Conserved and Divergent Features of pH Sensing in Major Fungal Pathogens

Purpose of Review For human fungal pathogens, sensory perception of extracellular pH is essential for colonisation of mammalian tissues and immune evasion. The molecular complexes that perceive and transmit the fungal pH signal are membrane-proximal and essential for virulence and are therefore of interest as novel antifungal drug targets. Intriguingly, the sensory machinery has evolved divergently in different fungal pathogens, yet spatial co-ordination of cellular components is conserved. Recent Findings The recent discovery of a novel pH sensor in the basidiomycete pathogen Cryptococcus neformans highlights that, although the molecular conservation of fungal pH sensors is evolutionarily restricted, their subcellular localisation and coupling to essential components of the cellular ESCRT machinery are consistent features of the cellular pH sensing and adaptation mechanism. In both basidiomycetes and ascomycetes, the lipid composition of the plasma membrane to which pH sensing complexes are localised appears to have pivotal functional importance. Endocytosis of pH-sensing complexes occurs in multiple fungal species, but its relevance for signal transduction appears not to be universal. Summary Our overview of current understanding highlights conserved and divergent mechanisms of the pH sensing machinery in model and pathogenic fungal species, as well as important unanswered questions that must be addressed to inform the future study of such sensing mechanisms and to devise therapeutic strategies for manipulating them.


Introduction
In fungi the ambient pH of the extracellular niche governs the expression and functionality of multiple secreted and cell surface-associated gene products that must be nimbly moderated to maintain nutrient acquisition, cell wall homeostasis and cation tolerance [1][2][3]. In the mammalian host, fungi are often exposed to a wide range of pH values that vary according to the tissue niche and inflammatory milieu. Therefore, understanding the functionality of mechanisms that promote versatility under pH flux is crucial for understanding how fungi are pathogenic and for informing improved disease control, particularly in the invasive disease-causing species recently classified by the World Health Organisation as being of critical priority; Aspergillus, Candida and Cryptococcus species [4, 5•].
Fungal pH adaptation, including in pathogens, relies upon highly conserved, mostly fungus-specific molecular mechanisms that converge upon pH-responsive transcription factors named PacC in filamentous fungi [6][7][8] or Rim101 in yeasts [9]. Intriguingly, the sensory machinery, that functions upstream of transcription factor activation, has evolved divergently in different fungal pathogens, yet spatial coordination of components is conserved. Relative to founding mechanistic studies conducted in the model ascomycetes Aspergillus nidulans and Saccharomyces cerevisiae, we here compare the pH sensing machinery of different fungal pathogens, reviewing recent research and identifying interesting new questions that are raised. The resultant overview Shadab Farhadi Cheshmeh Morvari and Bethany L. McCann contributed equally to this work. of current understanding is intended to inform future study of the sensing mechanisms and therapeutic strategies for manipulating them.
In the model ascomycete A. nidulans, the 760 amino acid (AA) AnPalH has a periplasmic N-terminal moiety, seven hydrophobic membrane-spanning domains and a long hydrophilic cytosolic terminus [10]. Negrete-Urtasun et al. (1999) confirmed that at alkaline ambient pH, the plasma membrane (PM) spanning AnPalH is required for AnPacC processing [10]. In A. nidulans and Aspergillus fumigatus, null mutants of An/afPacC exhibit morphological defects, alkaline and cation sensitivity and attenuation of virulence in murine models of invasive lung infection [6,7].
In S. cerevisiae, transient degradation of ScRim21 abolished pH signalling by suppressing proteolytic activation of ScRim101. Similar to AnPalH, the predicted ScRim21 (590 AA) structure consists of seven transmembrane domains with an extracellular N-terminus and a cytosolic C-terminus [13].
Deletion of RIM21, encoding the (529 AA) CaRim21 in C. albicans, revealed the loss of function phenotype including alkaline and cation sensitivity. Additionally, the loss of RIM21 resulted in an inability to transition from yeast to hyphae, a virulence trait goverened by CaRim101, suggesting loss of CaRim101 activation in the absence of CaRim21 [14]. In both S. cerevisiae and C. albicans, null mutants of Rim21 exhibit defects in alkaline growth and sporulation [9].
