Candida albicans' inorganic phosphate transport and evolutionary adaptation to phosphate scarcity

Phosphorus is essential in all cells structural, metabolic and regulatory functions. For fungal cells that import inorganic phosphate (Pi) up a steep concentration gradient, surface Pi transporters are critical capacitators of growth. Fungi must deploy Pi transporters that enable optimal Pi uptake in pH and Pi concentration ranges prevalent in their environments. Single, triple and quadruple mutants were used to characterize the four Pi transporters we identified for the human fungal pathogen Candida albicans, which must adapt to alkaline conditions during invasion of the host bloodstream and deep organs. A high-affinity Pi transporter, Pho84, was most efficient across the widest pH range while another, Pho89, showed high-affinity characteristics only within one pH unit of neutral. Two low-affinity Pi transporters, Pho87 and Fgr2, were active only in acidic conditions. Only Pho84 among the Pi transporters was clearly required in previously identified Pi-related functions including Target of Rapamycin Complex 1 signaling and hyphal growth. We used in vitro evolution and whole genome sequencing as an unbiased forward genetic approach to probe adaptation to prolonged Pi scarcity of two quadruple mutant lineages lacking all 4 Pi transporters. Lineage-specific genomic changes corresponded to divergent success of the two lineages in fitness recovery during Pi limitation. In this process, initial, large-scale genomic alterations like aneuploidies and loss of heterozygosity were eventually lost as populations presumably gained small-scale mutations. Severity of some phenotypes linked to Pi starvation, like cell wall stress hypersensitivity, decreased in parallel to evolving populations fitness recovery in Pi scarcity, while that of others like membrane stress responses diverged from these fitness phenotypes. C. albicans therefore has diverse options to reconfigure Pi management during prolonged scarcity. Since Pi homeostasis differs substantially between fungi and humans, adaptive processes to Pi deprivation may harbor small-molecule targets that impact fungal growth and virulence. Author SummaryFungi must be able to access enough phosphate in order to invade the human body. Virulence of Candida albicans, the most common invasive human fungal pathogen, is known to decrease when one of the proteins that brings phosphate into the fungal cell, called Pho84, is disabled. We identified three more proteins in C. albicans that transport phosphate into the cell. We found that Pho84 plays the largest role among them across the broadest range of environmental conditions. After eliminating all 4 of these transporters, we let two resulting mutants evolve for two months in limited phosphate and analyzed the growth and stress resistance of the resulting populations. We analyzed genomes of representative populations and found that early adaptations to phosphate scarcity occurred together with major changes to chromosome configurations. In later stages of the adaptation process, these large-scale changes disappeared as populations presumably gained small-scale mutations that increased cells ability to grow in limited phosphate. Some but not all of these favorable mutations improved resistance of evolving populations to stressors like membrane- and cell wall stress. Pinpointing distinct mutation combinations that affect stress resistance differently in populations adapting to scarce phosphate, may identify useful antifungal drug targets.