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Autophagic Punctum
Virulence 1:6, 1-4; November/December 2010; © 2010 Landes Bioscience
This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Beyond ergosterol
Linking pH to antifungal mechanisms
Yong-Qiang Zhang and Rajini Rao*
Department of Physiology; The Johns Hopkins University School of Medicine; Baltimore, MD USA
M
any antifungal drugs including the
highly successful azoles target the
fungal-specific sterol, ergosterol, yet the
molecular identity of cellular pathways
mediating antifungal activity remained
obscure. A recent study on the requirement of ergosterol in vacuolar H+-ATPase
(V-ATPase) function uncovered a critical role for ion homeostasis downstream
of azole inhibition of ergosterol biosynthesis. Here we review the functional
link between regulation of V-ATPase by
fungal membrane lipid components and
discuss contributions of V-ATPase function to pathogenicity. The importance
of pH homeostasis in virulence highlights its potential as target in antifungal
chemotherapy.
Key words: pH homeostasis, V-ATPase,
ergosterol, vacuole, Candida albicans,
yeast
Abbreviations: abbreviations
Submitted: 09/07/10
Accepted: 09/29/10
Previously published online:
www.landesbioscience.com/journals/
virulence/article/13802
Correspondence to: Rajini Rao;
Email: [email protected]
Addendum to: Zhang YQ, Gamarra S, GarciaEffron G, Park S, Perlin DS, Rao R. Requirement
for ergosterol in V-ATPase function underlies
antifungal activity of azole drugs. PLoS Pathog
2010; 6:e1000939.
www.landesbioscience.com
Sterols are major lipid components in
membranes. Besides plasma membrane,
where the bulk of sterols reside, sterols
have been found in intracellular organelles
including peroxisomes, mitochondria,
vacuoles/lysosome, ER and trans-Golgi
network. The presence of sterols in membranes modulates their thickness, fluidity
and permeability. Their preferential association with sphingolipids to form ‘raft’
like domains confers membrane heterogeneity and facilitates functions of raftassociated proteins. Fungal cells contain a
unique sterol, ergosterol. Deletion of genes
responsible for early steps in the ergosterol
biosynthetic pathway is lethal, demonstrating that ergosterol plays an essential
role in fungal cells. In the battle against
pathogenic fungi, ergosterol provides the
most common and effective target for
several classes of antimycotics, including azoles, allylamines, morpholines and
polyenes.1-3 Yet despite this importance,
the specific cellular pathways impacted
by ergosterol in mediating cytotoxicity of antifungal agents remain obscure.
Recently, we uncovered a plausible cellular mechanism for azole toxicity that links
ergosterol to pH homeostasis.4
The pleiotropic phenotypes of erg
mutants defective in the late steps of
ergosterol biosynthesis offer insight into
specific cellular processes modulated by
sterols. Thus, erg mutants are defective in
plasma membrane fusion and pheromone
signaling,5 in lipid raft association of beta1,3-glucanosyltransferase Gas1p,6 and
in plasma membrane targeting of tryptophan permease Tat2p and the marker
protein Fus1-Mid2-GFP.7,8 Ergosterol is
also important for endocytosis as demonstrated by defects seen in erg2Δerg6Δ and
erg3Δerg6Δ double mutants.9 These clearly
demonstrate that sterols are critical for
plasma membrane biogenesis and function.
In addition, erg mutants exhibit defects in
vacuolar fusion10 and in mitochondrial
morphogenesis and maintenance11 pointing to a critical role in organelle biogenesis. Some erg mutants are also associated
with ion-related phenotypes. For example,
growth of erg24Δ is dependent on external
Ca 2+ and is enhanced by Mg2+ 12 and erg6Δ
shows enhanced uptake of Li+ and Na+
ions.13 The mechanistic basis for many of
these phenotypes, nevertheless, is poorly
understood. Clearly our understanding of
the biochemical and cellular role of ergosterol is still limited.
We initially observed a striking overlap
between multiple erg phenotypes and those
associated with vma mutants, lacking the
vacuolar H+ pumping ATPase. Thus, in a
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Figure 1. Ergosterol removal alters V-ATPase
conformation. Vacuolar vesicles were
isolated from log phase cells of S. cerevisiae
strain BY4742 as described previously4 and
incubated on ice with or without methylbetacyclodextrin for 40 minutes. Vesicles were
then spun down and endogenous proteolysis
was analyzed by immunoblotting against
V-ATPase subunits Vph1 and Vma2.
