The opportunistic fungal pathogen Candida albicans has emerged as the 4th most common cause of hospital-acquired bloodstream infections, and a major cause of catheter and device infections, sepsis, and potentially devastating fungal endocarditis. Despite its clinical significance, diagnosis and treatment are limited by an incomplete understanding of Candida pathogenesis. A key determinant of C. albicans virulence is its ability to form hyphae or filaments. Studies from decades ago reported that cytosolic alkalinization, which is largely regulated by the plasma membrane ATPase Pma1p, immediately precedes filamentation. This association remains largely unexplained. Prior work has indicated that the vacuolar ATPase (V-ATPase) proton pump, a multi-subunit complex responsible for vacuolar acidification, influences cytosolic pH and filamentation. Although C. albicans mutants lacking subunits Vma2p or Vma3p lose nearly all V-ATPase function and are defective in cytosolic alkalinization and filamentation, a vph1 mutant with similarly defective V-ATPase function and cytosolic alkalinization, has only a modest reduction in filamentation. Thus, failure to alkalinize the cytosol by itself is not sufficient to inhibit filamentation; therefore there must be additional mechanisms that contribute to regulation of intracellular pH (pHi) and filamentation. This project will therefore examine the key hypotheses that: (i) pHi homeostasis is mediated by Pma1p and other proton transporters, which contribute to filamentation and (ii) regulation of pHi homeostasis is a key requirement for filamentation and virulence. Aim 1 is to determine the contribution of Pma1p and the cation-proton transporters Nhx1p, Vnx1p, and Vcx1p to C. albicans biology and pathogenesis. We will generate Pma1p functional mutants and nhx1, vnx1, and vcx1 null mutants to assess the specific role of each transporter. Aim 2 is to define the biology of cytosolic and organellar pH homeostasis in regulation of filamentation. We will assay cytosolic and organellar pH (Golgi, vacuole) and examine how pH changes in these compartments during filamentation. Using this set of proton transport mutants and a pH-sensitive green fluorescent protein, we will determine the contribution of each transporter to pHi regulation and filamentation. Our long-term goals are to understand the mechanisms governing pHi homeostasis, and identify fungal-specific inhibitors of proton transporters for discovery of new antifungal therapies.
|Effective start/end date||07/01/2016 → 06/30/2018|