Abstract
We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus C. albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.
Highlights
Candida albicans normally occurs as a relatively harmless organism in the human microbiome (Koh, 2013); C. albicans infection can be triggered by various perturbations in homeostasis, such as compromised immune defense or breaks in the epithelium–blood barriers due to injury or surgery
We developed a fluorescence method that allows in vivo realtime monitoring of the activity of C. albicans’ drug efflux pumps, Cdr1p, and Cdr2p, using a 3,3 -dipropylthiadicarbocyanine (diSC3(3)) probe (Szczepaniak et al, 2015)
As indicated by Plášek et al (2012) and Plášek and Gášková (2013), diS-C3(3) is a suitable probe to monitor real-time changes in plasma membrane potential; here, we are the first to describe this use in C. albicans
Summary
Candida albicans normally occurs as a relatively harmless organism in the human microbiome (Koh, 2013); C. albicans infection can be triggered by various perturbations in homeostasis, such as compromised immune defense or breaks in the epithelium–blood barriers due to injury or surgery. The risk of infection is increased in diabetic patients (Perlroth et al, 2007), possibly due to the dramatic effects of glucose on C. albicans’ metabolism (Brown et al, 2014) that increase virulence and drug resistance (Rodaki et al, 2009; Ene et al, 2012; Mandal et al, 2014). Due to C. albicans’ multiple mechanisms to adapt to and resist drugs, new experimental approaches must be developed to define the in vivo and/or real-time behaviors of individual cells (Brown et al, 2014). Many of C. albicans’ adaptations and resistance mechanisms are related to the supramolecular structure formed by the cell wall and plasma membrane. It is very important to develop methods to directly monitor drug transporters and plasma membrane-related activity in C. albicans
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