What is calcineurin?
Calcineurin is a highly conserved serine-threonine specific protein phosphatase and
was named based on its discovery as a calcium- and calmodulin-binding protein that
is highly abundant in the central nervous system (calci+neurin) [1]. Calcineurin is
a heterodimer comprised of a catalytic A subunit (CNA) and a regulatory B subunit
(CNB) [2,3]. In the basal state, CNB is bound by 2 Ca2+ ions and calcineurin is kept
inactive through an autoinhibitory domain (AID) that loops into the active site of
CNA [2] (Fig 1). Upon calcium influx, CNB binds to 2 additional Ca2+ ions causing
structural changes in the CNA-CNB heterodimer. Finally, the heterodimer is associated
with another calcium-binding protein, calmodulin, which itself is primed via structural
changes through binding of 4 Ca2+ ions. Calmodulin binding triggers the release of
the AID from the catalytic site of the enzyme rendering calcineurin fully active.
Once activated, calcineurin binds to its substrates by docking onto 2 short-linear
motifs, PxIxIT and LxVP, and dephosphorylates substrates to govern their subcellular
localization and functions [3].
10.1371/journal.ppat.1011445.g001
Fig 1
Calcineurin signaling in fungal pathogens. Calcineurin signaling is initiated upon
calcium influx and binding of calcium-calmodulin (CaM) to the calcineurin complex
(CNA and CNB). The binding of CaM releases an autoinhibitory domain that occupies
the active site in the catalytic subunit CNA converting calcineurin from an inactive
“OFF” configuration to a fully active “ON” state. Upon activation, calcineurin dephosphorylates
its substrates, which include a conserved transcription factor, Crz1, and several
others that remain unidentified in most fungal species. Through these targets, calcineurin
governs stress responses, morphological transitions, and virulence in fungal pathogens
of humans and plants. As described in the right-side panel, calcineurin directs yeast–hyphal
dimorphic transitions in some fungi, whereas it is required for vegetative yeast or
hyphal growth in other fungal species. In some cases, calcineurin orchestrates the
development of specialized structures, such as formation of appressoria in M. oryzae,
that are required for infection and virulence. Calcineurin activity can be inhibited
by FK506 and cyclosporine (CsA), both of which are immunosuppressive drugs when bound
to FKBP12 or cyclophilin A (Cyp). The figure was created with BioRender.com.
How important is calcineurin for fungal pathogens?
Calcineurin plays key roles in fungi in response to calcium influx and is the only
known calcium-responsive phosphatase. Calcineurin has been studied in several fungal
pathogens of both humans and plants and plays crucial roles in stress adaptation and
pathogenicity (Fig 1 and Table 1). In the human fungal pathogen, Candida albicans,
calcineurin is required for survival in serum and plays an important role during hyphal
growth required to cause infections [4–6]. Additionally, loss of function or inhibition
of calcineurin also results in enhanced susceptibility to antifungal drugs in widespread
clinical use such as fluconazole. Calcineurin plays a crucial role in the hyphal growth
and virulence of Aspergillus fumigatus, a filamentous fungal pathogen [7]. The catalytic
subunit, CNA, localizes to the active sites of hyphal growth and septum formation,
playing an important role in growth and septation in A. fumigatus [8]. Inhibiting
calcineurin activity also results in hypersensitivity to other cell wall inhibitors
in this fungus suggesting a major role for calcineurin in proper cell wall synthesis
and repair [9].
10.1371/journal.ppat.1011445.t001
Table 1
Calcineurin functions in fungal pathogens.
Fungal pathogen
Calcineurin functions
References
Human pathogens
Aspergillus fumigatus
Hyphal growth, cell wall integrity, septation, cation homeostasis, antifungal drug
resistance, and virulence
[7–9,22]
Candida albicans
Hyphal growth, ER stress response, cell wall integrity, azole tolerance, growth in
serum, and virulence
[4–6,20,34,37]
Candida glabrata
Thermotolerance, ER stress response, cell wall integrity, azole tolerance, growth
in serum, and virulence
[38,39]
Cryptococcus neoformans
Thermotolerance, cell wall integrity, antifungal drug tolerance, postmating dimorphic
transition from yeast to hyphae, and virulence
[10,11,21,24]
Mucor circinelloides
Vegetative dimorphic transition from yeast to hyphae, hyphal growth, antifungal drug
resistance, and virulence
[14,40]
Paracoccidioides brasiliensis
Vegetative dimorphic transition from hyphae to yeast, yeast and hyphal growth, and
calcium homeostasis
[13]
Talaromyces marneffei
Hyphal growth, conidiation and conidia germination, cell wall integrity, osmotic stress
response, survival in macrophages, and virulence
[16]
Trichosporon asahii
Thermotolerance, cell wall integrity, ER stress response, hyphal formation, and virulence
[15]
Plant pathogens
Botrytis cinerea
Conidiation, cation homeostasis, cell wall integrity, and virulence
[41]
Magnaporthe oryzae
Mycelial growth, conidiation, appressorium formation, and virulence
[17,23,42]
Ustilago maydis
Postmating dimorphic transition from yeast to hyphae, and virulence
[18]
Ustilago hordei
Thermotolerance, cell wall integrity, cation homeostasis, pH stress, and virulence
[43]
Functions listed in bold indicate roles that are known to be either completely or
partially independent of Crz1.
