellular replication of SM and MC. Phagocytosis of MC was significantly reduced compared with SM cells both in vitro and in vivo (Table 3). Next, we investigated whether the MC and SM GXMs differed in their ability to inhibit phagocytosis. Both SM and MC GXM significantly inhibited phagocytosis of acapsular C. neoformans cells (Cap67) mutant cells (P=0.001, ANOVA). However, the addition of 500μg/ml MC GXM 1 hour before phagocytosis (Figure 3a) reduced the phagocytosis of Cap67 cells significantly more than SM GXM (63% vs. 24% for MC and SM GXM, P = 0.003, t test). The same effect was seen when MC GXM was added to Cap67 mutant cells at the time of phagocytosis (40% vs. 7% for MC and SM GXM, P=0.04, t-test). Hence, MC GXM was a more effective inhibitor of phagocytosis.

Figure 3

Effects of SM and MC GXM on phagocytosis in vitro and in vivo. (a) Inhibition of phagocytosis of Cap67mutant cells by SM and MC GXM. Phagocytosis was significantly more inhibited (63% vs. 23%, P = 0.003, t test) by 500 μg MC GXM than SM GXM. Control is PI of Cap67 cells without GXM. (b) Formation of vacuoles in Mφ’s of SMmice are the result of digested C. neoformans. (c) In contrast, intracellular persistence of C. neoformans is observed in Mφ’s of MCmice.

Table 3

Comparison of phagocytosis of SM and MC cells in different Mφ’s

Histological examination of SM-infected lungs revealed vacuolated alveolar Mφ’s containing digested yeast cells (Figure 3b); in contrast, predominantly undigested encapsulated yeast cells were noted in alveolar Mφ’s of MCmice (Figure 3c). This may reflect a difference in intracellular survival in vivo; however, we could not document this with our killing assays in vitro (data not shown).

MC cells elicit a greater inflammatory response than SM cells. Consistent with survival differences, the inflammatory tissue response elicited by SM and MC cells differed. At day 14, lungs of SMmice exhibited moderate inflammatory changes with cellular infiltrates composed primarily of lymphocytes and only a few Mφ’s (Figure 4, a and b). By day 28, the cellular infiltrates had increased within the alveolar spaces with little concomitant lung damage (Figure 4c). By day 60, granuloma formation had begun and yeast cells were surrounded by organized tissue (Figure 4d), indicating successful containment of the infection. In contrast, at day 14, MC-infected lungs exhibited extensive cellular infiltrates beyond the peribronchial regions, which were predominantly composed of Mφ’s and neutrophils polymorphonuclear neutrophils (PMNs) with only a few lymphocytes (Figure 4, e and f). Near the time of death (day 28), the inflammatory response increased and resulted in extensive destruction of alveolar membranes (Figure 4g). The alveolar spaces were filled with yeast cells surrounded by large deposits of polysaccharide and Mφ’s (Figure 4h). Lung weights, which reflect the magnitude of the inflammatory response, GXM deposition, and organ edema, were also higher in MCmice compared with SMmice (Table 4). Over time, the fungal organ burden increased in MCmice but decreased in SMmice (Table 4). In summary, these data show that MC induced a greater and more destructive inflammatory response than SM.

Figure 4

Inflammatory immune response in SM- and MCmice. (a−d) Histological analysis of SM-infected lungs. (a and b) At day 14, moderate inflammation (H&E, ×20; a) with few Mφ’s (Mac 3, ×20; b). (c) At day 28, minimal destruction of lung tissue (H&E, ×50). (d) At day 60, granuloma formation and containment of C. neoformans (arrow; Mucicarmine [MUC] stain, ×50). (e−h) In contrast, in MCmice at day 14, severe inflammation (H&E, ×20; e) composed of many Mφ’s (Mac 3, ×20; f). At day 28, (g) extensive destruction of alveolar membranes (arrow; H&E, ×50), and (h) persistence of C. neoformans (arrow; MUC stain, ×50).

