Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy.

0.67 MB·English

by  Hayek, Summer R.

REVIEWARTICLE
published:27January2014
doi:10.3389/fphar.2014.00004
Advances in targeting the vacuolar proton-translocating
ATPase (V-ATPase) for anti-fungal therapy
SummerR.Hayek1,SamuelA.Lee2,3 andKarlettJ.Parra1*
1DepartmentofBiochemistryandMolecularBiology,SchoolofMedicine,UniversityofNewMexicoHealthSciencesCenter,Albuquerque,NM,USA
2DepartmentofInternalMedicine,SchoolofMedicine,UniversityofNewMexicoHealthSciencesCenter,Albuquerque,NM,USA
3SectionofInfectiousDiseases,NewMexicoVeteransHealthcareSystem,Albuquerque,NM,USA
Editedby: Vacuolar proton-translocating ATPase (V-ATPase) is a membrane-bound, multi-subunit
GeorgeTegos,UniversityofNew enzyme that uses the energy of ATP hydrolysis to pump protons across membranes.
Mexico,USA
V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for
Reviewedby:
generation of the membrane potential that drives secondary transporters and cellular
ParthaKrishnamurthy,Universityof
metabolism. V-ATPase is highly conserved across species and is best characterized in
KansasMedicalCenter,USA
GeorgeDiallinas,Universityof themodelfungus Saccharomycescerevisiae. However, recentstudiesinmammalshave
Athens,Greece identifiedsignificantalterationsfromfungi,particularlyintheisoformcompositionofthe14
*Correspondence: subunitsandintheregulationofcomplexdisassembly.Thesedifferencescouldbeexploited
KarlettJ.Parra,Departmentof
for selectivity between fungi and humans and highlight the potential forV-ATPase as an
BiochemistryandMolecularBiology,
anti-fungal drug target. Candida albicans is a major human fungal pathogen and causes
SchoolofMedicine,Universityof
NewMexicoHealthSciencesCenter fatalityin35%ofsystemicinfections, evenwithanti-fungaltreatment.Thepathogenicity
MS084670,Albuquerque,87131 ofC.albicanscorrelateswithenvironmental,vacuolar,andcytoplasmicpHregulation,and
NewMexico,USA
V-ATPaseappearstoplayafundamentalroleineachoftheseprocesses. Geneticlossof
e-mail:[email protected]
V-ATPaseinpathogenicfungileadstodefectivevirulence,andacomprehensivepictureof
themechanismsinvolvedisemerging.Recentstudieshaveexploredthepracticalutilityof
V-ATPaseasananti-fungaldrugtargetinC.albicans,includingpharmacologicalinhibition,
azole therapy, and targeting of downstream pathways.This overview will discuss these
studiesaswellashypotheticalwaystotargetV-ATPaseandnovelhigh-throughputmethods
foruseinfuturedrugdiscoveryscreens.
Keywords:fungalV-ATPase,vacuolarprotonpump,vacuolaracidification,pHhomeostasis,C.albicansvirulence,
anti-fungaltarget
V-ATPasePUMPS:STRUCTURE-FUNCTIONAND an intricate mechanism that uses relative rotation of subunits
MECHANISMOFCATALYSIS in V and V (Sun-Wada etal., 2003; Forgac, 2007). During
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VACUOLARH+-ATPase(V-ATPase)PUMPSARELARGEMULTI-SUBUNIT catalysis, hydrolysis of ATP within the protuberant structure of
MOLECULARMOTORSTHATCOUPLEACTIVETRANSPORTOFPROTONS V1 drives rotation of a central stalk (the rotor’s shaft) located
WITHATPHYDROLYSISTOACIDIFYINTRACELLULARCOMPARTMENTS nearthecatalyticsites.Therotatingcentralstalkisconnectedto
V-ATPase pumps generate and sustain the distinctive organelle a hydrophobic ring of proteolipid-like subunits in Vo (c-ring).
pH gradient of the endomembrane system that is necessary for During rotation, each subunit of the c-ring has one essen-
Golgi,endosomal,vacuolar,andlysosomalfunctions(Kane,2006; tial glutamate residue that accepts a proton from the cytosol
Forgac,2007). Redistributionof protonsfromthecytosoltothe andtransfersittotheorganelle’slumenagainstaconcentration
lumen of acidic organelles by V-ATPase pumps is essential for gradient.
