UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall

13 Pages·2017·2.31 MB·English

RESEARCH ARTICLE
crossm
UDP-4-Keto-6-Deoxyglucose, a Transient
Antifungal Metabolite, Weakens the
Fungal Cell Wall Partly by Inhibition of
UDP-Galactopyranose Mutase
D
LiangMa,aOmarSalas,bKyleBowler,bMaorBar-Peled,b,cAmirSharona o
w
DepartmentofMolecularBiologyandEcologyofPlants,TelAvivUniversity,TelAviv,Israela;Complex
n
CarbohydrateResearchCenter,UniversityofGeorgia,Athens,Georgia,USAb;DepartmentofPlantBiology, lo
UniversityofGeorgia,Athens,Georgia,USAc a
d
e
ABSTRACT Can accumulation of a normally transient metabolite affect fungal biol- d

ogy? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in Received28August2017 Accepted13 fr
o
October2017 Published21November2017
conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected m
CitationMaL,SalasO,BowlerK,Bar-PeledM,
in living cells, because it is quickly converted to UDP-rhamnose by the enzyme SharonA.2017.UDP-4-keto-6-deoxyglucose,a h
t
UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found transientantifungalmetabolite,weakensthe tp
that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP- fungalcellwallpartlybyinhibitionofUDP- ://
galactopyranosemutase.mBio8:e01559-17. m
KDG to levels that were toxic to the fungus due to destabilization of the cell https://doi.org/10.1128/mBio.01559-17. b
wall. Here we show that these negative effects are at least partly due to inhibi- EditorJosephHeitman,DukeUniversity io
.
tion by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which re- Copyright©2017Maetal.Thisisanopen- a
s
versibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP- accessarticledistributedunderthetermsof m
theCreativeCommonsAttribution4.0 .
Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM Internationallicense. o
r
enzyme with a K of 221.9 (cid:2)M. Deletion of the ugm gene resulted in strains with AddresscorrespondencetoMaorBar-Peled, g
weakened cell waills and phenotypes that were similar to those of the er deletion [email protected],orAmirSharon, o/
[email protected]. n
strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in A
the Δugm strain and reduced in the Δer strain, while overexpression of the ugm p
r
gene in the background of a Δer strain restored Galf levels and alleviated the phe- il
2
notypes. Collectively, our results show that the antifungal activity of UDP-KDG is due ,
2
to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and 0
1
that the rhamnose metabolic pathway serves as a shunt that prevents accumulation 9

of UDP-KDG to toxic levels. These findings, together with the fact that there is no b
y
Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as
g
antifungaldrugs. u
e
s
IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. t
We showed that production of the minor sugar rhamnose is used primarily to neu-
tralize the toxic intermediate compound UDP-KDG. This surprising finding highlights
a completely new role for minor sugars and other secondary metabolites with unde-
termined function. Furthermore, the toxic potential of predicted transition metabo-
lites that never accumulate in cells under natural conditions are highlighted. We
demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme,
thereby affecting production of Galf, which is one of the components of cell wall
glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide
sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our re-
sultssuggestthatUDP-KDGcouldserveasatemplatetodevelopantifungaldrugs.
KEYWORDS Botrytiscinerea,UDP-4-keto-6-deoxyglucose,UDP-galactopyranose
mutase,fungalcellwall,fungalnucleotidesugarmetabolism,galactofuranose,
metabolicintermediate
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Maetal.
Thefungalcellwallisarobustyetflexiblestructurecomposedofdifferentpolysac-
charide structures, including chitin, glucans, mannans, and a range of different
proteinsthatareoftenglycosylated(1,2).Thecellwallisvitalforfungalsurvivaland
proper development because it provides the fungal cell with mechanical strength,
whichcontributestotheabilityofthefungustowithstandosmoticchangesandother
stresses(3,4).Thevitalroleofthefungalwallandtheabsenceofprimarywallelements
inmammalsmakethefungalwallaprimetargetforantifungaldrugs.Indeed,several
antifungal drugs target the wall, including polyoxins and echinocandins that target
synthesisofchitinandbeta-1,3-glucan,respectively(5).
The biosynthesis and remodeling of fungal cell walls involve concerted actions
ofenzymesthatusearangeofactivatednucleotidesugarsasdonorstosynthesize
specificglycancomponentsthatarenecessaryforcellwallformation.Forexample,
the synthesis of chitin is mediated by chitin synthases that utilize UDP-N-
D
acetylglucosamineasasubstratetosynthesizelinearchitin(2),UDP-glucoseisutilized
o
by glucan synthases to produce various glucans (2), and GDP-mannose is used by w
n
mannosyltransferases to produce a range of proteins that are heavily decorated with lo
mannose residues (manno-proteins) (6, 7). Other nucleotide sugars common in most a
d
fungiareUDP-galactopyranose(UDP-Galp)andUDP-galactofuranose(UDP-Galf),which e
d
areusedforthesynthesisofdiversetypesofpolysaccharides(8)andglycoconjugates
f
r
(9). In addition to the abundant sugars that are found in the cell walls of all fungal o
m
species, several types of minor UDP-sugars are found only in certain species, for

h
exampleUDP-rhamnose(10,11),UDP-xylose(12),andUDP-glucuronicacid(13). t
t
Recently,twogenesinvolvedinthesynthesisofUDP-4-keto-6-deoxyglucose(UDP- p
:
/
KDG) and UDP-rhamnose were identified (14) and analyzed in two plant-pathogenic /m
fungi,BotrytiscinereaandVerticilliumdahliae(10,11).Deletionofthesecondgene(er) b
thatencodestheenzymeUDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase(ER) io
.
a
resulted in accumulation of UDP-KDG and reduced the ability of both fungi to infect s
m
plants (10, 11). Interestingly, deletion of the B. cinerea er gene (bcer) also caused
.
developmentaldefectsthatwereassociatedwithweakeningofthecellwall;however, o
r
g
themechanismleadingtowalldefectsandreducedvirulenceremainedunclear. /