Table 4

Comparison of CFU, lung weights, and GXM tissue content of infected lungs

Analysis of CMI. MCmice recruited four times more cells to the lung than SMmice (Figure 5a). Analysis of infiltrating cells by morphology, FACS, and immunohistochemistry showed that SMmice recruited a higher percentage of lymphocytes and a lower percentage of PMNs and Mφ’s compared with MCmice (Figure 5b). Accordingly, in lung cell suspensions we found alveolar Mφs’s attached to lymphocytes in SMmice (Figure 5c) and to PMNs in MCmice (Figure 5d). Because successful clearance of C. neoformans infection in BALB/c mice is dependent on a functional T cell response, we compared lymphocyte subpopulations in infected lungs and footpad DTH response to CneAg in SM- and MCmice. The FACS analysis of digested lung cells on day 14 and day 28 documented no difference in percentage of CD4⁺ and CD8⁺ lymphocytes for SM- and MCmice and predominance of CD4⁺ lymphocytes (data not shown). The DTH response was greater (P < 0.001, ANOVA) in MCmice than in SMmice (Figure 5e), confirming that both SM and MC can elicit an effective immune response in the footpad.

Figure 5

Analysis of inflammatory immune response. (a) Cell recruitment (RC) to lungs was four times higher in MCmice at day 25 than in SMmice (P < 0.001, t test). (b) Morphological analysis demonstrated a higher percentage of PMNs (P = 0.016 at day 14, P = 0.001 at day 25) and Mφ’s (P = 0.02 at day 25) but a lower percentage of lymphocytes (P = 0.003 at day 14 and P = 0.0018 at day 25, t test) in MCmice than in SMmice. (c and d) Stained cytospins (×20) of SMmice (c) showing Mφ’s surrounded predominantly by lymphocytes (arrow), and of MCmice (d) surrounded predominantly by PMNs (arrow). (e) DTH testing reveals increased footpad swelling (FPS) in MCmice compared with SMmice at day 14 and day 25 (P < 0.001, ANOVA).

Lung cytokine and chemokine expression differs in SM- and MC- infected mice. Cytokine and chemokine levels were measured by ELISA in lung homogenates from SM-, MC-, and sham-infected mice at days 1, 3, and 14. In two separate experiments, the tissue concentration of all of the cytokines and chemokines studied was significantly upregulated in SM- and MC-infected compared with sham-infected mice (P < 0.02, ANOVA). Significantly higher protein levels were detected in SM- compared with MCmice for IL-2, IL-4, IL-10, and TNF-α at day 14 after infection, whereas comparable tissue levels were determined for IL-6, IL-12, and IFN-γ (Table 5). The same trend was also noted at earlier times after infection but did not reach statistical significance (data not shown). Expression of MIP-1α and MCP levels was three to four times higher in MC- than in SMmice, and no difference was found for KC.

Table 5

Cytokine and chemokine expression profiles at day 14 in infected lungsA

This study demonstrates phenotypic switching for a fungus during experimental infection. The result of this switch is a MC phenotype that produces a larger GXM that reduces phagocytic efficacy by alveolar Mφ’s. Our data provide strong evidence that in vivo switching enhances the virulence of C. neoformans by eliciting a more vigorous and qualitatively different inflammatory response that cannot effectively clear the infection but rather promotes lung tissue destruction and rapid demise.

Proof that phenotypic switching occurs in vivo has heretofore been difficult to obtain, because it is not feasible to induce an infection with a single cell. We infected mice with a small inoculum (10³), which was capable of causing chronic infection but unlikely to contain one MC cell (in vitro switching rate [0.5 × 10^–4]) (25). Application of the Poisson formula indicated that the likelihood of MC colony recovery from SMmice as a result of mixed infection was exceedingly low (P = 1.1 × 10^–12). Two other observations were consistent with in vivo switching: (a) mouse-to-mouse variation (ranging from 0.1% to 100%) that would not be expected if the source of variant phenotypes were the inoculum; and (b) recovery of MC colonies exclusively from the brain in two mice, which indicates phenotypic switching in vivo after dissemination.