organelle pH homeostasis. In fungi, V-ATPase also contributes ImportantchangesoccurredasV-ATPaseevolvedfromS.cere-
to cytosolic pH regulation (Martinez-Munoz and Kane, 2008). visiae to humans. For example, the c-ring of all fungal species
(cid:2) (cid:2)(cid:2)
GeneticandpharmacologicinactivationofV-ATPasepumpsalters contains a combination of three subunits (Voc, Voc, Voc )
(cid:2)
intracellular and extracellular pH. It disturbs numerous cellu- whereas mammalian c-rings lack the Voc subunit. Phylogenic
lar processes including protein processing and sorting, protein analysisbyFinniganetal.(2012)suggeststhatthefungalVocand
(cid:2)
secretion, receptor-mediated endocytosis, vesicular membrane Voc subunitsevolvedfromageneduplicationinanancestralgene
trafficking, zymogen activation, and autophagy (Kane, 2006; thatwascommontoallfungalspecies.Intriguingly,thefunctions
(cid:2)
Forgac,2007). ofVocandVoc infungi,particularlythebindingcapabilitiesof
The vast majority of structural and mechanistic data on eachsubunit,appeartohavedegeneratedfromthecommonances-
eukaryotic V-ATPases available have been collected in Saccha- tor (Finnigan etal.,2012). This example of constructive neutral
romycescerevisiae.V-ATPaseprotontransportrequiresstructural evolutionsuggeststhatthecomplexityof theV-ATPasemachine
and functional coupling of a peripheral domain (V ) with a may have been driven in part by loss-of-function processes
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membrane-embeddeddomain(V ;Figure1).Couplinginvolves (Doolittle,2012).
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Hayeketal. TheV-ATPasemodelforanti-fungaltargeting
In general, the complexity of V-ATPase increased as species we also refer to S. cerevisiae and other fungi throughout this
evolvedfurtherfromfungi. Mammalsdevelopedmultipletissue review.
and membrane specific isoforms for most V and V subunits
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(Figure 1; Marshansky and Futai, 2008; Sun-Wada and Wada, CandidaalbicansISTHEPRIMARYHUMANFUNGAL
2010;Toeietal.,2010).Incontrast,onlyonesubunit(V a)ofV- PATHOGEN
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ATPaseinbuddingyeastspeciessuchasS.cerevisiaehasfunctional Candidaalbicansisthemostfrequentlydiagnosedfungalpathogen
homologues(Manolsonetal.,1994),andnon-buddingfungisuch andisthefourthleadingcauseofhospital-acquiredbloodstream
asNeurosporacrassacontainonlysinglesubunitisoforms(Chavez infectionsinNorthAmerica(Klotzetal.,2007).C.albicansisnor-
etal.,2006). mallyaharmlesscommensalintheoralcavity,digestivetract,and
The sequence conservation between human and S. cerevisiae genitalregionofhealthypeoplebutisalsoassociatedwithsuper-
subunitsis31–41%identityand51–60%similarity,dependingon ficialinfections. C.albicans canenterthebloodstreamfollowing
thesubunitandisoform(Rahmanetal.,2013).Thisrelativelylow tissue damage or the formation of fungal biofilms on medical
sequenceconservationmayexplaindifferencesinbindingaffinity implants,leadingtosepsis,andorganfailure. Theseseverecases
between V-ATPase subunits from human (Rahman etal., 2013) ofsystemiccandidiasisaremostcommoninpatientsundergoing
andS.cerevisiae(OotandWilkens,2012);itmayalsofinetuneV- immunosuppressivetherapyorthatareotherwiseimmunocom-
ATPaseactivityanddetermineregulatorymechanismsinresponse promised(PfallerandDiekema,2007).Critically,patientmortality
to diverse cellular signals and environments. Although infor- ratescanreach35%evenwithanti-fungaltreatment(Hornetal.,
mation describing the topological arrangements of the human 2009).
V V complex is not available, the V-ATPase overall structure InvasiveinfectionduetoC.albicansisamultifactorialprocess
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anditssophisticatedmechanismof rotationalcatalysisarelikely thatreliesonnumerousvirulencefactorstocontrolpathogenesis.
conservedfromS.cerevisiae. C.albicanscanexistaseitheraunicellularyeastorafilamentous
This review focuses on fungal V-ATPases with an emphasis hyphae.Thismorphologicaldimorphismcontributestovirulence,
on the human fungal pathogen Candida albicans. We summa- as the yeast form is considered nonpathogenic while the hyphal
rize our current understanding of the roles of V-ATPase in forminducesdamageandinvasionofhosttissue(Sudbery,2011).
pathogenicity as well as its antifungal drug targeting potential. C.albicansalsosecretesserineaspartylproteinasesandlipasesthat
Because V-ATPase subunit structure and composition, assem- are involved in nutrient acquisition, host cell degradation, and
bly and regulation, and multiple downstream cellular functions immuneevasion(Nagliketal.,2003).OtherC.albicansvirulence
are best studied in the fungus S. cerevisiae (Kane, 2006, 2007), pathways include iron acquisition from hemoglobin, protection
FIGURE1|V-ATPasesubunitcompositionandmechanismofcatalysis. theV1Acatalyticsubunitsdrivesrotationofashaft(subunitsD,F)that
TheV-ATPaseprotonpumpacidifiesthelumenoforganellesinthe penetratesV1andisboundtoarotatingproteolipidringstructureinVo
endomembranesystemofalleukaryoticcells.V-ATPasehas14subunitsthat formedbysubunitsc(3−4)c’c”(c-ring)thattogetherwithsubunitVoaforms
formtwodomains,V1andVo.V1(clearandgraysubunits,A3B3CDE3FG3H) thepathforprotontransport.WiththeexceptionofsubunitVoa,whichexists
hydrolyzesATPatthecytosolicsideofthemembrane,andVo(bluesubunits, intwoisoforms(Vph1pandStv1p),eachV-ATPasesubunitisencodedbya
ac3−4c’c”de)translocatesprotons.Transportofprotonsagainsta singlegeneinfungi.Incontrast,multipleisoforms(twotofour)existformost
concentrationgradiententailsarotationalmechanism.Hydrolysisof3ATPin subunitsofthemammalianV-ATPase.SubunitVoc(cid:2)isabsentinmammals.
