Review Article Regulated Cell Death as a Therapeutic Target for Novel Antifungal Peptides and

21 Pages·2017·1.76 MB·English

Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2018, Article ID 5473817, 20 pages
https://doi.org/10.1155/2018/5473817
Review Article
Regulated Cell Death as a Therapeutic Target for Novel Antifungal
Peptides and Biologics
1,2,3,4 5,6 7
Michael R. Yeaman , Sabrina Büttner, and Karin Thevissen
1DivisionofMolecularMedicine,LosAngelesCountyHarbor-UCLAMedicalCenter,Torrance,CA90502,USA
2DivisionofInfectiousDiseases,LosAngelesCountyHarbor-UCLAMedicalCenter,Torrance,CA90502,USA
3LosAngelesBiomedicalResearchInstituteatHarbor-UCLAMedicalCenter,Torrance,CA90502,USA
4TheDavidGeffenSchoolofMedicineatUCLA,LosAngeles,CA90024,USA
5DepartmentofMolecularBiosciences,TheWenner-GrenInstitute,StockholmUniversity,Stockholm,Sweden
6InstituteofMolecularBiosciences,UniversityofGraz,Graz,Austria
7CentreofMicrobialandPlantGenetics(CMPG),KULeuven,Leuven,Belgium
CorrespondenceshouldbeaddressedtoMichaelR.Yeaman;[email protected]
Received 13 December 2017; Accepted 7 March 2018; Published 26 April 2018
AcademicEditor:AlessandraRicelli
Copyright©2018MichaelR.Yeamanetal.This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Theriseofmicrobialpathogensrefractorytoconventionalantibioticsrepresentsoneofthemosturgentandglobalpublichealth
concerns for the 21st century. Emergence of Candida auris isolates and the persistence of invasive mold infections that resist
existing treatment and cause severe illness has underscored the threat of drug-resistant fungal infections. To meet these
growing challenges, mechanistically novel agents and strategies are needed that surpass the conventional fungistatic or
fungicidal drug actions. Host defense peptides have long been misunderstood as indiscriminant membrane detergents.
However, evidence gathered over the past decade clearly points to their sophisticated and selective mechanisms of action,
including exploiting regulated cell death pathways of their target pathogens. Such peptides perturb transmembrane potential
andmitochondrialenergetics,inducingphosphatidylserineaccessibilityandmetacaspaseactivationinfungi.Thesemechanisms
are often multimodal, affording target pathogens fewer resistance options as compared to traditional small molecule drugs.
Here, recent advances in the field are examined regarding regulated cell death subroutines as potential therapeutic targets for
innovative anti-infective peptides against pathogenic fungi. Furthering knowledge of protective host defense peptide
interactions with target pathogens is key to advancing and applying novel prophylactic and therapeutic countermeasures to
fungalresistanceandpathogenesis.
1. Significance of Fungal Infections [7–9].Additionally,Candidaspeciesareamongthemostcom-
moncausesofcatheter-relatedfungalinfections[10].Despite
1.1. Medical Burden of Fungal Infection. In the last two modestadvancesinantifungaltherapy,attributablemortality
decades, Candida species have emerged as the third most of candidemia remains approximately 40% [11]. The emer-
common pathogen of nosocomial septicemia, accounting gence of highly antifungal-resistant species such as C. auris
for 5–10% of all hospital-acquired bloodstream infections compounds these concerns [12]. Likewise, life-threatening
[1–3]. Overall incidence of candidemia now surpasses inci- infectionscausedbyAspergillus,Rhizopus,andMucorspecies
dences of bacteremia due to Escherichia coli or Klebsiella areseenwithincreasingincidenceasthenumbersofpatients
species[4,5].Furthermore,Candidaspeciesarethemostcom- having immunosuppression in settings of hematopoietic or
mon cause of deep-seated fungal infections in patients who solid organ transplant, cytotoxic cancer chemotherapy, and
haveextensiveburns[6]orhaveundergonetransplantation relatedconditionsincrease[13,14].Collectively,thesefungal
2 OxidativeMedicineandCellularLongevity
infections have unacceptably high mortality rates that may opportunistic fungal infections are increasingly common in
exceed50%,evenwithgold-standardantifungaltherapy. transplantation[26].
(5)Autoimmunity.Autoimmunediseasesaffect5–10%ofthe
1.2. Urgent Need for Innovative Solutions. Convergence of
population worldwide [27], translating to hundreds of mil-
increasing populations at risk of serious fungal infections,
lionsoflivesimpacted.Autoimmunediseasesareburgeoning
emerging resistance to conventional antifungal agents, and
globally[28],andasagroupareequivalentinprevalenceto
apaucityofdevelopmentofmechanisticallynovelantifungal
heart disease, and twice that of cancer [29, 30]. Immune-
therapeuticsportendsasignificantpublichealthconcern.
modifyingtherapiesusedtotreatautoimmunediseaseshave
significantrisksforlife-threateninginfection,includingthose
1.2.1.BurgeoningPopulationsatRisk.Populationsofindivid- causedbyopportunisticandpathogenicfungi[31,32].
ualsatriskofseverefungalinfectionsarerapidlyexpanding
in scope and number worldwide and are related to several
1.2.2. Resistance to Existing Antimycotics. Fungal infections
trends with respect to the global population demographics,
areoftendifficulttotreatforseveralreasons:(1)fewselective
includingthefollowing:
targets in these eukaryotic pathogens as compared to the
(1) Aging. Immune waning and senescence have significant human host; (2) increasing resistance to conventional anti-
fungalagentswhichhaveadvancedlittleinthepast50years;
negative impacts on host defense against infection [15, 16].
and (3) the lack of mechanistically novel antifungal agents
Infectionisnowtheattributablecauseofmortalityinnearly
developed for clinical application. As a result, an additional
one-thirdofallindividualsaged65yearsorolder[17].Esti-
matesprojectthatby2050,thenumberofpersons≥65years concern is the emergence of resistance to antifungal agents,
of age will reach 1.5 billion, and those aged≥80 years will whicharecommonlyusedtopreventandtreatdisseminated
candidiasis. Azole-resistant Candida strains are being iso-
reach 395 million [18, 19]. Thus, along with risk factors
lated with increasing frequency, even in patients without
imposed by age-related comorbidities, the growing cohort
ofagingindividualsportendssignificantincreasesinoppor- AIDS[1,2].Althoughalimitednumberofantifungaldrugs
have been developed, experience with antibacterial agents
tunisticandpathogenicfungalinfections.