Many transporters and glycosyltransferases can accommodate multiple substrates o
n
and could be potentially inhibited by analogues of their nucleotide sugars (15–20). A
GiventhestructuralsimilarityofUDP-KDGwithmanynucleotidesugars,wepostulated p
r
that the toxicity of UDP-KDG might be due to inhibition of glycosyltransferases that il
2
utilize nucleotide sugars to assemble glycans and glycoconjugates. It is also possible ,

2
thatUDP-KDGaffectsenzymesthatareinvolvedinconversionofonenucleotidesugar 0
1
to another. Accordingly, we searched for UDP-sugar-utilizing enzymes that could be 9
putative targets of UDP-KDG. Here we report on the identification of the UDP- b
y
galactopyranose mutase (UGM) enzyme, which reversibly converts UDP-galacto-
g
pyranose to UDP-galactofuranose, as a target of UDP-KDG. We show that UDP-KDG u
e
inhibitsB.cinereaUGM,whichresultsinreducedamountsofcellularandwallGalf,and s
t
weakeningofthefungalcellwall.
RESULTS
InhibitionofUDP-galactopyranosemutasebyUDP-KDG.Apossiblemechanism
by which UDP-KDG affects cell wall integrity is inhibition of UDP-sugar-utilizing en-
zymes that are involved in the synthesis of other UDP-sugars like UDP-glucose, UDP-
galactose (UDP-Gal), and UDP-N-acetylglucosamine. To test this possibility, we sub-
jected the Botrytis cinerea genome to a BLAST search for homologues of proteins
involved in nucleotide sugar metabolism. Among several candidate genes that were
found, we selected BC1G_09363 (NCBI), which potentially encodes UDP-galacto-
pyranosemutase(UGM),forfurtheranalysis.ThisgenewasexpressedinEscherichiacoli,
and enzymatic activity of the recombinant protein was determined. Figure S1 in the
supplemental material shows that the BC1G_09363 protein is active as a UDP-
galactopyranose mutase, transforming UDP-galactopyranose to UDP-galactofuranose
in the presence of NADPH and flavin adenine dinucleotide (FAD) (Fig. S1C). This
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FIG1 UDP-KDGinhibitsB.cinereaUDP-galactopyranosemutase.Kineticanalyseswereperformedwith /
m
increasingUDP-Galpconcentrations(0.1,0.5,1.0,1.5,2.0,and3.0mM).Fortheinhibitionassays,the
b
reactionsremainedthesameasforthekineticanalyseswiththeadditionofvariousconcentrationsof io
UDP-KDG(1,10,and100(cid:2)M). .
a
s
m
.
o
interconversion catalysis was further determined by tandem mass spectrometry (MS/ rg
/
MS)analyses(Fig.S1GtoI);hence,thisgeneproductwasnamedUDP-galactopyranose o
n
mutase. Kinetic analyses of enzyme activity showed that the K of the enzyme to
m A
UDP-Galpis395(cid:2)MandthatitwasstronglyinhibitedbyUDP-KDGwithaKivalueof p
221.9(cid:2)M(Fig.1). ril
bcugm and bcer deletion strains share similar phenotypes. To investigate 2
,

whethertheeffectsofUDP-KDGaremediatedbyinhibitionofB.cinereaUGM(BcUGM), 2
0
wegeneratedbcugmdeletionmutantsandcomparedtheirphenotypestothoseofa 1
9
previouslyreportedΔbcerstrain,whichdoesnotproduceUDP-rhamnosebutaccumu-
b
lates UDP-KDG (10). The following strains were used in the analyses: bcugm deletion y

g
strainΔbcugm2b1(abbreviatedΔugm)strain,bcerdeletionstrainΔbcer9a2(abbreviated u
Δer)strain,bcerbcugmdoubledeletionstrainΔbcerΔbcugm1c(abbreviatedΔerΔugm) e
s
strain, bcugm overexpression in the background of Δer strain Δbcer::OEbcugm5a (ab- t
breviated Δer::OEugm) strain, and bcugm complementation strain Δbcugm(cid:2)bcugm6a
(abbreviatedΔugm(cid:2)ugm)strain.
(i) Mycelial growth. Colonies of the Δugm strain developed irregular mycelial
clumpsandhadreducedradialgrowthratecomparedwithwild-typecolonies(Fig.2A
andB).BothofthesephenotypeswerealsoobservedintheΔerstrain;however,they
weremoreseverethanintheΔugmstrain,asdemonstratedbymore-intenseformation
of mycelial clumps and more-pronounced growth retardation. Furthermore, these
phenotypeswereevenmoresevereintheΔerΔugmmutantstrain,inwhichboththe
bcerandbcugmgenesweredeleted(Fig.2AandB).
(ii) Sclerotium formation. After 11 days of incubation in dark, the Δugm strain
producedaberrantsclerotiathatweremuchsmallerandlessmelanizedthansclerotia
produced by the wild-type strain (Fig. 2C). Similar to the growth-associated changes,
thedefectsinsclerotiumformationintheΔerstrainweremorepronouncedthaninthe
Δugmstrain.
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FIG2 TheΔugmstrainandtheΔerstrainsharesimilardevelopmentalandgrowthabnormalities.Cultureswereinitiatedbyplacinga5-(cid:2)l io.
dropletcontaining500sporesinthecenterofeachplate.(A)FungiwereculturedinCDmediumundercontinuouslightfor3days. a
s
Cultureswereinspectedusingastereomicroscope,andimageswerecaptured.Photographsshowhyphalorganizationofthewild-type m
(wt)strain,Δugmstrain,Δugm(cid:2)ugmcomplementationstrain,Δerstrain,andΔerΔugmdoubledeletionstrain.(B)FungalgrowthinCD .
o
medium. Data are the mean radii plus standard deviations (SD) (error bars) for three 3-day-old colonies. Values that are statistically
r
significantlydifferent(P(cid:3)0.01bytwo-tailedStudent’sttest)fromthewild-typevaluesareindicatedbyanasterisk.(C)Fungiwere g
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culturedonPDAmediumincompletedarknessfor11days.Photographsshowsclerotiaformationofthewt,Δugm,andΔerstrains. o
n

