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5 Surviving the Storm: How Candida Species Master Adaptation…
127
Fig. 5.4 Maintenance and adaptation to pH (a) external pH is sensed via the Rim101 pathway in
Candida. Two transmembrane proteins, Rim9 and Rim21, upon encountering neutral to alkaline
pH, cause hyperphosphorylation arrestin-like protein Rim8, causing endocytosis of transmembrane complex and simultaneous recruitment of endosomal sorting complex for transport (ESCRT)
I, II, III.Rim20 and Rim13, a signalling protease, are also recruited, which causes proteolytic
cleavage of the inactive C-terminal region of Rim101, which then migrates to the nucleus to act as
a master regulator for various cellular processes. Rim101 positively regulates Phr1 and negatively
regulates Phr2. These two proteins modulate cell wall transglycosidase and alkalinization-induced
hyphae formation. (b) Cytosolic alkalinization is regulated via H+ ATPase Pma1p, which extrudes
H+ ions out of the cell, causing increased pH. (c) Under alkaline pH, Rim101 also upregulates Cht2
chitinase, which cleaves β-glucan into short chains, causing their masking in the cell wall. (d)
Cytosolic alkalinization also contributed via Stp2, which regulated AAP.Amino acid is utilized as
a carbon source, leading to the conversion of NH3 to NH
+
and the release of H+ ions, causing an
4
increase in cytosolic pH. (e) Calcium signalling also helps in pH adaptation. Crz1 transcription
factor works along with the Rim101 pathway and positively regulates Phr1
128
A. Rana et al.
These low pH conditions trigger changes in cell wall architecture, exposing
chitin and β-glucan to the immune system, leading to the release of proinammatory cytokines. pH-dependent cell wall remodelling occurs via CHT2 chitinase via a Rim101 pathway. In C. albicans under alkaline environment, Rim101
is expressed which upregulates the expression of CHT2 chitinase that cleaves
the β-glucan chains into short chitin microbrils, causing them to embed or
masked onto the cell wall. However, under acidic pH, Rim101 remains unexpressed, leading to the downregulation of chitinase, resulting in long chitin and
β-glucan chain on the cell wall. This heightens pathogen exposure to neutrophils
and macrophages, leading to the hyperactivation of the immune system. Similar
pH-dependent cell wall remodelling was observed in C. tropicalis. However, in
C. krusei alkaline pH resulted in β-glucan unmasking (Sherrington etal. 2017)
(Fig.5.4).
Cytosolic pH regulation in fungi is mainly controlled by plasma membrane
H+-ATPase, Pma1p. This proton pump is constitutively expressed, and at the time
of hyphae formation, it is heterogeneously distributed. It is present in more
amount at the tip of the germinating hyphae. H+-ATPase Pma1p helps extrudes the
H+ ions outside of cell maintaining ion balance, neutral to alkaline pH and electrochemical gradient for nutrient uptake. Pmap1p constitute 20–40% of the plasma
membrane forming important structural component. C-terminal truncation from
18 to 38 amino acids causes decreases in H+-ATPase Pma1p activity and cytosolic
alkalinization. Cell in which H+-ATPase Pma1p activity was reduced up to 75%
showed high cytosolic acidication (pH ≤5.5) (Rane et al. 2019). H+-ATPase
Pma1p is regulated by glucose as increase in glucose concentration also causes
sharp increase in cytosolic alkalization. Pma1p C-terminal truncation also reduces
glucose induced pH homeostasis (Rane etal. 2019). Pma1 mutant shows increased
susceptibility to weak acids, low extracellular pH, low temperature and osmotic
pressure, and reduced virulence in a mouse model of disseminated candidiasis
(Becker etal. 2010) (Fig.5.4).
C. albicans neutralizes the acidic environment in phagosome while escaping
from macrophage by utilizing transcription factor of amino acid permease (AAP)
genes STP2, which helps in amino acid derived pH neutralization. STP2 mutant
fails to form hyphae under acidic environment; however, under neutral pH, it shows
yeast to hyphal transition (Vylkova and Lorenz 2014). For phagosomal alkalinization, C. albicans utilizes amino acid as a carbon source, releases ammonia (NH3),
and converts it into ammonium (NH
+
) via consuming H+ ions. The accumulation of
4
sufcient proton results in pH neutralization and subsequent hyphal formation and
phagocytic escape (Westman etal. 2018) (Fig.5.4).