Our data show that a greater likelihood of death occurred in mice with evidence of in vivo switching to the MC phenotype (Figure 2c). Hence, an infection dose (10⁴ CFU) that would not be expected to be lethal for the majority of mice became more virulent by phenotypic switching (31, 32). Because both SM and MC are intrinsically virulent isolates, death also occurred in the absence of switching. A failure of host defense that leads to an increased fungal burden is often invoked to explain enhanced cryptococcal virulence. Thus, the finding that phenotypic switching to MC augments the virulence of a C. neoformans strain is important, because it indicates that the pathogen can generate intrinsically more virulent variants in vivo.

The enhanced virulence of MC may be explained by the change in molecular weight for GXM, the predominant capsular polysaccharide and a key virulence factor. GXM is an immunomodulator (33) and can alter cytokine and chemokine production by monocytes and PMNs in vitro (34–39). GXM can be immunosuppressive in vivo (40), directly inhibit phagocytosis (6, 41), and delay the processing of phagocytosed C. neoformans (42). We found that phagocytosis of MC was impaired compared with SM cells (Table 3). This inhibition was more pronounced with MC than with SM GXM when phagocytosis of acapsular yeast cells was examined (Figure 3a) at GXM concentrations that were comparable to lung tissue concentrations during chronic infection (Table 4). Interestingly, previous studies using polysaccharides with variable molecular weights demonstrated that their ability to inhibit phagocytosis was a function of size, with the larger polysaccharide being more effective at inhibiting phagocytosis (43, 44). Whereas those studies used polysaccharides derived from different C. neoformans isolates, our data show that through phenotypic switching one individual C. neoformans isolate can alter the size of its polysaccharide and gain a biological advantage during infection. Unlike a hypervirulent C. neoformans mutant with an inactivated regulatory protein (PKR 1) of the capsule, there were no differences in capsule size under inducing conditions between the SM parent and the more virulent MC variant (Table 1) (9). Our findings are similar to those described for Pseudomonas aeruginosa, which also switches to a MC colony phenotype with an altered polysaccharide (45). Notably, the MC and more viscous polysaccharide of C. neoformans may create a more effective biofilm that protects it from host effector cells.

Successful phagocytosis is required for effective CMI, and proliferation of lymphocytes in response to C. neoformans correlates with the magnitude of phagocytosis (46, 47). Therefore, we hypothesized that impaired phagocytosis affects the host-pathogen interaction and alters the CMI. Our investigations found qualitative changes in effector cells with increased numbers of Mφ’s and PMNs in MC- compared with SM-infected lungs (Figure 5b). Although other groups have reported the association of minimal inflammatory responses with a failure to clear cryptococcal infection, we have found that an enhanced inflammatory response was associated with rapid demise in MCmice (32, 48).

Alveolar Mφ’s constitute the primary phagocytic cell in pulmonary cryptococcosis, and human autopsy studies indicate that PMNs are recruited primarily in response to tissue damage resulting from infection (49). Our findings suggest that impaired phagocytosis leads to upregulation of chemokines MCP and MIP-1α, which in turn leads to an increased influx of Mφ’s. We propose that, analogous to other chronic inflammatory lung diseases, such as pneumoconiosis (50), overactivation of alveolar Mφ’s results in the release of inflammatory mediators that promote destruction of lung tissue. In support of this concept, SMmice exhibited higher levels of IL-10, a potent downregulator of proinflammatory cytokines in vitro (51, 52) and in vivo (53). Although a significant increase in the examined proinflammatory cytokines was not documented in this study, there was a trend toward higher IFN-γ levels in MCmice. Taken together, the differences in cell recruitment and cytokine expression may have changed the immune response from a protective, effective immune response elicited by SM to a damage-driven inflammatory response elicited by MC.