S. cerevisiae and C. albicans express a second plasma membrane (PM)-associated 7TMD protein, required for Rim signalling, Dfg16. In both species, like Rim21, Dfg16 is required for the yeast to hyphal switch under alkaline conditions. It has been speculated that Dfg16 and Rim21 act as two components of a heterodimeric receptor [15] although direct proof of this hypothesis is difficult to attain due to the poor tractability of structural studies, a problem that might be soon overcome by the advent of higher throughput CryoEM studies [16•].
There are no homologues, in Cryptococcus neoformans, having sequence similarity to An/AfPalH and Sc/CaRim21. or Sc/CaDfg16. However, CnRra1, a 7TMD protein, was identified in 2015, as being the most upstream component required for both the proteolytic processing and nuclear localisation of CnRim101. Like null mutants of AfPalH and Sc/CaRim21 in the ascomycetes, null mutants of CnRra1 suffer alkaline and cation tolerance defects [12 •, 17].

Other Membrane-Proximal pH-Sensing Components
In S. cerevisiae, C. albicans and A. nidulans, an arrestin-like protein AnPalF or Sc/CaRim8 plays an integral role in pH sensing. There is no identified homologue of PalF/Rim8 in Cryptococci [17].
AnPalF interacts with two regions of the cytoplasmic terminus of AnPalH [18]. This interaction is conserved in A. fumigatus, and S. cerevisiae, confirmed in both instances by yeast two-hybrid analyses between AfPalF and AfPalH and ScRim8 and ScRim21 respectively [19,20].
In the absence of AnPalH, AnPalF does not become ubiquitinated, a critical, pH-dependent post-translational modification required for the recruitment and engagement of downstream components of the pH adaptation mechanism [21]. Covalent attachment of a single ubiquitin moiety to the AnPalF C-terminus (PalF-Ub) in the AnPalH null background bypasses the requirement for AnPalH to promote proteolytic activation of AnPacC [22][23][24][25][26].
ScRim8 constitutively interacts with ScRim21 through its arrestin domain(s) and is ubiquitinated at its C-terminus by the ScRsp5 ubiquitin ligase through interaction with the PXY motif located at the C-terminus of ScRim8 [27,28]. ScRim8 ubiquitination is critical for the binding of ScRim8 to ScVps23 (ESCRT-I) in a pH-dependent manner; however, ubiquitination does not occur in a pH-regulated manner [28]. Moreover, via immunoblot analysis, it has been shown that ScRim8 ubiquitination is not dependent on ScRim21 or ScDfg16 but is dependent on the expression of ScVps23 [28]. Thus, ScRim8 ubiquitination likely regulates pH signalling by recruiting downstream molecules to the plasma membrane [29].
CaRim8 is also subject to pH-dependent post-translational modification, becoming hyper-phosphorylated in response to extracellular alkalinisation [30•]. Mutants lacking either CaRim21, CaDfg16 or CaRim9 remain able to hypo-phosphorylate CaRim8 suggesting that phosphorylation is not dependent on the presence of these proteins. At acidic pH, hypo-phosphorylated CaRim8 interacts with non-phosphorylated CaRim21 and is constitutively trafficked to the vacuole, thereby moderating the functionality of pH adaptation via re-localisation of essential pH sensing components [28, 30•]. Although the level of phosphorylation of CaRim8 is pH-dependent, phosphorylation occurs in a casein kinase 1 (CK1)-dependent manner under both acidic and alkaline pH conditions at a Ser/Thr-rich region but requires localisation  [32]. There is no Rim9/PalI homologue in the Cryptococcus species. AnPalI is homologous with ScRim9 and based on hydrophilicity is also predicted to be a membrane-spanning protein [32,33]. The deletion of AnpalI, in A. nidulans, leads to partial loss-of-function phenotypes under alkaline pH, with significantly diminished levels of processed AnPacC [3,10,34]. Thus, based on both phenotypic and AnPacC processing data, it can be concluded that AnPalI contributes to pH signalling but is somewhat dispensable. The deletion of Carim9 significantly impacts the cellular responses to alkaline pH, via a complete loss of proteolytic activation of CaRim101 [14].