predictsthatresistancetonovelantifungaldrugswillemerge
(2) Cancer. Infection is a significant risk factor for morbid- as their use increases [33]. Multiple factors have led to the
continuous increase of reported antifungal resistance in the
ity in cancer patients and is a leading cause of attributable
laboratoryandinclinicalfailuresduetoresistanceinhuman
death in malignancy [16]. Beyond systemic impacts due to
cancer itself, cytotoxicchemotherapy renderspatients func- infection.Forexample,thefactthatwithfewexceptions,the
tionally immunosuppressed, thereby affecting key cell- antifungalagentsused inclinicalmedicinetodayarelargely
mediated effectors such as granulocytes and macrophages the same as those used for the prior several decades. Thus,
[20]. Further, hospitalization, long-term in-dwelling cathe- asis thecase forantibacterial resistance, exposing countless
terization,andchronicexposuretoantibioticsenhancetheir generations of fungal organisms to such agents necessarily
risks of fungal infection [21]. Projections by the World affords a survival advantage to those pathogens capable of
Health Organization and others estimate a dramatic resistance.KeythemesofconcernincludeCandidaparapsi-
increase in cancer incidence and prevalence in the next losis,C.krusei,andC.glabrataclinicalisolatesthatroutinely
two decades [22]. exhibit reduced susceptibility to multiple antifungal agents
[12]. While perhaps less common in incidence than infec-
(3) Surgery. With other healthcare-associated risk factors, tions due to C. albicans, these fungal pathogens can often
infections due tosurgicalprocedures are risinginincidence represent serious or life-threatening bloodstream and inva-
andassociatedwithworseoutcomes[23].Moreover,obesity, siveinfections.Moreover,theadventofpan-resistantspecies
diabetes, smoking, hypertension, coronary artery disease, of Candida, such as C. auris, is among the most significant
andchronicobstructivepulmonarydisease[24]arepreand publichealth concerns related to fungal infections in recent
postsurgical risks of infection. Compounding this issue is memory [12]. In addition, infections due to opportunistic
the wide use of broad-spectrum antibiotic prophylaxis in or pathogenic molds remain an urgent issue. Infections
relationtosurgery,whichtogetherwithimmunesuppression caused by Aspergillus, Rhizopus, Mucor, and related molds
canincreaserisksforopportunisticfungalinfections. are angioinvasive and destructive, often leading to necrotic
and irreversible tissue damage. Infections caused by these
(4) Transplant. Successful transplantation almost always Mucoromycotina, and those due to Cryptococcus, are typi-
requires prolonged or even life-long immune suppressive cally treated with cytotoxic levels of amphotericin B and
therapy. It follows that infectious complications remain a typicallyresistlesstoxicazolesorechinocandins[33].Echi-
leading cause of morbidity and mortality in settings of nocandinsarethenewestclassofantifungalsintroducedfor
organ or hematologic transplantation [25]. Opportunistic humantherapyinthelastdecade.However,recentdevelop-
fungal pathogens are among the most dangerous etiologies mentofechinocandinresistancehasbeenreportedviapoint
of these infections and can emerge when the host is ren- mutationsinFKSgenesthatencodetheechinocandintargets
dered immunocompromised by regimens necessary for (reviewed in [34]), supporting the urgent need to identify
engraftment.Inpartduetotheriseofantifungalresistance, novelantifungalagents.
OxidativeMedicineandCellularLongevity 3
1.2.3. Paucity of Antifungal Development. Amphotericin B existing colonies or abscesses, biofilm maturation and
wasoriginallydiscoveredinthe1950s,andhasbeenusedclin- survival in the face of antifungal agents, and many other
icaltotreatfungalinfectionsforapproximately50years.Over processes necessary for survival and pathogenesis. Thus,
thatsamespanoftime,remarkablefewmechanisticallynovel thereissoundrationalesupportingfungalRCDasaninno-
antifungalcompoundshavereachedregulatoryapprovalfor vative and mechanistically novel target of next generation
clinical use. These include 5-flucytosine (1963), the azoles antifungal therapeutics.
fluconazole(1990)andvoriconazole(2002),andtheechino- While the sense of an apoptotic program in unicellular
candincaspofungin(2003),posoconazole(2006),andanidu- organismsmightnotbeasobviousasinmetazoans,emerg-
lafungin (2007) [33]. Thus, the pharmacopeia of approved ing data depict scenarios in which the death of damaged or
antifungalagentsforbloodstreamorotherinvasiveinfections old cells favors the survival of a clonal population [37].
isessentially encompassedbyjustthreemechanisticclasses: RCDhasbeenshowntooccurduringphysiologicalscenarios
polyenes, azoles, and echinocandins. While echinocandins such as unsuccessful mating [38], differentiation of a yeast
are technically considered to have cyclic-peptide-like struc- colony [39], or replicative and chronological aging [40–42].
tures,thescopeofthediscussionhereinfocusesonnoncyclic Whilereplicativeagingreferstothenumberofcelldivisions
hostdefenseandsyntheticpeptidecompounds.Likewise,the an individual mother cell can undergo, chronological aging
manyfactorscontributingtoasignificantreductionofindus- reflects the time nondividing cells stay viable in stationary
try investment in developing novel antifungal agents is phase.Inbothagingscenarios,apoptosisservestheelimina-
beyondthescopeofthisdiscussion.Sufficeittosaythereis tion of cells that may be old, irreversibly injured, or which
nowanurgentneedtodiscoveranddevelopstructurallyand have dysfunctions that are detrimental to the larger fungal
mechanistically new antifungal agents to meet the growing cellpopulation. Aside from this, the apoptotic program can
threatoflife-threateninginfectionsduetothesepathogens. also be hijacked by competing fungal populations, for
example via secretion of killer toxins that trigger cell death
2. Regulated Cell Death in Fungi insusceptiblestrains[43].
In aggregate, the RCD subroutines can be induced by a
An important new area of discovery with great potential to plethora of different stimuli and scenarios, ranging from
reveal conceptually as well as mechanistically novel targets signalingmoleculesnecessary forsurvival andreproduction
for next-generation antifungal therapeutics is regulated cell to existing environmental and host threats, including anti-
death (RCD), previously referred to as programmed cell fungalagents.Therelativedependenceondistinctmolecular
death (PCD). For the last two decades, the occurrence of players and events for activation of specific RCD pathways
RCD in unicellular eukaryotes has been a topic of intensive appears to be subject to conditional contexts in which the
study,andfungihavebeenshowntosuccumbtoseveraldis- organism encounters a specific stress signal [44]. Based on
tinct subroutines of regulated cell death. The majority of theirmolecularandbiochemicalcharacteristics,theapopto-
these studies have been carried out in the well-established tic mode of cell death has been subdivided into extrinsic
model system Saccharomyces cerevisiae. According to the andintrinsicapoptosis[35].Whileextrinsicapoptosisisini-
classification of cell death modalities suggested by the tiatedbyactivationofspecifictransmembranereceptorsthat
NomenclatureCommitteeonCellDeath2018andtherecent subsequently trigger cellular demise, intrinsic apoptosis can
guidelines for cell death nomenclature in yeast [35, 36], S. be induced by a variety of cellular stresses that all converge
cerevisiae and likely other fungi can undergo at least three onmitochondria-mediatedcelldeathprocesses.Whileyeast
distinct types of RCD: intrinsic apoptosis (formerly type I does most probably not succumb to the typical extrinsic
PCD), autophagic cell death (formerly type II PCD), and apoptosis, intrinsic apoptosis does exist and represents
regulatednecrosis(formerlytypeIIIPCD).Whileconsider- the best studied RCD scenario in yeast so far. Morpholog-
able efforts have historically been focused on gaining a ically, this mode of cell death is characterized by hallmark
greater mechanistic and biological understanding of these features, including systematic DNA fragmentation, nuclear
RCDpathwaysinS.cerevisiae,recentstudieshavealsopro- condensation, exposure of phosphatidylserine at the outer
vided new insights into RCD in human pathogenic fungi, leafletoftheplasmamembrane,andaccumulationofreactive
includingCandidaandAspergillusspecies.Here,wesumma- oxygenspecies.