SMmice manifested effective CMI that led to granuloma formation, whereas MCmice did not. In BALB/c mice, successful clearance of C. neoformans infection is ultimately dependent on a Th1 response, which has been associated with the ability to mount a DTH response against CneAg (31, 32). Despite a failure to clear pulmonary infection, MCmice were still able to elicit a strong DTH response to CneAg in the footpad (Figure 5e). A dichotomy between relative degrees of DTH and pulmonary resistance has been reported previously (32). In this study, it most likely reflects a failure of MCmice to locally activate lymphocytes in the inflamed lungs despite an increased number of recruited cells including CD4⁺ and CD8⁺ lymphocytes. In support of this concept, we observed differences in cytokine profiles in MC-infected lungs. Despite increased cell recruitment in MC-infected lungs, we found significantly lower levels of cytokines that are critical for the development of protective T cell immunity (54, 55), namely, IL-2, IL-4, and TNF-α (Table 5). Because most of these cytokines are produced by more than one cell type, we cannot determine which cells contributed to the observed levels. Nonetheless, as antigen-presenting cells, alveolar Mφ’s play a pivotal role in orchestrating CMI, and, as such, they directly or indirectly control the production of cytokines such as TNF-α, IL-12, and IFN-γ (49, 56–58). In this regard, C. neoformans–laden monocytes can interact with T cells and promote the secretion of proinflammatory cytokines (38), and capsular polysaccharide can inhibit T cell proliferation by downregulating IL-2 production (59). Based on our results, we propose a model whereby altered GXM size in MC cells renders them more resistant to phagocytosis and impairs antigen presentation and lymphocyte activation. This in turn causes an upregulation of chemokines, which then increases recruitment of cells and promotes a damage-driven noneffective immune response that leads to rapid demise.

Phenotypic switching has been thoroughly studied in Candida albicans (19, 20) and, most recently, in Candida glabrata (21). In Candida the newly generated colony types are also associated with changes in virulence when reinoculated into animals. The switching C. albicans strain 3153A and C. neoformans strain 24067a exhibit an association, but no clear correlation, between karyotype changes and switching (23, 60). The variant RC-2 described here has a stable chromosome length change of one chromosome that differs from the original 24067 pattern (13) but does not manifest karyotype changes with switching. Similarly, no changes were detected in RFLP patterns of genomic DNA hybridized with CNRE when SM and MC variants were compared. Although the mechanism(s) responsible for switching in C. neoformans is not known, this process may occur without DNA rearrangement. In Candida, epigenetic mechanisms such as SIR2-mediated silencing and HDA1-mediated acetylation of histones play a role in regulating switching frequencies (60, 61).

In summary, C. neoformans provides an excellent model system to study the contribution of phenotypic switching to virulence, because chronic infections can be established by innoculation with a relatively low number of C. neoformans cells. This allowed us to demonstrate that phenotypic switching occurs in vivo, and that it affects the outcome of infection. Enhanced virulence in our model was the result of a dysfunctional immune response to an altered polysaccharide capsule as a consequence of phenotypic switching. The importance of the occurrence of phenotypic switching in vivo is that it regulates key virulence factors and leaves the host at risk for progression without the change of host immunity.

We thank D. Fries for help with the Poisson analysis, A. Rosas for assistance with phagocytosis assays, J. Nosanchuk for measuring cell charge, and L. Pirofski for critical review of the manuscript. This work was supported in part by NIH (RO-1-AI33774, AI3342, HL59842 to A. Casadevall, and KO8-AI01570 to B.C. Fries), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 99/00185-1 to C.P. Taborda), and Howard Hughes Medical Institute (postdoctoral fellowship to B.C. Fries).

See the related Commentary beginning on page 1577.

