Host Carbon Dioxide Concentration Is an Independent Stress for Cryptococcus neoformans That Affects Virulence and Antifungal Susceptibility

OBSERVATION

Cryptococcus neoformans is one of the most important human fungal pathogens and causes meningoencephalitis (CME). Recent estimates indicate that 223,000 new cases of CME occur each year with an annual mortality of 181,000 (1); the majority of CME disease affects people infected with HIV (2). Cryptococcus species are environmental yeasts that occupy a variety of niches, and therefore, Cryptococcus must transition from an environmental niche to the mammalian host to cause disease (3). Most strains isolated from the environment are much less virulent in animal models than strains isolated from human patients. For example, Litvintseva and Mitchell (4) found that only one out of 10 environmental strains caused mortality in a murine model of cryptococcosis by 60 days while 5/7 clinical strains caused lethal infection by 40 days. In vitro, all of the strains grew at 37°C and generated comparable levels of capsule and melanin (4).

Recently, Mukaremera et al. systematically characterized a set of genetically similar strains associated with highly variable patient outcomes (5). The virulence trends observed in patients were recapitulated in the inhalational murine model, providing important validation of the model as predicative of clinical virulence. Like the work of Litvintseva and Mitchell (4), extensive phenotyping of the strains did not reveal an in vitro phenotype that correlated with virulence (5). These data strongly indicate that uncharacterized virulence properties beyond the “big three” of host body temperature tolerance, melanization, and capsule formation play an important role in determining the virulence potential of a given cryptococcal strain (6).

We hypothesized that the host environment may contain additional stresses under which clinical/pathogenic isolates are fitter than environmental/nonpathogenic isolates. One dramatic difference between terrestrial and host environments is the concentration of carbon dioxide (CO₂): ambient air is ∼0.04% CO₂ while the CO₂ concentration in mammalian tissues is 125-fold higher (5%). In ambient air, Cryptococcus, like many yeasts, expresses carbonic anhydrase (Can2) which catalyzes the generation of essential HCO₃⁻ under low CO₂ concentrations. At 5% CO₂ in the host, CAN2 expression is repressed presumably due to sufficient levels of HCO₃⁻ produced by dissolved CO₂. Consequently, CAN2 is dispensable for growth at 5% CO₂ and virulence in a murine host (7). Host CO₂ concentrations, however, have a profound effect on C. neoformans biology because they induce capsule formation (8). Indeed, our hypothesis that host concentrations of CO₂ are a significant stress to C. neoformans is supported by the fact that Granger et al. noted that the growth of cultures slowed considerably after being shifted to host CO₂ concentrations to induce capsule formation (9). In addition, C. neoformans strains lacking calcineurin, a key stress response regulator, are hypersensitive to CO₂ (10). Finally, Bahn et al. found that elevated concentrations of CO₂ inhibit C. neoformans mating (7). Taken together, tolerance of host CO₂ concentrations seemed to us a potentially important independent trait of C. neoformans strains that cause disease in mammals.

To test the hypothesis that CO₂ tolerance may play a role in distinguishing between clinical and environmental strains of C. neoformans, we examined the CO₂ fitness of a set of 12 strains that had been previously characterized in the mouse pulmonary infection model by Litvintseva and Mitchell (4). The clinical and reference strain H99 as well as three other clinical strains grew similarly in the presence and absence of CO₂ on RPMI medium buffered to pH 7 with morpholinepropanesulfonic acid (MOPS) while all but one of the environmental strains displayed a growth defect in 5% CO₂ (Fig. 1A). The serotype D reference strain JEC21 is also CO₂ sensitive relative to H99 and the clinical isolates. Of 10 additional clinical strains isolated from patients at Duke University (gift of John Perfect), 8 had similar growth at ambient and host CO₂ concentrations (see Fig. S1 in the supplemental material). Of the 12 strains that were examined by Litvintseva and Mitchell in animal models (4), only those that were CO₂ tolerant caused disease by 60 days. The only environmental strain to be CO₂ tolerant was also the only one to cause disease.