Calcium signalling also plays a critical role through a CRZ1 transcription factor
in pH adaption. Under alkaline pH, CRZ1 along with Rim 101 pathway acts parallelly via the calcineurin pathway. CRZ1 is also known to positively regulate the
expression of PHR (Jiang etal. 2023). Rim 101 also collaborates with CRZ2 in
calcineurin-independent pathway to repress lamentation under acidic pH. Both
CRZ1 and CRZ2, in conjunction with calcineurin, are required for growth under
acidic environment (Kullas etal. 2007) (Fig.5.4).
5 Surviving the Storm: How Candida Species Master Adaptation…
129
5.9 The Role ofHeat Shock Proteins inThermotolerance
Hsps acts as molecular chaperones, playing a vital role in preventing protein misfolding and aggregation by directly interacting with their client proteins. In Candida
spp., Hsps involved in diverse role in basic physiological process as well as virulence (O’Meara and Cowen 2014). They are associated with various signalling pathways such as calcium-calcineurin, Ras1-cAMP-PKA, MAPK, and cell cycle
signalling. Various factors involved in these signalling cascades are client protein of
Hsps when thermal perturbance, oxidative stress, and various other environmental
cues causes protein misfolding and aggregation which triggers phosphorylation of
heat shock factor (Hsf1) (Burnie etal. 2006; Cuéllar-Cruz etal. 2014). Phosphorylated
Hsf1in turn induces the expression of Hsp encoding genes by interacting with heat
shock elements (HSEs) in their promotor region (Nicholls etal. 2009).
Heat shock proteins can be classied according on their molecular weight and
falling into two categories. They are termed as high molecular weight Hsp and low
molecular weight Hsp. Hsp with high molecular weight is ATP-dependent and
includes Hsp104, Hsp90, Hsp70, Hsp78, and Hsp60. On the other hand, low molecular weight Hsp is also known as small heat shock protein (sHsps) and are ATPindependent. In C. albicans, four Hsps are encoded: Hsp30/31, Hsp21, Hsp12, and
Hsp10 (Mayer et al. 2012). These Hsps play a critical role in ensuring the proper
folding and stability of proteins, particularly when cells face challenges like high
temperatures and other stressors.
5.9.1 Role ofHigh Molecular Weight Heat Shock Proteins
inCandida Thermotolerance
Hsp104 in known to be involved in thermotolerance. Homozygous mutants of
Hsp104 exhibit patchy and loose hyphal growth rather than intertwined with each
other, resulting in defective biolm formation (Fiori et al. 2012). Another wellstudied chaperones Hsp90in yeast and its downstream fate are mainly controlled
via various posttranslational modications like S-nitrosylation, phosphorylation,
and acetylation. The C-terminal domain of Hsp90 comprises of ten phosphorylation
sites that facilitate conformational switching after interacting with client protein
(Soroka etal. 2012). C. albicans Hsp90 also have acetylation site at lysine 30 and
271 (Li etal. 2017). The inhibitor of histone deacetylation such as trichostatin A
(TSA) abolishes Hsp90 mediated azole resistance (Robbins etal. 2012). Inhibiting
Hsp90 either through pharmacologically means or genetic manipulation has been
shown to inhibit biolm maturation and dispersal (Robbins etal. 2012). Additionally,
Hsp90 is involved in cell cycle progression and apoptosis and exhibits a synergistic
effect with uconazole against uconazole-resistant C. albicans (Dai etal. 2012;
Senn etal. 2012; Robbins et al. 2012). Hsp70 also have a conserved N-terminal
domain with ATP-binding and peptide binding sites (Craig etal. 1993). In C. albi-
cans, Hsp70 family proteins have two members stress-70 subfamily A and B (Ssa
and Ssb). The proteins are primarily expressed on cell surfaces and acts as a
130
receptor for antimicrobial peptide like salivary histatin5 (Hst5) (Li etal. 2003). Ssa1
and Ssa2 are involved in host cell endocytosis, hence play a crucial role in virulence
(Sun etal. 2010). C. albicans Hsp60 mRNA expression increases upon incubation
at 35°C, indicating its involvement in thermotolerance (Raggam etal. 2011).