The cellular content of ScDfg16 is reduced in the absence of ScRim9 but not in the absence of ScRim21 [13,35].
Transcription of Rim21, Dfg16 and Rim9 in neither S. cerevisiae nor C. albicans is pH-regulated. However, in both species following neutral-alkaline pH shifts which result in the proteolytic activation of Rim101, transcription of Rim8 is rapidly reduced. Reduction in Sc/CaRim8 transcription therefore results in a negative feedback loop that acts to prevent further transduction of alkaline pH response signals [30 •]. Obara et al. (2012) investigated the localisation, physical interaction and interdependency of pH sensing proteins in S. cerevisiae, proposing that ScRim21 functions as a pH sensor, with ScDfg16 and ScRim9 being required to maintain the stability/total cellular quantity of ScRim21, presumably by facilitating its PM delivery and localisation [13].

pH-Sensing Protein Complexes: Assembly and Subcellular Localisation
GFP-tagged ScRim21, ScDfg16 and ScRim9 proteins were primarily detected at the PM, with some detection at intracellular membranes. Interestingly, localisation of ScRim21, ScDfg16 and ScRim9 was significantly altered in mutants lacking two out of three components, where PM localisation of ScRim21 is undetectable in the Scrim9 or Scdfg16 null isolates. Interaction between ScRim21, ScDfg16 and ScRim9 was confirmed using co-immunoprecipitation pull-down assays [13].
In A. nidulans subcellular localisation studies carried out at acidic pH of AnPalH-GFP, expressed under the control of an over-expressing promoter alcA p , confirmed that AnPalH localises at the PM, but it predominantly accumulates in cytosolic compartments [21,34]. Given the likelihood of aberrant localisation when the pH sensor is expressed to physiological excess, a subsequent analysis via co-overexpression of both AnPalH-GFP and AnPalI-HA 3 , at stoichiometrically equivalent levels, resulted in the predominant localisation of AnPalH at the PM. Similar to the situation in S. cerevisiae, therefore, AnPalI likely has a role in assisting localisation of AnPalH at the PM [21,34]. AnPalF is also involved in assisting AnPalH PM localisation, as co-overexpression of AnPalH-GFP and AnPalF resulted in the localisation of AnPalH at the PM [21]. These findings also suggest that under acidic pH, AnPalH, AnPalI and AnPalF are interdependent components of a complex that is required for the correct localisation of the pH sensing machinery. Maintenance of this complex at the PM has not been investigated, as construction of the required strain, which co-overexpresses AnPalH, AnPalI and AnPalF at stoichiometrically equivalent levels, would likely deplete components of the downstream ESCRT machinery, adversely affecting AnPacC activation [36]. Thus, in A. nidulans, no subcellular localisation studies, with physiologically relevant or stoichiometric overexpression of one or more components, have been carried out at alkaline pH.
In C. neoformans, CnRra1 is localised to the PM, in a manner dependent on (i) integrity of the CnRra1 C-terminus [37] and (ii) extracellular pH (2). GFP-tagged truncated CnRra1 (CnRra1-296 T-GFP), which lacks the majority of the C-terminus, but retains a highly charged region, immediately downstream to the final TMD, is functional and exhibits similar localisation patterns to the full-length version of CnRra1 at both pH 4 and pH 8 with punctate structures forming at the cell surface in lower pH conditions and an increase in endomembrane staining at pH 8. More severe truncation of the C terminus results in loss of proteolytic activation of CnRim101 presumably via mislocalisation of the protein to intracellular and "perinuclear punctate structures" at both acidic and alkaline pH [2, 12 •, 37].

