Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 835081 8 pageshttpdxdoiorg1011552013835081

Research ArticleAnti-Candida Properties of Urauchimycins from ActinobacteriaAssociated with Trachymyrmex Ants

Thais D Mendes12 Warley S Borges34 Andre Rodrigues15 Scott E Solomon6

Paulo C Vieira4 Marta C T Duarte7 and Fernando C Pagnocca15

1 Center for the Study of Social Insects Sao Paulo State University (UNESP) 13506-900 Rio Claro SP Brazil2 EMBRAPA Agroenergy Parque Estacao Biologica 70770-901 Brasılia DF Brazil3 Chemistry Departament Federal University of Espırito Santo (UFES) 29075-910 Vitoria ES Brazil4 Chemistry Department Federal University of Sao Carlos (UFSCar) 18052-780 Sao Carlos SP Brazil5 Department of Biochemistry and Microbiology Sao Paulo State University (UNESP) 13506-900 Rio Claro SP Brazil6Department of Ecology and Evolutionary Biology Rice University Houston TX USA7Division of Microbiology Center for Chemistry Biology and Agriculture Research (CPQBAUNICAMP)13081-970 Paulınia SP Brazil

Correspondence should be addressed to Fernando C Pagnocca pagnoccarcunespbr

Received 12 November 2012 Revised 29 January 2013 Accepted 2 February 2013

Academic Editor Manish Bodas

Copyright copy 2013 Thais D Mendes et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

After decades of intensive searching for antimicrobial compounds derived from actinobacteria the frequency of isolation of newmolecules has decreased To cope with this concern studies have focused on the exploitation of actinobacteria from unexploredenvironments and actinobacteria symbionts of plants and animals In this study twenty-four actinobacteria strains isolated fromworkers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species Results revealed thatseven strains inhibited the tested Candida species Streptomyces sp TD025 presented potent and broad spectrum of inhibition ofCandida and was selected for the isolation of bioactive molecules From liquid shake culture of this bacterium we isolated therare antimycin urauchimycins A and B For the first time these molecules were evaluated for antifungal activity against medicallyimportant Candida species Both antimycins showed antifungal activity especially urauchimycin B This compound inhibited thegrowth of all Candida species tested with minimum inhibitory concentration values equivalent to the antifungal nystatin Ourresults concur with the predictions that the attine ant-microbe symbiosismay be a source of bioactivemetabolites for biotechnologyand medical applications

1 Introduction

The increased resistance of microorganisms to antibiotics is aproblem of public health [1]The increasing number of fungalspecies that can infect humans particularly immunocompro-mised individuals further reinforces this concern A limitednumber of antifungal agents are commercially available whencompared to antibacterial drugs This scenario motivates thesearch for new bioactive compounds in various biologicalsystems using several approaches including metagenomicsand microbial genome-mining

Actinobacteria are widely known for their ability to pro-duce bioactive secondary metabolites especially compoundswith antimicrobial activity These bacteria are responsible forproducing two-thirds of the commercially available antibi-otics [2 3] Most actinobacteria species explored commer-cially were isolated from the soil However after decades ofbioprospecting actinobacteria from this environment it isbecoming more difficult to obtain strains producing novelbioactivemetabolites [4]Thus many companies have turnedthe search for microbial producers of novel antifungal com-pounds to other environments such as hydrothermal vents

2 BioMed Research International

marine environments tropical rain forests and microbialsymbionts associated with plants and animals hosts [5 6]For example the occurrence of actinobacteria associatedwithmarine sponges and the fact that such strains produce com-poundswith antimicrobial activity confirms this potential [7ndash9] In addition endophytic actinobacteria are also exploredfor their capacity to produce antimicrobial compounds [10ndash12]

Several studies have focused on the association betweenactinobacteria and insects from an ecological perspective[13ndash25] On the other hand few studies have focused onthe multitude of chemical compounds that are involvedin such interactions [26] The best studied example is thesymbiosis between actinobacteria and fungus-growing ants(Hymenoptera Formicidae tribe Attini) In this associationthe actinobacteria are found on the antsrsquo integument andproduce antimicrobial compounds that help the ants tosuppress the microfungus Escovopsis sp [13 14] This fungusis considered a specialized parasite of the ant cultivar andcauses negative impacts to the ant colony [27]

Actinobacteria isolated from the integument of attineants are generally classified in the genus Pseudonocardia andStreptomyces Bioactive molecules have already been isolatedand characterized from actinobacteria isolated from severalattine genera [26]Pseudonocardia isolated fromAcromyrmexoctospinosus and Apterostigma dentigerum are known to pro-duce several compounds like (i) dentigerumycin a complexcompound active against Candida albicans and Escovopsis[28] (ii) a nystatin-like antifungal [29] (iii) the novel quinonepseudonocardones AndashC active against the malaria causalagentPlasmodiumberghei [30] (iv) the already known antibi-otics 6-deoxy-8-O-methylrabelomycin and X-14 881 bothactive against Bacillus subtilis and P berghei [30] In additionto Pseudonocardia actinobacteria in the genus Streptomycesare also found on the integument of Acromyrmex workersand were shown to produce (i) candicidin active againstEscovopsis sp [29 31ndash34] (ii) antimycins active againstEscovopsis sp [32ndash34] and (iii) actinomycin D actinomycinX2 and valinomycin that are active against B subtilis [32]

Poulsen [35] suggested that the attine ant-microbe asso-ciation is little explored regarding the search for newantimicrobials The author highlights the various symbioticassociations between attine ants and microorganisms as apromising source for drug discovery especially those withantimicrobial activity Here we explored the antimicrobialpotential of actinobacteria isolated from the integument ofTrachymyrmex fungus-growing ants and demonstrate theaction against differentmedically importantCandida speciesWe also report two previously described urauchimycins froma Streptomyces strain and emphasize the newly discoveredanti-Candida activity of these compounds