A. Rana et al.
5.9.2 Low Molecular Weight Hsp/Small Heat Shock Proteins
Low molecular weight Hsps or small Hsps undergo a transition from oligomeric to
multimeric form and interact with misfolded protein to halt the aggregation process
when exposed to thermal or other environmental cues. During this process, sHsps
hold the client protein and then major Hsps disaggregate and refold them (Cashikar
et al. 2005; Eyles and Gierasch 2010; Haslbeck et al. 1999; Narberhaus 2002).
HSP12 overexpression sensitize C. albicans to ketoconazole, itraconazole, and u-
conazole (Fu etal. 2012). Conversely, the HSP21 deletion reduces thermotolerance
and enhances sensitivity to oxidative stress. These small Hsps are associated with
the regulation of glycogen, glycerol, and trehalose homeostasis in response to
increased temperature, contributing to impair virulence in a systemic candidiasis
mouse model (Mayer etal. 2012).
5.10 Metabolic Strategies ofCandida spp.
forPathogenic Survival
Candida spp. has evolved various virulence traits to thrive within the host’s challenging
and nutritionally depleted environments. Among these traits, metabolic exibility is a
key factor in their survival but is less studied as a pathogenic feature. C. albicans is
extensively studied as the primary model, it is imperative to understand that other
Candida spp. might share similar or different metabolic characteristics contributing to
their pathogenicity. Here, we will discuss Candida’s metabolic virulence and the strat-
egies, molecules, and factors behind its metabolic exibility. Nitrogen and carbon are
major nutrients. Iron and zinc are equally vital elements for Candida spp. to establish
infection and ensure their survival within the host. These important metabolic factors
and how they might act as pathogenic factors are summarized further in Table5.1.
5.10.1 Amino Acid/Nitrogen Metabolism
Nitrogen serves as an important nutrient assimilated by fungal pathogens for survival. Still, in the host niche, usually the preferred sources of nitrogen like ammonium sulphate and some amino acids like glutamine, glutamate etc., are absent;
hence, the Candida spp., like C. albicans and C. glabrata etc, are known to harbor
various mechanisms and have evolved numerous regulators to acquire the nitrogen
from host systems. Nitrogen is indispensable for various critical processes, including DNA and RNA synthesis.
5 Surviving the Storm: How Candida Species Master Adaptation…
131
(continued)
Limjindaporn etal. (2003)
References
Liao etal. (2008)
mice model of systemic
of systemic Candidiasis
candidiasis
Dabas and Morschhauser
Vylkova and Lorenz
Miramon and Lorenz
(2014)
(2016)
Defective phagosomal
escape and
host macrophage
Regulates amino acid
(2008), Feng etal. (2016)
Shows positive
correlations with drug
avirulence in mice model
Liberates amino acids from
permeases
Ghosh etal. (2009),
Danhof and Lorenz (2015)
Defects in hyphal
formation, alkalization
pumps like MDR1
proteins in the absence of
nitrogen
and host phagosomal
regulated by STP2
Navarathna etal. (2012)
Defects in escape from
host phagosomes and
escape
as a nitrogen source
Amorim-Vaz etal. (2021)
Avirulence in mice
defects in mice kidney
colonization
model and Galleria
mellonella model
factor
Genes Metabolic pathway Species Function Virulence of mutants
1. GAT1 Nitrogen C. albicans Regulator of NCR pathway Reduced virulence in
S. no.
Table 5.1 Metabolic regulators involved in pathogenicity of Candida spp.
4. STP2 Nitrogen C. albicans Component of SPS system.
3 SSY1 Nitrogen C. albicans Component of SPS system Defects in escape from
2. GLN3 Nitrogen C. albicans Regulator of NCR pathway Avirulent in mice model
5. SAP2 Nitrogen C. albicans Activated by STP1
6. ATO5 Nitrogen C. albicans Ammonium transporter
7. DUR1,2 Nitrogen C. albicans Required for urea utilization
8. GCN4 Nitrogen C. albicans Master stress transcription
132
A. Rana et al.
Silao etal. (2019)
References
Defective hyphal
formation and escape
from phagosomes
pathway
Schrevens etal. (2018)
Askew etal. (2009)
Reduced virulence of
tye7gal4 in both Galleria
mellonella and mice
macrophages. Reduced
virulence in mice
model
glycolytic genes
Ramirez-Zavala etal.