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Hayeketal. TheV-ATPasemodelforanti-fungaltargeting
againstreactiveoxygenspecies,expressionofadhesionmolecules, Kane,2008;HuangandChang,2011).Together,thesedatasuggest
and formation of biofilms. Together, these pathways facilitate thatV-ATPasemaycontributetonumerousvirulencepathwaysin
hostcellinvasionandprotectagainstthehostimmuneresponse C.albicans,includingvacuolarfunctionandduringthegermtube-
(Karkowska-Kuletaetal.,2009). to-hyphaemorphologicaltransition.Next,wesummarizestudies
thathaveexaminedtheroleofV-ATPaseinfungalpathogenesis
CandidaalbicansPATHOGENICITYCORRELATESWITHpH usinggeneticloss-of-functionstudies.
REGULATION,SUGGESTINGTHATV-ATPaseMAYPLAYA
FUNDAMENTALROLEINFUNGALVIRULENCE
CandidaalbicansVIRULENCEISREGULATEDBYpHATTHE GENETICLOSSOFV-ATPaseINPATHOGENICFUNGI
EXTRACELLULAR,VACUOLAR,ANDCYTOPLASMICLEVEL AFFECTSVIRULENCE
Extracellular pH controls the morphological dimorphism of C. GENETICSTUDIESINVARIOUSPATHOGENICFUNGIHAVE
albicans.Thenon-pathogenicyeastformofthefungusgrowspref- ESTABLISHEDALINKBETWEENV-ATPaseACTIVITY,VACUOLAR
erentiallyunderacidicenvironmentalconditionswhileincreasing ACIDIFICATION,ANDFUNGALVIRULENCE
extracellular pH triggers hyphal growth and increased virulence Hiltyetal.(2008)removedVMA1(theV AsubunitofV-ATPase)
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(Sudbery,2011).C.albicanshasdevelopedmechanismsthatallow fromtheHistoplasmacapsulatumgenomeanddemonstratedthat
ittorapidlyrespondtoenvironmentalpHchanges,andmutants V Aisrequiredforironsequestration,replicationinmacrophages,
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lacking the ability to sense extracellular pH display reduced andgrowthasamold.Thevma1mutantswerealsoavirulentina
virulence(Davis,2009). mousemodelofhistoplasmosis(Hiltyetal.,2008).InCryptococ-
Vacuolar pH is important for numerous aspects of C. albi- cusneoformans,lossoftheV asubunitviadeletionofVPH1leads
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cansphysiologyandvirulence.Preservationofaprotongradient todefectiveproductionofcapsule,laccase,andurease,threevir-
across the vacuolar membrane is critical for general cellular ulencefactorsrequiredforC.neoformansinfectivity. Thesevph1
metabolism, including receptor-mediated endocytosis, intracel- mutants also displayed defective in vivo virulence in a murine
lular membrane trafficking, pro-hormone processing, protein modelofmeningo-encephalitis(Ericksonetal.,2001).Thesefind-
degradation, uptake of small molecules, and storage and detox- ingslendcredencetotheideathatV-ATPaseplaysacriticalrolein
ificationofmetabolitesandions(Vesesetal.,2008).Additionally, themaintenanceoffungalvirulence.
both activation and secretion of the proteinase and lipase vir- In contrast, studies inAspergillus fumigatus suggest that host
ulence factors and activity of the enzymes themselves require V-ATPase plays an important protective role during immune
optimalvacuolarpH(Nagliketal.,2003). NumerousC.albicans defense against fungal pathogens. A. fumigatus is the primary
mutantsthatdisplayabnormalvacuolaralkalinizationalsodisplay causative agent of life-threatening invasive bronchopulmonary
reduced filamentation and defective in vivo virulence (Bruck- aspergillosis. While host V-ATPase is critical for phagolysoso-
mann etal., 2000; Jia etal., 2002; Eck etal., 2005; Poltermann mal acidification and pathogen killing under non-pathogenic
etal.,2005;Palanisamyetal.,2010;Zhangetal.,2010;Patenaude conditions, infective variants of A. fumigatus prevent acidi-
etal.,2013;Raneetal.,2013).Theazoleclassofanti-fungaldrugs fication of phagolysosomes and allow for A. fumigatus ger-
alsofunctionsinpartthroughdisruptionofvacuolaracidification mination and immune escape (Ibrahim-Granet etal., 2003).