rizethebasicconceptsandsignificantadvancesinthisregard. The fundamental molecular machinery governing apo-
ptotic cell death appears to be evolutionary conserved,
2.1. Biological Roles of Regulated Cell Death in Fungi. The and numerous counterparts of mammalian key effectors
signals, mechanisms, and pathways through which eukary- of apoptosis have been identified in yeast in the past two
otic cells functionally age, wane, and ultimately meet death decades [45–47]. For instance, these include orthologues
isequallyimportanttotheirorigin,development,andmatu- of the apoptosis-inducing factor (Aif1) and Endonuclease
ration.Thus,RCDhasevolvedasameansforpopulationsof G (Nuc1). Whenreleased from mitochondriaupon anapo-
cells to most efficiently—and least disruptively—censor ptotic insult and translocation to the nucleus, they cause
those individual cells that either do not actively contribute DNA fragmentation and subsequent cell death [48, 49].
to the larger microbial community or that detract from it. For Endonuclease G, this process involves the mitochon-
Example settings in which RCD is particularly meaningful drial adenine translocator, implying the formation and
in this regard include reproduction, establishment of colo- actuation of a mitochondrial permeability transition pore
nies, quorum sensing and metastatic dissemination from in yeast apoptosis [48].
4 OxidativeMedicineandCellularLongevity
Mitochondrialfeaturessuchashyperpolarization,oxida- causing the dissipation of mitochondrial transmembrane
tive burst and generation of reactive oxygen species (ROS), potential,cytochromeCrelease,and cell death[57]. Several
dissipation of mitochondrial transmembrane potential, additionalproteaseshavebeenassociatedwithyeastapopto-
releaseofcytochromeC,lossofcytochromeCoxidaseactiv- sis.Amongthese,theHtrA2/Omi-orthologousserineprote-
ity, or mitochondrial fragmentation have all been shown to ase Nma111 is involved in oxidative stress-induced cell
contribute to apoptotic death to varying extents depending death [58] and responsible for the proapoptotic cleavage of
onrespectivetriggersandscenarios[50].Evenso,variations the yeast inhibitor-of-apoptosis protein Bir1 [59]. Likewise,
onthesethemeshavebeenobservedamongdifferingfungal the serine carboxypeptidase Kex1 is involved in cell death
organisms, including opportunistic yeast and pathogenic execution upontreatmentwithhypochloriteandaceticacid
fungi,asdiscussedbelow. aswellasdefectsinN-glycosylation[60,61].
Numerous endogenous and exogenous triggers have
2.2. Regulated Cell Death Subroutines in Saccharomyces been reported to initiate intrinsic apoptotic cell death path-
cerevisiae. Although S. cerevisiae is not a typical human ways in yeast, through metacaspase-dependent as well as
pathogen,theemergenceofopportunisticS.cerevisiaeinfec- metacaspase-independent pathways [45–47]. Among the
tionshasbeenreportedinpatientswithchronicdisease,can- endogenous triggers are for instance DNA damage and
cer, and immunosuppression [51]. Moreover, considerable replication stress [62], defects in N-glycosylation [61],
insights into RCD have been gained through studies using chronological or replicative aging [41, 42], perturbations in
thisorganismasamodel.Thefollowingdiscussionconsiders cytoskeleton dynamics [63], and impaired mRNA stability
the genetic and mechanistic aspects of RCD in S. cerevisiae [64]. Likewise, a diverse set of exogenous stimuli for yeast
in this light, particularly given many of the RCD determi- apoptosis has been identified, including treatment with low
nants and pathways have homologous systems in high doses of acetic acid and hydrogen peroxide, hyperosmotic
priority human pathogens, including Candida species and stress, heat, high salt concentrations, UV irradiation, heavy
pathogenic molds. metalssuchasiron,copper,andmanganese,ceramide,ami-
odarone, aspirin, diverse antitumor agents, and many more
2.2.1.IntrinsicApoptosis(TypeIPCD).Intrinsicapoptosisis [45, 46, 65]. Relevant to pathogenesis, specific host defense
perhaps the most well-known of RCD pathways, through factorshavebeenshowntoactviainductionoftheintrinsic
which cellular constituents are systematically degraded by pathwayofapoptosisinvariousfungi,includingS.cerevisiae.
caspase-dependentor-independentmechanisms.Compara- Forinstance,peptidesfromthedermaseptinfamilyproduced
ble to mammalian intrinsic apoptosis, apoptosis in yeast is by amphibians trigger Aif1-dependent apoptosis [66], and
accompaniedbyreleaseofcytochromeCfrommitochondria, theplant-deriveddefensepeptideosmotinkillsbyactivation
thusimpairingoxidativephosphorylation.However,thepar- ofRas-dependentapoptosis[67].Interestingly,thismodeof
ticipation of cytosolic cytochrome C in the formation of cell death requires the binding of osmotin to the plasma
apoptosomes, as is observed in mammalian cells, has not membrane receptor Pho36 and might thus constitute a
yetbeendemonstratedinyeast. variant of extrinsic apoptosis (also see Section (4), below).
Todate,oneorthologueofmammaliancaspaseshasbeen Homologuesofmammalianproteinsintegraltothemito-
identifiedinS.cerevisiae:themetacaspaseYca1(aliasMca1) chondrialpathwayofintrinsicapoptosishavebeenshownto
[52]. Despite different cleavage specificities of caspases befunctionalinyeastandtoactthroughconservedcelldeath
(targeting aspartate-X dyads) and metacaspases (targeting machinery. For instance, heterologous expression of the
lysine-X or arginine-X dyads), these proteases share a humanproapoptoticproteinBaxinyeastcausesoutermito-
common evolutionary origin and are integral to cell death chondrialmembranepermeabilization,cytochromeCrelease,
execution. Notably, the protein TSN (Tudor staphylococcal andcelldeath[45].Simultaneousheterologousexpressionof
nuclease)hasbeenestablishedasthefirstcommonsubstrate thehumanantiapoptoticregulatorsBcl-2orBcl-XLprevents
ofcaspasesandmetacaspases,arguingforstringentfunctional Bax-induceddeathandenhancesyeastresistancetotheapo-
conservation despite phylogenetic distance [53]. Approxi- ptotic stimuli H O and acetic acid [50]. Thus, the intrinsic
2 2
mately 40% of the apoptotic scenarios investigated in pathway of apoptosis in yeast can be complemented by
S. cerevisiae to date depend at least in part on the presence humanorothermammalianhomologues,againhighlighting
ofYca1asexecutorofcellulardestruction[54].Thefactthat thefunctionalconservationoftheapoptoticprogram.