Chew etal. (2019a),
Barelle etal. (2006),
Ramirez and Lorenz
escapes and attenuation
of virulence in mice
Glyoxylate cycle enzyme Defective macrophage
C. glabrata
(2017)
(2007)
Decreased tness in mice
model of gastrointestinal
model
Candidiasis
CCR pathway
Lagree etal. (2020)
Defective hyphae
formation, biolm
formation, sensitivity to
cell wall inhibitors and
reduced damage of host
CCR pathway
Xu etal. (2014)
and avirulence in mice
macrophages
model
Nitrogen C. albicans Regulators of proline
PUT2
Genes Metabolic pathway Species Function Virulence of mutants
9. PUT1 and
S. no.
Table 5.1 (continued)
(proline
catabolism
pathway
mutants)
10 MUP1 Nitrogen C. albicans Methionine permease Defective escape from
Carbon C. albicans Transcription factor of
GAL4
11. TYE7 and
12. ICL1 Carbon C. albicans
13. SAK1 Carbon C. albicans Activates SNF1 kinase of
Carbon C. albicans Downstream effectors of
MIG2
14 MIG1 and
15 CFL1 Iron C. albicans Ferric reductase enzyme Defective lamentation
5 Surviving the Storm: How Candida Species Master Adaptation…
Ramanan and Wang
References
Cheng etal. (2013),
Srivastava etal. (2014),
mice model (C. albicans
Chakraborty etal. (2020)
and C. glabrata)
Altered morphology and
biolm formation (C.
(2000), Srivastava etal.
(2014)
virulence in systemic
candidiasis
parapsilosis)
2. Defect in kidney
Hsu etal. (2011)
colonization of mice
Defective virulence in
Kim etal. (2008), Nobile
etal. (2009)
mice model
Defective lamentation,
biolm formation
Xu etal. (2015)
Defective virulence in
mice model
133
Multicopper oxidase Reduced virulence in
C. glabrata
C.
parapsilosis
Iron C. albicans
Genes Metabolic pathway Species Function Virulence of mutants
CaFET34
S. no.
16 FET3
Iron permease protein 1. Compromised
C. glabrata
17 FTR1 Iron C. albicans
regulating iron acquisition
genes
factor
transcription factor
18 HAP43 Iron C. albicans Transcription factor
19 CSR1(ZAP1) Zinc C. albicans Zinc-specic transcription
20 SUT1 Zinc C. albicans Activator of CSR1/ZAP1
134
A. Rana et al.
Though there are numerous sources of nitrogen in human systems, including
amino acids, urea, peptides, and proteins, the amino acids are easier to assimilate.
Notably, certain amino acids can serve as a dual role as carbon and nitrogen, highlighting their signicance in Candida’s metabolic strategies (Vylkova and Lorenz
2014; Vylkova etal. 2011; Wong etal. 2008).
5.10.1.1 Amino Acid Sensing Pathway
Usually, in the host niche, the preferred sources of nitrogen, like ammonium sulphate or amino acids like glutamine and glutamate, are rare, so there are various
mechanisms utilized by fungal pathogens to utilize and acquire nitrogen from the
host niche. The major extracellular sensing amino acid pathways are NCR, TOR,
and SPS-mediated amino acid sensing (SPS pathway). This pathway regulates the
expression of downstream effectors that regulates the metabolism and virulence of
pathogens like C. albicans in human host (Zhang etal. 2018; Ries et al. 2018).
These pathways are majorly studied in the model yeast S. cerevisiae.
1. Nitrogen Catabolite Repression (NCR) is an essential regulatory pathway that
orchestrates the utilization of alternative nitrogen sources without preferred
options, ensuring the efcient allocation of resources. This intricate mechanism
has been extensively studied in the model organism S. cerevisiae, shedding light
on its complexity. Four GATA transcription factors govern NCR where ScGat1
and ScGln3 emerge as positive regulators, while Dal80 and Gzf3 act as negative
regulators. These GATA factors compete for binding to target genes having
GATAAG sequence, thus exerting their inuence on the expression of various
permeases and regulatory enzymes during nitrogen starvation (Hofman-Bang
1999; Magasanik and Kaiser 2002; Tudzynski 2014). In the presence of pre-
ferred nitrogen sources, Gat1 and Gln3 remain sequestered within the nucleus, a
process orchestrated by the Ure2 protein, which predominantly phosphorylates
Gln3 and maintains its nuclear residence (Georis etal. 2009, 2011; Ljungdahl
and Daignan-Fornier 2012; Silao and Ljungdahl 2021).
