The primary target organ of Cryptococcus gattii is different from that of Cryptococcus neoformans in a murine model

Abstract

Cryptococcosis is caused by the opportunistic pathogen Cryptococcus neoformans or by the primary pathogen Cryptococcus gattii. Epidemiological studies suggest that patients infected with C. gattii mainly present with pulmonary disease, while those infected with C. neoformans commonly manifest meningoencephalitis. We compared the pathogenesis of the two species using the C. neoformans H99 and C. gattii R265 strains in a murine inhalation model. C. neoformans grew faster in the brain and caused death by meningoencephalitis, while C. gattii grew faster in the lungs and caused death without producing fulminating meningoencephalitis. Despite the consistent failure to recover R265 cells from blood, a fraction of the R265 population was detected in the extrapulmonary organs, including the brain. Upon intravenous (i.v.) inoculation of 10(4) cells via the tail vein, however, C. gattii produced severe meningoencephalitis, demonstrating that C. gattii cells can efficiently cross the blood-brain barrier. Interestingly, i.v. inoculation with five cells caused brain infection in only 10% of C. gattii-infected mice, compared to 60% of mice infected with C. neoformans. In mice that had been initially inoculated via the pulmonary route and subsequently challenged intravenously, a protective effect was observed only in mice infected with C. gattii. C. neoformans cells grew 10 to 100 times faster than C. gattii cells in blood or serum collected from naive mice. The paucity of meningoencephalitis upon inhalation of C. gattii, therefore, may be partly due to an unknown factor(s) in the host's blood coupled with immune protection that reduces dissemination to the brain and fosters lung infection.

Importance: While Cryptococcus neoformans is the most common cause of fatal meningoencephalitis, especially in HIV patients, Cryptococcus gattii causes disease mainly in non-HIV patients. Clinical studies revealed that most patients infected with C. gattii VGII strains have lung infections with minimal brain involvement. Despite extensive clinicopathological studies on cryptococcosis in animal models, only a few have included C. gattii. We compared the pathogenesis of the two species in mice using an inhalation model. Similar to infection in humans, even though C. gattii can cross the blood-brain barrier, it failed to cause fatal meningoencephalitis but caused fatal lung infection. We show that growth of C. gattii in mouse blood is significantly slower than that of C. neoformans and that a secondary protective phenomenon, though weak, manifests itself only in C. gattii infection. Our study provides a model for understanding the clinicopathological differences between these two closely genetically related pathogens.

FIG 1

Heavy brain fungal burden is found only in mice infected with H99, not in those infected with R265. C57BL/6 mice were inoculated by intrapharyngeal (i.p.) aspiration of 50,000 yeast cells/mouse. At least 3 mice were sacrificed at each time point for fungal load determination. (A) Survival curves were obtained from a study of 10 mice per group, which was independent of the time course study. (B to F) CFU in each organ at the indicated time points. Error bars show 2 standard deviations (SD). *, P < 0.05.

FIG 2

R265 causes severe brain infection in an intravenous (i.v.) model. BALB/c mice were inoculated with 50,000 cells/mouse of H99 or R265 via the i.v. route. At least 3 mice each were sacrificed at each time point for the determination of the fungal tissue load. Survival curves (A) were generated from the results obtained with 10 mice per group, which are independent of the time course study (B and C). Error bars show 2 SD. *, P < 0.05.

FIG 3

H99 is more virulent than R265 when mice are inoculated with very low numbers of cells i.v. Eight to ten BALB/c mice were inoculated i.v. with approximately 5 or 50 cells of H99 or R265, and the survival of mice was monitored.

FIG 4

Lower growth of R265 than H99 in murine blood and serum. Blood or serum was collected from naive C57BL/6 mice. Yeast cells were diluted and incubated in 100 µl of whole blood, serum, or RPMI with 5% CO₂ at 37°C for the indicated period, and numbers of CFU were recorded. Error bars show 1 SD. *, P < 0.05.

FIG 5

Preintrapulmonary infection slightly prolongs the survival of mice subsequently infected with R265 via i.v. inoculation. Five to eight BALB/c mice per group were used. Mice were first inoculated i.p. with approximately 50 cells of H99 or R265. After 2 weeks, the preinfected and naive mice were injected i.v. with approximately 500 or 5,000 cells of H99 or R265. The survival of the mice was recorded.