caspase-likeactivitieshavebeendetectedevenincellslacking
Yca1 indicates that additional proteases with caspase-like 2.2.2.AutophagicCellDeath(TypeIIPCD).Autophagyrep-
activity contribute to RCD in yeast [55]. One such protease resents thedegradationpathway ineukaryoticcellsthrough
may be the separin Esp1, a highly conserved protease that which the bulk of intracellular molecular turnover occurs,
facilitates sister chromatid separation during metaphase to includingbreakdownofawiderangeofcytoplasmicmaterial
anaphasetransitionviacleavageofScc1(aliasMcd1),asub- such as aggregated proteins, organelles, and in some cases,
unit of the cohesion complex [56]. Esp1 belongs to the CD pathogendeterminants.Duringmacroautophagy,cargodes-
clan(superfamily)ofcysteineproteases,agroupofproteases tinedfordegradationissequesteredintodouble-membraned
thatalsoincludescaspases,transamidases,andbacterialgin- vesiclestermedautophagosomesandtargetedtothevacuole
gipains [56]. During hydrogen peroxide-induced apoptosis, for subsequent degradation and recycling. Discovered more
cleavage of Scc1 by Esp1 yields a C-terminal Scc1-fragment thanhalfacenturyago,autophagyisnowrecognizedasacat-
that relocalizes from the nucleus to the mitochondria, abolicprocessrequiredforthecoordinatedregulationofcell
OxidativeMedicineandCellularLongevity 5
development,infectioncontrol,aging,andotherphysiologi- acetyltransferasesblocksregulatednecrosisaswell[77].Fur-
calandpathophysiologicalfates,includingcelldeath.Similar thermore, peroxisomal function, perturbation of vacuolar
to other RCD subroutines, the fundamental autophagic function, and pH homeostasis as well as excess palmitoleic
mechanisms and molecules are conserved across the evolu- acid and elevated levels of free fatty acids have been associ-
tionary spectrum from microbes to man. Indeed, most of ated with regulated necrotic cell death [78–82]. In counter-
theautophagy-related(ATG)genesandpathwayshavebeen balance, high levels of the vacuolar protease Pep4, the yeast
identifiedandcharacterizedinyeastcellsundernutrientlim- orthologueofmammaliancathepsinD,preventbothapopto-
iting and other stress conditions [68, 69]. While autophagy tic and necrotic death during yeast chronological aging.
mostlyrepresentsaprosurvivalandlongevity-ensuringpro- Notably, the inhibition of apoptosis requires the proteolytic
gram, excessive autophagy can also contribute to cell death, activity of native Pep4, while the suppression of necrosis is
inparticularduringdevelopment[70].Autophagiccelldeath facilitatedbytheshortpropeptideversionofPep4[83].
is characterized by increased numbers of autophagosomes
alongwithaberrantproteinandorganelleturnover.Evidence 2.3. Regulated Cell Death in Candida Species. Candida
suggests that autophagic cell death occurs in yeastcells, but albicans represents the main model system to study fungal
the precise contribution of distinct autophagic processes to pathogenicityandvirulence,andRCDofC.albicanshasbeen
cell death execution is relatively unexplored [46]. Even so, observed upon exposure to various antifungals and other
data supporting this concept are emerging. Heterologous stressors (Figure 1). Candida species have been shown to
expression of mammalian p53 in yeast causes cell death undergoRCDwithtypicalcharacteristicsofintrinsicapopto-
accompaniedbyanupregulationofautophagy,anddeletion sis, whereas other forms of RCD such as autophagic cell
ofATG1andATG5partlyrestoressurvival[71].Inaddition, death and regulated necrosis remain largely unexplored in
selective leucine starvation causes a mode of death that this organism [84]. Among the triggers for such apoptotic
requiresthepresence ofessentialautophagyregulatorssuch pathwaysareforinstanceaceticacidandhydrogenperoxide
as Atg8 [72]. Indications for an involvement of excessive [85], antifungal peptides from plants [86, 87], and other
autophagy targeting mitochondria (mitophagy) derive from sources[88,89],aswellasavarietyofbotanicalssuchasperi-
studies showing that cells lacking the mitophagy regulator llaldehyde,honokiol,baicalin,andcinnamaldehyde[90–93].
Uth1 are no longer susceptible to cell death induced by Inaddition,clinicalantifungalagents,includingcaspofungin
expression of mammalian Bax [73]. However, as Bax also andmicafungin,twoechinocandinsthatdisturbcellwallbio-
causesmitochondria-mediatedapoptosisinyeast,theprecise genesis,canpromoteRCDinC.albicans[94,95].Inmostof
contributionsofmitophagytoBax-mediatedyeastcelldeath these scenarios, apoptoticdeathof Candida is accompanied
requiresfurtherinvestigation. byanaccumulationofreactiveoxygenspeciesandmitochon-
drialdysfunction[91,92,96].
2.2.3.RegulatedNecrosis(TypeIIIPCD).Historically,necro- As in S. cerevisiae, one metacaspase (Mca1) has been
sishasbeenpredominantlyconsideredtorepresentapurely identifiedinC.albicanstodate,andMca1-dependentaswell
coincidentalmode of cell death upon extreme, biochemical, as -independent apoptotic cell death scenarios have been
immunological, or mechanical insult that results in mem- detected[84].C.albicanscellslacking Mca1aremoreresis-
brane rupture, swelling of organelles, and indiscriminant tant to oxidative stress-induced cell death than parental
spillingofcellcontentintoitssurroundings.Inrecentyears, organisms[97].Ofnote,increasedcaspaseactivityhasbeen
amorespecificviewofnecrosishasemerged,representinga observedinrelationtoinductionofapoptosisinC.albicans
morefine-tunedandregulatedmechanismwithimplications exposed to other stimuli, for instance the quorum sensing
ininflammatoryresponsesinavarietyofphysiologicalcon- molecule farnesol [98, 99], the plant-derived macrocyclic
ditions[74,75].Inmammaliancells,thecascadeofmolecu- bisbibenzyl plagiochin E [96] or silibinin [100]. In con-
lar events and signaling pathways that ultimately leads to trast, cell death resulting from caspofungin is executed
necrotic RCD often involves proteases such as calpains and through a Mca1-independent manner but instead requires
cathepsins,aswellasthekinasesRIP1andRIP3[74].While Aif1 [94, 101].
most of our knowledge of regulated necrosis comes from Amphotericin B, an antifungal agent in clinical use for
mammalian systems, studies demonstrating this form of more than 30 years, triggers apoptotic death of C. albicans
RCD existing in yeast have been published [76]. During [85, 102] and inhibits biofilm formation of C. albicans,
chronological aging, a portion of dying cells exhibit typical C. krusei, and C. parapsilosis [103]. Its fungicidal effects are
hallmarks of necrosis, including loss of membrane integrity accompanied by increased caspase activities, and concomi-
andnucleocytosolicredistributionofNhp6A,theyeastcoun- tant exposure to caspase inhibitors provides cytoprotection.