In C. albicans, both Gat1 and Gln3 are essential for the utilization of alternative nitrogen sources and exhibit both independent and overlapping functions
during nitrogen scarcity (Dabas and Morschhauser 2007; Limjindaporn etal.
2003). Whereas in C. glabrata, Gat1, Gln3, and Ure2 are analogous to their
counterparts in S. cerevisiae, they bear little resemblance regarding protein
sequence. In C. glabrata, Gln3 assumes a central role in NCR, while Gat1’s
prominence becomes evident when Gln3 and Ure2 are conspicuously absent
(Perez-Delos Santos and Riego-Ruiz 2016).
2. SPS (Ssy1/Ptr3/Ssy5) Sensing Pathway—The plasma membrane-localized SPS
system is a crucial sensor for detecting extracellular amino acids within the surrounding media. In S. cerevisiae, the SPS system has been primarily studied.
Comprised of key components such as the amino acid sensor Ssy1, the scaffold
protein Ptr3, and the protease Ssy5, this intricate system helps in activating multiple
AAP genes responsible for facilitating amino acid uptake. The SPS system effectively regulates two transcription factors, STP1 and STP2, which feature inhibitory
5 Surviving the Storm: How Candida Species Master Adaptation…
N-terminal domains, causing their localization within the cytoplasm. However, in the
presence of external amino acids, Ssy1 initiates a cascade by activating the Ssy5
protease. Ssy5, in turn, cleaves the N-terminal domains of STP1 and STP2, enabling
their translocation to the nucleus. In this nuclear realm, these factors then activate
many SPS-regulated genes, including AAPs, those are essential for amino acid transport and utilization. Subsequently, these AAPs acquire their native conformation
with the assistance of an endoplasmic reticulum (ER) chaperone known as Shr3in
S. cerevisiae (Silao and Ljungdahl 2021; Ljungdahl etal. 1992; Kota etal. 2007).
While the components of the SPS sensor in C. albicans exhibit homology to those
in S. cerevisiae, a notable difference emerges in the downstream roles of STP1 and
STP2 (Brega et al. 2004; Martinez and Ljungdahl 2004; Miramon and Lorenz
2016). STP1, for instance, is subject to NCR regulation and is responsible for acti-
vating major broad-spectrum aspartyl proteases and oligopeptide transporters
(OPTs) (Dabas and Morschhauser 2008; Martinez and Ljungdahl 2005). Conversely,
STP2 maintains constitutive expression and oversees AAPs, similar to the role of
STP1 and STP2 in S. cerevisiae (Martinez and Ljungdahl 2005).
Transceptors can also facilitate the sensing of external amino acids, the major
one being the ScGap1a sensor of all -amino acids, citrulline and some polyamines
and -amino acids (Jauniaux and Grenson 1990; Uemura etal. 2005). C. albicans
possess about six ScGAP1 homologs with CaGap1, CaGap2, and CaGap6 seem to
have transceptor activity (Kraidlova etal. 2011).
In addition to the regulatory pathways discussed above, the TOR and GAAC
pathways also play integral roles in nitrogen acquisition. The changes in intercellular amino acid levels activate the Tor kinases, which mediate intercellular amino
acid metabolism and nitrogen source acquisition (Zhang etal. 2018; Loewith and
Hall 2011). The CaTor1 is known to regulate NCR and AAPs, the detailed mechanism of which is yet unknown (Bastidas etal. 2009). During nutrient starvation,
Gcn2 kinase causes a decrease in global translation, which mediates the expression
of transcription factor Gcn4, the primary regulator of the GAAC (general amino
acid control) pathway (Hinnebusch 2005; Rana etal. 2021). Activated Gcn4 in
S. cerevisiae regulates around 30 amino acid biosynthesis genes, representing
around 12 major pathways and purine biosynthesis (Hinnebusch 2005). In C. albi-
cans, Gcn4, in addition to its metabolic functions, also inuences the morphogenetic response during amino acid starvation (Tripathi etal. 2002).