2 Material and Methods

21 Actinobacteria Isolation and Identification TwelveTrachymyrmex colonies were collected in different Brazilianbiomes (see Table S1 in Supplementary Material availableonline at httpdxdoiorg1011552013835081) Colonies

were carefully excavated in order to reach the first fungusgarden chamber Fungus garden with the tending workersand brood was sampled using an alcohol-flamed spoon andstored in sterile plastic containers All containers were keptin a cooler during transport to the laboratory where theywere maintained at 25∘C

From each colony we randomly selected four workersfor actinobacteria isolation Then the propleural plateswere scraped with a sterile needle under a low powerstereomicroscope All ants used in the present study had avisible whitish covering on the propleural plates Scrapingswere plated on SCN agar (in g sdot Lminus1 100 starch 03 casein20 KNO

3 20NaCl 20 K

2HPO4 005MgSO

4sdot 7H2O

002 CaCO3 001 FeSO

4sdot 7H2O and 180 agar supplemented

with 005 Nystatin) [36] After scraping the entire bodyof all workers was inoculated on SCN agar All plateswere incubated at 25∘C for 30 days From each sampledTrachymyrmex colony one representative strain wasselected from each morphotype obtained The strains weresubcultured in YMA (in g sdot Lminus1 30 yeast extract 30 maltextract 50 peptone 100 glucose 180 agar) and stored atminus80∘C in 15 glycerol

We used a molecular approach to provide taxonomicaffiliation to actinobacteria strains Genomic DNA wasextracted following the method of Sampaio et al [37] Wecarried out 16S rDNA PCR with the universal primers27F (51015840-AGAGTTTGATCATGGCTCAG-31015840) and 1492R (51015840-TACGGTTACCTTGTTACGACTT-31015840) [38] Reactions wereconducted in a final volume of 25 120583L and contained 1 120583Lof diluted DNA template (1 10) 20120583L of each primer(10mM) 25 120583L of 10X buffer 10 120583L of MgCl

2(50mM)

40 120583L of dNTPs (125mM each) 02 120583L of Taq polymerase(5U120583L) and 123 120583L of ultrapure water Amplicons werecleaned up with GFX PCR DNA and Gel Band Purifica-tion Kit (GE Healthcare) Forward and reverse sequenceswere generated using the same primers along with aninternal primer U519F (51015840-CAGCMGCCGCGGTAATWC-31015840) Sequences were generated using BigDye Terminator v31Cycle Sequencing Kit (Life Technologies) in an ABI 3130sequencer andmanually edited inBioEdit v 713 [39] Contigswere compared with those available in the databases NCBI-GenBank (httpblastncbinlmnihgov) and RibossomalDatabase Project (RDP httprdpcmemsuedu) Sequencesgenerated in the present study were deposited in NCBI-GenBank (accessions KC480554-KC480557)

A phylogenetic analysis was carried out in order to deter-mine the taxonomic affiliation of strain TD025 Sequencesof closest relatives were retrieved from the NCBI-GenBankand the RDP Project and aligned in ClustalW Phylogeneticreconstruction was performed using the neighbor-joiningalgorithm implemented in PAUP v 40 [40] Genetic dis-tances were calculated using the Kimura 2-parameter modelof nucleotide substitution [41] Robustness of the relation-ships was estimated from 1000 bootstrap pseudoreplicates

22 Organic Extracts and Antifungal Assays All actinobac-teria were grown in Erlenmeyer flasks (250mL) containing50mL of modified MPE medium (in g sdot Lminus1 50 soy flour

BioMed Research International 3

200 glucose 50 of NaCl 40 CaCO3) [42] Each flask was

inoculated with five mycelium disks (1 cm in diameter) cutfrom a previously grown culture and then incubated at28∘C for two days on an orbital shaker at 150 rpm Fromthis culture 10mL was inoculated in two Erlenmeyer flasks(250mL) containing 100mL of the same medium and incu-bated for five days on the same conditions After incubationthe fermented broth was separated from the mycelium bycentrifugation and partitioned three times with ethyl acetate(EtOAc) The organic solvent was evaporated under vacuumand the EtOAc extracts were diluted in RPMI-1640 culturebroth containing 10 DMSO and used in the antimicrobialassays

The antimicrobial activity of the extracts was evalu-ated against the yeasts Candida albicans CBS 562 Candidadubliniensis CBS 7987 Candida glabrata CBS 138 Candidakrusei CBS 573 Candida parapsilosis CBS 604 and Candidatropicalis CBS 94 The minimum inhibitory concentration(MIC) was determined using the microdilution methodaccording to the M27-A2 standard of the Clinical and Lab-oratory Standards Institute [43]

23 Isolation and Characterization of the Bioactive Com-pounds of Strain TD025 Theactinobacteria strain that exhib-ited both a broad antifungal spectrum and lower MIC valueswas strain TD025 In order to identify the compoundsresponsible for the observed results the strainwas cultured in5 L of modifiedMPEmedium and the extracts were obtainedas described