terpart of mammalian high mobility group box-1 protein Interestingly, simultaneous pharmacological inhibition of
HMGB1 [77]. This necrotic, age-associated cell death is histone deacetylation enhances amphotericin B-induced
inhibited by spermidine, a natural polyamine whose levels apoptosis of established Candida biofilms [103]. Further-
decline during the aging process. Spermidine inhibits cell more, earlier studies connected apoptotic death in C. albi-
death via an epigenetic process that involves histone H3 cans to increased Ras signaling [104] and defects in
deacetylation and induction of autophagy. Thus, the ability glutathione synthesis [105]. More recently, human lactofer-
of distinct pharmacological and genetic interventions to rin has been reported to induce apoptosis in C. albicans via
modulatethisprocessarguesthatnecroticcelldeathishighly bindingandinhibitionoftheplasmamembraneH+-ATPase
regulated. In this respect, deletion of distinct histone Pma1,whicheventuallyresultsindisturbedionhomeostasis,
6 OxidativeMedicineandCellularLongevity
PSerine Ceramides
Host cell
CW
6
Regulated
N CM cell death
4
RCD
2 Mito Nucleus
Pathway
1
3 5
N Candida Cytoplasm
Defense
peptide
0 0.5 1 2 Time (hr)
Figure 1: Model of host defense peptide mechanisms versus C. albicans. (1) Host cells activated by C. albicans deploy prestored and
upregulate nuclear- (N-) encoded host defense peptides that directly interact with C. albicans to (2) target electronegative cell wall
components (e.g., glycosylceramide or all specific cell proteins); (3) permeabilization of the cytoplasmic membrane during or following
entry into the cytoplasm; (4) target the electronegative phospholipid composition and transnegative potential (Δψ) of mitochondria
(Mito); (5) perturb mitochondrial functions essential to cell cycle and trafficking, as well as de-energization and respiratory decoupling
activationofcaspaseand/ormetacaspasepathwayresponses;(6)combinedeffectsofcellenvelopedamageandmitochondrialdysfunction
invokes the regulated cell death response which corresponds to hallmark features of apoptosis, including phosphatidylserine (PS)
expression. This integrated model is supported by recent publications [115, 125, 131]. It should be understood that different antifungal
peptidesmayexertdifferentmechanismsoradifferentmechanisticsequence.Forexample,inthecaseofplantdefensins,thesequenceof
membrane perturbation and ceramide accumulation has not yet been resolved. It could well be that ceramide accumulation is a first
consequenceofinteractionwithglucosylceramides(e.g.,step2;aswithRsAFP2).Alternatively,membraneperturbationcouldpotentially
beaconsequenceoftheinductionofRCDandhence,onlyappearsatstep6.
mitochondrialdysfunction,anddeath[106].Moreover,host a process of caspase-independent apoptosis in this organ-
cells seem to be able to utilize the apoptotic program of ism. Early studies also suggest that Mucor species have
fungalpathogens asadefensesystem. Forexample, interac- explicit RCD responses to stress. For instance, the HMG-
tion with macrophages induces metacaspase activation and CoA reductase inhibitor lovastatin can inhibit posttrans-
apoptosisinC.albicans[107].Inthisprocess,distinctmeta- lational modification of proteins, including prenylation.
caspase substrates involvedinglycolysisandproteinquality Following exposure to lovastatin, M. racemosus arrested
control weredecreased [107]. sporangiospore germination,yielding profoundcytoplasmic
It should be understood that a given fungal organism condensation and DNA fragmentation [110]. More recent
mayemploydistinctapoptoticpathways,dependingonmul- studiesreportedthatspecificsesterterpene-typemetabolites,
tiple factorsin context. For example, cell density may affect including ophiobolins A and B, can induce apoptosis in
nutritionalavailabilityoractivatequorumsensingpathways Mucor species [111]. The calcineurin pathway governs key
leading to apoptosis [108]. Thus, the effects of farnesol or virulenceandantifungalresistance pathwaysinRhizopusas
othersignalswithrespecttoquorumsensing,biofilmforma- well as in other pathogenic molds such as Mucor. Interest-
tion, and cell death may vary depending on the microenvi- ingly, whenexposed tothecalcineurininhibitor tacrolimus,
ronmental conditions in context of infection and the thefungistatictriazoleagentsposaconazoleanditraconazole
strategiesoffungalgrowthcharacteristicstherein(e.g.,yeast becamefungicidalforR.oryzae[112].Thiseffectwasaccom-
versushyphae). paniedbyDNAfragmentation,phosphatidylserineexternal-
ization, ROS accumulation, and activation of caspase-like
2.4.RegulatedCellDeathinPathogenicMolds.Comparedto functions. From these examples, there is considerable evi-
Saccharomyces or Candida species, less is known regarding dence supporting the view that caspase-dependent and
RCD in Aspergillus species (Ascomycetes) or their Mucor- -independent pathways of RCD exist in pathogenic molds
omycotina cousins, Rhizopus or Mucor. However, recent andyeasts.
data point to parallels in RCD among pathogenic fungi.
Farnesol-induced quorum-sensing mechanisms may exist 3. Regulated Cell Death as an Antifungal
in Aspergillus, ultimately yielding RCD [109]. For example, Peptide Target
in A. nidulans, farnesol induces the expression of an
apoptosis-inducing factor (AIF-) like mitochondrial oxido- Host defense peptides (HDPs) of different structural scaf-
reductase, mitochondrial ATPase inhibitor, and cyto- folds occurring naturally or engineered have now been
chrome C peroxidase. As a result, ROS accumulation and shown to activate fungal RCD by way of key mechanisms,
mitochondrial fragmentation is observed, consistent with including perturbation of quorum sensing, mitochondrial
OxidativeMedicineandCellularLongevity 7
Table1:Mainclassesofhostdefensepeptides(HDPs)showntoinduceRCD.
MainclassesofRCD-inducingantifungalpeptides∗
ModeofactionapartfromRCDinductionand
Name Source
mitochondrialdysfunction
A.Helicalandextendedpeptides
Periplanetasin-2 Cockroach(Periplanetaamericana) Lipidperoxidation,caspaseactivation
Centipedes
Scolopendin Metacaspaseactivation
ClassChilopoda
Metacaspaseactivation,inhibitionofmembrane
Lactoferrin Bovine/human
H+-ATPasePma1
B.γ–corecontainingpeptides
Interactionwithfungal-specificglucosylceramide,
PlantdefensinRsAFP2 Radish(Raphanussativus) inductionofcellwallstress,ceramideaccumulation,
septinmislocalization,metacaspaseindependent
InteractionwithPAandPIphospholipids,
PlantdefensinHsAFP1 Coralbell(Heucherasanguinea) accumulationatbudsandsepta,internalization,
pHdependentactivityinvitro
Plantdefensin-likepeptideApDef-1 Adenantherapavonina Cellcycledysfunction,metacaspaseactivation
DysfunctionalmitochondrialΔψandcytochrome
InsectdefensinCoprisin DungbeetleFamilyScarabaeoidea
Crelease
Fungaldefensin-likepeptideNFAP Neosartoryafischeri Cellwalldysfunction,accumulationofnucleiat
brokenhyphaltips
Fungaldefensin-likepeptideAFP Aspergillusgiganteus Cellwallperturbation
NeutrophildefensinhNP-1 Human Membranepermeabilizationanddepolarization
BetadefensinhβD-2 Human RCDmodulatedbyBcr1andSsd1proteinsinC.albicans
Perturbmembraneenergeticsandinhibitmacromolecular
Kinocidins(e.g.,CXCL4,CXCL8) Mammalian synthesis;pH-relatedactivityinvitro;RCDmodulatedby
Bcr1andSsd1proteinsinC.albicans
∗NotethatHistatin-5andplantdefensin-likepeptideLpDef1werenotintegratedinthetableastheirpotentialinductionofRCDisstillunderinvestigation.
function,autophagyormitophagy,disruptionofreplication C from mitochondria, with activation of caspases, DNA
or reproductive mechanisms, and/or interference with cell fragmentation, and eventual cell death. Therefore, by
cycle and aging. A particularly attractive aspect of targeting inducing insurmountable oxidative stress, it appears that
RCD in developing novel antifungal agents relates to periplanetasin-2 evokes intrinsic apoptosis in C. albicans.
the potential minimization of unintended consequences of
inflammation that may accompany death of fungal cells 3.1.2. Scolopendin. Scolopendins are recently identified
exposedtocytotoxicagentsthatinduceunregulatednecrotic antimicrobialpeptidesofcentipedes[89].Aprototypescolo-
death.Thefollowingdiscussionfocusesonselectedexamples pendin was found to induce apoptosis in C. albicans, as
fromrecentstudiesthatofferinsightsintotheRCD-inducing evidencedbymitochondrialdysfunction,ROSaccumulation,
mechanismsofhostdefensepeptidesandhowtheymightbe cytochrome C release, deenergization, phosphatidylserine
exploited as novel antifungal agents and strategies. Recent externalization,chromatincondensationandfragmentation,
and prior evidence is considered from studies of RCD andcelldeath.TheseRCDmechanismswereassociatedwith
mechanisms differentially induced by distinctive classes of metacaspaseactivation.
antifungalHDPs(summarizedinTable1).