A
p
Pathogenicity.InfectionofbeanleaveswithsporesoftheΔugmstrainresultedin
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well-developedlesionsat3dayspostinfection.Theselesionswereintermediateinsize il
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comparedtothelargerlesionsoftheleavesinoculatedwiththewild-typestrainand ,
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thesmallerlesionscausedbytheΔerstrain(Fig.3). 0
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The similarity of the phenotypes for the Δugm and Δer strains supports the possi- 9

bilitythatUDP-KDGinhibitsBcUGMinvivo.Themore-severeappearanceofallofthe b
y

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FIG3 MildlyreducedvirulenceintheΔugmstrain.Bean(Phaseolusvulgaris)leaveswereinoculatedwith7.5-(cid:2)ldropletsofaspore
suspensioncontaining105spores/ml.Inoculatedplantswereincubatedinahumidchamberat22°Cwithlight.(A)Typicallesions3dpi.
(B)Averagelesionsize3dpi.DataarethemeansplusSDforeightlesions.Valuesthatarestatisticallysignificantlydifferent(P(cid:3)0.01)by
two-tailedStudent’sttestfromthewild-typevaluesareindicatedbyabracketandasterisk.
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UDP-KDGInhibitsFungalUDP-GalactopyranoseMutase
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FIG4 TheΔugmstraindisplayscellwalldefects.Colonieswereinitiatedbyplacinga5-(cid:2)ldropletcontaining500sporesinthecenterofeachplate,andthe n
plateswereincubatedat22°C.PDAplatescontain500(cid:2)g/mlCongored(CR)or250(cid:2)g/mlcalcofluorwhite(CW)ascellwallstressors.(A)Colonyspreading lo
at3dpi.(B)Averagecolonyradiimeasuredat3dpi.Dataarethemeanradii(cid:4)SDforthreecolonies.Valuesthatarestatisticallysignificantlydifferent(P(cid:3) a
0.01bytwo-tailedStudent’sttest)fromthewild-typevaluesareindicatedbyanasterisk. d
e
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f
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examinedphenotypiccharacteristicsintheΔerstrainindicatesthatUDP-KDGpossibly

h
hasadditionaltargetsotherthanBcUGM,ahypothesisthatisfurthersupportedbythe
t
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evenmoreseverephenotypeoftheΔerΔugmdoublemutant. p
:
The(cid:2)ugmstrainhascellwalldefects.Totestforpossiblealterationsofthecell //m
wallintheΔugmstrain,fungiwereculturedonmediacontainingthecellwallstressors b
calcofluorwhite(CW)andCongored(CR)thatperturbcellwallassemblybybindingto io
.
a
chitin(21,22).GrowthoftheΔugmstrainwasmarkedlyreducedonmediacontaining s
either500(cid:2)g/mlCRor250(cid:2)g/mlCW,concentrationsthatonlyslightlyinhibitgrowth m
.
ofthewild-typestrain(Fig.4).TheseresultsshowthattheΔugmstrainhasaweakened o
r
g
cellwall,thushighlightingtheimportanceofUGMforcellwallintegrity.Theeffectsof
/

thecellwallstressorsongrowthoftheΔerorΔerΔugmstrainswereevenstrongerthan o
n
ongrowthoftheΔugmstrain,whichisinaccordancewiththemore-severedevelop- A
mental defects observed in these strains. Overexpression of the bcugm gene in the p
r
Δbcer background (Δer::OEugm strain) partially compensated for the defects, further il
2
supportingthenotionthatUGMisinhibitedbyUDP-KDGintheΔerstrain(Fig.4). ,