(Zhangetal.,2010). Notably, work by Slesiona etal. demonstrated that less virulent
Finally, cytoplasmic pH contributes to filament formation Aspergillus variants can be made virulent via pharmacological
during C. albicans virulence. Germ tube formation, the pre- inhibition of host V-ATPase resulting in loss of phagolysoso-
cursor step to hyphal formation, requires alkalinization of the mal acidification (Slesiona etal., 2012). These findings sug-
cytoplasm (Stewart etal., 1988). In fungi, cytosolic pH is reg- gest that fungal pathogens may inactivate host V-ATPase path-
ulated via the Pma1p plasma membrane proton transporter, ways during immune evasion and suggest that the balance of
which pumps protons out of the cell and into the extracellu- pathogenversushostV-ATPaseactivityiscriticalfordetermining
larspacetomaintainaneutral-to-alkalinecytosolandanacidic virulence.
extracellular environment (Monk etal., 1991). The importance StudiesspecificallyexaminingvirulenceinC.albicanshavefur-
of Pma1p activity and cytosolic alkalinization in C. albicans thersolidifiedtheimportanceofV-ATPaseinthisprocess.Inthe
virulence is illustrated by studies showing that Pma1p activ- iron-deplete conditions of host tissue, C. albicans must extract
ity and expression is upregulated during filamentation (Kaur ironfromhemoglobinforsurvival.Weissmanetal.(2008)demon-
(cid:2)
and Mishra, 1991; Monk etal., 1993). Furthermore, C. albicans stratedthatnullmutantsofvma11(subunitV c)aredeficientin
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mutantsthatcannotproperlyalkalinizetheircytosolinresponse ironacquisition.UponlossofVMA7(subunitV F)inC.albicans,
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to filamentation cues are avirulent (Stewart etal., 1988, 1989; Poltermann etal. (2005) noted defects in in vitro filamentation
Mahantyetal.,1990). andinvivovirulenceduringsystemiccandidiasis.Theauthorsfur-
Thecentralimportanceof pHregulationinC.albicans viru- therconnectedthesedefectstobiochemicalphenotypesincluding
lencemakesV-ATPaseanattractivetargetforanti-fungaltherapy vacuolaralkalinization,pH-dependentgrowth,andsensitivityto
(Parra,2012).InadditiontoitscriticalroleinvacuolarpHhome- metal ions (Poltermann etal., 2005). Recently, our laboratories
ostasis (Kane, 2006; Tarsio etal., 2011), V-ATPase is a known demonstratedthatinduciblelossofVMA3(subunitV cofthec-
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regulatorofPma1pactivityinS.cerevisiae.V-ATPasemutantsdis- ring)inC.albicansresultsinalkalinevacuoleswithfissiondefects,
playabnormallyacidifiedcytosolduetoalackofproperlylocalized leading to reduced protease and lipase secretion, defective fila-
or functional Pma1p (Perzov etal., 2000; Martinez-Munoz and mentation,andineffectivemacrophagekilling(Raneetal.,2013).
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Hayeketal. TheV-ATPasemodelforanti-fungaltargeting
Unpublished studies from our laboratories have yielded similar CURRENTUTILITYOFV-ATPaseASANANTI-FUNGALDRUG
phenotypesfollowingtheinduciblelossofVMA2(subunitV B). TARGET
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InC.albicans,theVoasubunitistheonlysubunitwithinthe The recent emergence of multidrug resistant strains of C. albi-
enzyme complex that is encoded by multiple isoforms: VPH1 cans has made the development of novel classes of anti-fungal
localizes V-ATPase to vacuoles while STV1 traffics V-ATPase to drugs paramount (Hameed and Fatima, 2013). V-ATPase is an
theGolgiandendocyticorganelles(Patenaudeetal.,2013;Raines attractive target for drug discovery, given the numerous lines
etal., 2013). This difference was recently exploited to study the of genetic evidence supporting a critical role for V-ATPase in
contribution of organelle-specificV-ATPase to C. albicans viru- C. albicans virulence. We next consider the feasibility of V-
lence. BothourlaboratoriesandPatenaudeetal.(2013)showed ATPase as an anti-fungal drug target via an overview of three
thatlossofVPH1butnotSTV1leadstovacuolaralkalinization, currently available methods that utilizeV-ATPase-related mech-
abnormalvacuolarmorphology,anddefectivemetalionseques- anisms: pharmacological inhibition of V-ATPase, azole therapy,
tration(Rainesetal.,2013).Ourstudynotedthatalthoughthese andPma1pinhibition.