The fermented broth (5 L) was separated from the cells bycentrifugation and portioned three times with ethyl acetate(EtOAc) The organic solvent was evaporated under vacuumThe crude extract a dark green oil (140 g) was separatedby means of column chromatography using silica gel 60eluted with n-hexaneEtOAc as the elution gradient yielding8 fractions All fractions were submitted to antimicrobialassays against C albicans following the procedure describedabove The most active fraction (675mg) was subjected topreparative TLC (thin layer chromatography) eluted withn-hexaneEtOAc (7 3) two times yielding 3 subfractionsThe subfractions obtained were submitted to antimicrobialassays against C albicans The most active subfraction wassubmitted to semipreparative HPLC separations carried outin a Shimadzu (LC-6AD apparatus Japan) multisolventdelivery system Shimadzu SPD-M10Avp Photodiode ArrayDetector and an Intel Celeron computer for analytical systemcontrol data collection and processing (software Class-VP)The separation was carried out using VP 25010 NUCLEOSIL120-5 C18 column eluted with acetonitrilewateracetic acid(50 50 001) at a flow rate of 3mL sdotminminus1 yielding com-pounds 1 and 2The isolated molecules were characterized by1H and 13C NMR spectroscopic experiments recorded on aBRUKER DRX-400 spectrometer with CDCl

3as solvent and

TMS as internal standard

24 Minimum Inhibitory Concentration andMinimum Fungi-cide Concentration of Bioactive Compounds After isola-tion and determination of the structure of the targeted

compounds they were evaluated for antimicrobial activityfollowing the method described previously Besides the MICdetermination we also evaluated the minimum fungicidalconcentration (MFC)TheMFCwas determined by inoculat-ing Sabouraud dextrose medium with 10 120583L of the contentsof each of the wells where there was growth inhibition ofyeast theMFCwas defined as the lowest concentration of thesubstance capable of preventing the onset of colony formingunits

3 Results and Discussion

31 Actinobacteria Isolation and Identification Several acti-nobacteria colonies were observed after incubation of iso-lation plates We selected just one morphotype of each perant colony rendering a total of 24 strains ouf of 12 Tra-chymyrmex spp nests (Table S1) Four actinobacteria generawere identified and Streptomyces was the most abundanttaxon (Table 1) corresponding to 6667 of the strains Itis assumed that the main actinobacteria associated with theintegument of attine workers is the genus Pseudonocardia[14ndash16 20] However several authors have demonstrated theisolation of actinobacteria other than Pseudonocardia on theintegument of attine ants [17ndash19 21 22 29 31]Theprevalenceof Streptomyces and absence of Pseudonocardia among ourisolates may be due to the culture medium used [44] TheSCN medium is suitable for the isolation of fast-growingactinobacteria but according to other authors [13ndash16] the useof a low-nutrient medium such as chitin agar may providethe recovery of Pseudonocardia strains

In eight out of 12 ant colonies we obtained more thanone actinobacteria morphotype (Table S1) From colonyCTL080820-02 the two morphotypes isolated from twodifferent workers were identified as the same actinobacteriaspecies (Tables S1 and 1) On the other hand differentactinobacteria species were isolated in the seven remainingcolonies We also observed the occurrence of different acti-nobacteria strains in a single worker (Tables S1 and 1) Thisresult demonstrates the diversity of actinobacteria present onthe integument of these ants (Table S1 nests SES080911-04and SES080924-01)

The 16S rDNA sequence of strain TD025 showed 99similarity with sequences of several species of the genusStreptomyces deposited in the databases For a better charac-terization we performed a phylogenetic analysis (Figure 1)The result suggests a differentiated phylogenetic position forstrain TD025 when compared with the remaining sequencesThis preliminary analysis allowed us to assign this strainas belonging to the genus Streptomyces with S cirratus asthe closest relative strain (Figure 1) However more refinedphylogenetic analyses along with morphological and physi-ological studies are necessary to ensure the identification ofTD025 to the species level

32 Screening for Antifungal Activity Our screening forantifungal activity revealed that seven out of 24 extracts(2916) inhibited the growth of at least one Candidaspecies C albicans was the most sensitive yeast and was


Table 1 Actinobacteria identification according to 16S rDNA sequencing

Isolate Id bp1 NCBI-GenBank closest relative Coverage Accession TD016 1251 Nocardia neocaledoniensis DSM 44717 100 100 JF797311TD017 1248 Nocardia neocaledoniensis DSM 44717 100 100 JF797311TD018 1184 Streptomyces zaomyceticus NRLL B-2038 99 100 NR044144TD019 1342 Streptomyces alivochromogenes NBRC 3404 99 100 AB184761TD020 1167 Streptomyces sannanensis NBRC 14239 99 99 NR041160TD021 1250 Streptomyces mauvecolor NBRC 13854 100 99 NR041154TD022 1254 Streptomyces lydicus CGMCC 41412 100 100 JN566018TD023 1246 Streptomyces sp QZGY-A17 100 100 JQ812074TD025 1333 Streptomyces sp QLS92 100 99 JQ838121

Streptomyces cirratus 100 99 JQ222143TD027 1342 Streptomyces alivochromogenes NBRC 3404 99 100 AB184761TD028 1353 Actinoplanes ferrugineus 100 99 AB048221TD030 1255 Streptomyces sp CA13 100 99 AB622252TD032 1263 Streptomyces chartreusis NBRC 12753 100 100 NR041216TD033 1278 Streptomyces griseoplanus 99 100 HQ699516TD034 1283 Amycolatopsis decaplanina DSM 44594 100 100 NR025562TD035 1320 Streptomyces atriruber NRLL B-24676 100 99 FJ169330TD045 1183 Amycolatopsis equina 100 99 HQ021204TD047 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251TD049 1260 Streptomyces luteogriseusNBRC 13402 100 99 NR041128TD050 1261 Streptomyces rubiginosohelvolus NBRC 12912 100 100 NR041093TD051 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251TD053 1265 Streptomyces kunmingensis NBRC14463 99 98 AB184597TD055 1173 Amycolatopsis albidoflavus NBRC100337 100 100 AB327251TD058 1257 Streptomyces globisporus KCTC 9026 100 100 HQ9955041bp base pair

inhibited by seven extracts with MIC ranging between 10and 1000 120583g sdotmLminus1 (Table 2) The yeasts C glabrata and Ctropicalis were the most resistant strains being inhibited byone and two actinobacteria extracts respectively with MICvalues of 1000120583g sdotmLminus1 (Table 2)