1. Mitchell, T, Perfect, J. Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995. 8:515-548.
   View this article via: PubMed Google Scholar
2. Kwon-Chung, K, Polacheck, J, Popkin, T. Melanin lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol 1982. 150:1414-1421.
   View this article via: PubMed Google Scholar
3. Kwon-Chung, KJ, Edman, JC, Wickes, BL. Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 1992. 60:602-605.
   View this article via: PubMed Google Scholar
4. Cox, GM, et al. Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. Mol Microbiol 2001. 39:166-175.
   View this article via: PubMed CrossRef Google Scholar
5. Odom, A, et al. Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 1997. 16:2576-2589.
   View this article via: PubMed CrossRef Google Scholar
6. Bulmer, GS, Sans, MD. Cryptococcus neoformans. III. Inhibition of phagocytosis. J Bacteriol 1968. 95:5-8.
   View this article via: PubMed Google Scholar
7. Granger, DL, Perfect, JR, Durack, DT. Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide. J Clin Invest 1985. 76:508-516.
   View this article via: JCI PubMed CrossRef Google Scholar
8. Perfect, JR. Fungal virulence genes as targets for antifungal chemotherapy. Antimicrob Agents Chemother 1996. 40:1577-1583.
   View this article via: PubMed Google Scholar
9. D’Souza, CA, et al. Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol Cell Biol 2001. 21:3179-3191.
   View this article via: PubMed CrossRef Google Scholar
10. Alspaugh, JA, Perfect, JR, Heitman, J. Cryptococcus neoformans mating and virulence are regulated by the G- protein alpha subunit GPA1 and cAMP. Genes Dev 1997. 11:3206-3217.
    View this article via: PubMed CrossRef Google Scholar
11. Cherniak, R, Morris, L, Belay, T, Spitzer, E, Casadevall, A. Variation in the structure of glucuronoxylomannan in isolates from patients with recurrent Cryptococcal meningitis. Infect Immun 1995. 63:1899-1905.
    View this article via: PubMed Google Scholar
12. Currie, B, et al. Sterol compositions and susceptibilities to amphotericin B of environmental Cryptococcus neoformans isolates are changed by murine passage. Antimicrob Agents Chemother 1995. 39:1934-1937.
    View this article via: PubMed Google Scholar
13. Franzot, SP, et al. Microevolution of a standard strain of Cryptococcus neoformans resulting in differences in virulence and other phenotypes. Infect Immun 1998. 66:89-97.
    View this article via: PubMed Google Scholar
14. Chen, LC, Casadevall, A. Variants of a Cryptococcus neoformans strain elicit different inflammatory responses in mice. Clin Diagn Lab Immunol 1999. 6:266-268.
    View this article via: PubMed Google Scholar
15. Fries, BC, Casadevall, A. Serial isolates of Cryptococcus neoformans from patients with AIDS differ in virulence for mice. J Infect Dis 1998. 178:1761-1766.
    View this article via: PubMed CrossRef Google Scholar
16. Speed, B, Dunt, D. Clinical and host differences between infections with the two varieties of Cryptococcus neoformans. Clin Infect Dis 1995. 21:28-34.
    View this article via: PubMed Google Scholar
17. Reznick, DN, Shaw, FH, Rodd, FH, Shaw, RG. Evaluation of the rate of evolution in natural populations of guppies (Poecilia reticulata). Science 1997. 275:1934-1937.
    View this article via: PubMed CrossRef Google Scholar
18. Deitsch, KW, Moxon, ER, Wellems, TE. Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections. Microbiol Mol Biol Rev 1997. 61:281-293.
    View this article via: PubMed Google Scholar
19. Slutsky, B, et al. “White-opaque transition”: a second high-frequency switching system in Candida albicans. J Bacteriol 1987. 169:189-197.
    View this article via: PubMed Google Scholar
20. Slutsky, B, Buffo, J, Soll, DR. High-frequency switching of colony morphology in Candida albicans. Science 1985. 230:666-669.
    View this article via: PubMed CrossRef Google Scholar
21. Lachke, SA, Srikantha, T, Tsai, LK, Daniels, K, Soll, DR. Phenotypic switching in Candida glabrata involves phase-specific regulation of the metallothionein gene MT-II and the newly discovered hemolysin gene HLP. Infect Immun 2000. 68:884-895.
    View this article via: PubMed CrossRef Google Scholar
