In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

Abstract

Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

“…aeruginosa ) did not yield satisfactory signal‐to‐noise ratios. We attributed this lack of reliable fluorescent signal to two main reasons: (i) deficient (and potentially stochastic) expression of the gene(s) encoding the pH indicator(s) and (ii) some of the currently available ratiometric fluorescent protein indicators have been tailored for eukaryotic systems (Tournu et al ., ; Reifenrath and Boles, ). In order to overcome these issues, we constructed a broad‐host‐range vector for the calibrated expression of a variant of the pH‐sensitive pHluorin2 indicator protein (Mahon, ).…”

Role of major facilitator superfamily transporter Qdr2p in biofilm formation by Candida glabrata

“…24 Other pHi measurement could be considered for future work, such as utilising a pH-sensitive fluorescent protein pHluorin (PHL) which could provide more stable and specific pHi reading in the highly dynamic intracellular pH condition. 44,45 This work is the first study (to our knowledge) to investigate the role of C glabrata QDR2 not only in biofilm formation, but also in the maintenance of pHi. Moreover, the Cg∆qdr2 mutant showed increased sensitivity towards some stresses, such as H 2 O 2 , citric acid and Zn +2 ( Figure 5).…”

“…Nevertheless, pHi measurement using fluorescent staining might have limitations, such as the dye ability to enter the cells and leaked out of the cells . Other pHi measurement could be considered for future work, such as utilising a pH‐sensitive fluorescent protein pHluorin (PHL) which could provide more stable and specific pHi reading in the highly dynamic intracellular pH condition …”

Presenting a codon-optimized palette of fluorescent proteins for use in Candida albicans

“…Another interesting feature of this tool is the possibility of using a codon usage set by tabulating highly expressed genes. This method was applied in the previously published optimization and application of pHluorin2 in C. albicans, where they selected RPL29, RPL32, RPL39, ACT1 and ENO1 to form the reference codon usage table 22 . We further refer to this optimization as OPT High.…”