Except for strain TD034 identified as Amycolatopsisdecaplanina (Table 1) the other actinobacteria exhibitingantimicrobial activity were identified as belonging to thegenus Streptomyces This genus is recognized as the largestproducer of antibiotics because from approximately 3000known antibiotics obtained from actinobacteria the genusStreptomyces contributes with 90 of this total [45]

The extracts of Streptomyces sp TD025 and Streptomycescrystallinus TD027 showed activity against all yeast strainsexcept for C glabrata These extracts were effective againstC albicans and C krusei and showed low activity againstC tropicalis (Table 2) More interestingly both strains wereisolated from the same colony but from independent workers(Table 1) Because lower MICs were obtained for the extractof Streptomyces sp TD025 this strain was selected to verifythe chemical compounds responsible for the antimicrobialactivity

33 Bioactive Compounds of Streptomyces sp TD025 Chro-matographic procedures revealed that EtOAc extract fromTD025 contains two compounds (1 and 2 Figure 2)

Compounds 1 and 2 exhibited typical NMR data ofurauchimycins (Figures S1 and S2) Their NMR data are inagreement with those previously reported by Imamura et al[46] Although these urauchimycins (1 and 2) have alreadybeen isolated they have never been tested on various speciesof Candida as carried out in the present study

The 13C NMR spectrum of 1 showed 22 carbon signalsfour carbonyls (120575 1790 120575 1706 120575 1698 and 120575 1587) threequaternary sp2 carbons (120575 1506 120575 1274 and 120575 1128) threemethine aromatic carbons (120575 1248 120575 1206 and 120575 1190) twosp3 methylene group (120575 356 and 120575 305) three sp3 methinegroups (120575 540 120575 501 and 120575 324) three oxymethinic groups(120575 771 120575 763 and 120575 709) and four methyl groups (120575 184120575 184 120575 151 and 120575 114) Two carboxylic carbons 120575 1706and 120575 1739 showed correlations with different hydrogens ofthe structure showing a dilactone system of nine memberstypical of the antimycin class

The 1H NMR spectrum showed a singlet at 120575 850assigned to a hydrogen bounded to a carbonyl group andthree aromatic hydrogens at 120575 855 (dd J 81 and 12Hz) 120575 724(dd J 81 and 12Hz) and d 692 (t J 81 Hz) suggesting a 123trisubstituted aromatic ring The substance was identified asurauchimycin A by comparison with the literature data [46]

The 1H and 13C NMR spectram of 2 were very similar tothose observed for compound 1 Differences were observed inchemical shifts of the hydrogen of the methyl and methylene


100

73

65

74

98

100

100

86

58

58

68

71

0001

Streptomyces sp (JQ838121)

Streptomyces cirratus (JQ422143)

Streptomyces sp (EF093114)

Streptomyces spiroverticillatus (NR041214)

Streptomyces griseobrunneus (AB249912)

Streptomyces albolongus (NR041144)

Streptomyces flavogriseus (NR028988)

Streptomyces griseolus (NR041207)

Streptomyces cinereorectus (NR041173)

Streptomyces badius (NR043350)

Streptomyces rubinosohelvolus (NR041093)

Streptomyces globisporus (NR044145)

Streptomyces crystallinus (NR041177)

TD025 (KC480562)

Figure 1 Phylogenetic relationships of strain TD025 (in bold) isolated from the integument of Trachymyrmex spThe phylogeny was inferredfrom 16S rDNA sequences retrieved from the NCBI-GenBank using the neighbor-joining algorithm and the Kimura 2-parameter model ofnucleotide substitution Numbers in parentheses correspond to GenBank accessions Numbers on branches indicate the bootstrap supportafter 1000 pseudoreplicates The scale bar denotes the number of substitutions per site

groups of the side chain Based on published data [46]compound 2 was identified as Urauchimycin B an isomer ofcompound 1

Urauchimycins belong to the antimycin class a groupof well-known antifungals Antimycins act by inhibiting theelectron flow in the mitochondrial respiratory chain [47]Antimycins have been previously identified in Streptomycesisolated from the integument of attine ants [32ndash34] Schoe-nian and colleagues [32] detected the well-know antimycinsA1ndashA4 in 50 of the actinobacteria identified as Streptomycesisolated from workers of several Acromyrmex species Thesedata along with the rare antimycins identified in the presentstudy indicate that this chemical class is often producedby Streptomyces associated with attine ants Compoundsbelonging to this class may have an important role in theattine ant-microbe association

Another antifungal compound widely distributed inStreptomyces associated with attine ants is candicidin[31ndash34] which was not detected in Streptomyces sp TD025 Itis possible that candicidin was lost in one of the purificationsteps of the AcOEt extract or it is not produced by this strain

Urauchimycins A and B were previously isolated fromStreptomyces sp Ni-80 isolated from a marine sponge inUrauchicove Irimore Japan These substances were thefirst antimycins having an odd number of carbons and abranching side chain [46] Imamura et al [46] suggested thatsuch structures are the result of an evolution of actinobacteriain the marine environment which could have resulted in achange in their secondary metabolism

In 2006 two new urauchimycins were described urau-chimycinC isolated from Streptomyces sp B1751 frommarinesediment and urauchimycin D isolated from Streptomycessp AdM21 from soil [48] In the study by Imamura andcoworkers [46] the urauchimycins A and B inhibited themorphological differentiation of C albicans up to a con-centration of 10 120583g sdotmLminus1 Urauchimycins C and D showedno inhibitory activity against C albicans Mucor miehei andbacteria [48]