2
Caspofunginisanantifungaldrug(asemisyntheticechinocandin)thatinhibitsthe 0
activityof(1¡3)-(cid:3)-D-glucansynthaseandtherebydisturbstheintegrityofthefungal 19
cellwall(23).TotestforpossiblesynergismbetweeninhibitionofUGMandcaspofun- b
y
gin,weculturedthestrainsonmediaandevaluatedtheirsensitivitytocaspofunginby
g
usingEteststrips(24).At5dayspostinoculation(dpi),thewild-typestraindeveloped u
e
confluentgrowthinthepresenceofcaspofunginandtheMICwas0.38(cid:2)g/ml.TheMIC s
t
fortheΔugmstrainwassignificantlyless(0.047(cid:2)g/ml),andaclearzoneofinhibition
was observed (Fig. 5). Similarly, the Δer strain also showed a wider zone of inhibition
comparedtothewildtype,withacaspofunginMIC(0.032(cid:2)g/ml)lowerthanthatofthe
Δugmstrain(Fig.5).
Collectively,theseresultsprovideevidencethatlossorinhibitionofBcUGMaffects
sensitivityofthefungustocellwall-targetingcompounds,confirmingthatthecellwall
integrityofbothstrainsiscompromised.
Galactofuranoseformationisinhibitedinthe(cid:2)erstrain.UGMenzymescatalyze
the interconversion of UDP-Galp and UDP-Galf. Since UDP-Galf is the donor for Galf
residues,comparisonofGalfcontentinglycoproteinscanbeusedtoestimatethelevel
ofUDP-Galf.Consequently,ifBcUGMisinhibitedbyUDP-KDG,theamountofthesugar
residue Galf that is incorporated into cell wall glycans should be reduced in the Δer
strain.FungalcellwallglycoproteinsusuallycontainGalfinbothN-linkedglycansand
O-linked glycans, which play important roles in protein stability, secretion, and local-
ization(9).InordertodeterminetherelativeGalfcontent,weextractedbothcellwall
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FIG5 TheΔugmandΔerstrainsaremoresusceptibletocaspofungin.Eteststripsimpregnatedwitha
gradientofcaspofunginwereplacedonGamborg-glucoseagarplatescontaining104spores/mlofthe
differentstrainsandculturedfor5daysat22°C.Thezoneofinhibitionisoutlinedbytheblackdashed
line,andtheMICisreadasthepointwheretheellipseofthezoneofinhibitioninterceptstheteststrip
(blackarrow).
D
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proteinsandtotalcellularproteinsfromdifferentfungalstrainsanddetectedtheGalf lo
epitopes by Western blotting with anti-Galf monoclonal antibody L10-1 (25). Protein a
d
bands that mainly varied in size between 48 and 200 kDa were recognized in total e
d
cellular proteins and cell wall protein samples extracted from the wild-type strain
f
r
(Fig.6AandB).NosignalwasdetectedinsamplesoftheΔugmstrain,althoughequal o
m
amountsofproteinswereloaded.Thelackofantibodyreactionwithproteinsextracted

h
fromtheΔugmstrainconfirmsthefollowing.(i)Thegenestudiedencodesafunctional t
t
UGM.(ii)NoalternativemetabolicroutescanformUDP-Galf.(iii)UDP-Galfisthesugar p
:
/
donor for the synthesis of Galf-containing glycoproteins in B. cinerea. Our working /m
modelsuggeststhatUDP-KDGaccumulationintheΔerstrainhasaninhibitoryeffecton b
io
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FIG6 Galactofuranose(Galf)formationisreducedintheΔerstrain.(A)WesternblottodetectGalfcontentintotalcellularproteins.Onehundredmicrograms
ofisolatedproteinswasloadedforeachstrain.(Top)Anti-Galf((cid:4)-Galf)L10-1monoclonalantibodieswereappliedtodetectGalfcontent.(Bottom)Antiactin
monoclonalantibodywasappliedasacontrolforproteinamountintotalcellularproteinsamples.(B)WesternblottodetectGalfcontentincellwallproteins.
Fiftymicrogramsofisolatedproteinswasloadedforeachstrain.(Top)Anti-GalfL10-1monoclonalantibodieswereappliedtodetectGalfcontent.Signalsof
samplesonasingleblotmembraneweredetectedusingaMicroChemiimagingsystem(DNR;Bio-ImagingSystems,Israel).Thepositionsofmolecularmass
markers(inkilodaltons)ofamergedproteinladderintheunlabeledleftmostlaneareindicatedtotheleftofthegel.(Bottom)ThePonceauSproteinstaining
wasusedforaloadingcontrol.(C)Comparisonofbcugmexpressioncomparedtobcgpdhexpression.
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UDP-KDGInhibitsFungalUDP-GalactopyranoseMutase
overall formation of UDP-Galf. Accordingly, the Δer strain should contain reduced
amounts of Galf in the glycoproteins. In agreement with this scenario, the anti-Galf
signalsintheΔerstrainweresignificantlyreducedintotalcellularproteins(Fig.6A)as
wellasincellwallproteins(Fig.6B)comparedwiththeGalfamountsinproteinextracts
fromthewild-typestrain.Analysisofgeneexpressionshowed1.5-fold-higherexpres-
sionlevelofthebcugmgeneintheΔerstrain(Fig.6C),rulingoutthepossibilitythat
reduction of Galf level in glycoproteins is due to reduced levels of bcugm gene
expression. Collectively, these results support our biochemical analyses that the re-
duced UGM enzymatic activity in the Δer strain results from inhibition of BcUGM by
UDP-KDG.
DISCUSSION
Nucleotide sugars are precursors of primary metabolic pathways yielding diverse
D
structuresofglycoproteins,polysaccharides,andglycolipids(10).Thefungalcellwallis
o
composedprimarilyofglycans,includingstructuralsugarpolymers,suchaschitinand w
n
glucans,andarangeofglycoproteinsthataredecoratedwithvarioustypesofsugars. lo
The roles of specific sugars within large polymers or glycoproteins are not easy to a
d
decipher,becausethesugarmayactasastructuralelement,itmayacttostabilizean e
d
enzymethatisglycosylated,oritmayhavearoleasarecognitionepitope.Inaprevious
f
r
study, we elucidated a two-step metabolic pathway of UDP-rhamnose production in o
m
B. cinerea (10). The first enzyme (UDP-glucose-4,6-dehydratase [DH]) converts UDP-

h
glucosetoproducetheintermediatemetaboliteUDP-KDG,whichisquicklyconverted t
t
toUDP-rhamnosebyER(seeFig.S2Ainthesupplementalmaterial).Aloss-of-function p
:
/
strain in which the entire UDP-rhamnose pathway was deleted was indistinguishable /m
fromthewild-typestrain,andwecouldnotinferanyharmfulconsequenceforthelack b
of rhamnose in the fungus. However, blocking of the second step of the pathway by io
.
a
deletionofthebcergeneledtomultipledevelopmentaldefects,whichwereassociated s
m
withweakeningofthecellwall.Weshowedthatthedefectswereduetoaccumulation
.
of the intermediate metabolite UDP-KDG, which as far as we can tell is not used for o
r
g
metabolism other than UDP-rhamnose formation. Hence, the rhamnose metabolic /

pathwaymightserveasashuntthatpreventsaccumulationofUDP-KDGtotoxiclevels o
n
byconvertingittoUDP-rhamnose. A
In humans, certain metabolic disorders are caused by the accumulation of toxic p
r
intermediate compounds when the product of an enzyme cannot be used by a il
2
downstreamenzymeinthemetabolicnetwork(26).Forexample,classicalgalactosemia ,

