The role of a pleiotropic drug resistance (PDR) transporter in the cryptococcal-host interactions

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PROJECT SUMMARY / ABSTRACT Cryptococcus neoformans is one of the most common fungal pathogens, responsible for over 200,000 deaths yearly, mostly in the immunocompromised. Unfortunately, due to medical advances such as chemother- apy or steroid treatments, this population of susceptible individuals is increasing. Despite the increasing burden of cryptococcal disease, there are only three clinically available therapeutics, and they are plagued by cost, toxicity, and recently, antifungal resistance. Making the situation even more challenging, the pathogenesis of Cryptococcus is incompletely understood, limiting the number of potential targets or processes that could be used as intervention points. All of this has resulted in a mortality rate that ranges from ~20% in the US and Europe, to >71% in Africa and Southeast Asia. Therefore, there is a clear need to improve the current anticryp- tococcal drugs and to better understand the fungal-host interactions, which could lead to novel and more effective treatments. This application addresses both of these needs. The fungal-host interactions, particularly the inter- actions with the lung macrophages, will determine the outcome of the infection. While investigating the interactions between cryptococci and macrophages, we identified an uncharacterized fungal gene that affects phagocytosis by macrophages. Analysis of the amino acid sequence of this gene, which we are calling PDR6, shows that it contains all the features of ABC transporters of the PDR family, which are usually efflux pumps. Consistently, this mutant is hypersensitive to fluconazole, the mainstay treatment for cryptococcosis, and shows an altered antifungal profile relative to wild-type. Notably, infection of a murine model with this mutant results in attenuation and altered progression of the disease. This proposal is organized to study these two phenotypes: altered antifungal responses (Aim 1) and altered virulence and host interactions (Aim 2). Hence, we are tackling the two general problems that are currently preventing an appropriate management of this disease. In Aim 1 we will elucidate the function of Pdr6 and determine how it affects the sensitivity of the cell to fluconazole and other antifungals. In Aim 2 we will identify the Pdr6-dependent changes in the fungal cell and the host that result in altered host interactions and attenuated virulence. These two aims, although independent, are complimentary, and will be performed in parallel. Completion of the aims proposed will result in a significant advancement of both (1) our understanding of ABC transporter functions in general, and specifically in Cryptococcus, and (2) our understanding of fungal-host interactions and their contribution to virulence. These results will synergize with the other projects in the lab, and will open multiple new directions of research that will be used for future grant applications. Interestingly, close homologs of PDR6 are present in many pathogenic fungi, but are absent from the model non-pathogenic ones, hence the discoveries here will have broad impact in the fungal field.