The study of antimicrobial activity of urauchimycins Aand B was restricted to C albicans in the work by Imamuraand colleagues [46] The reisolation of these molecules in thepresent study allowed a better evaluation of its spectrum of


Table 2 Minimum inhibitory concentrations (120583gsdotmLminus1) of actinobacteria extracts towards different medically important Candida species

Isolate ID C albicans C dubliniensis C glabrata C krusei C parapsilosis C tropicalisCBS 562 CBS 7987 CBS 138 CBS 573 CBS 604 CBS 94

TD016 lowast lowast lowast lowast lowast lowast



TD019 60 900 lowast 40 80 1000TD020 lowast lowast lowast lowast lowast lowast


TD022 800 lowast 1000 lowast lowast lowast


TD025 40 700 lowast 15 125 1000TD027 60 1000 lowast 15 200 lowast



TD032 1000 lowast lowast lowast lowast

TD033 10 800 lowast 125 200 lowast











lowastMinimum inhibitory concentration gt 1000120583gsdotmLminus1

NHH

O

HN

OO

O

O

OOH

OH

1

NHH

OHN

OO

O

O

O

2

OHOH

Figure 2 Chemical structures of compounds isolated from Streptomyces sp TD025 (1) urauchimycin A (2) urauchimycin B

activityTheurauchimycins from Streptomyces sp TD025 pre-sentedMIC values equivalent to the reference antifungal nys-tatin for C albicans and C glabrata (Table 3) UrauchimycinB showed inhibitory activity against all Candida strains eval-uated showing MIC similar to those provided by nystatin

Urauchimycin B showed a broad spectrum of activityagainst Candida spp with MIC values equivalent to theantifungal nystatin which indicates the potential for medicaluse For many years antimycins were used for the treatmentof human infections but due to its mechanism of actionand associated side effects its use in human treatment wasdiscontinued [47] However with the pressing need for new

antifungal agents that complement or substitute for the scarceproducts available on the market it is interesting and nec-essary to determine the toxicity presented by urauchimycinB to assess whether it can be used as an antifungal agent forhumans and animals In addition evaluation of the isolatedcompound againstCandida species resistant to commerciallyavailable antifungal agents should be performed to confirmthe potential of this relatively unexplored antifungal

Here we show that Trachymyrmex ants one attine genusunderstudied with respect to its microbial symbionts har-bor antimicrobial-producing actinobacteria As observed byother authors [28ndash33] the present study demonstrates that


Table 3 Minimum inhibitory concentration (MIC) and minimumfungicide concentrations (MFC) (120583gsdotmLminus1) of Urauchimycins A andB obtained from Streptomyces sp TD025 in comparison with theantifungal Nystatin

Candida species Urauchimycin A Urauchimycin B NystatinMIC MFC MIC MFC MIC MFC

C albicans 1 lowast 1 3 1 2C dubliniensis 800 lowast 2 3 1 2C glabrata 2 156 2 2 1 1C krusei 156 156 2 3 2 3C parapsilosis lowast lowast 2 2 1 2C tropicalis lowast lowast 2 2 4 4lowast

gt1000120583gsdotmLminus1

actinobacteria of attine ants are able to produce antifungalcompounds active against other fungal species and not onlyagainst the specific fungal parasite Escovopsis

Moreover our study corroborates previous work [35] thatsuggests the attine ant-microbe association is a promisingsource of microorganisms that produce active metabolitesThe few recent studies that focused on the chemical charac-terization of bioactive compounds produced by actinobacte-ria associated with attine ants support the potential isolationof novel molecules with biological activity [28ndash32] Thusan exploration program of isolation of bioactive moleculesfrom actinobacteria from attine ants certainly will resultin the discovery of novel compounds with activity againstmicroorganisms that are potentially pathogenic to humans

4 Conclusion

As suggested by Poulsen [35] we found that the integumentof Trachymrymex ants is a potential source for the isolationof actinobacteria that produce bioactivemoleculesThe isola-tion of Urauchimycins A and B enabled for the first time theevaluation of their activity against various Candida speciesUrauchimycin B showed a broad spectrum of activity andMIC values equivalent to the reference antifungal nystatinToxicity studies and in vivo activity should be carried outin order to verify the potential use of this molecule in thetreatment of fungal infections

Acknowledgments

The authors thank FAPESP (Fundacao de Amparo a Pesquisado Estado de Sao Paulo) CNPqINCT (ConselhoNacional deDesenvolvimento Cientıfico e Tecnologico) and NSF IRFP(United States National Science Foundation InternationalResearch Fellowship Program no 07012333) for financialsupport The authors also thank CAPES (Coordenacao deAperfeicoamento de Pessoal de Ensino Superior) for provid-ing a scholarship to the first authorThis work was conductedunder collecting permit number 14789-1 issued by the ldquoInsti-tuto Brasileiro do Meio Ambiente e dos Recursos NaturaisRenovaveisrdquo (IBAMA) and the ldquoInstituto Chico Mendes deConservacao da Biodiversidaderdquo (ICMBio) The authors also

thank two anonymous referees for helpful comments on thispaper

References

[1] J Travis ldquoReviving the antibiotic miraclerdquoNature vol 264 no5157 pp 360ndash362 1994

[2] S Miyadoh ldquoResearch on antibiotic screening in Japan overthe last decade a producing microorganism approachrdquo Actino-mycetologica vol 7 no 2 pp 100ndash106 1993

[3] G L Challis and D A Hopwood ldquoSynergy and contingencyas driving forces for the evolution of multiple secondarymetabolite production by Streptomyces speciesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 24 pp 14555ndash14561 2003