2
happens with infants who lack galactose-1-phosphate uridyl transferase activity (27), 0
1
leading to the accumulation of galactose-1-phosphate, which functions as a potent 9
competitiveinhibitorofphosphoglucomutasetodisrupttheglycolyticpathway(28).In b
y
microorganisms,accumulationofcertainmetabolicintermediatescanalsobringabout
g
toxiceffects.Forinstance,accumulationofsugar-phosphatesduetodisruptionofsugar u
e
metabolismhavebeenshowntobehighlytoxicinMycobacteriumtuberculosis(29,30). s
t
In the fungal system reported herein, deletion of bcer led to defects that result from
accumulation of the toxic intermediate metabolite UDP-KDG, while lack of rhamnose
had no effect on the fungus. Searching the JGI MycoCosm database (http://genome
.jgi.doe.gov/programs/fungi/index.jsf) using B. cinerea DH (BcDH) as a query revealed
thatthe100highestrankingfungiforblastptophitsalsocontainedBcERhomologues,
further indicating that the er gene tends to coexist with the dh gene, probably as a
safeguardtopreventaccumulationofUDP-KDG.
Given the structural similarity of UDP-KDG with other nucleotide sugars, we rea-
soned that UDP-KDG might interfere with certain nucleotide sugar-utilizing enzymes.
Amongseveralpotentialtargets,wefoundthatUDP-KDGinhibitsBcUGM.Itispossible
that the inhibition is due to the nature of UDP-KDG to exist in 4-hydrated form
(Fig.S2B),butfurtherstudies,includingcocrystallographyoftheBcUGMwithUDP-KDG
willberequiredtodeterminethenatureoftheinteractionofBcUGMwithUDP-KDG.
To evaluate whether inhibition of BcUGM by UDP-KDG is associated with the
developmental defects of the Δer strain, we produced bcugm deletion strains and
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comparedtheirphenotypeswiththephenotypeoftheΔerstrain.Overall,thepheno-
typic characteristics of the Δugm strain resembled those of the Δer strain, including
growth inhibition, developmental changes, impaired virulence, and weakening of the
cellwall;however,thephenotypicchangesoftheΔugmstrainweremilderthanthose
oftheΔerstrain.CoupledwithinvitroinhibitionofBcUGMbyUDP-KDG,theseresults
suggestedthatinvivoinhibitionofBcUGMbyUDP-KDGaccountsatleastpartlyforthe
developmental defects of the Δer strain. The consistently weaker effect in the Δugm
strain suggests that UDP-KDG might have additional targets other than BcUGM. This
possibility was further supported by the phenotypic characteristics of the Δer Δugm
doubledeletionstrainthatweremoreseverethanthephenotypiccharacteristicsofthe
ΔerstrainandthemilderphenotypiccharacteristicsintheΔer::OEugmdoubledeletion
mutant.
SinceUGMfacilitatestheconversionofUDP-GalptoUDP-Galf,whichisthedonorfor
D
theGalfincorporatedintoglycansandglycoconjugates,thelevelsofGalfareindicative
o
ofUGMactivityinthecell.WeestimatedtherelativelevelsofGalfinthemutantsby w
n
Western blot analysis using monoclonal antibodies that recognize Galf-containing lo
epitopes. In accordance with the expected inhibition of BcUGM, Galf levels were a
d
markedlyreduced(butnotabolished)intheΔerstrain.Transcriptlevelsofthebcugm e
d
gene in the Δer strain were slightly higher than in the wild-type strain, further
f
r
confirming that reduced Galf levels are due to inhibition of the BcUGM enzyme. In o
m
addition,GalfcontentintheΔer::OEugmstrainwashigherthanthatintheΔerstrain,

h
indicating that the inhibition of Galf production by UDP-KDG can be mitigated by t
t
overexpressing bcugm. Taken together, these results show that UDP-KDG inhibits the p
:
/
enzymaticactivityofBcUGMnotonlyinvitrobutalsoinvivo,resultingindecreasedGalf /m
levelsandaweakeningofthefungalcellwall. b
Galf-containing structures are important for survival and pathogenicity in many io
.
a
pathogenicmicroorganisms.TheseGalf-containingstructuresincludethelipopolysac- s
m
charides(LPS)OantigensofGram-negativebacteria,extracellularorcapsularpolysac-
.
charides of a variety of Gram-positive and Gram-negative bacteria, surface glycocon- o
r
g
jugatesofsomeprotozoanparasites,aswellasfungalcellwallandsecretedmolecules /

(8,31).BecauseGalfproductiondependsonUGM,changesordefectsinUGMactivity o
n
leadtochangesinGalfthatcanaffectfungalgrowthandvirulence.Forexample,the A
Aspergillus niger Δugm mutant exhibits increased sensitivity to cell wall-perturbing p
r
agents (32), and in Aspergillus nidulans, the ugm gene is necessary for conidiation, il
2
hyphalgrowth,andmorphogenesis,anditalsoaffectsantifungaldrugsensitivity(33, ,