biochemical defects contributed to reduced protease and lipase
secretion,thevph1mutantsdisplayedonlymodestfilamentation PHARMACOLOGICALINHIBITIONOFV-ATPase
defectsandwild-typelevelsofbiofilmformationandmacrophage V-ATPaseinhibitorshavebeenusedforover20yearstostudythe
killing (Raines etal., 2013). Our results suggest that vacuolar functionandmechanismofV-ATPaseactivityinorganismsrang-
acidification is dispensable during certain C. albicans virulence ing from fungi to humans. As of 2009, eight types of V-ATPase
pathways. Furthermore, Stv1p-containing V-ATPase complexes inhibitors had been described, including the best characterized
may play novel roles in pathogenesis (Figure 2). In contrast, plecomacrolide class (Huss and Wieczorek, 2009). The pleco-
the vph1 mutant cells used in the Patenaude etal. (2013) study macrolides include bafilomycin and concanamycin, which are
displayed defective filamentation, reduced damage to epithelial antibiotics that bind to theV c subunit of V-ATPase to prevent
o
and macrophage host cells, and avirulence during systemic in c-ringrotationandinterferewithATPhydrolysisandprotontrans-
vivo infection. These results suggest that vacuolar acidification port simultaneously (Bowman etal., 1988; Drose etal., 1993).
isessentialforallformsofC.albicansvirulence.Theexplanation Notably, both bafilomycin and concanamycin have been shown
for these disparate results remains to be determined but likely toinhibitV-ATPasefromC.albicans(CalvertandSanders,1995;
centers around differences in strain background or methodol- Chanetal.,2012). However,noneof themoleculesbelongingto
ogy.Nonetheless,thesestudiesraisethefascinatingpossibilitythat theaforementionedeightclassesofV-ATPaseinhibitorsarecur-
organelle-specificV-ATPaseactivitycanbemodulatedtocontrol rentlyusedtotreatC.albicansinfectioninaclinicalsetting;many
specificC.albicansvirulencepathways. cannot differentiate between the fungal target and mammalian
FIGURE2|V-ATPaseinnon-vacuolarorganelles(Stv1p-containing unpublishedresults).VMA3deficient:Vacuolaralkalinizationanddefective
complexes)playsayetunknownroleinC.albicansvirulence.Wild-type: Pma1pactivityoccuratlevelsequaltothatofVPH1deficientcellswhenall
Whenfunctional,V-ATPase-mediatedacidificationofvacuoles,Golgi,and V-ATPasefunctionismissing(Stv1p-andVph1-containingV-ATPase
secretoryvesiclesmaintainsorganellepHandsupportstrafficofPma1pto complexes;Raneetal.,2013andunpublishedresults).However,VMA3
thecellsurfaceforprotoneffluxandmaintenanceofanalkalinecytosol.VPH1 deficientC.albicansexhibitsgrowthdefectsatneutralpHandseverely
deficient:C.albicansgrowsnormallyatneutralpHwhenonly reducedfilamentationundertheseconditions.Wethereforehypothesizethat
Vph1p-containingV-ATPasecomplexes(vacuolarmembrane)aremissing. thepresenceofStv1p-containingV-ATPaseinnon-vacuolarorganelles
Onlymodestfilamentationdefectsareobvious,despitetheconcomitant maintainsvirulenceinthefaceofdefectivevacuolarandcytoplasmicpH
vacuolaralkalinizationanddefectivePma1pactivity(Rainesetal.,2013and homeostasis.Pink=acidic/acidified,blue=alkaline/alkalinized.
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Hayeketal. TheV-ATPasemodelforanti-fungaltargeting
hostV-ATPase(BowmanandBowman,2005;HussandWieczorek, These studies by Zhang etal. (2010) have identified a new
2009). regulatorypathwayinvolvedinV-ATPasefunctionaswellaseluci-
Recently,ourlaboratorydevelopedahigh-throughputscreen- datedanovelmechanismunderlyingazoletoxicityinC.albicans.
ing method to identify newV-ATPase inhibitors in S. cerevisiae From a therapeutic standpoint, these studies provide proof-of-
(Chan etal.,2012). In this method, S. cerevisiae cells are trans- principle that anti-V-ATPase agents could work in the clinical
formedwiththegeneforpHluorin,apH-sensitiveversionofGFP setting.Importantly,drugstargetingV-ATPasemayactmechanis-
(Brettetal.,2005).ThefluorescenceintensityofpHluorincanbe ticallysimilartoazoleswhilebypassingthemultidrugresistance
usedtoidentifymoleculesthatacidifythecytosolofS.cerevisiae thathasplaguedcurrentanti-fungaltherapies.