[4] M Goodfellow and H P Fiedler ldquoA guide to successful bio-prospecting informed by actinobacterial systematicsrdquo Antonievan Leeuwenhoek vol 98 no 2 pp 119ndash142 2010

[5] A C W Waugh and P F Long ldquoProspects for generating newantibioticsrdquo Science Progress vol 85 no 1 pp 73ndash88 2002

[6] J Clardy M A Fischbach and C T Walsh ldquoNew antibioticsfrom bacterial natural productsrdquo Nature Biotechnology vol 24no 12 pp 1541ndash1550 2006

[7] T K Kim A K Hewavitharana P N Shaw and J A FuerstldquoDiscovery of a new source of rifamycin antibiotics in marinesponge actinobacteria by phylogenetic predictionrdquo Applied andEnvironmental Microbiology vol 72 no 3 pp 2118ndash2125 2006

[8] R Gandhimathi M Arunkumar J Selvin et al ldquoAntimicrobialpotential of sponge associated marine actinomycetesrdquo Journalde Mycologie Medicale vol 18 no 1 pp 16ndash22 2008

[9] E J Choi H C Kwon J Ham and H O Yang ldquo6-Hydrox-ymethyl-1-phenazine-carboxamide and 16-phenazinedim-ethanol from a marine bacterium Brevibacterium sp KMD003 associated with marine purple vase spongerdquo Journal ofAntibiotics vol 62 no 11 pp 621ndash624 2009

[10] B Bieber J Nuske M Ritzau and U Grafe ldquoAlnumycin anew naphthoquinone antibiotic produced by an endophyticStreptomyces sprdquo Journal of Antibiotics vol 51 no 3 pp 381ndash382 1998

[11] U F Castillo G A Strobel E J Ford et al ldquoMunumbicinswide-spectrum antibiotics produced by Streptomyces NRRL30562 endophytic on Kennedia nigriscansrdquo Microbiology vol148 no 9 pp 2675ndash2685 2002

[12] D Ezra U F Castillo G A Strobel et al ldquoCoronamycinspeptide antibiotics produced by a verticillate Streptomyces sp(MSU-2110) endophytic onMonstera sprdquoMicrobiology vol 150no 4 pp 785ndash793 2004

[13] C R Currie J A Scottt R C Summerbell and D MallochldquoFungus-growing ants use antibiotic-producing bacteria tocontrol garden parasitesrdquoNature vol 398 no 6729 pp 701ndash7041999

[14] C R Currie J A Scott R C Summerbell and D MallochldquoCorrigendum fungus-growing ants use antibiotic-producingbacteria to control garden parasitesrdquo Nature vol 423 no 6938p 461 2003

[15] M J Cafaro M Poulsen A E F Little et al ldquoSpecificity inthe symbiotic association between fungus-growing ants andprotective Pseudonocardia bacteriardquo Proceedings of the RoyalSociety B vol 278 no 1713 pp 1814ndash1822 2011


[16] M J Cafaro and C R Currie ldquoPhylogenetic analysis of mutual-istic filamentous bacteria associated with fungus-growing antsrdquoCanadian Journal of Microbiology vol 51 no 6 pp 441ndash4462005

[17] C Kost T Lakatos I BottcherW R ArendholzM Redenbachand R Wirth ldquoNon-specific association between filamentousbacteria and fungus-growing antsrdquo Naturwissenschaften vol94 no 10 pp 821ndash828 2007

[18] U G Mueller D Dash C Rabeling and A RodriguesldquoCoevolution between attine ants and actinomycete bacteria areevaluationrdquo Evolution vol 62 no 11 pp 2894ndash2912 2008

[19] U G Mueller ldquoSymbiont recruitment versus ant-symbiont co-evolution in the attine ant-microbe symbiosisrdquoCurrent Opinionin Microbiology vol 15 no 3 pp 269ndash277 2012

[20] M Poulsen M Cafaro J J Boomsma and C R CurrieldquoSpecificity of themutualistic association between actinomycetebacteria and two sympatric species of Acromyrmex leaf-cuttingantsrdquoMolecular Ecology vol 14 no 11 pp 3597ndash3604 2005

[21] T D Zucchi A S Guidolin and F L Consoli ldquoIsolationand characterization of actinobacteria ectosymbionts fromAcromyrmex subterraneus brunneus (Hymenoptera Formici-dae)rdquoMicrobiological Research vol 166 no 1 pp 68ndash76 2010

[22] R Sen H D Ishak D Estrada S E Dowd E Hong and U GMueller ldquoGeneralized antifungal activity and 454-screening ofPseudonocardia and Amycolatopsis bacteria in nests of fungus-growing antsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 106 no 42 pp 17805ndash178102009

[23] M Kaltenpoth W Gottler G Herzner and E Strohm ldquoSym-biotic bacteria protect wasp larvae from fungal infestationrdquoCurrent Biology vol 15 no 5 pp 475ndash479 2005

[24] M Kaltenpoth W Goettler C Dale et al ldquoCandidatusStreptomyces philanthi an endosymbiotic streptomycete in theantennae of Philanthus digger waspsrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 6 pp1403ndash1411 2006

[25] J J Scott D C Oh M C Yuceer K D Klepzig J Clardy andC R Currie ldquoBacterial protection of beetle-fungusmutualismrdquoScience vol 322 no 5898 p 63 2008

[26] R V vander Meer ldquoAnt interactions with soil organisms andassociated semiochemicalsrdquo Journal of Chemical Ecology vol38 no 6 pp 728ndash745 2012

[27] C R Currie ldquoPrevalence and impact of a virulent parasite on atripartitemutualismrdquoOecologia vol 128 no 1 pp 99ndash106 2001