Importance of Endocytosis for Fungal pH Sensing
Whilst the evidence for fungal pH sensing complexes to localise to the PM is compelling, the spatial and functional convergence of pH sensing complexes with components of the endocytic machinery might differ by fungal species.
Epifluorescence microscopy and pulldown studies indicated that the ESCRT machinery of A. nidulans may be recruited to punctate sites at the cytosolic side of the PM. AnVps23, the ubiquitin-binding vacuolar protein sorting (VPS), ESCRT1 component, is conserved and universally required for proteolytic activation of pH-responsive transcription factors in pathogenic fungi [2,27]. AnVps23 was co-immunoprecipitated exclusively with ubiquitinated AnPalF; additionally, AnVps23 localised to punctate inner leaflet sites of the PM in an AnPalF-dependent manner [38].
In A. nidulans, using SynA as a surrogate marker, the endocytosis of the pH signalling complex was assessed via a secretory V-SNARE internalisation assay; maintenance of SynA at the PM is indicative of inhibition of endocytosis. In endocytosis-deficient mutants, no alteration in the level of AnPacC activation was detectable [36]. Under conditions where AnPalH localisation to the PM is not stably maintained, i.e. in the absence of, or in strains without stoichiometrically equivalent expression of AnPalI or AnPalF, recycling endocytosis of AnPalH is provoked. Under physiologically relevant levels of expression of AnPalH, it seems that AnPalF stabilises the PM localisation whilst also promoting the recruitment of downstreamacting pathway components [36].
As in A. nidulans, inhibition of endocytosis did not affect the activation of the ScRim101 pathway in S. cerevisiae, and the endocytosis of ScRim21 is considered to turn over stimulated ScRim21 following successful signal transduction [29]. In Saccharomyces, ScRim components downstream of ScRim21, accumulate at the PM in a ScRim21-dependent manner following alkaline stresses. ScSnf7/ScVps32, a highly conserved and abundant component of ESCRTIII, universally required in the proteolytic processing of pH-responsive transcription factors [2,27] localises in both the PM and the late endosome under alkaline pH; only PM localisation of ScSnf7 is essential for ScRim101 signalling [29]. Co-overexpression of ScRim8 and ScVps23 results in the accumulation of both ScRim8 and recruited ScVps23 at the PM, under acidic conditions [28].
In response to environmental alkalinisation, CnRra1 localises first to endocytic vesicles, then to endomembranes such as the perinuclear endoplasmic reticulum or intracellular vesicles. CnRra1 is maintained within membranes via CnNap1 (nucleasome adaptor protein 1) [37]. Acidification of previously alkaline environments results in the recycling of CnRra1 from internal membranes to punctate PM loci; this recycling also occurs, following the successful activation of CnRim101. This endocytosis of CnRra1 is clathrin-dependent, whereby clathrin coating of CnRra1 vesicles results in the recruitment of ESCRT complexes and downstream-acting CnRim pathway components. Pitstop-2-mediated inhibition of clathrin-dependent endocytosis results in a decrease in Rim101 nuclear localisation [37]. This indicates that clathrin-mediated endocytosis of CnRra1 is essential for the activation of CnRim101.

The C-Terminus of PalH/Rim21/CnRra1 Plays Crucial Roles in Both the Localisation and Function of the pH Sensor
The C-terminus of ScRim21 starts from amino acid 301 and ends at amino acid 533 [39]. ScRim21C is enriched in charged amino acids and interacts with the inner leaflet of the PM, Nishino and colleagues showed that GFP-ScRim21C was primarily located at the plasma membrane at acidic pH; external alkalisation resulted in the disassociation of GFP-Rim21C from PM and localisation to the cytosol and the nucleus at pH 8. Following re-acidification of the environment to pH 4.5, GFP-Rim21C localises to the PM within 5 minutes [40 •]. To determine whether charged amino acid clusters located in ScRim21C are important for ScRim21 functionality, site-directed mutagenesis of ScRim21C conducted in a strain lacking the full-length ScRim21 revealed that three consecutive Glu residues (353-355) of an EEE motif were essential. In this situation, the postulated reason for the lack of ScRim101 activation is aberrant recruitment of a downstream Rim component, ScRim20.