2
34).Inaddition,disruptionofugminAspergillusfumigatusresultsinweakenedcellwall 0
1
and likely attenuated virulence in mice (25, 35). Similarly, disruption of bcugm in 9
B.cinerearesultedinweakenedcellwallandotherdevelopmentaldefects.Sincethere b
y
is no Galf in mammals and higher plants, Galf biosynthetic pathways have been
g
exploredasattractivetargetsforthedevelopmentofantibacterialdrugs(36–41).The u
e
amino acid sequence conservation between prokaryotic and eukaryotic UGM is less s
t
than 15% (8), indicating that fungal and bacterial UGM enzymes are quite divergent.
Because of these structural differences, it is expected that substrate binding mecha-
nisms also differ in the fungal and bacterial UGM (42), and therefore, inhibitors of
bacterialUGMarenotexpectedtobeefficientinfungi.Besides,alloftheseinhibitors
wereidentifiedbyinvitroenzymaticassays,whichmaynotpredicttheirentryintoand
trueefficacyinlivingcells.SinceUDP-KDGinhibitsBcUGMbothinvitroandinvivo,it
mightbepossibletodevelopantimetabolitesthatmimicUDP-KDGasaUGM-targeting
antifungaldrug.Anotherwaymightbetoidentifycompoundsthatinhibitnucleotide-
rhamnosesynthase(NRS)/ERenzymeasawaytoforceaccumulationoftoxicinterme-
diates.
Although the Δugm strain phenocopied the Δer strain in various aspects, the
phenotype differed in some specific features such as reduced spore size and delayed
spore germination that were not observed in the Δer strain (Fig. S3). Considering the
differencefromtheΔugmstrain,theremaininglowlevelsofGalf,orthespecificsalvage
pathway stimulated by severe cell wall defects in the Δer strain, may underlie the
November/December2017 Volume8 Issue6 e01559-17 mbio.asm.org 8
®
UDP-KDGInhibitsFungalUDP-GalactopyranoseMutase
uniquecharacteristicsoftheΔugmstrain.Ontheotherhand,theΔerstraindisplayed
a more-debilitated phenotype than the Δugm strain did, meaning that BcUGM is not
sufficienttoaccountforalloftheadverseeffectsbroughtaboutbyUDP-KDG.Inorder
to test whether UDP-KDG can inhibit other cell wall-synthesizing enzymes besides
UGM,bothinvitroandinvivoinhibitionassayshaveyettobeperformed.
Drugsynergycanresultwhendrugstargetthesameorfunctionallyrelatedpathway
butthroughdifferenttargets(43).ThehighersensitivityoftheΔugmandΔerstrainsto
cell wall inhibitors suggested that a UGM-targeting drug might be applied in combi-
nationwithantifungaldrugsthattargetothercellwall-synthesizingpathways,anotion
that was supported by its potentiating the sensitivity of the mutants to caspofungin.
Becausecaspofungininhibitstheenzyme1,3-(cid:3)-D-glucansynthase,thecombinationof
deletion of bcugm with caspofungin resulted in a synergistic effect. Interestingly,
despitepartialinhibitionofBcUGMintheΔerstrain,thesensitivityoftheΔerstrainto
D
caspofunginwasslightlyhigherthanthatoftheΔugmstrain,possiblyduetoinhibition
o
of multiple targets by UDP-KDG. These results demonstrate the antifungal activity of w
n
UDP-KDG, but more research will be necessary to determine the true potential of lo
UDP-KDGasaleadmoleculeforantifungaltherapies. a
d
e
MATERIALSANDMETHODS d

Fungiandmedium.Botryotiniafuckeliana(deBary)Whetzel((cid:5)Botrytiscinerea)strainB05.10and fr
o
derivedtransgenicstrainswereculturedonpotatodextroseagar(PDA)(Acumedia,Lansing,MI)at22°C m
withcontinuousfluorescentlightsupplementedwithnear-UVlight.Routinecultureorgrowthassays
h
werecarriedoutinpotatodextrosebroth(PDB)(Acumedia,Lansing,MI),CzapekDox(CD)medium(0.3% t
t
NaNO,0.05%KCl,0.05%MgSO ·7HO,0.001%FeSO ·7HO,0.1%KHPO ·3HO,2%sucrose[pH7.3]), p
3 4 2 4 2 2 4 2 :
orGBmedium(GamborgB5includingvitaminmixture;DuchefaBiochemie,Haarlem,theNetherlands) //
m
supplementedwith2%glucose(GB5-Glc).
Cloningandexpressionofbcugm.Bacterialcodon-optimizedB.cinereageneBC1G_09363(poten- b
io
tially encodes UDP-galactopyranose mutase [BcUGM]) was synthesized (GenScript). For recombinant
.
proteinproduction,thegenewasPCRamplifiedandclonedintopET28b-Tevexpressionplasmid(44). a
s
PCRamplificationprimersincludedanextra15-nucleotidesequenceatthe5=endwithhomologytothe m
cloningsiteofthepETplasmid(seeTableS1forthesequencesofallprimers).PrimersGalpMutasePIPE .
o
SandGalpMutasePIPEASwereusedtoamplifythebcugmgene.Forthevector,primersZL169and r
g
KB_T7t were used to amplify the pET28b-TEV which when expressed gives a fusion protein with /