genome-wide screen of hypersensitivity to
amiodarone, an antifungal agent known
to disrupt cellular Ca 2+ and H+ homeostasis, we recovered multiple vma and erg
mutants.14 A closer examination revealed
that erg mutants as exemplified by erg24Δ
(lacking C-14 sterol reductase), resembled
vma mutants in hypersensitivity to Zn2+
toxicity, inhibitors of the Ca 2+ -activated
phosphatase calcineurin (FK506) and
a cell wall disrupting agent (calcofluor
white). More significantly, erg mutants
exhibited the classical vma phenotype:
an inability to grow in alkaline medium,
consistent with an inability to acidify the
vacuole. Measurement of vacuole pH in
situ confirmed that erg mutants failed to
acidify vacuoles, essentially indistinguishable from vma mutants lacking a functional H+ pump. Finally, purified vacuoles
showed severe impairment of H+ pumping
and ATP hydrolysis rates in erg24Δ. Taken
together, these findings indicated a critical
requirement for ergosterol in V-ATPase
function.4
Three classes of antifungals (azoles,
morpholines and allylamines) target
enzymes catalyzing early reactions in the
2
ergosterol biosynthesis pathway. The most
widely used antifungal fluconazole targets
the lanosterol demethylase Erg11p, immediately upstream from Erg24p. We found
that fluconazole treatment of S. cerevisiae
or Candida albicans phenocopied deletion
of downstream ERG genes in eliciting
vacuolar alkalinization and inhibition of
V-ATPase. Strikingly, feeding S. cerevisiae
with exogenous ergosterol restored both
fungal growth and vacuolar acidification
in fluconazole treated cells.
Several lines of evidence suggest direct
modulation of V-ATPase by ergosterol.
First, ergosterol depletion in erg24Δ or by
fluconazole treatment (20 μg/ml, 6 h) did
not alter V-ATPase expression or its localization on vacuolar membrane.4 Second,
ergosterol depletion did not dissociate V1
and Vo domains, a mechanism by which
glucose rapidly regulates V-ATPase activity. Upon acute removal of ergosterol from
purified vacuolar vesicles by methyl-betacyclodextrin, both V1 and V0 domains
remained
membrane-associated
yet
V-ATPase activity was lost. Interestingly,
V-ATPase subunits exhibited distinct
proteolytic patterns following ergosterol
extraction, including enhanced degradation of specific fragments and appearance
of novel peptide bands (Fig. 1). This indicates that V-ATPase adopts a different
conformation in the absence of ergosterol
that may correlate with loss of activity.
Membrane protein function is tightly
coupled to membrane lipid composition. Indeed, association of V-ATPase
with cholesterol has been documented in
mammalian cells15,16 and incorporation
of cholesterol into membranes stimulated V-ATPase activity in osteoclasts.17
Phosphatidylinositol phosphates, sphingolipids and cardiolipin have also been
reported to regulate V-ATPase function, albeit by distinct mechanisms. In
renal epithelial cells, alteration in phosphatidylinositol phosphate composition
affected V-ATPase assembly and trafficking.18 Cardiolipin is located in mitochondrial membrane and its effect on V-ATPase
suggests functional cross-talk between
vacuoles and mitochondria.19 The role of
sphingolipids in yeast V-ATPase regulation has been characterized in detail.
It was shown that depletion of sphingolipids with C26 acyl group disrupted
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structural integrity of V1–Vo assembly
and disabled V1 domain function.20 This
is in contrast to our findings that ergosterol depletion did not impair V-ATPase
assembly or domain integrity, although it
may have altered protein conformation.
Thus, although intimately associated in
membranes, ergosterol and sphingolipids
have distinct effects on protein function.
Ergosterol has rigid planar rings whereas
sphingolipids display flexible long hydrocarbon chains. This structural difference
may dictate their preferential interaction with distinct protein motifs, thereby
modulating protein function by different
mechanisms.
The importance of the V-ATPase in
fungal physiology and virulence cannot be
underestimated. The V-ATPase maintains
acidic pH within the vacuole to activate
lysosomal enzymes for protein processing
and degradation, and generates a proton
motive force across endomembranes for
the transport, sequestration and detoxification of metabolites, ions and drugs.21,22
Besides the vacuole, V-ATPase is broadly
distributed in the endomembrane system,
including the Golgi, secretory vesicles, early
and late endosomes23 where its function is
critical for endocytosis, vesicle trafficking,
uncoupling of ligand-receptor complexes,
and pH-driven exocytosis. Impairment of
V-ATPase function, thus, could affect surface expression and secretion of proteins
such as superoxide dismutase (Sod5p)
and lipases, which play important roles
in facilitating infection.24,25 In addition,
Ca 2+ rich, alkaline serum conditions of the
mammalian host present a hostile environment for fungal cells. Defective pH and
Ca 2+ -homeostasis would render fungal
pathogens susceptible to detrimental ion
fluxes encountered in the host. Therefore,
disruption of V-ATPase activity downstream from antifungal drug inhibition
of ergosterol biosynthesis should cripple
multiple cellular functions required for
pathogenesis. Indeed, both erg24 -/- and
vma7-/- null mutants of C. albicans were
shown to be avirulent in mouse models
of Candidiasis.26,27 Thus, while ergosterol
depletion may well impact additional cellular targets that remain to be elucidated
at a molecular level, loss of V-ATPase
function is sufficient to account for loss of
virulence.