In Cryptococcus neoformans, mutation or inhibition of calcineurin renders cells inviable
at 37°C, and avirulent in mice [10]. In addition, calcineurin is also required for
hyphal growth in C. neoformans, which is essential for completing the sexual cycle
and producing infectious spores [11]. Microscopy studies demonstrated that calcineurin
re-localizes to septation sites as well as processing-bodies (P-bodies) and stress
granules during 37°C heat stress [12]. Calcineurin has also been found to play essential
roles in virulence in several other human fungal pathogens including Mucor circinelloides,
Paracoccidioides brasiliensis, Talaromyces marneffei, and Trichosporon asahii [13–16].
Magnaporthe oryzae is a serious threat to global food security and is responsible
for approximately a 30% loss in rice productivity each year. M. oryzae infects plant
leaves through specialized structures called appressoria. Calcineurin is required
for the formation of appressoria and therefore, it is essential for pathogenesis [17].
Calcineurin also plays a critical role in the pathogenicity of Ustilago maydis, a
plant fungal pathogen that infects corn [18]. U. maydis undergoes a postmating morphological
transition from yeast to hyphal growth that is essential for virulence. Mutation of
calcineurin results in the production of multi-budded yeast cells that fail to form
hyphae and are thus unable to cause infections. Overall, calcineurin directs stress
responses and morphological changes in each of these pathogens and thus serves as
a globally conserved virulence factor.
What are calcineurin’s substrates?
One of the most conserved calcineurin targets in fungi is Crz1, a zinc-finger transcription
factor, which is a homolog of NFAT in humans [19]. Upon dephosphorylation by activated
calcineurin, Crz1 translocates into the nucleus and regulates the transcription of
target genes involved in stress responses. While Crz1 is known to perform roles in
the calcineurin pathway in several fungal species, its activity fails to account for
all of calcineurin’s functions. For example, Crz1 in C. albicans plays roles in pH
sensitivity, hyphal growth, and drug tolerance but is not required for virulence,
unlike calcineurin [20]. Similarly, Crz1 deletion does not lead to the same level
of thermal sensitivity as deletion or inhibition of calcineurin in C. neoformans [21].
In addition, Crz1 is not required for hyphal growth and sporulation in this species,
in contrast to calcineurin. While both calcineurin and the Crz1 homolog CrzA play
an important role in the virulence of A. fumigatus, CrzA deletion does not phenocopy
the severe hyphal growth defect of the calcineurin mutant, and accordingly, calcineurin
plays a more prominent role in cell wall biosynthesis compared to the more restricted
role of Crz1 [22]. Interestingly, the Crz1 homolog in M. oryzae is not required for
appressorium formation [23], but Crz1 mutation still reduces rice infection rates.
Crz1 in U. maydis remains to be identified. Overall, these studies demonstrate that
Crz1 is a conserved and key downstream effector of calcineurin in most fungal pathogens,
but also emphasizes that additional substrates of calcineurin must also play critical
roles in stress responses.
Surprisingly, aside from Crz1, most calcineurin targets in pathogenic fungi remain
understudied and poorly characterized, except for some potential candidates identified
in C. neoformans. A phosphoproteome study in this fungus identified several calcineurin
targets, in addition to Crz1 [24]. Many of these substrates localize to P-bodies/stress
granules and are predicted to play important roles in RNA processing. Additionally,
substrates involved in septation and vesicle trafficking were also identified, in
accord with previous localization studies [12,25]. Analysis of calcineurin substrates
revealed that both Crz1 and P-body/stress granule targets contribute to thermal stress
adaptation and virulence but their mutation confers only intermediate phenotypes in
comparison to wild-type and calcineurin mutants [24]. While Crz1 controls the expression
of several cell wall biosynthesis genes in a calcineurin-dependent manner, thus regulating
cell wall integrity, the roles of calcineurin in P-bodies/stress granules are not
well understood but may involve RNA metabolism. Interestingly, combined mutation of
Crz1 and P-body targets does not recapitulate the phenotypes of calcineurin deletion,
suggesting that additional substrates may be important for calcineurin functions [24].
A recent study revealed that Crz1 also localizes to stress granules and septa upon
heat stress, similar to calcineurin [26]. Interestingly, the study found that Crz1
localizes to stress granules prior to its translocation to the nucleus hinting at
the possibility that calcineurin might be dephosphorylating Crz1 at stress granules.