N-terminalsixhistidinesfollowedbyatobaccoetchvirus(TEV)recognitionaminoacidsequence. o
EscherichiacoliRosetta2(DE3)pLysSstrainscontainingpET28b-TEV_BcUGMorpET28b-TEV_BcDH(a n

control plasmid) were cultured overnight at 37°C with shaking at 250 rpm in 5 ml LB medium A
supplementedwith50(cid:2)g/mlkanamycinand35(cid:2)g/mlchloramphenicol.Eachculturewastransferredto pr
a flask containing 250 ml LB medium supplemented with the same antibiotics, and the flasks were il
2
incubated(37°C,250rpm)untilcelldensityreachedanopticaldensityat600nm(OD )of0.6to0.8.
Isopropyl-(cid:3)-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of6100mM, and the , 2
culturesweregrownfor20h(18°C,250rpm).Thecellswerecollectedbycentrifugation(6,000(cid:6)g, 0
1
10 min at 4°C) and washed with 20 ml cold double-distilled water (DDW), and each cell pellet was 9
resuspendedin10-mlcoldlysisbuffer(50mMTris-HCl[pH7.5],50mMNaCl,10%glycerol,1mMEDTA, b
0.5mMphenylmethylsulfonylfluoride[PMSF],and1mMdithiothreitol[DTT]).Thecellswerelysedby y

sonicationinice-coldwaterusingaMisonixS-4000sonicator(Misonix,Inc.,Farmingdale,NY)equipped g
u
with1/8-in.microtipprobe(12cycleswith1cycleconsistingofa20-spulseat30%amplitudeand30s e
off).Aftersonication,thesampleswerecentrifuged(6,000(cid:6)gfor15minat4°C),andthesupernatant s
wasrecoveredandcentrifugedagainat20,000(cid:6)gfor30minat4°C.Theresultantsupernatantwas t
subjectedtoNicolumnpurificationusingaFast-FlowNi2(cid:2)-Sepharosecolumn(GEHealthcare)(2mlof
resinpackedinapolypropylenecolumn[1-cminnerdiameterby15cm]).Recombinantproteinswere
elutedwithelutionbuffercontainingincreasedconcentrationsofimidazole(10to250mM)andanalyzed
bymeasurementoftheirspecificactivityandbySDS-PAGEfollowedbyCoomassiebluestaining.
Enzyme activities and inhibition assay. Activity of recombinant BcUGM was tested in a 50-(cid:2)l
reactionsolutioncontaining50mMTris-HCl(pH7.5),1mMNADPH,1mMflavinadeninedinucleotide
(FAD),and1mMUDP-galactopyranose.Theinitialreactionswereperformedat30°Cfor1handthen
inactivatedby98°Cfor5min.Anequalvolumeofchloroformwasadded,thetubeswerecentrifugedfor
5 min at 14,000 (cid:6) g, and 30 (cid:2)l of the upper aqueous phase was removed and mixed with 57 (cid:2)l
acetonitrileand3(cid:2)lof0.5Mammoniumacetate(pH4.35).Aportion(25(cid:2)l)ofthismixturewasused
forhydrophilicinteractionliquidchromatography(HILIC)byhigh-performanceliquidchromatography
withaUVdetector(HPLC-UV)andanother25(cid:2)lwasanalyzedbyliquidchromatographycoupledto
tandem mass spectrometry (LC-MS/MS) (10). The amount of product formed was determined by
HPLC-UV.
TriplicateBcUGMassayswereperformedforkineticanalyseswithincreasingUDP-Galpconcentra-
tions(0.1,0.5,1,1.5,2,and3mM).Reactionswerecarriedoutat30°Cfor30minwith20(cid:2)lpurified
BcUGMwithafixedamountofFAD,NADPH,andTris-HCl(pH7.5).TheamountofUDP-Galfwasanalyzed
byHPLC-UVandverifiedbyliquidchromatographyandmassspectrometrywithelectrosprayionization,
November/December2017 Volume8 Issue6 e01559-17 mbio.asm.org 9
®
Maetal.
iontrap,andtimeofflighttechnologies(LC-MS-ESI-IT-TOF).Kineticswerecalculatedbyfittingenzyme
kineticcurvesusingtheGraphPadPrism7software.
Fortheinhibitionassays,thereactionmixturesremainedthesameasforthekineticanalyseswiththe
additionofvariousconcentrationsofUDP-KDG(1,10,and100(cid:2)M).ToobtainUDP-KDGcompound,a
large-scalereactionwasperformedwithB.cinereaUDP-glucose-4,6-dehydratase(BcDH)thatconverts
UDP-glucosetoUDP-KDG.This1-mlreactionconsistedof50mMTris-HCl(pH7.5),1mMUDP-glucose,
1mMNAD(cid:2),andBcDH(14).Thereactionwasconductedfor2handheatinactivated,andtheproduct
wasseparatedbyHPLC-UVandverifiedbyLC-MS-ESI-IT-TOF.Calculationofinhibitionwasperformed
usingGraphPadPrism7.
Generation of B. cinerea mutant strains. B. cinerea ugm homologue (bcugm) (GenBank acces-
sion number XM_001551972) was identified by a BLAST search with Aspergillus fumigatus UDP-
galactopyranosemutase(GenBankaccessionnumberAJ871145).TheNCBIB.cinereaB05.10genome
sequence(GenBankaccessionnumberXM_001551972)andflankingregionwasusedtodesignprimers
(TableS1).
B. cinerea bcugm deletion strains (Δbcugm strains) were generated by replacing the entire open
readingframe(ORF)ofthebcugmgenewithahygromycinresistancecassetteconsistingofanAspergillus
D
nidulans oliC promoter (PoliC), a hygromycin phosphotransferase gene (hph), and bcacr (B. cinerea
o
enoyl-[acylcarrierprotein]reductase)terminator(Tacr)(45).Adeletioncassettewasgeneratedbyadding w
500bpeachofthe5=and3=flankingregionsofthebcugmgeneoneithersideofthehphcassetteby n
overlapPCR(Fig.S4).Adoubledeletionstrainofthebcerandbcugmgenes(ΔbcerΔbcugmstrain)was lo
generatedbydeletingthebcugmORFwithanourseothricinresistancecassette(nr)inthebackground a
d
ofaΔbcerstrain(shortforΔbcer9a2strainasmentionedinreference10.Thebcugm-nrdeletionconstruct e
wasassembledinthesamewayasdescribedforthehphdeletioncassette(Fig.S5). d