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Another critical consequence of disabling V-ATPase is inhibition of filamentous growth, as was observed for the
vma7-/- strain of C. albicans in the presence of serum or liquid Spider medium
(with mannitol).26 Fluconazole treatment
and ERG3 deletion have both been shown
to inhibit filamentation,28,29 which can
now be explained, at least in part, due to
impaired V-ATPase function. The ability
to transition from yeast to hyphae is a critical virulence factor for polymorphic pathogenic fungi, such as C. albicans.30 To infect
their hosts, pathogenic fungi first have to
penetrate host epithelial barriers. Once in
the bloodstream, fungal cells must survive
the attack by macrophages and dendritic
cells and subvert innate immune system.
A recent report shows that C. albicans
cells invade oral and gastrointestinal tractepithelia in hyphal form.31 Another study
shows that wild-type C. albicans cells are
more capable than a filamentation-defective mutant (efg1Δ/cph1Δ) in blocking
phagosomal maturation and acidification.32 Consistent with these findings, we
observe that vma7-/- mutants of C. albicans
fail to colonize Caco-2 epithelial models
in culture and are efficiently eliminated by
murine J774A.1 macrophage cells (Fig. 2).
Given the significance of filamentation in
virulence, the molecular basis underlying
the requirement for V-ATPase function in
filamentation is a critical area in the study
of fungal pathogenesis.
The requirement of ergosterol for
V-ATPase function suggests that combining azole drugs with other agents that
can trigger harmful ion fluxes would have
synergistic antifungal effect. The antiarrhythmia drug amiodarone elicits a surge
of cytosolic H+ and Ca 2+ that triggers ion
stress, cell cycle arrest and fungal death,33,34
and exhibits synergism with azoles in
vitro.14,35,36 We showed that pretreatment
with fluconazole exacerbated amiodarone
induced H+ and Ca 2+ surges in S. cerevisiae
and impaired vacuole function in C. albicans.4 Consistent with increased ion stress
from the drug combination, we observed
lysis of C. albicans cells in vitro (Fig. 3)
and significant reductions in microbial
kidney burden in a murine Candidiasis.35
Maintaining different organelles at
distinct pH is critical for multiple cellular events in all eukaryotic cells. The
Figure 2. C. albicans vma7-/- mutant cells are eliminated by macrophages and fail to colonize
epithelial cells. Murine macrophage cells (J774A.1) and human colorectal epithelial c ells (Caco-2)
were grown in DMEM with 10% fetal bovine serum to confluence (37ºC, 5% CO2). Log phase C.
albicans WT (CNC44) and vma7Δ/vma7Δ cells (VHU4)26 were inoculated into mammalian cultures
at multiplicity of infection of 1/1000 and incubated for 20 hours. Note the inability of vma-/- strains
to form hyphae and colonize host cells.
Figure 3. Combination of fluconazole and amiodarone induces lysis of C. albicans cells. Early log
phase cells of C. albicans SC5314 were grown in YPD or YPD plus 3.3 μg/ml fluconazole for 6 hours
at 30ºC. Cells were collected and incubated in YPD plus 5% fetal bovine serum with or without
amiodarone (10 μM; AMD) and fluconazole (3.3 μg/ml; Fluc) for 2 hours at 37ºC. Fewer cells and
increased lysis can be observed in the presence of both amiodarone and fluconazole.
www.landesbioscience.comVirulence
3
importance of pH homeostasis in fungal
pathogenicity is illustrated by the avirulence of V-ATPase null mutants. Future
studies of other proteins involved in ion
homeostasis will provide in-depth understanding of the role of intracellular ion
traffic in specific aspects of pathogenicity,
such as secretion of virulence factors, filamentation, biofilm formation, subverting
host innate immunity, and adaptation of
parasitic life styles. Meanwhile, the success of azole drugs and the realization of
their effect on V-ATPase function demonstrate the effectiveness of targeting pH
homeostasis in fungal pathogens. The
therapeutic potential of combining agents
that weaken maintenance of pH homeostasis and those disrupt ion fluxes remain
to be further explored in the future.
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