Sterol-Response Pathways Mediate Alkaline Survival in Diverse Fungi

Abstract

The ability for cells to maintain homeostasis in the presence of extracellular stress is essential for their survival. Stress adaptations are especially important for microbial pathogens to respond to rapidly changing conditions, such as those encountered during the transition from the environment to the infected host. Many fungal pathogens have acquired the ability to quickly adapt to changes in extracellular pH to promote their survival in the various microenvironments encountered during a host infection. For example, the fungus-specific Rim/Pal alkaline response pathway has been well characterized in many fungal pathogens, including Cryptococcus neoformans. However, alternative mechanisms for sensing and responding to host pH have yet to be extensively studied. Recent observations from a genetic screen suggest that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. This work explores interactions among mechanisms of membrane homeostasis, alkaline pH tolerance, and Rim pathway activation. We find that the sterol homeostasis pathway is necessary for growth in an alkaline environment and that an elevated pH is sufficient to induce Sre1 activation. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol but is not dependent on Rim pathway signaling, suggesting that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response and pathogenesis. IMPORTANCE The work described here further elucidates how microorganisms sense and adapt to changes in their environment to establish infections in the human host. Specifically, we uncover a novel mechanism by which an opportunistic human fungal pathogen, Cryptococcus neoformans, responds to increases in extracellular pH in order to survive and thrive within the relatively alkaline environment of the human lung. This mechanism, which is intimately linked with fungal membrane sterol homeostasis, is independent of the previously well-studied alkaline response Rim pathway. Furthermore, this ergosterol-dependent alkaline pH response is present in Candida albicans, indicating that this mechanism spans diverse fungal species. These results are also relevant for novel antimicrobial drug development as we show that currently used ergosterol-targeting antifungals are more active in alkaline environments.

Internalization of the host alkaline pH signal in a fungal pathogen

“…In this mutant background, Rra1 is localized to endomembranes in both activating (pH 8) and inactivating conditions (pH 4) (Fig 4A and 4B). However, Rim signaling is still intact in the sre1 Δ mutant background as demonstrated previously by normal processing of the Rim101 transcription factor in response to elevated pH [26]. Together these data support that Rra1 membrane puncta are not essential for alkaline-induced Rim signaling.…”

“…We previously identified a Rim-independent mechanism of the fungal alkaline pH response in which the Sre1 transcription factor and its downstream effectors in the ergosterol biosynthesis pathway are activated in response to alkaline pH [26]. Other work has also demonstrated that the sre1 Δ mutant has depleted levels of ergosterol in the plasma membrane and altered abundance of sterol-rich domains, affecting the localization of membrane-associated proteins [27–29].…”

“…Furthermore, treating wildtype cells with Filipin III does not lead to decreased growth at alkaline pH despite similar disruption of cell surface puncta. Wildtype C. neoformans cells were able to grow at a range of increasing pH growth conditions (pH 4,5,6,7, and 8) despite high concentrations of Filipin III (62.5 ug/mL) [10 ug/mL for microscopy experiments in [26]]. These results indicate that Sre1-mediated ergosterol and membrane homeostasis is essential for Rra1 localization in plasma membrane puncta at low pH, but that this localization is not necessary for Rim pathway activation.…”

“…It has also been determined that Rim21 localizes to portions of the membrane that are devoid of cortical ER, eliminating MCL microdomains (Sterol transporter regions) as potential resting sites [66]. This is also an important distinction based on our previous studies that identified the sterol-mediated alkaline response as a Rim pathway-independent alkaline response process in C. neoformans [26].…”

Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

“…Research on C. neoformans helps us better understand this pathogen, contributes to the development of treatments for C. neoformans infections, and advances our understanding of fungal pathogens in general. RNA-Seq has been used extensively in C. neoformans studies to elucidate regulatory networks of protein-coding genes and mRNA structure and function ( Chang et al 2014 ; Chen et al 2014 ; Janbon et al 2014 ; Gish et al 2016 ; Chow et al 2017 ; Brown et al 2018 , 2020 ; Burke et al 2018 ; Wallace et al 2020 ; Yu et al 2020 ). While there is intense interest in the role of ncRNA in higher eukaryotes such as humans, relatively little work has explored the implications of ncRNA in fungi, with focus largely on model fungi such as Saccharomyces cerevisiae ( Bird et al 2006 ; Bumgarner et al 2009 , 2011 ; Gelfand et al 2011 ; Parker et al 2018 ) and Schizosaccharomyces pombe ( Ding et al 2012 ; Atkinson et al 2018 ).…”