3.1.3. Lactoferrin. Lactoferrin is one of the most exten-
3.1.HelicalandExtendedPeptides.Perhapsthemostwidely sively studied HDPs. In early work, Andres et al. showed
recognized structural class of peptides having antifungal that this peptide induces apoptotic cell death in C. albicans
activityarethoseexhibitingα–helicalorextendedstructures. via K+ channel-mediated K+ efflux [114]. More recently,
Examples of this group of molecules for which evidence of this same group has shown lactoferrin to induce apoptosis
RCDhasbeenreportedarediscussedbelow. by inhibiting the membrane H+-ATPase Pma1, leading to
subsequent mitochondrial dysfunction [106]. Interestingly,
3.1.1. Periplanetasin-2. This peptide has recently been Yount et al. previously noted that toxicity of human
isolated from the cockroach Periplaneta americana and beta-defensin 2(hβD-2) and crotamine toxin corresponded
studiedintermsofitsmechanismofactionagainstC.albicans to common structure-activity relationships promoting tar-
[113].Thesyntheticamideversionofthepeptideledtolipid geting of eukaryotic K+ channels, including that of C. albi-
peroxidation, accumulation of ROS, externalization of cans [115]. Human lactoferrin has also been found to
phosphatidylserine,dissipationofΔψ,andlossofcytochrome triggercaspase-dependentcelldeathinSaccharomyces[116].
8 OxidativeMedicineandCellularLongevity
3.1.4. Histatin-5. It remains unclear whether the antifungal GCS1 gene encoding glucosylceramide synthase [128]. Sub-
peptidehistatin-5,foundinhumansalivaandoralsecretions, sequently,Thevissenetal.demonstratedthatRsAFP2isnot
induces apoptosis in C. albicans. Investigations by Vylkova internalized in the cell per se, but associates with the cell
etal.showedthathistatin-5evokesosmoticstressresponses envelope,resulting incellwall stress, septin mislocalization,
[117], and ensuing studies by Sun et al. demonstrated it to and accumulation of ceramides in C. albicans [129]. The
perturbATPasefunctionsinC.albicans[118].Priortothose latter effects are likely responsible for the induction of apo-
studies, Helmerhorst et al. demonstrated that histatin-5 ptosis;however,theexactmechanismyieldingRCDrelative
exertsitsantifungalactivitythroughtheformationofreactive to the RsAFP2-induced ceramide accumulation is hitherto
oxygen species [119] and also showed histatin-5 to target unknown.Thus,multiplelinesofevidencesuggestthispep-
fungal energetic systems and to cause mitochondrial dys- tideinducesRCDbywayoftargetingmitochondrialandcell
function [120]. Collectively, such findings strongly implied cyclefunctions.
RCD-like mechanisms. However, studies by Wunder et al. TheseantifungaleffectsofRsAFP2werealsofoundtobe
reportedthathistatin-5doesnotexertitsantifungalmecha- synergistic with caspofungin in mitigating the pathogenic
nismsviaapoptosis[121]. consequences of C. albicans biofilms [130]. In the case of
HsAFP1,theidentityofthefungal-specificmembranerecep-
3.2. γ–Core Peptides. Cysteine-stabilized (CS) HDPs across torhasnotyetbeenelucidated.Itsfungicidalactionexploits
the phylogenetic continuum share a 12–18 residue multidi- theoxidativerespiratorychaininC.albicanstocausehyper-
mensional structure-function feature known as the γ–core accumulation of ROS among other phenotypic markers of
[115, 122]. While this structure-function signature is apoptoticdeath. Interestingly,genesassociated withconfer-
conserved across a vast evolutionary distance, accessory ringsusceptibilitytothispeptidewerethosemediatingmito-
sequenceshaveadaptedtooptimizefunctionsindistincthost chondrialresponseandrelatedstress-inducedfunctions[87].
anatomicandmicrobiologicniches.Forinstance,thisfunda- Very recent evidence indicates that HsAFP1 can bind to
mentalmotifisconservedinpeptidesofthedefensin,CSαβ, phosphatidic acid (PA) and to a lesser extent, to various
and many other distinct peptide families originating from phosphatidylinositolmoieties[131].Specifically,thispeptide
bacteria, fungi, plants, insects, and humans [123], but the accumulates at the cell surface of yeast cells with intact
accessory domains have undergone evolutionary radiation. membranes,mostnotablyatthebudsandsepta,andissub-
Examples of apoptosis-inducing γ–core peptides that have sequently internalized. Further, PA was found to play an
antifungalefficacyareconsideredbelow. influential role in the internalization of HsAFP1, as yeast
expressingreducedPAlevelsinternalizedlessofthispeptide
3.2.1. Plant Defensins RsAFP2 and HsAFP1. As with all (Thevissen, unpublished data). However, additional as yet
defensin-family polypeptides of plants, RsAFP1, RsAFP2, unknown fungal-specific processes and targets also appear
and HsAFP1 are cysteine-stabilized, cationic peptides con- to be playing a role in HsAFP1 internalization. Likewise, in
taining a multidimensional γ–core motif [122, 124]. Plant the case of the plant defensin NaD1, cell wall components
defensins are in general characterized by broad-spectrum havebeenimplicatedinitsinternalization[132,133].Micro-
antifungal activity and typically have minimal toxicity to bicidal effects resulting from induction of RCD, such as
plant or human cells in vitro [125]. Originating from the energy perturbation and membrane permeabilization, are
commonradishplantfamilymemberRaphanussativusand presumably secondary effects that ensue following HsAFP1
thecoralbellHeucherasanguinea,therespectiveplantdefen- internalization[131].