In A. nidulans, the C-terminal domain of AnPalH contains two high-affinity AnPalF-binding sites, one directly adjacent to TM7 at residues 349 to 384, and then residues 654-760. To determine if the region between the two AnPalF binding sites is essential for functional signalling, a strain (AnpalH654) was constructed where AAs 385 to 653 were substituted by a "synthetic linker consisting of a Gly-Ala pentamer". Unlike the ΔAnpalH mutant, the AnpalH654 variant was able to grow under alkaline conditions, and processing of AnPacC was maintained, albeit slightly weaker than WT. Therefore, the region between the two identified AnPalF binding sites is not essential for pH signalling (54). Residues 349-385 of the AnPalH C-terminus are sufficient to interact with AnPalF in two-hybrid assays (45). The importance of clusters of charged AAs in the C-terminal domain of AnPalH has not been explored, and neither have there been any published studies on the localisation or functionality of C-terminal mutants of AnPalH.
Comparison of the ScRim8 binding site of ScRim21 (residues 327-533) with the first AnPalF binding site of AnPalH revealed the presence of a conserved Trp-Glu-Trp motif (1). In A. nidulans, the Trp 349 -Glu 350 -Trp 351 motif is located on the interface between the C-terminus of TM7 and the cytosolic terminus. The removal of E350 and W351 results in a complete loss of function phenotype upon exposure to alkaline pH, suggesting that this motif is critical for AnPalH-AnPalF interactions. Additionally, a mutation in a conserved Leu 368 located within the first AnPalF binding site of the cytosolic terminus of AnPalH impaired binding of AnPalF to AnPalH [40•]. The effects of mutations in the conserved Trp-Glu-Trp motif and Leu have not been studied in ScRim21.
CnRra1C is enriched in arginine and lysine residues that are crucial for the PM localisation of the protein. When expressed in a ΔCnRra1 null, CnRra1-296 T-GFP complements the loss of function, through maintenance of a highly, positively charged region directly downstream to the TMD region; however, the CnRra1-273 T-GFP (2), which lacks these charged residues, is unable to localise to the PM or to overcome the loss of function phenotype. This highly charged region, therefore, is essential for localisation and functionality, although the mechanisms by which this occurs are not yet fully understood.
To date, a detailed analysis of the mechanistic and functional roles of the C-terminal domain of CaRim21 is lacking.

Biophysical Determinants of pH Signalling Activation
The composition of lipids is different between the inner (cytoplasmic) and the outer (extracellular) membranes of the fungal PM, resulting in an asymmetric distribution of phospholipids, with negatively charged phosphatidylserine (PS) confined to the inner leaflet. Lipid asymmetry is generated and mediated by "ATP-dependent inward (flip) and outward (flop) trans-bilayer movements of lipid molecules", catalysed by flippases and floppases, respectively.