22. Goldman, DL, Fries, BC, Franzot, SP, Montella, L, Casadevall, A. Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents. Proc Natl Acad Sci USA 1998. 95:14967-14972.
    View this article via: PubMed CrossRef Google Scholar
23. Fries, BC, Goldman, DL, Cherniak, R, Ju, R, Casadevall, A. Phenotypic switching in Cryptococcus neoformans results in changes in cellular morphology and glucuronoxylomannan structure. Infect Immun 1999. 67:6076-6083.
    View this article via: PubMed Google Scholar
24. Schwan, TG, Hinnebush, BJ. Bloodstream- versus tick-associated variants of a relapsing fever bacterium. Science 1998. 280:1938-1940.
    View this article via: PubMed CrossRef Google Scholar
25. Ebert, D. Experimental evolution of parasites. Science 1998. 282:1432-1435.
    View this article via: PubMed CrossRef Google Scholar
26. Nosanchuk, JD, Casadevall, A. Cellular charge of Cryptococcus neoformans: contributions from the capsular polysaccharide, melanin, and monoclonal antibody binding. Infect Immun 1997. 65:1836-1841.
    View this article via: PubMed Google Scholar
27. Spitzer, ED, Spitzer, SG. Use of a dispersed repetitive DNA element to distinguish clinical isolates of Cryptococcus neoformans. J Clin Microbiol 1992. 30:1094-1097.
    View this article via: PubMed Google Scholar
28. Taborda, CP, Casadevall, A. Immunoglobulin M efficacy against Cryptococcus neoformans: mechanism, dose dependence, and prozone-like effects in passive protection experiments. J Immunol 2001. 166:2100-2107.
    View this article via: PubMed Google Scholar
29. Huffnagle, GB, et al. Leukocyte recruitment during pulmonary Cryptococcus neoformans infection. Immunopharmacology 2000. 48:231-236.
    View this article via: PubMed CrossRef Google Scholar
30. Murphy, JW, Schafer, F, Casadevall, A, Adesina, A. Antigen-induced protective and nonprotective cell-mediated immune components against Cryptococcus neoformans. Infect Immun 1998. 66:2632-2639.
    View this article via: PubMed Google Scholar
31. Huffnagle, GB, Yates, JL, Lipscomb, MF. Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4+ and CD8+ T cells. J Exp Med 1991. 173:793-800.
    View this article via: PubMed CrossRef Google Scholar
32. Huffnagle, GB, Yates, JL, Lipscomb, MF. T cell-mediated immunity in the lung: a Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice. Infect Immun 1991. 59:1423-1433.
    View this article via: PubMed Google Scholar
33. Vecchiarelli, A. Immunoregulation by capsular components of Cryptococcus neoformans. Med Mycol 2000. 38:407-417.
    View this article via: PubMed CrossRef Google Scholar
34. Vecchiarelli, A, et al. Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes. Infect Immun 1995. 63:2919-2923.
    View this article via: PubMed Google Scholar
35. Retini, C, et al. Capsular polysaccharide of Cryptococcus neoformans induces proinflammatory cytokine release by human neutrophils. Infect Immun 1996. 64:2897-2903.
    View this article via: PubMed Google Scholar
36. Vecchiarelli, A, et al. Purified capsular polysaccharide of Cryptococcus neoformans induces interleukin-10 secretion by human monocytes. Infect Immun 1996. 64:2846-2849.
    View this article via: PubMed Google Scholar
37. Lendvai, N, Qu, XW, Hsueh, W, Casadevall, A. Mechanism for the isotype dependence of antibody-mediated toxicity in Cryptococcus neoformans-infected mice. J Immunol 2000. 164:4367-4374.
    View this article via: PubMed Google Scholar
38. Vecchiarelli, A, Retini, C, Pietrella, D, Monari, C, Kozel, TR. T lymphocyte and monocyte interaction by CD40/CD40 ligand facilitates a lymphoproliferative response and killing of Cryptococcus neoformans in vitro. Eur J Immunol 2000. 30:1385-1393.
    View this article via: PubMed CrossRef Google Scholar
39. Mozaffarian, N, Casadevall, A, Berman, JW. Inhibition of human endothelial cell chemokine production by the opportunistic fungal pathogen Cryptococcus neoformans. J Immunol 2000. 165:1541-1547.
    View this article via: PubMed Google Scholar
40. Mody, CH, Syme, RM. Effect of polysaccharide capsule and methods of preparation on human lymphocyte proliferation in response to Cryptococcus neoformans. Infect Immun 1993. 61:464-469.
    View this article via: PubMed Google Scholar
41. Kozel, TR, Mastroianni, RP. Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. Infect Immun 1976. 14:62-67.