sinsRsAFP2andHsAFP1aremodelsforstudyingapoptotic
mechanisms of action versus C. albicans. Aerts et al. were 3.2.2.PlantDefensin-LikePeptides.Theseedsofthelegumi-
perhaps the first to demonstrate that a plant defensin nousplantAdenantherapavoninaarethesourceofarecently
(RsAFP2) can induce intrinsic apoptosis in C. albicans, identifiedpeptidethatexertsapoptoticmechanismsofaction
independentofthemetacaspaseMca1[86].Thisobservation against fungi [134]. This defensin-like plant polypeptide
was followed by a report on the induction of apoptosis of termedApDef-1causescellcycledysfunctionandconcomi-
C.albicansbyanotherplantdefensin,HsAFP1[87].Interest- tantintracellularROSaccumulationandchromatinconden-
ingly,HsAFP1wasthefirstplantdefensinforwhichadirect sation prior to metacaspase activation and cell death by
interaction with a fungal-specific lipid-based receptor was intrinsic apoptosis. It is not yet known if ApDef-1 triggers
demonstrated[126],howeveratthattime,thisreceptorcould theseeffectsthroughgeneralstressresponsesorifprototypic
not be identified. Later studies by Thevissen and coworkers fungal pathogens such as C. albicans or pathogenic molds
demonstrated that RsAFP2 interacts with fungal-specific are susceptible to this mechanism of action. The LpDef1
glucosylceramides (GlcCer) [127]. The GlcCer constituents peptide recently isolated from the seeds of Lecythis pisonis
are present in the cell membrane and wall of susceptible causestheaccumulationofROSandmitochondrialdysfunc-
fungi. As RsAFP2 cannot interact (or does so to a substan- tion inC.albicans,pointing towards RCDasmechanism of
tially lesser extent) with structurally distinct GlcCer from action[135].
plantorhumancells[127],theinductionofRCDisselective
to organisms that contain the fungal-specific sphingolipid 3.2.3. Insect Defensin Coprisin. Coprisin is an antimicrobial
receptor.ThisrelationshipexplainswhyRsAFP2isnotactive peptide from the dung beetle with features of the broader
against the emerging fungal pathogen Candida glabrata, as insect defensin family [136, 137]. In its 43 amino acid
this organism does not produce GlcCer due to a lack of the form, there are two Cys-disulfide bonds that stabilize its
OxidativeMedicineandCellularLongevity 9
characteristic CS–αβ structure. Lee et al. reported this form immuneeffectors, such as leukocytes [123, 146, 147].These
ofcoprisintoexertenergy-andsalt-dependentmechanisms host defense peptides have common structural configura-
ofRCDinC.albicans[138].Theseactivitiesincludeaccumu- tions comprising three modular domains: (1) N-terminal
lationofintracellular ROS,dysfunctional mitochondrialΔψ domain containing the chemokine cysteine motif; (2) inter-
and cytochrome C release, phosphatidylserine externaliza- posing γ–core domain; and (3) C-terminal microbicidal
tion,andmetacaspaseactivation,leadingtoapoptoticdeath helical domain [147, 148]. All mammalian kinocidins are
ofC.albicans. characterized by this structural pattern. Human kinocidins
representingallfourconventionalchemokinecysteinearray
3.2.4. Fungal Defensin-Like Peptides. Along with higher structuregroups(C,CC,CXC,andCX C)havebeenidenti-
3
eukaryotic organisms, fungi themselves appear to have fied and demonstrated to have direct microbicidal activity
exploited RCD over an evolutionary timespan as a means against human pathogens [147], and congeners of these
to protect themselves from other competing fungal organ- moleculeshavebeenengineered forenhancedantimicrobial
isms.Forexample,theNeosartoryafischeriantifungalprotein activity[149,150].Thetwopredominantgroupsofkinocidins
(NFAP)isabasic,cysteine-rich,extracellularantifungalpro- aredistinguishedasα(CXC)orβ(CCandother).Examplesof
teinwithstructuralsimilaritytodefensins[139].Thispeptide α–kinocidins(CXC)includeplateletfactor-4(PF-4;CXCL4)
is characterized by a β–barrel topology, constituting five and platelet basic peptide (PBP; CXCL7), interleukin-8
highly twisted antiparallel β–strands, stabilized by disulfide (CXCL8), monokine induced by interferon-γ (MIG-9;
bridges. However, in contrast to its β–defensin relatives, CXCL9), interferon-γ inducible protein-10kDa (IP-10;
NFAP contains a hydrophobic core [140]. Homologues of CXCL10),andinterferon-inducibleTcellα-chemoattractant
this peptide have also been isolated from Aspergillus (I-TAC9; CXCL11) [123, 147, 151]. Specific examples of
giganteus (Aspergillus giganteus antifungal protein (AFP)), β–kinocidins (CC and other subgroups) include monocyte
Aspergillus clavatus (Aspergillus clavatus antifungal protein chemoattractant protein-1 (MCP-1; CCL2), macrophage
(AcAMP)),andAspergillusniger(Aspergillusnigerantifungal inflammatoryprotein-1(MIP-1;CCL3),RANTES(regulated
protein(ANAFP))[141,142].Specifically,NFAPisproduced upon activation, normal T cell expressed/secreted; CCL5),
by the N. fischeri NRRL 181 isolate (anamorph: Aspergillus and lymphotactin (CL1). The importance of these concepts
fischerianus). Heterologous expression of the nfap gene in and roles for host defense peptides having more than just
the NFAP-sensitive A. nidulans revealed the induction of antimicrobialactivityarereviewedelsewhere[146,152].
intrinsic apoptosis, as well as damage and dysfunction of Kinocidin holoproteins and their modular peptide
the cell wall, the destruction of chitin filaments, and the domainsexertdirectantifungalactivitieswhicharecondition-
accumulationofnucleiatthebrokenhyphaltips[143]. ally dependent. For example, kinocidins CXCL1 (GRO-a),
CXCL8 (IL-8), CCL5 (RANTES), and CL1 (lymphotactin)
3.2.5. Human Defensins. Human neutrophil defensin-1 exert significant candidacidal efficacy at pH5.5 but little
(hNP-1) and human beta-defensin 2 (hβD-2) are among or no activity at pH7.5 in vitro [147]. The fungicidal effect
the most predominant of the host defense peptides elabo- of the CXCL8 holoprotein was observed with as little as
rated within neutrophils and expressed by the integument 1nmol/ml, with complete sterilization of a 6 log CFU
(epithelial barriers), respectively. Notably, hβD-2 has been inoculum of C. albicans exposure to 5nmol/ml of this pro-
showntoperturbmitochondrialenergeticsandinducephos- tein. Importantly, the hemipeptide of CXCL8 containing
phatidylserineaccessibilityinC.albicans[115].Suchresults the γ–core and microbicidal helix accounted for all of
indicate that these peptides exert their candidacidal mecha- the antifungal activity of the holoprotein. For example,
nisms at least in part via a RCD response involving mito- the isolated microbicidal helix of CXCL8 at 0.5nmol/ml
chondrial targeting. These effects were influenced by pH exerted sterilization of a 6 log inoculum of this organism
and occurred in relationship to altered cell membrane per- in the same solution phase assay in vitro. Interestingly,
meability. Similar mechanisms of anticandidal activity have whereasthekinocidinstypicallyexertstronganti-C.albicans
been observed for hNP-1. Extending on these findings, two activityatpH5.5,theirefficacyisconsiderablylessatpH7.5.
geneshavenowbeenidentifiedinHDPresistanceinC.albi- This pattern of activity is opposite to that of these peptides
cansandS.cerevisiae.Ganketal.showedthatthegeneSSD1 versus bacteria, which generally is substantially greater at
isintegraltotheabilityofthesefungitosurviveinthefaceof pH7.5thanpH5.5invitro.
human defensins [144]. Moreover, an ssd1 null C. albicans Mechanisms of kinocidin antifungal action have been
strain was significantly less virulent in a mouse model of studiedusingC.albicansclinicalisolatesandgeneticmutants.