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FIG 1

In vitro CO₂ tolerance correlates with cryptococcal virulence in insect and mammalian hosts. (A) Clinical and environmental C. neoformans isolates were cultured on RPMI medium buffered to pH 7 with MOPS at 37°C in ambient air (∼0.04% CO₂) or in 5% CO₂. The information about the genotype of the strains and the median survival days of mice infected by these strains intranasally was obtained from a previous study (4). (B) Competition assay of isolate A4-34-6 and mCherry-labeled H99 cultured on RPMI medium buffered to pH 7 with 165 mM MOPS in ambient air or in 5% CO₂. P < 0.00001, Student’s t test. (C) Cell suspensions of H99 (A), JEC21 (D), and XL1462 (AD hybrid) at equal concentrations were spotted onto RPMI medium and incubated at 37°C in ambient air or in 5% CO₂. (D and E) CD-1 mice were inoculated with H99, CO₂-tolerant environmental strain A1-84-14, and CO₂-sensitive strain A1-38-2 (5 to 7 animals per group) by tail vein injection. Five animals were sacrificed at 5 days for brain fungal burden, and 10 animals per group were monitored for morbidity until day 21 (D); asterisks indicate statistically significant difference between indicated groups by Student’s t test of log-transformed data. Survival curves were analyzed by Kaplan-Meier/log rank test (E). (F) The great wax moth Galleria mellonella larvae in the final-instar larval stage (10 to 15 per group) were injected with the indicated clinical or environmental isolates via the last left proleg. The survival rate of the infected larvae over days postinoculation is shown. NS, nonsignificant; ****, P < 0.0001 compared to H99 by log rank test.

To obtain a more quantitative measure of the in vitro fitness advantage of a CO₂-tolerant strain over a CO₂-sensitive strain, we carried out a competition experiment in which the mCherry-labeled H99 strain and unlabeled, environmental strain A4-34-6 were cocultured as a 1:1 mixture in ambient air or 5% CO₂; the ratio of H99 to A4-34-6 was determined by microscopy. A4-34-6 has a 5-fold fitness defect relative to H99 in ambient air, and that defect is increased to 50-fold in 5% CO₂ (Fig. 1B). To determine if CO₂ tolerance is recessive or dominant, the previously generated diploid AD hybrid was compared to its parental strains serotype A H99 and serotype D JEC21 (11). The diploid AD hybrid is CO₂ tolerant, indicating that the trait is dominant (Fig. 1C). Consistent with other yeasts (12), CO₂ is fungistatic and dose dependent (data not shown). Because CO₂ and HCO₃⁻ are substrates in reactions of central carbon metabolism (12), we wondered if increasing the glucose concentration of RPMI from 0.2% to 2% would affect CO₂ sensitivity; however, it had no effect on the growth of the sensitive strains (Fig. S1).

As reported by Litvintseva and Mitchell (4), all clinical strains caused lethal infections in mice within 40 days while the only environmental strain to cause a lethal infection within 60 days was the CO₂-tolerant strain A1-84-14 (Fig. 1A). To determine if the virulence differences between the CO₂-tolerant and CO₂-sensitive strains were dependent on the infection model, the environmental CO₂-tolerant and CO₂-sensitive strains were compared in both the intravenous model of disseminated murine cryptococcosis and the Galleria mellonella model. Five days postinfection (Fig. 1D), the fungal brain burden was 2 log₁₀ CFU/g lower in the CO₂-sensitive environmental strain than the CO₂-tolerant environmental strain, while the CO₂-tolerant environmental strain was similar to the reference strain H99. Consistent with the pulmonary infection model data, the median survival of mice infected with the CO₂-tolerant environmental strain is modestly longer than that of mice infected with the highly virulent H99 reference strain (Fig. 1E). Mice infected with the CO₂-sensitive strain, in contrast, were asymptomatic for an additional week. The fungal burden for the CO₂-sensitive-strain-infected mice had increased 1.7 log₁₀ over the 16 days (Fig. S3), indicating that the environmental strain replicated very slowly in the brain. Finally, G. mellonella larvae infected with CO₂-sensitive strains showed prolonged survival relative to larvae infected with CO₂-tolerant strains (Fig. 1F). Taken together, these data strongly support a correlation between CO₂ tolerance and virulence in multiple models of cryptococcal infection.