    View this article via: PubMed Google Scholar
42. Collins, H, Bancroft, G. Encapsulation of Cryptococcus neoformans impairs antigen-specific T cell responses. Infect Immun 1991. 59:3883-3886.
    View this article via: PubMed Google Scholar
43. Small, J, Mitchell, T. Strain variation in antiphagocytic activity of capsular polysaccharide from Cryptococcus neoformans serotype A. Infect Immun 1989. 57:3751-3756.
    View this article via: PubMed Google Scholar
44. Yasuoka, A, Kohno, S, Yamada, H, Kaku, M, Koga, H. Influence of molecular sizes of Cryptococcus neoformans capsular polysaccharide on phagocytosis. Microbiol Immunol 1994. 38:851-856.
    View this article via: PubMed Google Scholar
45. Mathee, K, et al. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 1999. 145:1349-1357.
    View this article via: PubMed Google Scholar
46. Syme, RM, Bruno, TF, Kozel, TR, Mody, CH. The capsule of Cryptococcus neoformans reduces T-lymphocyte proliferation by reducing phagocytosis, which can be restored with anticapsular antibody. Infect Immun 1999. 67:4620-4627.
    View this article via: PubMed Google Scholar
47. Syme, RM, Spurrell, JC, Ma, LL, Green, FH, Mody, CH. Phagocytosis and protein processing are required for presentation of Cryptococcus neoformans mitogen to T lymphocytes. Infect Immun 2000. 68:6147-6153.
    View this article via: PubMed CrossRef Google Scholar
48. Blackstock, R, Buchanan, KL, Adesina, AM, Murphy, JW. Differential regulation of immune responses by highly and weakly virulent Cryptococcus neoformans isolates. Infect Immun 1999. 67:3601-3609.
    View this article via: PubMed Google Scholar
49. Feldmesser, M, Kress, Y, Novikoff, P, Casadevall, A. Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect Immun 2000. 68:4225-4237.
    View this article via: PubMed CrossRef Google Scholar
50. Vanhee, D, Gosset, P, Boitelle, A, Wallaert, B, Tonnel, AB. Cytokines and cytokine network in silicosis and coal workers’ pneumoconiosis. Eur Respir J 1995. 8:834-842.
    View this article via: PubMed Google Scholar
51. de Waal Malefyt, R, Abrams, J, Bennett, B, Figdor, CG, de Vries, JE. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 1991. 174:1209-1220.
    View this article via: PubMed CrossRef Google Scholar
52. Levitz, SM, Tabuni, A, Nong, SH, Golenbock, DT. Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide. Infect Immun 1996. 64:945-951.
    View this article via: PubMed Google Scholar
53. Huaux, F, et al. Role of interleukin-10 in the lung response to silica in mice. Am J Respir Cell Mol Biol 1998. 18:51-59.
    View this article via: PubMed Google Scholar
54. Huffnagle, GB, et al. Afferent phase production of TNF-alpha is required for the development of protective T cell immunity to Cryptococcus neoformans. J Immunol 1996. 157:4529-4536.
    View this article via: PubMed Google Scholar
55. Buchanan, KL, Murphy, JW. Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. Infect Immun 1993. 61:2854-2865.
    View this article via: PubMed Google Scholar
56. Vecchiarelli, A, et al. Role of human alveolar macrophages as antigen-presenting cells in Cryptococcus neoformans infection. Am J Respir Cell Mol Biol 1994. 11:130-137.
    View this article via: PubMed Google Scholar
57. Levitz, SM. Macrophage-Cryptococcus interactions. Immunol Ser 1994. 60:533-543.
    View this article via: PubMed Google Scholar
58. Levitz, SM, et al. Variables affecting production of monocyte chemotactic factor 1 from human leukocytes stimulated with Cryptococcus neoformans. Infect Immun 1997. 65:903-908.
    View this article via: PubMed Google Scholar
59. Vecchiarelli, A, et al. Encapsulation of Cryptococcus neoformans regulates fungicidal activity and the antigen presentation process in human alveolar macrophages. Clin Exp Immunol 1994. 98:217-223.
    View this article via: PubMed Google Scholar
60. Perez-Martin, J, Uria, JA, Johnson, AD. Phenotypic switching in Candida albicans is controlled by SIR2 gene. EMBO J 1999. 18:2580-2592.
    View this article via: PubMed CrossRef Google Scholar
61. Klar, AJ, Srikantha, T, Soll, DR. A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans. Genetics 2001. 158:919-924.
    View this article via: PubMed Google Scholar