followingV-ATPaseinhibition.WescreenedthePrestwickChem-
ical Library (a collection of 1120 off-patent drug compounds) Pma1pINHIBITION
using this method and identified alexidine dihydrochloride and GiventheinterplaybetweenV-ATPaseandPma1pinS.cerevisiae
thonzonium bromide as novel and specific V-ATPase inhibitors (Perzov etal., 2000; Martinez-Munoz and Kane, 2008; Huang
(Chan etal., 2012). Cells treated with these drugs showed a V- and Chang, 2011) and C. albicans (unpublished results), anti-
ATPase-specificpH-sensitivegrowthphenotype(vmaphenotype) fungaltherapiesthatrelyonPma1pinhibitionmayaffectpathways
exclusive of fungi species. These inhibitors belong to a unique downstreamofV-ATPasefunction.Indeed,Bowmanetal.(1997)
classofV-ATPaseuncouplersthatinhibitprotontransportwith- demonstratedthat64of66mutationsthatrescuedconcanamycin
outaffectingATPhydrolysis.Wealsoconfirmedthatdisulfiram, A-mediatedV-ATPaseinhibitioninN.crassawerelocalizedtothe
acompoundidentifiedpreviouslybyahigh-throughputscreen- PMA1gene.Thesyntheticorganoseleniumcompoundebselenisa
ingmethodthatidentifiesdrugsthatalkalinizevacuoles(Johnson knowninhibitorofPma1p(Chanetal.,2007;Billacketal.,2009).
etal.,2010),inhibitsV-ATPaseactivityviathepHluorinscreening Ebselen exhibits anti-fungal activity against wild-type strains of
method. C. albicans (Soteropoulos etal., 2000; Bouhafs and Jarstrand,
Alexidine dihydrochloride, thonzonium bromide, and disul- 2002;Wojtowiczetal.,2004)aswellasfluconazole-resistantstrains
firam also inhibit V-ATPase in isolated C. albicans vacuoles. (Billack etal.,2009). These results again demonstrate the prece-
Alexidine dihydrochloride and thonzonium bromide also cause dencethattargetingcomponentsof theV-ATPasepathway,even
general cellular toxicity in intact C. albicans cells (Chan etal., via a downstream element such as Pma1p, can circumvent the
2012). Thesedatasuggestthatthesepre-existingdrugscouldbe drug resistance that has developed against presently available
repurposedasanti-fungaltherapies.Indeed,themajorityofcom- therapies.
poundsinthePrestwickChemicalLibraryhaveknownsafetyand
toxicityprofiles(Wermuth,2004),makingdrugrepurposingaless HYPOTHETICALAPPROACHESFORTARGETINGV-ATPase
dauntingtaskthandenovodrugdiscovery. THEDEVELOPMENTOFNEWANTI-FUNGALTREATMENTSAGAINST
V-ATPaseMUSTINVOLVENOVELSTRATEGIESFORIDENTIFYING
AZOLETHERAPY THERAPEUTICTARGETS
Azoles are a class of antifungal agents that are commonly used Selectivityforfungalpathogensoverinnocuoushosttissueisone
(cid:2)
to treat fungal infections in the clinical setting. Azoles inhibit criticaltargetingfactortoconsider.TheV c subunitofV-ATPase,
o
C-14α-demethylase, an enzyme necessary for the conversion of encodedbyVMA11,isapromisingcandidatetarget(Parra,2012).
(cid:2)
lanosteroltoergosterol,anessentialcomponentof fungalmem- TheV c subunitisfoundspecificallyinfungiandlacksamam-
o
branes(Chapmanetal.,2008).Atleastfourergosterolbiosynthesis malian homolog (Figure 1). Promisingly, previous studies have
genes(ERG3,ERG4,ERG5,andERG11)areaffectedbyazoles.The demonstrated that C. albicans null mutants of vma11 do not
generalmembranedisruptionresultingfromazoletreatmentalters properly acquire iron from hemoglobin, a critical virulence fac-
the activity of several membrane-bound transporters and chan- tor within host tissue (Weissman etal.,2008). Future structural
(cid:2)
nelsincludingthoseassociatedwiththetransportofaminoacids studies of the V c subunit will aid in the rational design of a
o
andothernutrients,chitinsynthesis,andmitochondrialoxidation fungal-specificanti-V-ATPasedrug.
(VandenBossche,1985;Barrett-Beeetal.,1991;Georgopapadakou MostfungalV-ATPasesubunitsareencodedbyasinglegene;
andWalsh,1996). the V a subunit is the only subunit encoded by two isoforms.
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Forsometime,itwasassumedthatazolescausedfungaltoxi- Mammalian V-ATPases are strikingly different, with seven dif-
cityprimarilythroughdamagetothecellmembraneresultingin ferentsubunitsdisplayingisoformvariation(Jefferiesetal.,2008;
cellpermeabilityandlysis(Chapmanetal.,2008).However,recent Figure 1). This drastic difference between fungi and mammals
workhasuncoveredacriticalroleforergosterolinV-ATPasefunc- could be exploited for fungal-specific drug development. For
tion.InS.cerevisiae,mutantsdefectiveinergosterolbiosynthesis example, theV C subunit exists as three isoforms in mammals.