CO₂ levels affect the composition and function of cellular membranes in a variety of biological systems, which is proposed to be one possible mechanism of CO₂-mediated growth inhibition (12, 13). The two most important anticryptococcal drugs, amphotericin B and fluconazole, affect membrane-related processes (14), and thus, we hypothesized that CO₂ may modulate antifungal susceptibility. The MIC was determined using Etest strips on solid agar RPMI medium buffered to pH 7 with 165 mM MOPS. The MIC for amphotericin B was not affected by CO₂ in any of the strains tested (≤2-fold change between ambient air and 5% CO₂ [Fig. 2A and Fig. S4B]). In contrast, the fluconazole MIC decreased approximately 8- to 10-fold (4 μg/ml to 0.5 μg/ml) for H99, CO₂-tolerant clinical isolates (C23 and C27), and environmental (A1-84-14) isolates in the presence of 5% CO₂ (Fig. 2B and Fig. S4B). The MICs of both itraconazole (Fig. 2B) and voriconazole (Fig. S4B) are decreased at 5% CO₂, indicating that the effect is not limited to fluconazole. Five percent CO₂ has no effect on the MIC of fluconazole against the C. albicans reference strain SC5314 (2 μg/ml). The effect of CO₂ is also not specific to ergosterol biosynthesis inhibitors in that the sphingolipid biosynthesis inhibitor myriocin is also more active at host CO₂ levels than in ambient air (Fig. 2B).

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FIG 2

CO₂ affects cryptococcal susceptibility to antifungals. (A) The CO₂-tolerant cryptococcal isolates, including the reference and clinical isolate H99, the clinical isolate C27, and the environmental isolate A1-84-14, are more susceptible to fluconazole in 5% CO₂ than ambient air, but amphotericin B susceptibility is unaffected. Suspensions of C. neoformans isolates were spread onto RPMI agar medium. Etest strips with fluconazole (FL) or amphotericin B (AP) were placed on top of the air-dried yeast lawn. The cells were then incubated at 37°C in ambient air or in 5% CO₂. (B) H99 is more susceptible to fluconazole, itraconazole, and myriocin in 5% CO₂ by disk diffusion assay. H99 cells were spread onto RPMI agar medium. Disks containing fluconazole (4 μg), itraconazole (4 μg), myriocin (0.8 μg), or DMSO (control) were air dried and placed on top of the yeast lawn. Cells were incubated at 37°C for 2 days in ambient air or in 5% CO₂. The size of the halo surrounding the disks correlates with cryptococcal susceptibility to the drugs.

The physiological mechanism underlying the differential sensitivity of Cryptococcus strains to host levels of CO₂ awaits further study. A variety of potential mechanisms for the fungistatic and bacteriostatic effect of CO₂ have been proposed, including altered membrane fluidity and inhibition of biosynthetic reactions involving CO₂ (12). Despite these outstanding mechanistic questions, the effects of host concentrations of CO₂ on C. neoformans virulence and antifungal susceptibility have two important implications. First, CO₂ tolerance appears to be an independent virulence feature of pathogenic strains of C. neoformans that correlates with variations in mammalian virulence. It will be interesting to expand this analysis to determine if CO₂ tolerance contributes to the variation in patient outcome (5). Based on fungal burden data reported by Litvintseva and Mitchell (4), the CO₂-sensitive strains cause a significantly lower lung burden than CO₂-tolerant strains while the brain burden for CO₂-sensitive strains is almost undetectable at postinfection day 60. Although the CO₂-sensitive strain has reduced virulence in the intravenous model, which rapidly establishes central nervous system (CNS) infection, it is able to replicate within the brain. Taken together, these preliminary and previously published data (4) indicate that CO₂ tolerance may play a more important role in dissemination from the lung than in replication within the brain.

Second, the profound effect of host CO₂ concentrations on in vitro azole activity identifies a potential limitation of current antifungal drug susceptibility testing conditions in predicting the outcomes of patients treated with azoles. Indeed, the lack of correlation between MICs generated by standardized antifungal susceptibility testing assays and clinical outcomes for cryptococcal infections has been well described, and a number of potential explanations for this discrepancy have been proposed (15). Our data suggest that CO₂ is likely to be an important factor in drug susceptibility. Although the mechanism of this effect will require additional investigation, it is unlikely that high CO₂ concentrations directly reduce ergosterol levels because strains with reduced ergosterol content typically have reduced susceptibility to amphotericin B, a drug that binds directly to ergosterol. Regardless of the mechanism, the use of host CO₂ concentrations may represent a simple adjustment that could improve the correlation between MIC and clinical outcome.