Can calcineurin serve as a drug target?
Calcineurin, due to its diverse and critical roles, is an attractive antifungal drug
target candidate. Due to its stimulatory role in human T-cells, it is an established
target of 2 immunosuppressive drugs, FK506 and cyclosporine (CsA), both of which inhibit
calcineurin as drug–protein complexes after binding to their respective immunophilin
targets, FKBP12, and cyclophilin A [2]. Both FK506 and CsA also exhibit broad antifungal
activity highlighting the potential of calcineurin as an antifungal drug target [27].
However, the conservation of calcineurin signaling between humans and fungi requires
alternative approaches to identify aspects for fungal-specific targeting. Recently,
structure-guided synthetic analogs of FK506 have been developed that demonstrate greater
relative specificity for fungal calcineurin than its human counterpart [28–30]. Strengthening
this proof-of-concept, these analogs retain antifungal activity with reduced immunosuppressive
activity signifying that this approach could yield drug candidate leads. Such an approach
of structure-guided synthesis of inhibitors can also be applied to target downstream
components of calcineurin, such as Crz1. Another approach could be to identify unique
structural and/or sequence features that differ between fungi and the host for drug
targeting. Interestingly, a filamentous fungal-specific phosphorylated serine proline-rich
(SPRR) domain was identified in A. fumigatus CNA homolog, which is important for regular
hyphal growth, revealing unique protein sequence features that could be exploited
for drug development [31]. These findings have revealed a hidden potential of calcineurin
inhibitors and suggest more extensive research involving structure-guided drug design
is warranted.
What remains to be learned about calcineurin in pathogenic fungi?
Fungal infections pose a significant challenge to both global human health and food
security [32,33]. This threat is further magnified by the increasing population of
immunocompromised people and increasing resistance of fungal pathogens to existing
treatment options. This scenario suggests that more research is needed to identify
novel fungal-specific drug targets that could lead to the development of fungicidal
drugs. Studying and characterizing calcineurin in detail in key fungal pathogens has
the potential to identify fungal-specific drug targets within this signaling network.
However, at present, there is a substantial gap between our knowledge of calcineurin’s
roles in fungi and how these are performed at a molecular level. Most research has
focused on the functions of calcineurin in cell wall integrity pathways and studying
the contributions of Crz1 as a downstream effector. While both of these factors are
important and necessary components of the signaling network, they are not sufficient
to fully explain the role of calcineurin in fungal pathogenesis. Therefore, more focused
and mechanistic approaches are required to critically dissect the roles of calcineurin
and its substrates to provide important breakthroughs.
Future studies should delineate a more complete repertoire of calcineurin substrates
in fungi. This may result in the identification of calcineurin effectors that are
required for virulence but might be specific to a fungal species. In addition, characterizing
these effectors in multiple species could also reveal a set of substrates or functions
that are specific to fungi and absent from humans. For example, calcineurin interactions
at septa appear to be conserved across fungi and thus reflect interactions with fungal-specific
targets [8,12]. In addition, such studies may also reveal the basis of the requirement
for calcineurin in specific host niches. For example, the requirement for calcineurin
in C. albicans virulence in mice depends on the mode of infection, where it is essential
for systemic infection, but not for pulmonary or vaginal infection [4,5,34]. Such
studies could help delineate specific host factors that contribute to the establishment
of infections revealing interesting infectious disease biology involving calcineurin
signaling. This might include delineating roles of calcineurin effectors in different
host niches allowing the development of effector-targeting drugs that might be condition
specific.
Another exciting aspect of calcineurin signaling that is understudied is the documentation
of kinases that phosphorylate calcineurin substrates and thereby regulate their function.
For example, the kinase responsible for phosphorylating Crz1 and required for its
nuclear export in fungal pathogens remains uncharacterized. Interestingly, 2 separate
kinases, Hrr25, and protein kinase A, are known to phosphorylate Crz1 in S. cerevisiae
suggesting that there may be more than 1 Crz1-regulating kinase in other fungi as
well [19,35]. Calmodulin is known to also activate several kinases in response to
calcium signaling [36] and some of these kinases may phosphorylate calcineurin’s substrates.
Recognizing and studying these negative regulators of calcineurin signaling will be
important and may lead to the identification of core calcineurin effectors that play
essential roles in fungal pathogenesis.
Calcineurin plays a fundamental role in virtually every fungal pathogen studied to
date and yet research into this pathway can be accelerated. Going forward, further
research into calcineurin signaling will be necessary to fully exploit the therapeutic
potential of this essential virulence network. Targeting calcium–calcineurin signaling
has the potential to significantly influence not only human health and global food
resources but also biodiversity because of its significance in a broad range of fungal
pathogens. As a true Achilles’ heel, calcineurin signaling can be aimed at and targeted
to counter the emerging threats of fungal diseases.