Lipid asymmetry and proton electrochemical gradients, generated by differing proton concentrations inside (pH 7.4) and outside (pH 4.5) of the cell, are paramount for controlling PM polarisation [40•]. External alkalisation collapses the proton electrochemical gradient, resulting in depolarisation of the PM. Therefore, one hypothesis is that ScRim21 senses change in ambient alkaline pH by detecting the depolarisation status of the PM through a lipid sensing motif found in its C-terminal tail (13). Alternatively, ScRim21 may be able to sense alterations in lipid asymmetry caused by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced membrane depolarisation, suggesting that ScRim21C detects changes in lipids (PS) at the inner leaflet of the PM, triggering pathway activation (13). Consistent with both of these hypotheses Obara et al. (2012) showed using cells that do not express the PS synthase Cho1 and thus do not produce PS, or are defective in Lem3-regulated phospholipid asymmetry that the ScRim101 pathway can become constitutively activated under such conditions, even in the absence of an alkaline signal (13). PM depolarisation induced by CCCP also triggers the activation of ScRim101 in a ScRim21-dependent manner, in the absence of alkaline stress (13). The ability of ScRim21C to sense alterations in lipid asymmetry was analysed by monitoring the subcellular localisation of ScRim21C variants in cells mutated for lipidsynthesis or asymmetry. ScRim21C completely disassociated from PM in both lem3∆ and pdr5∆ cells. Thus, it was concluded that the cytosolic terminus of ScRim21 can sense and respond to the alterations in lipid asymmetry. ScRim21C variants lacking an ERKEE motif which is adjacent to the EEE motifs showed that the EEE motif has a crucial role in sensing or responding to changes in lipid asymmetry. The ERKEE motif, in particular the positively charged RK sequence, is required for ScRim21C association to the PM, whilst the negatively charged EEE motif is required for disassociation from the PM. It is therefore postulated that these motifs work together, forming a sensor. It is postulated that dissociation from the plasma membrane initiates recruitment of proteins acting downstream of ScRim21, via posttranslational modification of ScRim8 (57). The flipping of three phospholipids: phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine decreased significantly at alkaline pH compared to neutral and acidic [41]. In addition, it has been indicated that alteration in the lipid asymmetry of the PM resulted in an accumulation of the downstream cysteine protease required to cleave ScRim101, ScRim20 at the PM [29].
In addition to ergosterol homeostasis being a requirement for PM localisation of CnRra1, the normal asymmetry between leaflets maintains CnRra1 protein localisation in sterol-rich domains of the PM. This interaction likely occurs through charged AA interactions between the PM and the C-terminal domain of CnRra1. Temporary dissociation of the C-terminal domain of CnRra1 driving the endocytosis of CnRra1 from the PM as a result of lipid asymmetry highlights that regulation of the PM composition has a significant role in the activation of CnRim signalling in Cryptococcus. In strains lacking the regulatory subunit, cdc50 of the type IV ATPases, the flippases that govern maintenance of PM asymmetry [42], defects in growth in alkaline environments are exhibited. These mutant strains also exhibit a delay in nuclear localisation and activation of Rim101 (2). Cdc50 actively restores normal membrane asymmetry following external pH-induced dysregulation of the PM, which results in the disassociation of the C-terminal domain of CnRra1, its endocytosis and subsequent activation of Rim signalling (2). In Rim101 null mutants, because of dysregulated phospholipid maintenance of the PM, CnRra1 has a decreased ability to recycle to the PM, potentially due to changes in the ability of CnRra1 to interact with the PM.

Conclusions
Adaptation to environmental pH is critical for the survival and proliferation of many clinically important fungi. The inability to adapt to pH flux often results in loss of fitness, virulence or viability. Such adaptations require precise governance of gene expression that is dependent upon transcription factor activation, itself dependent upon the conversion of an extracellular stimulus to an intracellular signal. In many model and pathogenic fungi, the integrity of a PM-associated complex of transmembrane proteins and cognate arrestins is essential for pH sensing; however, recent studies in C. neoformans have provided detailed examples of divergent sensing and signalling mechanisms. Although knowledge of how fungal pathogens sense environments has improved, there remains overt reliance upon understanding these mechanisms in model organisms. A selection of important unanswered questions is provided in Table 2.
Funding This work was funded by the MRC Centre for Medical Mycology at the University of Exeter (MR/N006364/2 and MR/ V033417/1). This research was carried out at the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). This work was also funded by the MRC project grants MR/M02010X/1, MR/S001824/1 and MR/L000822/1 and the BBSRC project grant BB/V017004/1 to EMB. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.

Conflict of Interest
The authors declare no competing interests.

Human and Animal Rights and Informed Consent
All reported studies/ experiments with human or animal subjects performed by the authors have been previously published and complied with all applicable ethical standards.
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