[28] D C Oh M Poulsen C R Currie et al ldquoDentigerumycin abacterial mediator of an ant-fungus symbiosisrdquo Nature Chemi-cal Biology vol 5 no 6 pp 391ndash393 2009

[29] J Barke R F Seipke S Gruschow et al ldquoA mixed communityof actinomycetes produce multiple antibiotics for the fungusfarming ant Acromyrmex octospinosusrdquo BMC Biology vol 8article 109 2010

[30] G Carr E R Derbyshire E Caldera et al ldquoAntibiotic andantimalarial quinones from fungus-growing ant-associatedPseudonocardia sprdquo Journal of Natural Products vol 75 no 10pp 1806ndash1809 2012

[31] S Haeder R Wirth H Herz and D Spiteller ldquoCandicidin-producing Streptomyces support leaf-cutting ants to protecttheir fungus garden against the pathogenic fungus EscovopsisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 12 pp 4742ndash4746 2009

[32] I I Schoenian M M Spiteller M J Manoj et al ldquoChemicalbasis of the synergism and antagonism in microbial communi-ties in the nests of leaf-cutting antsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no5 pp 1955ndash1960 2011

[33] R F Seipke J Barke C Brearley et al ldquoA single Streptomycessymbiont makes multiple antifungals to support the fungusfarming ant Acromyrmex octospinosusrdquo PLoS ONE vol 6 no8 Article ID e22028 8 pages 2011

[34] F R Seipke S Gruschow R J Goss and M I Hutchings ldquoIso-lating antifungals from fungus-growing ant symbionts using agenome-guided chemistry approachrdquo Methods in Enzymologyvol 517 pp 47ndash70 2012

[35] M Poulsen ldquoBiomedical exploitation of the fungus-growing antsymbiosisrdquo Drug News and Perspectives vol 23 no 3 pp 203ndash210 2010

[36] E Kuster and S TWilliams ldquoSelection of media for isolation ofstreptomycetesrdquo Nature vol 202 no 4935 pp 928ndash929 1964

[37] J P Sampaio M Gadanho S Santos et al ldquoPolyphasictaxonomy of the basidiomycetous yeast genus Rhodosporid-ium Rhodosporidium kratochvilovae and related anamorphicspeciesrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 2 pp 687ndash697 2001

[38] D J Lane ldquo16S23S RNAr sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematic E Stackebrandt andM Goodfel-low Eds pp 115ndash175 John Willey New York NY USA 1991

[39] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9597NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[40] D L Swofford PAUPlowast Phylogenetic Analysis Using Parsimony(lowast and other Methods) Version 4 Sinauer Associates Sunder-land Mass USA 2002

[41] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[42] S M Bomfim Isolamento de metabolitos antifungicos de Strep-tomyces sp UFPEDA 3347 endofito deMomordica charantia L(Curcubitaceae) [MS thesis] Universidade Federal de Pernam-buco Recife Brazil 2008

[43] CLSI (Clinical and Laboratory Standards Institute) ldquoNormaAprovada M27-A2rdquo Metodo de referencia para testes dediluicao em caldo para determinacao da sensibilidade deleveduras a terapia antifungica 2002

[44] C N Seong J H Choi and K S Baik ldquoAn improved selectiveisolation of rare Actinomycetes from forest soilrdquo Journal ofMicrobiology vol 39 no 1 pp 17ndash23 2001

[45] M GWatve R TickooMM Jog and B D Bhole ldquoHowmanyantibiotics are produced by the genus StreptomycesrdquoArchives ofMicrobiology vol 176 no 5 pp 386ndash390 2001

[46] N Imamura M Nishijima K Adachi and H Sano ldquoNovelantimycin antibiotics urauchimycins A and B produced bymarine actinomyceterdquo Journal of Antibiotics vol 46 no 2 pp241ndash246 1993

[47] C J Barrow J J Oleynek H H Sun et al ldquoAntimycinsinhibitors of ATP-citrate lyase from a Streptomyces sprdquo Journalof Antibiotics vol 50 no 9 pp 729ndash733 1997

[48] C B F Yao M Schiebel E Helmke H Anke and H LaatschldquoPrefluostatin and new urauchimycin derivatives produced byStreptomycete isolatesrdquo Zeitschrift fur Naturforschung B vol 61no 3 pp 320ndash325 2006


marine environments tropical rain forests and microbialsymbionts associated with plants and animals hosts [5 6]For example the occurrence of actinobacteria associatedwithmarine sponges and the fact that such strains produce com-poundswith antimicrobial activity confirms this potential [7ndash9] In addition endophytic actinobacteria are also exploredfor their capacity to produce antimicrobial compounds [10ndash12]

Several studies have focused on the association betweenactinobacteria and insects from an ecological perspective[13ndash25] On the other hand few studies have focused onthe multitude of chemical compounds that are involvedin such interactions [26] The best studied example is thesymbiosis between actinobacteria and fungus-growing ants(Hymenoptera Formicidae tribe Attini) In this associationthe actinobacteria are found on the antsrsquo integument andproduce antimicrobial compounds that help the ants tosuppress the microfungus Escovopsis sp [13 14] This fungusis considered a specialized parasite of the ant cultivar andcauses negative impacts to the ant colony [27]