135
5.11 Role ofNitrogen andAmino Acid Metabolism
Regulators inCandida Pathogenesis
Candida spp. pathogens can successfully infect the human host and survive in
almost all anatomically possible sites (Silao and Ljungdahl 2021). One of the reasons for this is its ability to utilize diverse host nitrogen sources like amino acids,
peptides, proteins, etc. (Silao and Ljungdahl 2021). The different regulators involved
in the nitrogen acquisition from the host also serve as potent pathogenicity factors.
136
A. Rana et al.
The major regulators of NCR in C. albicans are Gat1 and, Gln3 and their null
mutants show compromised survival in a murine model of disseminated candidiasis. In particular, the Gat1 mutant exhibits complete avirulence (Limjindaporn etal.
2003; Liao etal. 2008). The Gat1 predominately regulates the expression of STP1,
which, in turn, regulates SAP2 (secreted aspartyl protease) when the proteins are the
primary nitrogen source (Dabas and Morschhauser 2008). During nitrogen scarcity,
the SAPs are the virulent traits of numerous pathogenic fungi that degrade proteins
and release amino acids (Monod etal. 2002). C. albicans harbors around ten aspartic proteinases (SAP 1–10) that mediate virulent traits such as adhesion, biolm
formation, host penetration, and host immune modulation (Ries etal. 2018; Naglik
etal. 2004, 2008; Gropp etal. 2009). The SAPs have also been reported in different
Candida sp. like tropicalis, guilliermondii, and lusitaniae, with SAPP1 and SAPP2
of C. tropicalis having denite and important roles in pathogenicity (Singh etal.
2019; Parra-Ortega etal. 2009). STP2 plays a vital role in the utilization of amino
acids as alternativenitrogen sources by regulating the expression of AAPs, which
mediates the uptake of extracellular amino acids, and excess nitrogen gets excreted
as ammonia by ammonium transporter ATO5 (Vylkova etal. 2011; Danhof and
Lorenz 2015). The arginine metabolism releases ornithine, urea, and free ammonium. The ammonium is then utilized by urea amidolyase CaDur1,2 (under NCR
regulation) to CO2 and ammonia. This process aids in hyphal morphogenesis, a
potent virulent trait (Navarathna etal. 2011, 2012). Additionally, it catalyzes the
neutralization of environmental pH, and this neutralization aids in escaping from
host phagosomes, providing C. albicans the power to modulate the host environment. Macrophages are nutritionally poor but amino acids rich, and the mutants of
the SPS system, Stp2, and transporters like ATO5 have reduced hyphae formation.
They are incapable of escaping phagosomes as they show defective alkalization
(Miramon and Lorenz 2016; Danhof and Lorenz 2015; Vylkova and Lorenz 2014;
Lorenz etal. 2004; Ghosh etal. 2009). Conversely, a report by Westmann etal. challenges the notion that phagosomal alkalization in C. albicans is not a prerequisite to
escape from macrophage and further the avirulence of GDH2 mutants in both invitro and the invivo mice model conrms the fact. GDH2 catalyzes glutamate to
alpha-ketoglutarate and is responsible for the bulk of ammonia production, hence
alkalization (Westman etal. 2018; Silao and Ljungdahl 2021; Silao etal. 2020). The
Gcn4 transcription factor in C. albicans induces Efg1-dependent hyphal formation
during amino acid starvation, contributing to biolm formation and virulence as
shown from the invivo mice model of Candidiasis (Tripathi etal. 2002; AmorimVaz etal. 2021). The TOR kinase, in addition to regulation of NCR in C. albicans,
is also known to regulate the Gcn4 activation, morphogenesis, and conditional biolm formation (Ries etal. 2018). Moreover, in C. auris too, an inhibition of TOR
via rapamycin results in reduced biolm formation (Biswas etal. 2023b).
Furthermore, using some nonpreferred nitrogen sources strongly inuences pathogenesis in C. albicans. Proline, one of the most abundant amino acids, can serve as a
nitrogen and carbon source, with its uptake mediated by uncharacterized permease
CaPUT4 (Tebung etal. 2017). As it is present in collagen and mucin, proline pathway
mutants show defective macrophage escape as they require proline catabolism to