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display alkaline vacuoles and the vma phenotype (Zhang etal., Ifaninhibitorisdesignedtospecificallytargetadomainoffungal
2010).Surprisingly,theV-ATPaseholoenzymeisproperlyassem- V Cthatismissingfromoneormoreofthemammalianisoforms,
1
bledatthevacuolarmembranedespiteergosteroldepletion.The allcellularV-ATPaseactivitywillceaseinthefungalpathogenwhile
mutant phenotypes instead appear to result from reduced ATP the non-targeted mammalian isoforms compensate and main-
hydrolysis and proton pumping within the V-ATPase. Flucona- tainV-ATPaseactivityinthehostcell. Thisstrategywillrequire
zoletreatmentofC.albicanscellsalsoresultsinalkalinevacuoles high-resolutionstructuresofallfungalandmammalianisoforms
(Zhangetal.,2010),furthersolidifyingthatazolesfunctioninpart andisthereforealonger-rangepossibility.However,itremainsa
throughinhibitionofV-ATPase. fascinatingoptionforfungal-specificdrugtargeting.
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Hayeketal. TheV-ATPasemodelforanti-fungaltargeting
V-ATPase activity is regulated in part by reversible disassem- alkalinizationofthecytoplasmisageneraldeterminantoffungal
bly,theprocessbywhichtheV andV sectorsseparatefromone virulence (Stewart etal.,1988), and CapHluorin-mediated drug
1 o
another to prevent ATP hydrolysis and proton transport (Kane screensmayresultintherapiddiscoveryofnewanti-fungalthera-
andParra,2000;Kane,2006).Reversibledisassemblyistriggered piesthatfunctionindependentlyofV-ATPase.Previousstudiesin
primarilybyglucosedeprivation, althoughextracellularpHand S.cerevisiaeprovedthefeasibilityofmodifyingpHluorinfortar-
salt stress are thought to contribute to the process. In mam- getingtospecificcellularcompartmentssuchastheGolgi(Tarsio
mals, cell-specific regulatory pathways also control disassembly etal.,2011).CapHluorinwillfacilitatesimilarstudiestoexamine
inatissue-specificmanner(Kane,2012).DisassemblyinC.albi- the importance of organelle-specificV-ATPase function and pH
cans hasnotbeenstudied,butconservedsimilaritiesbetweenS. homeostasisinC.albicans.Importantly,theCapHluorinconstruct
cerevisiae and mammals suggest C. albicans will utilize similar optimizedfornon-canonicalcodonusewillenablecytosolicpH
pathways. Notably, the V a subunit and organelle environment measurementsnotjustinC.albicans,butinallfungalspeciesthat
o
appeartoregulatedisassemblyinS.cerevisiae;Vph1p-containing utilizetheCTGclade,includingthehumanpathogensC.dublin-
complexes disassociate upon glucose deprivation while Stv1p- iensis,C.tropicalis,C.parapsilosis,andC.lusitaniae(Paponetal.,
containing complexes are less sensitive (Kawasaki-Nishi etal., 2012).
2001). In contrast, mammalian cells contain fourV a isoforms
o
localizedtodifferenttissues.Thissuggeststhatdifferencesiniso- ACKNOWLEDGMENTS
form structure and interaction, localization, or susceptibility to Theauthorsgratefullyacknowledgesupport,inwholeorinpart,
environmental cues could be exploited for fungal-specific drug fromtheNationalInstitutesofHealth(NIH),Bethesda,Maryland,
developmentasdescribedpreviously.Alternatively,indirectlytar- USA,includingGrants5R01GM086495(toKarlettJ.Parra)and
geting the regulator proteins involved in disassembly may allow K12GM088021(toSummerR.Hayek).
forfungal-specificinhibitionofV-ATPaseactivity(Kane,2012).
KEYCONCEPTS
Finally, Pma1p is a fungal-specific protein with no known
mammalianhomologs.SimultaneoustargetingofbothV-ATPase VACUOLARH+-ATPase(V-ATPase)PUMPSARELARGEMULTI-SUBUNIT
and Pma1p should therefore disrupt both vacuolar and cyto- MOLECULARMOTORSTHATCOUPLEACTIVETRANSPORTOFPROTONS
plasmic pH homeostasis in fungi versus vacuolar pH alone in WITHATPHYDROLYSIS
mammalian cells. This dual approach may thereby prove more V-ATPaseactivityisrequiredforacidificationofintracellularcom-
detrimental to the fungal pathogen than the host cell. This partmentsandgeneratesandsustainsthepHgradientrequiredfor
methodisalsoadvantageousinthatitcanbeachievedeitherby functionoftheendomembranesystemorganelles.
creationofanovelV-ATPase/Pma1pdualinhibitororbyadmin-
CandidaalbicansISTHEMOSTFREQUENTLYDIAGNOSEDFUNGAL
istration of an anti-fungal drug cocktail consisting of presently
PATHOGEN
available therapies. Additionally, as Pma1p is a plasma mem-
Although it exists commensally with humans, it can cause
braneproteinwithaccesstotheextracellularspace, anti-Pma1p
life-threateningbloodinfectionsunderoptimalpathogeniccon-
drugs may function regardless of accumulation within the fun-
ditions. C. albicans has developed resistance to many currently
gal cell. This possibility is highly advantageous, as drug uptake
availableanti-fungaldrugs,andmortalityratescanreach35%even
often limits efficacy in fungi due to a lack of specific uptake
withpropertreatment. Thereisthereforeadireneedtodevelop
systems.
newanti-fungaltherapies.
