Characterization of the Cellular and Immunological Functions of an Atypical PDR-Type Transporter in the Human Fungal Pathogen Cryptococcus neoformans

Fungal infections kill close to 3.8 million people yearly, with the biggest burden in resource-limited regions. One of the leading causes of fungal infections is the environmental, opportunistic pathogen, Cryptococcus neoformans, which is also one of the leading causes of death in the HIV+ population. Moreover, with recent medical advances, the population at risk of developing disease from this fungus is increasing, hence C. neoformans represents a serious neglected health concern. Given that cryptococcal infection is becoming more widespread and that current therapies are inappropriate, C. neoformans is on the top priority group of the WHO’s list of fungal pathogens threatening human health, with a mortality rate as high as 81%. Hence, there is a clear need for a better understanding of the cryptococcal-host interactions that can lead to novel and more effective therapeutics. Previous clinical studies have correlated fungal uptake by macrophages with mortality, hence we performed a screen of 1,200 C. neoformans single-gene deletions for fungal regulators of phagocytosis. One of the mutants identified with increased uptake was missing the gene CNAG_06909. We have called this uncharacterized gene PDR6 due to similarities to Pleiotropic Drug Resistance genes in other fungi and hypothesized that it may play a role in the virulence of C. neoformans. Sequence analysis showed that PDR6 encodes a unique half-size ATP-binding cassette (ABC) transporter protein. Microbial ABC transporters often play a role in the efflux of substrates across a membrane and have specifically been implicated in the development of antimicrobial resistance. Consistently, we found that the pdr6? strain was hypersensitive to fluconazole (FLC), the most common antifungal drug. In fact, antifungal ETEST strips and broth dilution assays with other antifungals demonstrated that the pdr6? strain had altered antifungal profiles relative to WT control, particularly against the azole-class antifungals. Additionally, we found that the pdr6? strain has significantly less ergosterol in its plasma membrane when compared to the WT, which could explain the hypersensitivity to azoles, defects in biofilm formation, capsule shedding, and host recognition exhibited by this mutant. Cellularly, these results are consistent with a model where Pdr6 regulates ergosterol transport into the plasma membrane, affecting both azole resistance as well as host-fungal interactions. Given that PDR6 regulates the interaction with macrophages, which subsequently influences the outcome of the disease, we next characterized the immunological functions of PDR6 in vivo. We found that animals infected with the pdr6? strain lived significantly longer than those infected with the WT fungus. Although analysis of the colony forming units over the course of infection revealed reduced growth and dissemination of the pdr6? strain, histological analysis of pulmonary tissue identified increased damage and inflammation in the lungs of the pdr6?-infected mice at late time points when compared to the WT control. These findings were further supported by quantification of immune cells and pro-inflammatory cytokines. Additionally, survival was significantly extended in the pdr6?-infected mice when the immune response was dampened using the corticosteroid dexamethasone. Together, these data support our hypothesis that the pdr6?-infected mice succumb to infection because of pneumonia and an uncontrolled pro-inflammatory immune response in the lungs rather than the typical meningitis associated with the WT fungus. This highlights the importance of the biological function of Pdr6 in the progression and development of cryptococcal meningitis. Clinically, given its indirect involvement in antifungal resistance, PDR6 represents a promising and novel therapeutic target in treating cryptococcal meningitis. Furthermore, determining the cellular and immunological functions of PDR6 will not only open new avenues of investigation in the cryptococcal field, but also affect the PDR transporter field since these half-size, atypical transporters are conserved in evolution but their function has not been characterized.