Pulmonary Iron Limitation Induced by Exogenous Type I IFN Protects Mice from Cryptococcus gattii Independently of T Cells.

Cryptococcus neoformans causes deadly mycosis primarily in AIDS patients, whereas Cryptococcus gattii infects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence of C. gattii As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome of C. neoformans and C. gattii infections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. In C. neoformans-infected mice, pICLC activity was associated with C. neoformans containment and classical Th1 immunity. In contrast, pICLC activity against C. gattii did not require any immune factors previously associated with C. neoformans immunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly, C. gattii pICLC activity depended on ß-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggesting C. gattii pICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcus in vitro, suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection by C. gattii and C. neoformans but by distinct mechanisms; the C. gattii effect was mediated by iron limitation, while the effect on C. neoformans infection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for different Cryptococcus species suggests a possible mechanism by which HIV infection may select against C. gattii but not C. neoformansIMPORTANCECryptococcus neoformans and Cryptococcus gattii cause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species, C. gattii infects very few AIDS patients, while C. neoformans infection is an AIDS-defining illness, suggesting that the host response to HIV selects C. neoformans over C. gattii We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action against C. neoformans was due to activation of well-defined immune pathways known to deter C. neoformans, whereas these immune pathways were dispensable for pICLC treatment of C. gattii Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to control C. neoformans infection but is protective against C. gattii Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibited C. gattii, thus suggesting an entirely new mode of nutritional immunity activated by viral signals.

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Azole heteroresistance in Cryptococcus neoformans: emergence of resistant clones with chromosomal disomy in the mouse brain during fluconazole treatment.

We have previously reported that Cryptococcus neoformans strains are innately heteroresistant to fluconazole in vitro, producing minor, highly resistant subpopulations due to adaptive formation of disomic chromosomes. Using a mouse model, we assessed the emergence of heteroresistant clones in the brain during fluconazole treatment and found that the occurrence of heteroresistant clones in vivo with chromosomal disomy is strain dependent. Interestingly, emergence of heteroresistant clones in vivo was unrelated to the strain's MIC to fluconazole.