ANOVELHIGH-THROUGHPUTMETHODFORFUTURE
CandidaalbicansVIRULENCEISREGULATEDBYpHATTHE
V-ATPaseDRUGDISCOVERYINC.albicans
EXTRACELLULAR,VACUOLAR,ANDCYTOPLASMICLEVEL
Ourneedforanti-V-ATPasetherapiesgrowsincreasinglycritical
ExtracellularpHtriggersamorphologicalswitchtothepathogenic
as we improve our understanding of the function of V-ATPase
form of the fungus. Vacuolar acidification allows for activa-
infungalvirulence.However,directscreeningforanti-V-ATPase
tion/secretion of virulence enzymes. Cytoplasmic alkalinization
drugsinC.albicans istypicallyhinderedbythenon-availability
precedestheformationofgermtubesduringC.albicansfilamen-
of high-throughput screening tools in the pathogenic fungi. We
tation.
previouslydemonstratedthepracticalutilityofmeasuringcytoso-
licpHasasurrogateforV-ATPasefunctioninahigh-throughput GENETICSTUDIESINVARIOUSPATHOGENICFUNGIHAVE
screenforinhibitorsofS.cerevisiaeV-ATPase(Chanetal.,2012). ESTABLISHEDALINKBETWEENV-ATPaseACTIVITY,VACUOLAR
This method uses pHluorin, a pH-sensitive GFP construct that ACIDIFICATION,ANDFUNGALVIRULENCE
canbestablytransformed,therebyavoidingexpensiveandtime- StudiesinHistoplasmacapsulatum,Cryptococcusneoformans,and
consumingsingle-usedyes.Recently,Ullahetal.(2013) werethe Candida albicans have demonstrated that loss of all cellular V-
firsttosuccessfullyexpresspHluorininapathogenicfungus, C. ATPaseleadstoalkalinevacuolesandahostofinvitroandinvivo
glabrata.However,non-canonicalcodonuseprecludesthedirect virulencedefects.
applicationoftheexistingpHluorinconstructinC.albicans.
Our lab is currently working to create a pHluorin con- V-ATPaseISANATTRACTIVETARGETFORDRUGDISCOVERY
struct optimized for C. albicans (CapHluorin) that will enable Current anti-fungal therapies that involve the V-ATPase path-
easy,inexpensive,andhigh-throughputmeasurementof cytoso- way include direct pharmacological inhibition of V-ATPase, the
licpHforuseinscreeningforV-ATPaseinhibitors.Additionally, clinically prescribed azole class of drugs, and Pma1p inhibitors.
FrontiersinPharmacology|ExperimentalPharmacologyandDrugDiscovery January2014|Volume5|Article4|6
“fphar-05-00004” — 2014/1/24 — 16:34 — page 6 — #6
Hayeketal. TheV-ATPasemodelforanti-fungaltargeting
However, the emergence of multidrug resistant strains of C. albicans is a multifunctional protein that interacts with the putative vacuo-
albicansmakesadditionaldrugdiscoveryimperative. lar H+ -ATPase subunit Vma7p. Int. J. Med. Microbiol. 295, 57–66. doi:
10.1016/j.ijmm.2004.12.007
Erickson,T.,Liu,L.,Gueyikian,A.,Zhu,X.,Gibbons,J.,andWilliamson,P.R.
THEDEVELOPMENTOFNEWANTI-FUNGALTREATMENTSAGAINST
(2001).MultiplevirulencefactorsofCryptococcusneoformansaredependenton
V-ATPaseMUSTINVOLVENOVELSTRATEGIESFORIDENTIFYING VPH1.Mol.Microbiol.42,1121–1131.doi:10.1046/j.1365-2958.2001.02712.x
THERAPEUTICTARGETS Finnigan,G.C.,Hanson-Smith,V.,Stevens,T.H.,andThornton,J.W.(2012).
Suchstrategiesincludetargetingthefungal-specificV c(cid:2)subunit, Evolutionofincreasedcomplexityinamolecularmachine.Nature481,360–364.
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doi:10.1038/nature10724
utilizing differences in isoform composition and complex dis-
Forgac, M. (2007). VacuolarATPases: rotary proton pumps in physiology and
assembly between fungi and mammals, dual V-ATPase/Pma1p
pathophysiology.Nat.Rev.Mol.CellBiol.8,917–929.doi:10.1038/nrm2272
inhibition,andcreationofahigh-throughputscreeninC.albicans. Georgopapadakou,N.H.,andWalsh,T.J.(1996).Antifungalagents:chemother-
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Slesiona,S.,Gressler,M.,Mihlan,M.,Zaehle,C.,Schaller,M.,Barz,D.,etal.(2012). orreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensor
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differentstrategiesduringinteractionswithmacrophages.PLoSONE7:e31223. acceptedacademicpractice.Nouse,distributionorreproductionispermittedwhich
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