A bcugmoverexpressionconstructwaspreparedbyplacingthebcugmORFunderthestrongH2B f
r
promoterofB.cinerea(10)andflankedbyannrselectioncassette.TheB.cinerea-specificbcniiAsequence o
m
(46)wasincludedasahomologyregionforintegrationoftheconstruct(pNAH-ugm)tothebcniiAlocus

intheB.cinereagenome(Fig.S6).TheplasmidwaslinearizedwithApaIrestrictionenzymeandusedto h
t
transformtheB.cinereaΔerstraintogeneratetheΔbcer::OEbcugmstrainasdescribedpreviously(10). tp
A bcugm complementation cassette was prepared by assembling three PCR fragments using a :/
/
GibsonAssemblymastermixkit(NewEnglandBiolabs).Thethreefragmentsincludethebcugmgene m
withtheupstream500-bpregionasthepromoterandthedownstream340bpastheterminator,the b
PoliC-nrcassette,andtheTcel5a(B.cinereacel5a;GenBankaccessionnumberAY618929.1)termination io
signal(Fig.S7).ThisconstructwastransformedtoaΔbcugmstraintogeneratethebcugmcomplemen- .a
tationstrain(Δbcugm(cid:2)bcugmstrain). s
m
GenetictransformationofB.cinereawiththedifferentDNAconstructswasperformedaspreviously
.
described(10).Coloniesthatdevelopedontheselectionmediaweretransferredtoseparateplates.At o
r
least10isolateswereobtainedforeachtypeofstrain,andDNAwasextractedfromeachcolonyand g
/
analyzedbyPCRtoverifyintegrationoftheconstructatthedesiredlocus.Homokaryoticstrainswere o
obtained by single-spore isolation using a Sporeplay dissection microscope (Singer Instruments, UK). n

DerivedcolonieswereanalyzedbyPCRtoverifythatthestrainishomokaryoticatthedesiredlocus,and A
additionalroundsofsinglesporeisolationwereperformedincasesofimpurity.Atleastfourseparate p
r
single-sporeisolateswereobtainedforeachstrain. il
Cellwallintegrityassay.Sporesfrom9-day-oldPDAcultureswerecollectedusingsterileDDWand 2
,
filteredthroughtwolayersofMiracloth(Millipore,USA).Thesporeswerethendilutedto105spores/ml, 2
and5(cid:2)lofasporesuspensionofeachstrainwasappliedtothecenterofaPDAplatewithoutorwith 0
1
thespecifiedconcentrationofcellwall-inhibitingcompoundscalcofluorwhite(CW)orCongored(CR).
9
Theplateswereincubatedat22°C,andtheradiusofeachcolonywasmeasured3dayspostinoculation
b
(dpi).Eachexperimentwasrepeatedatleastfivetimeswiththreereplicationspertreatment. y
To measure the MIC of caspofungin, the filtered spores were mixed into 20 ml of 50°C GB5-Glc g
mediumtoafinalconcentrationof104/mlandimmediatelypouredin9-cm-diameterpetriplates.Next, u
e
theEteststrips(bioMérieux,France),containingacontinuouscaspofunginconcentrationgradientfrom s
0.002to32(cid:2)g/ml,wereplacedonthesurfaceoftheinoculatedagarmediaandincubatedat22°C.The t
EtestMICwasdefinedasthedrugconcentrationatwhichtheborderoftheellipticalzoneofcomplete
inhibitionintersectedthescaleontheantifungalteststrip.Assayswererepeatedthreetimes.
Pathogenicityassay.Pathogenicityassayswereperformedontheleavesof10-day-oldbeanplants
(Phaseolusvulgariscv.Dwarfsugar)aspreviouslydescribed(47).Fourleavesfromtwoplants(twoleaves
perplant)wereusedforeachtreatment.Leaveswereinoculatedwith7.5-(cid:2)ldropletsofsporesuspension
(105spores/mlinGB5-Glc),andtheradiusoflesionswasrecordedevery24h.Experimentswererepeated
atleastfivetimes.
Isolation of fungal proteins. (i) Cell wall proteins. Spores were collected from 9-day-old PDA
cultures,filteredthroughtwolayersofMiracloth,andthenaddedtoa50-mlErlenmeyerflaskwith10ml
PDB to a final concentration of 106 spores/ml. Samples were incubated on an orbital shaker with
agitation at 180 rpm for 24 h. Then, samples were centrifuged at 3,000 (cid:6) g for 10 min, and the
supernatant was removed. Mycelial pellets were washed sequentially with 20 ml of DDW, 20 ml of
washingsolutionA(1MNaCl,25mMEDTA,1mMPMSF),and20mloflysisbuffer(10mMTris-HCl
[pH7.4],10mMEDTA,1mMPMSF)toremoveproteinspresentinthemediumandproteinsthatare
looselyconnectedwiththehyphae.Thissequentialwashingcyclewasperformedthreetimesforeach
washingstep,thenthewashedmycelialpelletsweredriedbylyophilizationovernight,andthedried
pelletswerehomogenizedbyrepeatedhomogenizationusingaTissueLyser(Qiagen)operatingat30Hz
for3mineachtimeuntilcompletecellbreakage.
November/December2017 Volume8 Issue6 e01559-17 mbio.asm.org 10