Lack of an atypical PDR transporter generates an immunogenic Cryptococcus neoformans strain that drives a dysregulated and lethal immune response in murine lungs.

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for >150,000 deaths every year with a mortality rate as high as 81%. This high medical burden is due, in part, to an incomplete understanding of its pathogenesis. In a previous study, we identified a cryptococcal atypical pleiotropic drug resistance (PDR) transporter, PDR6, that regulated antifungal resistance and host interactions. Here, we follow-up on the role of PDR6 in cryptococcal virulence. In vivo, mice infected with the pdr6Δ strain display altered symptomatology and disease progression. Specifically, we observed a significant increase in the innate immune cell populations in the pdr6Δ-infected mice when compared to their WT-infected littermates. Furthermore, quantification of pulmonary cytokines/chemokines revealed a robust increase of pro-inflammatory cytokines in mice infected with the pdr6Δ mutant strain. Whereas antifungal treatment of pdr6Δ-infected animals did not affect survival, treatment with a corticosteroid significantly extended survival, highlighting the importance of a balanced/controlled host immune response. We determined that the hyper-inflammatory immune response occurs, in part, because the loss of the Pdr6 transporter indirectly alters the cryptococcal cell wall architecture and results in the increased exposure of chitin, ß-glucan, and other cryptococcal-specific pathogen associated molecular patterns. Taken together, this study provides clinical insights regarding cryptococcal pathogenesis while also providing additional functions of PDR-type ATP-binding cassette (ABC) transporters in pathogenic fungi.

An Atypical ABC Transporter Is Involved in Antifungal Resistance and Host Interactions in the Pathogenic Fungus Cryptococcus neoformans.

ATP-binding cassette (ABC) transporters represent one of the largest protein superfamilies. Functionally diverse, ABC transporters have been implicated in many aspects of microbial physiology. The genome of the human fungal pathogen Cryptococcus neoformans encodes 54 putative ABC transporters and most of them remain uncharacterized. In a previous genetic screen for fungal regulators of phagocytosis, we identified an uncharacterized gene, CNAG_06909, that modulates host interactions. This gene encoded a half-size ABC transporter of the PDR-type, and phenotypic studies of a strain with this gene deleted revealed an altered antifungal susceptibility profile, including hypersensitivity to fluconazole (FLC). This gene, which we named PDR6, localized to the endoplasmic reticulum (ER) and plasma membrane (PM), and when absent, less ergosterol was observed in the PM. Additionally, we observed that the pdr6Δ strain displayed a reduction in secreted polysaccharide capsular material. These changes to the cellular surface may explain the observed increased uptake by macrophages and the reduced intracellular survival. Finally, studies in mice demonstrated that Pdr6 function was required for the normal progression of cryptococcal infection. Taken together, this study demonstrates a novel dual role for PDR transporters in C. neoformans, which could represent a potential target for antifungal therapeutics. Furthermore, the atypical half-size transporter encoded by PDR6 is conserved in many fungal pathogens, but absent in model nonpathogenic fungi. Hence, this study provided a function for this unique group of fungal half-size PDR transporters that, although conserved, remain largely understudied. IMPORTANCE Conserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or anti-cancer drugs. Despite their importance, they are understudied in fungal pathogens. In the environmental fungus Cryptococcus neoformans, a leading cause of fungal infections, only a few ABC transporters have been studied. Here, we characterized an atypical, half-size, ABC transporter of the PDR-type, that affected both antifungal resistance and host-pathogen interactions. PDR-type transporters are only present in fungi and plants, and this subgroup of half-size transporters was conserved in fungal pathogens, yet their function was completely unknown. Because the current treatments for cryptococcal infection are suboptimal, understanding the mechanisms of antifungal resistance and the host interactions that drive the infection is critical to improving the management of this disease. Here, we provide insights into these important aspects of cryptococcal pathogenesis.