infection as compared to its wild-type counterpart. Subse- For example, a synthetic peptide congener (RP-1) designed
quent investigations by Jung et al. demonstrated that the on the microbicidal helices of mammalian CXCL4 (PF-4)
regulatory gene BCR1 also contributes to host defense family kinocidins caused invagination and permeabilization
peptide resistance in C. albicans likely via a pathway that of the cell membrane, condensation of cytoplasmic macro-
intersectsthatofSSD1[145].Ofnote,asyntheticβ–hairpin molecules,andlossofmitochondrialenergeticsinwild-type
peptide(RP-13)thatlacksaγ–coremotifdidnotexertantic- C. albicans [145, 153]. Gank et al. and Jung et al. demon-
andidalactivitiesidenticaltothoseofhNP-1orhβD-2. stratedthatSsd1andBcr1proteinsfunctioninapathwaythat
is integral to the survival of this organism in the face of
3.2.6.Kinocidins.Kinocidinsarechemokinesthatexertdirect low concentrations RP-1 and other host defense peptides
microbicidalactivitiesandpotentiatefunctionsofsynergistic [144,145].Togainfurthermechanisticinsights,C.albicans
10 OxidativeMedicineandCellularLongevity
wildtype,Δssd1/Δssd1null,SSD1complementedandforced successfully reached phase III clinical trials, and no host
overexpressionmutantswereexposedtoRP-1underdistinct defense peptide isolated from higher organisms or engi-
pH conditions simulating bloodstream (pH7.5) or abscess neered mimetic thereof has achieved regulatory approval
(pH5.5) contexts in vitro. Mechanisms of action were then foruseinclinicaltherapy[162,163].Evenlessprogresshas
evaluated using multiparametric flow cytometry assay beenmadewithrespecttodevelopmentofpeptidetherapeu-
devised to simultaneously assess six modes of activity tics targeting fungal infections. There are only few reports
(osmotic homeostasis; macromolecular condensation; cell thatdocumentinvivoefficacyofantifungalpeptides.Inthis
membranepermeability;mitochondrialenergetics;phospha- respect, Tavares et al. demonstrated efficacy of the native
tidylserinedisplay;andcaspase-like proteaseactivity)[153]. plantantifungalpeptideRsAFP2whenadministeredprophy-
SSD1 expression inversely correlated with antimicrobial lactically in a mouse candidiasis model [164]. Moreover,
peptide susceptibility, mitochondrial deenergization and combinedtherapyofPAF,thesmallantifungalproteinfrom
phosphatidylserineaccessibility.Moreover,SSD1expression Penicilliumchrysogenum,andamphotericinB(AMB),which
corresponded to mitigation of membrane permeability act synergistically in vitro, was more effective than either
(assessedbypropidiumiodidestaining,PI)andcaspase-like AMBorPAFtreatmentaloneinamousemodelforinvasive
protease induction and was greater at pH7.5 than pH5.5. pulmonary aspergillosis [165]. Aside from toxicologic and
Collectively, these results suggest that RP-1 and perhaps pharmacologic barriers (see below), one significant issue in
otherhostdefensepeptidesinduceRCDinC.albicanswhich thisrespectistherealitythatinvitroactivitydoesnotconsis-
can be modulated to some extent by Ssd1 protein. Similar tently translate to in vivo efficacy. As discussed above, the
studiesfocusingonΔbcr1/Δbcr1mutantsofC.albicansdem- challenge of context likely plays a major role in this regard.
onstrated that the Bcr1 protein functions downstream of For example, many published methods assess antimicrobial
Ssd1 to mediate low level resistance to RP-1 and perhaps peptide efficacy in austere buffer systems that lack relevant
other host defense peptides by fostering homeostatic mem- physiologic constituents or conditions. Complicating this
brane integrity and mitochondrial energetics in vitro [145]. issueistheobservationthateventhemostbioactivepeptides
Furthermore, a homozygous null mutant of SSD1 (Δssd1/ can exhibit little or no efficacy in complex media (e.g.,
Δssd1) was significantly less virulent in a murine model of Mueller-Hinton or Brain-Heart Infusion broth) often used
hematogenous candidiasis [144]. Thus, the Ssd1-mediated for standard MIC testing. Thus, the relevance of in vitro
pathway also plays an important role in the survival of susceptibility testing of anyantimicrobial peptide should be
C. albicans during infection in vivo. considered in relation to these limitations. A more relevant
but more cost- and labor-intensive method involves testing
3.3.OtherRegulatedCellDeath-InducingPeptides.Avariety peptide compounds in blood biomatrices, including whole
of other natural peptides and proteins have been shown to blood, plasma, and serum ex vivo [166]. This method has
induce apoptosis in yeast. Examples include osmotin [67], two importantadvances overassaysdone inbuffersystems:
melittin[154],theaspergillocidalpeptidePAFofPenicillium (1)peptidesmustovercomebindingtomolecularorcellular
chrysogenum [155], the surfactant protein WH1 fungin constituentspresentinthematrixand(2)additiveorsynergis-
produced by Bacillus amyloliquefaciens [156], a truncated ticfunctionsofapeptidewithotherhostdefensemechanisms
derivative of dermaseptin S3 [66], yeast pheromone [157], can be assessed, for example, potentiation of neutrophil
psacotheacin [158], and the killer toxins produced by the opsonophagocytosis and intracellular killing [146].
yeast Kluyveromyces lactis [159]. Other peptides for which Translationofinvitroactivitytoinvivoefficacyhasalso
evidence exists of apoptosis-related mechanisms of action beenlimitedwithrespecttoantifungalpeptideefficacy.Gank
arereviewedinDeBruckeretal.[160].Additionalexamples etal.correlatedinvitrohypersusceptibilityofC.albicansto
ofpeptidesthatexertantifungalactivitybutforwhichRCD hostdefensepeptideswithinvivohypovirulenceinamurine
hasnotbeensubstantiatedarereviewedin[161]. modelofhematogenousdissemination [144].Veryrecently,
Cools et al. demonstrated that truncated HsAFP1-based
4. Development of Peptide Anti-Infectives peptides spanning the γ–core were very active in combina-
Targeting Fungal RCD tion with caspofungin against biofilms in vitro. However,
translationtoinvivoconditionsfailedduetotheinteraction
Meritoriousattemptshavebeenmadetotranslatethepotent ofthetruncatedpeptideswithserumalbumins[167],which
andrapidinvitroantimicrobialactivitiesofnaturallyoccur- could not be resolved by their subsequent PEGylation or
ring host defense and related peptides of higher organisms cyclization. Interestingly, however, native HsAFP1 still dis-
intonovelanti-infective drugs.Foravarietyofreasons,this played activity in the presence of serum albumins, possibly
goal has proven quite elusive to date. Even so, exciting owing to its structured configuration. Hence, it is plausible
advances are emerging regarding structure, mechanism, that the use of native HDPs or their engineered congeners,
and development of peptide-based agents. In the following alone or in combination with standard antimycotics, bears
discussion, the main challenges experienced to date and great potential to combat fungal infections including those
opportunitiesonthehorizonarereviewed. resistanttocurrenttherapies.
4.1.InVitrotoInVivoTranslation.Therehavebeennumer- 4.2. Off-Target Effects and Toxicity. One key challenge to
ous examples of antibacterial peptide efficacy in animal therapeuticdevelopmentofpeptideantifungalagentsrelates
models of infection. However, few of these agents have to the undefined toxicity of certain candidates tested