Actinobacteria isolated from the integument of attineants are generally classified in the genus Pseudonocardia andStreptomyces Bioactive molecules have already been isolatedand characterized from actinobacteria isolated from severalattine genera [26]Pseudonocardia isolated fromAcromyrmexoctospinosus and Apterostigma dentigerum are known to pro-duce several compounds like (i) dentigerumycin a complexcompound active against Candida albicans and Escovopsis[28] (ii) a nystatin-like antifungal [29] (iii) the novel quinonepseudonocardones AndashC active against the malaria causalagentPlasmodiumberghei [30] (iv) the already known antibi-otics 6-deoxy-8-O-methylrabelomycin and X-14 881 bothactive against Bacillus subtilis and P berghei [30] In additionto Pseudonocardia actinobacteria in the genus Streptomycesare also found on the integument of Acromyrmex workersand were shown to produce (i) candicidin active againstEscovopsis sp [29 31ndash34] (ii) antimycins active againstEscovopsis sp [32ndash34] and (iii) actinomycin D actinomycinX2 and valinomycin that are active against B subtilis [32]

Poulsen [35] suggested that the attine ant-microbe asso-ciation is little explored regarding the search for newantimicrobials The author highlights the various symbioticassociations between attine ants and microorganisms as apromising source for drug discovery especially those withantimicrobial activity Here we explored the antimicrobialpotential of actinobacteria isolated from the integument ofTrachymyrmex fungus-growing ants and demonstrate theaction against differentmedically importantCandida speciesWe also report two previously described urauchimycins froma Streptomyces strain and emphasize the newly discoveredanti-Candida activity of these compounds

2 Material and Methods

21 Actinobacteria Isolation and Identification TwelveTrachymyrmex colonies were collected in different Brazilianbiomes (see Table S1 in Supplementary Material availableonline at httpdxdoiorg1011552013835081) Colonies

were carefully excavated in order to reach the first fungusgarden chamber Fungus garden with the tending workersand brood was sampled using an alcohol-flamed spoon andstored in sterile plastic containers All containers were keptin a cooler during transport to the laboratory where theywere maintained at 25∘C

From each colony we randomly selected four workersfor actinobacteria isolation Then the propleural plateswere scraped with a sterile needle under a low powerstereomicroscope All ants used in the present study had avisible whitish covering on the propleural plates Scrapingswere plated on SCN agar (in g sdot Lminus1 100 starch 03 casein20 KNO

3 20NaCl 20 K

2HPO4 005MgSO

4sdot 7H2O

002 CaCO3 001 FeSO

4sdot 7H2O and 180 agar supplemented

with 005 Nystatin) [36] After scraping the entire bodyof all workers was inoculated on SCN agar All plateswere incubated at 25∘C for 30 days From each sampledTrachymyrmex colony one representative strain wasselected from each morphotype obtained The strains weresubcultured in YMA (in g sdot Lminus1 30 yeast extract 30 maltextract 50 peptone 100 glucose 180 agar) and stored atminus80∘C in 15 glycerol

We used a molecular approach to provide taxonomicaffiliation to actinobacteria strains Genomic DNA wasextracted following the method of Sampaio et al [37] Wecarried out 16S rDNA PCR with the universal primers27F (51015840-AGAGTTTGATCATGGCTCAG-31015840) and 1492R (51015840-TACGGTTACCTTGTTACGACTT-31015840) [38] Reactions wereconducted in a final volume of 25 120583L and contained 1 120583Lof diluted DNA template (1 10) 20120583L of each primer(10mM) 25 120583L of 10X buffer 10 120583L of MgCl

2(50mM)

40 120583L of dNTPs (125mM each) 02 120583L of Taq polymerase(5U120583L) and 123 120583L of ultrapure water Amplicons werecleaned up with GFX PCR DNA and Gel Band Purifica-tion Kit (GE Healthcare) Forward and reverse sequenceswere generated using the same primers along with aninternal primer U519F (51015840-CAGCMGCCGCGGTAATWC-31015840) Sequences were generated using BigDye Terminator v31Cycle Sequencing Kit (Life Technologies) in an ABI 3130sequencer andmanually edited inBioEdit v 713 [39] Contigswere compared with those available in the databases NCBI-GenBank (httpblastncbinlmnihgov) and RibossomalDatabase Project (RDP httprdpcmemsuedu) Sequencesgenerated in the present study were deposited in NCBI-GenBank (accessions KC480554-KC480557)

A phylogenetic analysis was carried out in order to deter-mine the taxonomic affiliation of strain TD025 Sequencesof closest relatives were retrieved from the NCBI-GenBankand the RDP Project and aligned in ClustalW Phylogeneticreconstruction was performed using the neighbor-joiningalgorithm implemented in PAUP v 40 [40] Genetic dis-tances were calculated using the Kimura 2-parameter modelof nucleotide substitution [41] Robustness of the relation-ships was estimated from 1000 bootstrap pseudoreplicates

22 Organic Extracts and Antifungal Assays All actinobac-teria were grown in Erlenmeyer flasks (250mL) containing50mL of modified MPE medium (in g sdot Lminus1 50 soy flour







3as solvent and






















100

73

65

74

98

100

100

86

58

58

68

71

0001














TD025 (KC480562)


































NHH

O

HN

OO

O

O

OOH

OH

1

NHH

OHN

OO

O

O

O

2

OHOH













4 Conclusion


Acknowledgments



References
























































3as solvent and






















100

73

65

74

98

100

100

86

58

58

68

71

0001














TD025 (KC480562)


































NHH

O

HN

OO

O

O

OOH

OH

1

NHH

OHN

OO

O

O

O

2

OHOH













4 Conclusion


Acknowledgments



References































































100

73

65

74

98

100

100

86

58

58

68

71

0001














TD025 (KC480562)


































NHH

O

HN

OO

O

O

OOH

OH

1

NHH

OHN

OO

O

O

O

2

OHOH













4 Conclusion


Acknowledgments



References












































































NHH

O

HN

OO

O

O

OOH

OH

1

NHH

OHN

OO

O

O

O

2

OHOH













4 Conclusion


Acknowledgments



References

























































4 Conclusion


Acknowledgments



References


















































Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants

Documents

Transcript of Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants