research article a novel insight into dehydroleucodine...

Research ArticleA Novel Insight into Dehydroleucodine Mediated Attenuation ofPseudomonas aeruginosa Virulence Mechanism

S Mustafi1 M L Veisaga2 L A Loacutepez3 and M A Barbieri1245

1Department of Biological Sciences Florida International University Miami FL 33199 USA2Biomolecular Sciences Institute Florida International University Miami FL 33199 USA3Laboratory of Cytoskeleton and Cell Cycle Institute of Histology and Embryology Faculty of MedicineNational University of Cuyo 5500 Mendoza Argentina4Fairchild Tropical Botanic Garden 10901 Old Cutler Road Coral Gables FL 33156 USA5International Center of Tropical Botany Florida International University Miami FL 33199 USA

Correspondence should be addressed to M A Barbieri barbierifiuedu

Received 27 June 2015 Revised 9 September 2015 Accepted 30 September 2015

Academic Editor Paul M Tulkens

Copyright copy 2015 S Mustafi et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increasing resistance of Pseudomonas aeruginosa (P aeruginosa) to conventional treatments demands the search for noveltherapeutic strategies In this study the antimicrobial activity of dehydroleucodine (DhL) a sesquiterpene lactone obtainedfrom Artemisia (A) douglasiana was screened against several pathogenic virulence effectors of P aeruginosa In vitro minimuminhibitory concentration of DhL was determined against P aeruginosa strains PAO1 PA103 PA14 and multidrug resistant clinicalstrain CDN118 Results showed that DhL was active against each strain where PAO1 and PA103 showed higher susceptibility (MIC048mgmL) as compared to PA14 (MIC 096mgmL) and CDN118 (MIC 098mgmL) Also when PAO1 strain was grown inthe presence of DhL (MIC

50 012mgmL) a delay in the generation time was noticed along with significant inhibition of secretory

protease and elastase activities interruption in biofilm attachment phase in a stationary culture and a significant decline in Type IIIeffector ExoS AtMIC

50 DhL treatment increased the sensitivity ofP aeruginosa towards potent antibiotics Furthermore treatment

of P aeruginosa with DhL prevented toxin-induced apoptosis in macrophages These observations suggest that DhL activity wasat the bacterial transcriptional level Hence antimicrobial activity of DhL may serve as leads in the development of new anti-Pseudomonas pharmaceuticals

1 Introduction

Pseudomonas aeruginosa (P aeruginosa) is a Gram-negativeopportunistic pathogen with a high prominence of intrinsicantibiotic resistance [1] Resistant strains of P aeruginosaare commonly found as a secondary infection in immune-compromised patients with cystic fibrosis COPD AIDSand cancer and even among diabetics [2ndash4] leaving seri-ous blood stream infection with significant mortality andhealthcare costs [5] The success in establishing P aeruginosapathogenicity is largely due to formation of intractablebiofilm and secretion of myriads of virulent factors includingLasA protease LasB elastase pyocyanin pyoverdin Type IIIsecretion (T3S) effectors and alginate [6ndash9] Unfortunatelyselection of the most appropriate antibiotic is complicated

due to the ability of P aeruginosa to develop resistance tomultiple classes of antibiotics

P aeruginosa can develop resistance to antibiotics becauseof the low permeability of its outer membrane the con-stitutive expression of various efflux pumps [10] and thenaturally occurring chromosomal AmpC 120573-lactamase turn-ing it resistant toward penicillin G aminopenicillins andfirst- and second-generation cephalosporin [11] P aeruginosaeasily acquires additional resistancemechanismswhich leadsto serious therapeutic problems [12] Currently antipseu-domonas treatments include higher-than-usual doses of 120573-lactam fluoroquinolones and amino glycosides which pos-sess a high degree of toxicity and very low eradication rate[13]The dearth of successful antibiotics to completely controlP aeruginosa infectionmakes it crucial to finding alternatives

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 216097 11 pageshttpdxdoiorg1011552015216097

2 BioMed Research International

to currently available drugs Since the pathogenicity of Paeruginosa is regulated by several secretory-systemsmediatedcell-to-cell communications inhibition of this system cancause attenuation of virulence and protect against infection[14 15]

Artemisia the largest diverse genera of Asteraceae familypossesses medicinally valuable essential oils and secondarymetabolites [16 17] Many studies indicate antimicrobialactivity in Artemisia (A) spp [18ndash20] A douglasiana is welldocumented as preventive folk medicine in Argentina [21]Dehydroleucodine (DhL) a sesquiterpene lactone of the gua-ianolide group is one of the principal active secondary met-abolites in A douglasiana [21] DhL first isolated from Lid-beckia pectinata [22] possesses cytoprotective activity [23] aswell as antimicrobial activity against several pathogens [24ndash27]

In this study we tested the antibacterial activity of DhLand against P aeruginosa virulence At first we evaluated theminimum inhibitory concentration (MIC) and its synergisticeffect on other commercial antibiotics and thenwe examinedthe antipseudomonal properties of this phytochemical interms of arresting growth and attenuating virulent factorssuch as biofilm formation and secretion of Type III effectorselastase A and elastase B We also assessed the capacity ofthis phytocompound to inhibitP aeruginosa induced host celltoxicity

2 Material and Methods

21 Reagents Dehydroleucodine was extracted from A dou-glasiana (Voucher specimen 2012-1018-F Fairchild TropicalBotanical Garden Miami FL) as previously documented[21 28] DH-DhL was prepared as described previously [21]All chemicals and reagents were purchased from Sigma-Aldrich (St Louis MO) unless otherwise indicated Primaryand secondary antibodies used in immunoblotting werepurchased from Cell Signaling Technology Inc (DanversMA) Culture supplies were purchased from Invitrogen LifeTechnologies (Carlsbad CA) Prototypic P aeruginosa strainsused in this study are PAO1 clinical isolates PA103 and PA14(kindly provided by Dr Dara Frank University ofWisconsinMadison) and P aeruginosa strain PA103ΔUΔT expressingpUCP plasmid-encoded ExoS tagged with a hemagglutininepitope (ExoS-HA) (kindly provided by Dr Joan C OlsonWest Virginia University) In addition Staphylococcus aureus(ATCC 12600) was used in the LasA assay Cells weremaintained in Luria Broth (LB) (Life Technologies CarlsbadCA) or Agrobacterium (AB) minimum medium (Bio-WorldVisalia CA) to which glucose and casein amino acids [20wtvol] were added

22 Determination of Minimum Inhibitory Concentration(MIC) andMinimumBactericidal Concentration (MBC) Theminimum inhibitory concentration of DhL was determinedby micro dilution broth assay [12] for PA01 PA103 PA14 andCDN118 strains of P aeruginosa Serial doubling dilution ofDhL was made ranging from 224mgmL to 0224120583gmLTwenty (20) 120583L of standard inoculums (05 McFarland) of all

three P aeruginosa strains was introduced to desired volumeof growth medium and incubated at 37∘C for 18 hr Controlswere set up as follows (1) sterility control LB broth only (2)growth control (negative) LB broth + bacteria (3) positivecontrol LB broth with Gentamycin (4) vehicle control LBbroth + bacteria + DMSO MIC was interpreted as the leastconcentration with no observable turbidity Optical densityreadings (120582 = 600 nm) were taken using microplate readerat 0 and 18 hr MIC

90and MIC

50were determined as the

concentration of DhL that inhibited bacterial growth by 90and 50 respectively Results were reported as the MICMIC50 and MIC

90for growth at 18 hr after inoculation

Minimum bactericidal concentration (MBC) wasrecorded as a lowest extract concentration killing 999of thebacterial inocula after 24 h incubation at 37∘C Each experi-ment was repeated at least three times MBC values weredetermined by removing 100 120583L of bacterial suspension fromsubculture demonstrating no visible growth and inoculatingnutrient agar plates Plates were incubated at 37∘C for a totalperiod of 24 h The MBC is determined with the wells whosethe concentrations are ge MIC [29] According to the ratioMBCMIC we appreciated antibacterial activity If the ratioMBCMIC is 1 or 2 the effect was considered bactericidalbut if the ratio MBCMIC is 4 or 16 the effect was defined asbacteriostatic [30]

For each assay 012mgmL (MIC50) of DhL was added

to P aeruginosa culture at early log phase unless otherwisestated The effect of DhL on bacterial cell proliferation wasdetermined by monitoring the growth curve of P aeruginosastrain PAO1 Briefly an overnight culture (in LB medium) ofPAO1was diluted toOD

600005 in LBmedium (control) or LB

and DhL (012mgmL) and incubated at 37∘C while shakingThe OD

600was monitored at 30min intervals until OD

600

of approximately 17 was obtained (approximately 8 hr) AllOD600

measurements were verified at a 110 dilution forgreater accuracy

23 Determination of Antibiotic Synergy with DhL by theE-Test Strip Bacterial suspensions (PAO1) homogenized insterile saline were prepared from overnight fresh culturesto a McFarland standard of 05 and were spread with asterile cotton swab on 150mm Mueller-Hinton agar controland containing DhL (MIC

50) plates The agar plates were

allowed to stand for 15ndash20min at room temperature toallow any excess surface moisture to be absorbed beforeplacement of MIC test strips Antibiotic gradient strips (E-test Liofilchem Italy) containingGentamycin (concentrationrange 0016ndash256mgL) Ciprofloxacin (concentration range0002ndash32mgL) or Chloramphenicol (concentration range0016ndash256mgL) were then used the MHA plates with theover layered strips were incubated at 37∘C or 24 hr and thegrowth inhibition zones were measured Differences in MICvalues between the control and test plates were recorded(end points were determined according to themanufacturerrsquosinstructions)

The Muller Hinton agar (MHA) dilution method wasused to evaluate the Fractional Inhibitory ConcentrationIndex (FICI) of DhL with Chloramphenicol on PAO1 being

BioMed Research International 3

tested following previously established protocol [31 32]Eight serial twofold dilutions of DhL were prepared asdescribed before to obtain final concentration range of224mgmLndash0224120583gmL A series of twofold serial dilutionsof Chloramphenicol was also prepared similar to DhLAll antibacterial standards dilutions were mixed with theappropriate concentration of DhL compounds thus obtaininga series of the combinations of Chloramphenicol and DhLThe concentrations prepared corresponded to 1 12 14 18116 132 164 1128 and 1256 of MIC values The 96-wellplate containing 100 120583L of Mueller Hinton (MH) broth wasused For bacteria strain PAO1 three columns of eight wellsof 96-well plate were used Each well received the culturemedium + combination of DhL with Chloramphenicol +inoculum (10120583L of inocula) and INT (sigma-Aldrich StLouis MO) (50120583L 02mgmL) The plates were coveredand incubated at 37∘C for 24 hr All tests were performedin triplicate and the bacterial activity was expressed as themean of inhibitions produced Inhibition of bacterial growthwas judged by rose or yellow color The analysis of thecombination of DhL and Chloramphenicol was obtained bycalculating the Fractional Inhibitory Concentration Index(FICI) as follows FICI = (MICof the combination ofDhL andChloramphenicolMICa alone) + (MIC of the combinationof DhL and ChloramphenicolMICb alone) where MICais Minimal Inhibitory Concentration of DhL and MICb isMinimal Inhibitory Concentration Chloramphenicol TheFICI was interpreted as follows (1) a synergistic effect whenFICI le 05 (2) an additive or indifferent effect when FICI gt05 and lt1 and (3) an antagonistic effect when FICI gt 1

24 LasA and LasB Activities Staphylolytic activity of LasAwas determined spectrophotometrically by measuring thedecrease in absorbance at 595 nm (OD

595) due to lysis

of heat inactivated Staphylococcus aureus (S aureus) cells(03mgmL 002MTris-HCl pH 85) upon addition of PAO1culture supernatants [34] One hundred (100)120583L aliquot ofP aeruginosa culture supernatant with or without DhL (afternormalizing the supernatant total protein concentration)was added to 900 120583L of a boiled S aureus suspension TheOD595

was determined after 0 5 10 20 30 45 and 60minrespectively Activity was expressed as the change in theOD595

hr120583g protein One unit of activity was defined as theamount of enzyme that causesOD

595decrease of 1 absorbance

unitminElastolytic activity of LasB in PAO1 culture fluids was

determined by elastin Congo red (ECR) assay [35] One hun-dred (100) 120583L aliquot of the ABmedium culture supernatantsfrommid log phase with or without DhL treatment (concen-tration was normalized) was added to 900 120583L of ECR buffer(100mM Tris-HCl 1mM CaCl

2 pH 75) containing 20mg

ECR Tubes were incubated for 18 hr at 37∘C with rotationand then were placed on ice after 01mL of 012M EDTA wasadded Insoluble ECRwas removed by centrifugation and theOD495

was measured Absorption due to pigments producedby P aeruginosawas corrected for by subtracting theOD

495of

each sample that had been incubated in the absence of ECR

Cell-free AB medium alone and AB medium with DhL wereused as negative controls

25 Relative Gene Expression Analysis of LasR Total mRNAwas purified from mid log phase PAO1 (108 cellsmL)untreated or after DhL treatment using QIAGEN RNeasymini kit (QIAGEN Valencia CA) and quantified followingstandard techniques cDNA was synthesized from 1 120583g ofmRNA using iScript Supermix (Biorad Hercules CA) Realtime PCRwas performed in triplicate from each sample usinga BioradCFx96Real Time System (BioradHercules CA) andSsoAdvanced SYBR Green Supermix (Biorad Hercules CA)with the primers for LasR (51015840-GAAATGTGCCTTTCCGGC-ACAAC-31015840 and 51015840-AGGCCATAGCGCTACGTTCTTCTT-31015840) and gyrB (DNA-gyrase subunit B 51015840-TGCTGAAGG-GGCTGGATGCCGTACGCAAGC-31015840 and 51015840-TATCCA-CCGGAATACCGCGTCCATTGTCGC-31015840) Relative geneexpression was determined using the 2minusΔΔCT method [36]Mean CT of triplicate measures was computed for eachsample Sample mean CT of gyrB (internal control) wassubtracted from the sample mean CT of the respective geneof interest (ΔCT) The ΔCT of the sample with no treatmentwas selected calibrator and subtracted from the mean ΔCT ofeach experimental sample (ΔΔCT) 2

minusΔΔCT yields fold changein gene expression of the gene of interest normalized to theinternal control gene expression

26 Polyvinyl Chloride Biofilm Formation The effect of DhLon the attachment phase of biofilm formation was measuredby using the polyvinyl chloride biofilm formation assay [37]Briefly overnight cultures of PAO1 were resuspended in freshABmedium in the presence and the absence DhL After 24 hrof incubation at 30∘C the biofilms in the polyvinyl chloridemicrotiter plates were visualized by staining with a crystalviolet solution Plates were rinsed once to remove planktoniccells and then attached cells to the surface were quantified bysolubilizing the dye in ethanol by measuring the absorbanceat OD

546

27 Type III Secretion Assays Low calcium fractionationprotocol was adopted for the detection of Type III secre-tion effectors [8] Briefly P aeruginosa PAO1 PA103ΔUΔT(control) and PA103ΔUΔT expressing HA-ExoS were grownovernight in LB Bacteria were then subcultured 1 1000 inLB supplemented with 5mM EGTA with or without DhLand grown for 6 h at 37∘C with aeration Bacterial densitieswere determined at OD

600 Bacteria were sedimented by

centrifugation at 3220timesg for 15min at 4∘CCulture supernatant was collected and proteins were

precipitated with 5 trichloroacetic acid and washed oncewith ice-cold acetone Proteins were resuspended accordingto culture density and separated by SDS-PAGE Proteinsderived from precipitated PAO1 supernatant were run onSDS-PAGE and then either stained with Coomassie blueor transferred onto nitrocellulose membranes as describedbelow


28 Immunoblotting Analysis Samples (treated and non-treated with DhL) were resuspended in sample buffer andthen separated by SDS-PAGE transferred onto nitrocellu-lose membranes (Biorad Laboratories Hercules CA USA)and incubated with antibodies specific to HA-epitope tagfor 2 hr at room temperature Subsequently membraneswere incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies A Super Signal West Picochemiluminescent substrate kit (PierceThermo Fisher Sci-entific Inc Rockford) was used to visualize protein bandsBand densities fromWestern blots were quantified using theImage J64 software at a gray-scale amplification of 600 dpi

29 P aeruginosa Mediated Apoptosis of Macrophage CellsThe effect of DhL treatment on P aeruginosamediated apop-tosis of J774-Eclone mouse macrophage cell was monitoredas previously described [38] J774-Eclone cell monolayer wasplated from suspension culture 1 day prior to infection inDMEM supplemented with 5 FBS (DMEM-5FBS) Cellmonolayer (5 times 105 cells per well gt80 confluence) waswashed with phosphate-buffered saline (PBS) mixed witheither DhL treated or DhL-untreated PAO1 bacteria at amultiplicity of infection (MOI) of 20 and incubated for2 hr at 37∘C in a 5 CO

2incubator The cells were washed

with PBS to remove the nonadhering bacteria Fresh DMEMcontaining 5 FBS was supplemented with 400120583gmL ofGentamycin and then added to cells for an additional 2 hrto kill any unwashed P aeruginosa As positive control forthe apoptosis J774-Eclone cells were incubated with 01 120583Mof Staurosporine (Fischer Scientific Waltham MA) for 1 hrCells were washed once with PBS and stained with 1mgmLHoechst 33258 (Molecular Probes Inc Eugene OR) for10min in the dark as described in Section 2 Chromatincondensation was examined under the fluorescence micro-scope by using a DAPI (4 6-diamidino-2-phenylindole) filterafter stained cells were mounted onto slides using mountingmedium

210 Statistical Analysis All experiments were performedindependently in triplicate and each experiment was repeatedthree times Values represent the mean plusmn SEM of threeindependent experiments Data were analyzed by Mann-Whitney U test with a significance threshold value of 005by using the SPSS (Chicago IL) statistical software package

3 Results

We first examined the antibacterial effects of DhL againstseveral P aeruginosa strains (PAO1 PA103 PA14 and clinicalstrain CDN118 a multidrug resistant strain) with vary-ing concentrations of DhL ranging from 224mgmL to0224mgmL As displayed in Table 1 DhL inhibited 50of PAO1 and PA103 population at 012mgmL whereas048mgmL of DhL successfully inhibited 50 of PA14population The minimum inhibitory concentration that isthe lowest concentration of the DhL solution required tocompletely inhibit the growth of the P aeruginosa was048mgmL for PAO1 and PA103 as compared to 096mgmL

Table 1 Minimum inhibitory concentration against P aeruginosastrains was carried out previously [33]lowast It shows complete killing(MIC)lowast 50 of killing (MIC

50)lowast and 90 of killing (MIC

90)lowast

P aeruginosastrain Antimicrobial agent MIC MIC

90MIC50

(mgmL) (mgmL) (mgmL)

PAO1 DhL 048 024 012Gentamycin 0075 0064lowast 0032lowast

PA14 DhL 096 048 024Gentamycin 01 gt0064lowast gt0032lowast

PA103 DhL 048 024 012Gentamycin 0075 0064lowast 0032lowast

CDN118 DhL 098 056 028Gentamycin 01 gt0064lowast gt0032lowast

Table 2 Bacteriocidal (+) and bacteriostatic (minus) effect of DhL onseveral experimental strains of Pseudomonas aeruginosa

Pseudomonasaeruginosa strains Bacteriocidal (+) Bacteriostatic (minus)

PAO1 +PA103 +PA14 minus

Clinical strain minus

Bacteriocidal (MBCMIC = 1 or 2)Bacteriostatic (MBCMIC = 4 or 16)Results are means of number of colonies plusmn standard deviation

for the more virulent strain PA14 (Table 1) However forthe clinical strain DhL MIC was 028mgmL (Table 1) Thebactericidal and bacteriostatic effect of DhL was determinedby MBCMIC (Table 2) The antimicrobial effect of DhL isprominent against all strains of P aeruginosa tested herealthough strains PAO1 and PA103 seemed to be more suscep-tible to DhL than PA14 and clinical strain CDN118

We then determined whether DhL activity at MIC50

concentrationwas effective to slow down the bacterial growthin vitro in a time-dependent manner We found that the idealgrowth phases of PAO1 represented a log phase during thefirst 150min and then a stationary phase with OD

60017 that

remained until 360min (Figure 1) Treatment with DhL atMIC50concentration (012mgmL) declined the slope of log

phase that ended at 270min of culture which then turnedto stationary phase at a very low OD

600(ie 07) Noticeable

difference in generation time (50plusmn5)was observed between1 and 3 hr of growth (mid log phase) between treated anduntreated PAO1 (Figure 1) Treatment at MIC

90failed to

portray normal growth curve (data not shown)Several studies have suggested that combining plant- or

animal-derived natural compounds with antibiotics is a newstrategy for developing therapies against infections [39ndash41]We report that the antimicrobial activities of commercialantibiotics are enhanced by pretreating P aeruginosa with012mgmLDhLMICvalueswere decreased by the coactionsof DhL with gentamicin (aminoglycoside) Chloramphenicoland Ciprofloxacin (fluoroquinolone) Figure 2 To further


H O

O

O

(a)

PAO1

lowastlowastlowastlowast

lowast

lowast

lowast

lowastlowast

lowast

PAO1 + DhL

30 60 90 120 150 180 210 240 270 300 3300

Time (min)

0

02

04

06

08

10

12

14

OD600

(log

scal

e)

(b)

Figure 1 DhL inhibits growth of P aeruginosa (a) Chemical structure of DhL (b) P aeruginosa PAO1 strain was grown in the absence (I) andpresence (e) of 012mgmL DhL from early log phase to stationary phase (17 hr) at 37∘C with constant shaking Generation time (between1 and 3 hr) of PAO1 in presence of DhL (calculated from three independent experiments) was 64 plusmn 5min whereas it was 35 plusmn 5min in thecontrol PAO1 culture Data represent the mean of three independent experiments plusmn SEM

Treatment Gentamycin Chloramphenicol Ciprofloxacin

Control 10 10 0125DhL

038 038 0064MIC50 120 (120583gmL)

MIC (120583gmL)

(a)

10

(b)

038

(c)

Figure 2 Synergistic effect of DhL with systemic antibiotics (a) Table represents synergistic activity of subinhibitory concentration of DhLand antibiotics belonging to various classes determined by the E-test stripagar dilution method against PAO1 (b) A control plate (MHA)with Chloramphenicol E strip (c) A Chloramphenicol E-test plate (MHA) with DhL at a dilution of MIC

50concentration

confirm the combined effect of DhL and conventional antibi-otics we calculated the FICI for DhL and Chloramphenicolcombination on PAO1 (Table 3) Overall the observationsfrom both assays demonstrated significant antibacterial syn-ergy activities of DhL on P aeruginosa

Elastase A and elastase B (LasB) are effectors of TypeII secretion system in P aeruginosa and are major virulent

factors in lung infection often related to high death rate inacute pneumonia [42] The elastase properties of LasA andprotease activity of LasB play a major role in P aeruginosapathogenesis [43 44] LasB secretion is a prominent virulencefactor in cystic fibrosis lung infection [45] which is knownto degrade elastin and collagen in host [46] To investigatethe effect of DhL on LasA and LasB activities P aeruginosa


Table 3 FICa MIC of the combinationMICa alone FICb MIC of the combinationMICb alone FICI FICa + FICb FICI interpretationsynergistic effect when FICI le 05 additive effect when 1 gt FICI lt 05 and antagonistic effect when FICI gt 1

Microorganism MIC (120583gmL) Chloramphenicol FICa FICb FICI EffectPseudomonas aeruginosa PAO1 gt400 025 le003 le028 Synergistic

strain PAO1 was incubated with DhL at 012mgmL (MIC50)

as described in Section 2 PAO1 cultural supernatant showedan exponentially increasing staphylolytic effect indicatingprotease activity when compared to S aureus concentrationin LB as a control (Figure 3(a)) A significant decrease (89 plusmn3) in LasA activity (Figure 3(a)) was observed after 60minof incubation of S aureus substrate along with the DhLtreated P aeruginosa strain PAO1 Furthermore significantdecrease of 75 plusmn 5 in LasB activity was also observed whenPAO1 was grown in the presence of DhL (Figure 3(b)) Inparallel control studies DhL along with S aureus in absenceof PAO1 did not show any LasA activity as well as DhL and LBalone did not show elastase activity (data not shown) In bothexperiments PAO1 cultures grown in presence or absence ofDhL were normalized so that the total number of bacteriaremained the same

LasR is a transcription regulator of LasA and LasB gene[43 47] The mRNA expression level of LasR in PAO1 popu-lation from mid log phase reduced fivefold after DhL treat-ment (Figure 3(c)) These results indicated DhL effectivelyinhibited both LasA and LasB activity in PAO1 populationby controlling transcription regulator in live bacteria andthe absence of such virulent activities was not due to loss ofbacterial viability

P aeruginosa has the ability to form biofilms in whichcells are organizedinto layers and enmeshed in a matrixof mucoid polysaccharides [48] P aeruginosa biofilms arelinked to quorum sensing behavior [48 49] by using twodistinct acyl-homoserine lactone [3] Biofilm-grown bacte-rial cells show increased resistance to antibiotics [48] Todetermine whether DhL could prevent biofilm formationP aeruginosa strain PAO1 was incubated in the absence orin the presence of DhL and then the generation of biofilmswas quantified as described in Section 2 Results indicated asignificant decrease of 55plusmn5 in biofilm formation (Figure 4)in DhL treated PAO1 compared to the control suggesting aninhibitory effect of DhL on P aeruginosa biofilm formation

T3S system is a complex multiprotein apparatus thatfacilitates the secretion and translocation of effector proteinssuch as ExoS ExoT ExoU and ExoY from the cytoplasm ofbacterial cell to the cytoplasm of mammalian cells ActiveT3S system is significantly associated with acute infectionsextreme clinical outcomes anddeath in patients infectedwithP aeruginosa [50] ExoS and ExoT are bifunctional proteinswith Rho-GAP and ADP-ribosylation activity whereas ExoUis a potent phosphor lipase and ExoY is adenylate cyclase [9]In animal infection models mimicking P aeruginosa medi-ated acute human infections such as burn wounds acutepneumonia and corneal infection it was shown that T3S isan important virulence mechanism [51 52] To determinewhether DhL inhibits the T3S system-mediated secretion ofeffector proteins such as ExoS P aeruginosa strain PAO1

was incubated in the absence or in the presence of DhLunder T3S system-inducing conditions (see Section 2) Thesecreted effectors were analyzed on SDS-PAGE as describedin Section 2 DhL inhibited the secretion of ExoS in PAO1 byat least 95 (Figure 5 (top panel)) To further confirm thisresult presence of ExoS was screened in the secretion profileof PA103ΔUΔT HA-ExoS strain carrying HA-tagged ExoSin the absence or in the presence ofDhLThe secretion ofHA-tagged ExoS was probed with immunoblotting using specificanti-HA antibody These results indicated that DhL blockedthe secretion of HA-ExoS (Figure 5 (bottom panel))

Several Studies have suggested that T3S system effec-tors from P aeruginosa are responsible for inducing rapidapoptosis in macrophage cells and epithelial cells [49] Inan effort to support our previous observation antimicrobialactivity of DhL was tested in a cellular activity assay forT3S system effector translocation into mammalian cells [53]P aeruginosa PAO1 strain was added to J774-Eclone cellmonolayer at MOI of 20 1 in the absence or in the presenceof DhL as described in Section 2 After 2 hr of incubationat 37∘C cells were stained with Hoechst stain and apoptoticnuclei were observed under fluorescent microscope Thetotal percentage of apoptotic cell counted suggests that DhLtreated P aeruginosa PAO1 strain fails to induce apoptosissignificantly when compared to untreated PAO1 inducedapoptosis (control) Almost 87 plusmn 5 inhibition of apoptosiswas observed in PAO1 population previously treated withDhL (Figure 6) As a positive control of apoptosis we havetreated J774-Eclone cells with Staurosporine (see Section 2)Together these results demonstrate that DhL suppresses theT3S system-mediated secretion of effectors fromPAO1 as wellas the translocation of effectors into the target mammaliancells

4 Discussion

P aeruginosa represents a wide number of strains withstrong pathogenic capacity and has developed a number ofstrategies to affect and invade the host cell PAO1 strain ofP aeruginosa is the standard laboratory strain as well asgenetic reference strain [33] PA14 a ldquomultihostrdquo pathogenis capable of infecting animals (in a burned mouse model)plants insects and nematodes [54] Both strains were ableto initiate and maintain chronic infection in rat lung model[55] Exotoxin-Uproducing strain PA103 is a cytotoxic strainof P aeruginosa which is known to cause severe alveolarepithelial injury during infection [56] In this study we havedemonstrated that DhL is bacteriostatic for virulent strainsof Pseudomonas aeruginosa PA14 and CDN118 [57 58] butbacteriocidal for less virulent strains PAO1 and PA103 of Paeruginosa


PAO1

lowastlowastlowastlowastlowast

PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)

Figure 3 Effect of DhL on P aeruginosa strain PAO1 protease activities Staphylolytic LasA and elastolytic LasB activities were monitored inthe absence or in the presence of DhL (012mgmL) (a) To measure the proteolytic activity of LasA within the supernatant fraction of thePAO1 growth culture a staphylolytic assay was employed accounting the lysis of heat inactivated intact S aureus at OD

595as described in

Section 2 Proteolytic activity of PAO1 supernatant (PAO1 ◻) was significantly (asterisk indicates 119875 lt 005) inhibited when PAO1 were treatedwith DhL (PAO1 + DhL e) LB + S aureus was used as control (I) Data represent the mean of three independent experiments plusmn SEM (b)Levels of LasB elastolytic activity (within the PAO1 cultural supernatant with or without DhL treatment) were measured by Elastin Congored assay at OD

495as described in Section 2 PAO1 were grown in LB broth at 37∘C for 16 hr The cultures were adjusted to an OD

540of 35ndash

40 before harvesting to eliminate growth-related variations in elastolytic activity Significant (asterisk indicated 119875 lt 005) decrease in LasBactivity was observed in PAO1 when treated with DhL Data represent themean of three independent experimentsplusmn SEM (c) Relative mRNAexpression level of LasR gene at mid log phase from DhL treated P aeruginosa shows fivefold inhibition compared to untreated populationData represent the mean of three independent experiments plusmn SEM

When PAO1 strainwas treatedwith sub-MICdose ofDhL(MIC50

= 012mgmL) and inoculated in growth mediuma significantly reduced (50 plusmn 5) generation time wasobserved Such treatment also modified the susceptibility ofP aeruginosa toward potent antibiotics and lowered the MIC

systemic antibiotics A combination of sub-MIC levels ofDhL and antibiotics can help reduce undesirable side effectsfrom routine antibiotic dosage The FICI assay of DhL withconventional antibiotic on PAO1 strain showed significantsynergistic effect


lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay

Figure 4 Effect of DhL on P aeruginosa biofilm formation Over-night cultures of PAO1 were resuspended in fresh AB medium(control) and inABmedium supplementedwithDhL (see Section 2)in PVC tubes After 24 hr of incubation at 30∘C the biofilms on thewall of the tubes were visualized by staining with a crystal violetsolution Biofilm formation was quantified by measuring OD

546of

crystal violet-stained wells rinsed with ethanol Each column isthe mean of three individual experiments with two replicates pertreatment Significant (asterisk indicated 119875 lt 005) decrease inbiofilm formation was observed in PAO1 when treated with DhLData represent the mean of three independent experiments plusmn SEM

DhL treatment also inhibited severalvirulence factors inPAO1 at the threshold dose of 012mgmL PAO1 culturalsupernatant treated with DhL showed 89 plusmn 3 decrease inLasA activity and 75 plusmn 5 reduction in LasB activity Signifi-cantly low mRNA expression of transcription regulator LasRcorroborates with the activity status as well It is importantto note that the virulence mechanisms in P aeruginosa arehighly controlled by transcription factors and regulators It iswell documented that LasA and LasB transcription activationrequires LasR gene products [33]

DhL treated PAO1 culture failed to demonstrate success-ful initial attachment phase during the course of biofilmformation and prolonged observations revealed disruption inbiofilm formation compared to untreated PAO1 culture (datanot shown) Inhibitory effects ofDhLwere also poignant forPaeruginosa T3S system that successfully restrained secretionof important T3S effectors such as ExoS ExoT ExoY andExoUOverall the results indicated a prominent regulation ofP aeruginosa growth and virulence due to DhL treatment invitro Finally when DhL treated P aeruginosa was coculturedwith mouse macrophage cells the typical PAO1 inducedcellular apoptosis was successfully controlled reinforcing theactive inhibition of T3S system virulence which otherwiseleads to cytotoxicity

Although the exactmechanismofDhLmediated suppres-sion of P aeruginosa virulenceis still under investigation it

PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT

Figure 5 Effect of DhL on Type III secretion Top panel representsType III secretion profile of P aeruginosa strain PAO1 in presenceof DhL as observed with SDS-PAGE stained with Coomassie bluestain Type III null mutant (PA103 ΔUT) was used as a control Lane1 represents untreated PAO1 secretion profile and lane 2 representstreated PAO1 secretion profile Under Coomassie blue stain proteinbands were observed at sim53 kDa sim49 kDa and sim37 kDa indicatingExoT ExoS and ExoY respectively in PAO1 Type III secretion assaywhich were not shown in DhL treated PAO1 Type III secretion assayLanes 3 and 4 showing untreated and DhL treated control strain(PA103 ΔUΔT) with no visible Type III secretion profile Bottompanel represents immunoblot results of DhL treated PA103ΔUΔTHA-ExoS strain carrying HA-tagged ExoS as described in Section 2HA specific antibody was used in immunoblot (IB) to confirm thepresence of sim49KDa ExoS secretion Experiments were repeatedthree times to confirm the observation

is likely that DhL imparts a direct effect within the bacterialcells rather than extracellular substances and mucoid layersof biofilm Previous studies suggested that the 120572-methylene-120574-lactone moiety in sesquiterpene lactones like DhL exertsits biological activities by Michael-type additions attackingnucleophilesThemodified nucleophiles react reversibly withsulfhydryl groups in the cell such as transcription factors [5960] To confirm the functional properties of DhL we utilizeda DhL derivative which is a mixture of two epimers (11S)DH-DhL and (11R)DH-DhL [21] DH-DhL epimers which aredevoid of the highly reactive 120572-methylene-120574-lactone moietyfailed to show any significant antipseudomonalactivity atsimilar concentration to DhL (024mgmL)

Due to high mutation rate in bacterial genome P aer-uginosa is extremely difficult to eradicate from mucoidcolonies [61]Therefore alternatively antivirulence therapy toprevent P aeruginosa invasion holds strong grounds againstdevelopment of bacterial resistance [15 62] The success ofDhL against several virulence effects of P aeruginosa in thisstudy can lead to new treatment strategies and could also pavetheway to the reduction of the amount of antibiotics required


0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast

PAO1 PAO1 + DhL Stau

Figure 6 Effect of DhL on P aeruginosa induced apoptosis J774-Eclone cells were plated in monolayer (5 times 105 cells per well) onceadhered they were infected with PAO1 bacteria previously grownin LB-DhL (MIC

50 012mgmL) or only LB at a multiplicity of

infection of 20 and incubated for 2 hr at 37∘C in a 5CO2incubator

In a parallel experiment an equal number of J774-Eclone cells wassubjected to 1 120583M Staurosporine for positive control Cells werethen washed with PBS stained with Hoechst dye and subjectedto fluorescence microscopy (Section 2) Five fields were randomlysampled from each experimental population and all of the cellsstained with Hoechst dye in each field were counted up to 500in total The total number of apoptotic cells with condensed orfragmented nuclei was determined in the five sampled regionsand was expressed as follows percentage of apoptosis per samplenumber of apoptotic cellstotal number of cells times 100 Each columnis the mean of three individual experiments with two replicatesper treatment Significant (asterisk indicates 119875 lt 005) decreasein apoptotic cells was noticed when infected with PAO1 grown inpresence of DhL Data represent the mean of three independentexperiments plusmn SEM

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors acknowledge Florida International University forfinancial support They thank Dara Frank and Dr Joan COlson for reagents and P aeruginosa strains

References

[1] N Mesaros P Nordmann P Plesiat et al ldquoPseudomonasaeruginosa resistance and therapeutic options at the turn of thenew millenniumrdquo Clinical Microbiology and Infection vol 13no 6 pp 560ndash578 2007

[2] M Z R Gomes C R Machado M De Souza da Conceicaoet al ldquoOutbreaks persistence and high mortality rates of mul-tiresistant Pseudomonas aeruginosa infections in a hospital with

AIDS-predominant admissionsrdquo Brazilian Journal of InfectiousDiseases vol 15 no 4 pp 312ndash322 2011

[3] K Engler KMuhlemann C Garzoni H Pfahler T Geiser andC Von Garnier ldquoColonisation with Pseudomonas aeruginosaand antibiotic resistance patterns in COPD patientsrdquo SwissMedical Weekly vol 142 no 1 Article ID w13509 2012

[4] M Hogardt and J Heesemann ldquoMicroevolution of Pseu-domonas aeruginosa to a chronic pathogen of the cystic fibrosislungrdquo Current Topics in Microbiology and Immunology vol 358pp 91ndash118 2013

[5] HWisplinghoff H Seifert S M Tallent T Bischoff R PWen-zel and M B Edmond ldquoNosocomial bloodstream infectionsin pediatric patients in United States hospitals epidemiologyclinical features and susceptibilitiesrdquoPediatric InfectiousDiseaseJournal vol 22 no 8 pp 686ndash691 2003

[6] E Kessler M Safrin W R Abrams J Rosenbloom and D EOhman ldquoInhibitors and specificity of Pseudomonas aeruginosaLasArdquo The Journal of Biological Chemistry vol 272 no 15 pp9884ndash9889 1997

[7] K S McIver E Kessler and D E Ohman ldquoIdentification ofresidues in the Pseudomonas aeruginosa elastase propeptiderequired for chaperone and secretion activitiesrdquo Microbiologyvol 150 no 12 pp 3969ndash3977 2004

[8] V T Lee R S Smith B Tummler and S Lory ldquoActivitiesof Pseudomonas aeruginosa effectors secreted by the type IIIsecretion system in vitro and during infectionrdquo Infection andImmunity vol 73 no 3 pp 1695ndash1705 2005

[9] A R Hauser ldquoThe type III secretion system of Pseudomonasaeruginosa infection by injectionrdquo Nature Reviews Microbiol-ogy vol 7 no 9 pp 654ndash665 2009

[10] D M Livermore ldquoOf Pseudomonas porins pumps and car-bapenemsrdquo Journal of Antimicrobial Chemotherapy vol 47 no3 pp 247ndash250 2001

[11] P Nordmann and M Guibert ldquoExtended-spectrum beta-lactamases in Pseudomonas aeruginosardquo Journal of Antimicro-bial Chemotherapy vol 42 pp 128ndash131 1998

[12] S T Micek A E Lloyd D J Ritchie R M Reichley V J FraserandM H Kollef ldquoPseudomonas aeruginosa bloodstream infec-tion importance of appropriate initial antimicrobial treatmentrdquoAntimicrobial Agents andChemotherapy vol 49 no 4 pp 1306ndash1311 2005

[13] A R Hauser and P Sriram ldquoSevere Pseudomonas aeruginosainfections Tackling the conundrum of drug resistancerdquo Post-graduate Medicine vol 117 no 1 pp 41ndash48 2005

[14] M Hentzer H Wu J B Andersen et al ldquoAttenuation of Pseu-domonas aeruginosa virulence by quorum sensing inhibitorsrdquoThe EMBO Journal vol 22 no 15 pp 3803ndash3815 2003

[15] A Adonizio K-F Kong and K Mathee ldquoInhibition of quorumsensing-controlled virulence factor production in Pseudomonasaeruginosa by south Florida plant extractsrdquo AntimicrobialAgents and Chemotherapy vol 52 no 1 pp 198ndash203 2008

[16] E Choi H Park J Lee and G Kim ldquoAnticancer antiobesityand anti-inflammatory activity of Artemisia species in vitrordquoJournal of Traditional Chinese Medicine vol 33 no 1 pp 92ndash97 2013

[17] J-H Kim S-H Jung Y-I Yang et al ldquoArtemisia leaf extractinduces apoptosis in human endometriotic cells through regu-lation of the p38 and NF120581B pathwaysrdquo Journal of Ethnopharma-cology vol 145 no 3 pp 767ndash775 2013

[18] D Kalemba D Kusewicz and K Swiader ldquoAntimicrobialproperties of the essential oil of Artemisia asiatica NakairdquoPhytotherapy Research vol 16 no 3 pp 288ndash291 2002


[19] M Ramezani B S Fazli-Bazzaz F Saghafi-Khadem and ADabaghian ldquoAntimicrobial activity of four Artemisia species ofIranrdquo Fitoterapia vol 75 no 2 pp 201ndash203 2004

[20] A R Ahameethunisa and W Hopper ldquoAntibacterial activityof Artemisia nilagirica leaf extracts against clinical and phy-topathogenic bacteriardquo BMC Complementary and AlternativeMedicine vol 10 article 6 2010

[21] O S Giordano E Guerreiro M J Pestchanker J GuzmanD Pastor and T Guardia ldquoThe gastric cytoprotective effect ofseveral sesquiterpene lactonesrdquo Journal of Natural Products vol53 no 4 pp 803ndash809 1990

[22] F Bohlmann and C Zdero ldquoPolyacetylenic compounds on theconstituents of the tribe Arctotideaerdquo Chemische Berichte vol105 pp 1245ndash1257 1972

[23] T Guardia J A Guzman M J Pestchanker E Guerreiro andO S Giordano ldquoMucus synthesis and sulfhydryl groups incytoprotection mediated by dehydroleucodine a sesquiterpenelactonerdquo Journal of Natural Products vol 57 no 4 pp 507ndash5091994

[24] P A Barrera V Jimenez-Ortiz C Tonn O Giordano NGalanti and M A Sosa ldquoNatural sesquiterpene lactones areactive against Leishmania mexicanardquo Journal of Parasitologyvol 94 no 5 pp 1143ndash1149 2008

[25] A EVegaGHWendel AOMMaria andL Pelzer ldquoAntimi-crobial activity of Artemisia douglasiana and dehydroleucodineagainst Helicobacter pylorirdquo Journal of Ethnopharmacology vol124 no 3 pp 653ndash655 2009

[26] S D Brengio S A Belmonte E Guerreiro O S GiordanoE O Pietrobon and M A Sosa ldquoThe sesquiterpene lactonedehydroleucodine (DhL) affects the growth of cultured epi-mastigotes of Trypanosoma cruzirdquo Journal of Parasitology vol86 no 2 pp 407ndash412 2000

[27] W N Setzer B Vogler J M Schmidt J G Leahy and R RivesldquoAntimicrobial activity of Artemisia douglasiana leaf essentialoilrdquo Fitoterapia vol 75 no 2 pp 192ndash200 2004

[28] H A Priestap K A Abboud A E Velandia L A Lopez andM A Barbieri ldquoDehydro-leucodin a guaiane-type sesquiter-pene lactonerdquo Acta Crystallographica Section E StructureReports Online vol 67 no 12 2011

[29] T Rabe D Mullholland and J van Staden ldquoIsolation and iden-tification of antibacterial compounds from Vernonia colorataleavesrdquo Journal of Ethnopharmacology vol 80 no 1 pp 91ndash942002

[30] P Berche J-L Gaillard and S Richard ldquoInvasiveness andintracellular growth of Listeria monocytogenesrdquo Infection vol16 supplement 2 pp S145ndashS148 1988

[31] A Rosato C Vitali N De Laurentis D Armenise and M AMilillo ldquoAntibacterial effect of some essential oils administeredalone or in combination with Norfloxacinrdquo Phytomedicine vol14 no 11 pp 727ndash732 2007

[32] E J Wolfe V Mathur S Tomlanovich et al ldquoPharmacokineticsof mycophenolate mofetil and intravenous ganciclovir aloneand in combination in renal transplant recipientsrdquo Pharma-cotherapy vol 17 no 3 pp 591ndash598 1997

[33] S Mahajan-Miklos L G Rahme and F M Ausubel ldquoEluci-dating the molecular mechanisms of bacterial virulence usingnon-mammalian hostsrdquo Molecular Microbiology vol 37 no 5pp 981ndash988 2000

[34] E Kessler M Safrin J C Olson and D E Ohman ldquoSecretedLasA of Pseudomonas aeruginosa is a staphylolytic proteaserdquoThe Journal of Biological Chemistry vol 268 no 10 pp 7503ndash7508 1993

[35] M J Bjorn P A Sokol and B H Iglewski ldquoInfluence of ironon yields of extracellular products in Pseudomonas aeruginosaculturesrdquo Journal of Bacteriology vol 138 no 1 pp 193ndash2001979

[36] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[37] G A OrsquoToole and R Kolter ldquoInitiation of biofilm formationin Pseudomonas fluorescens WCS365 proceeds via multipleconvergent signalling pathways a genetic analysisrdquo MolecularMicrobiology vol 28 no 3 pp 449ndash461 1998

[38] J Jia Y Wang L Zhou and S Jin ldquoExpression of Pseudomonasaeruginosa toxin ExoS effectively induces apoptosis in hostcellsrdquo Infection and Immunity vol 74 no 12 pp 6557ndash65702006

[39] S Hemaiswarya A K Kruthiventi and M Doble ldquoSynergismbetween natural products and antibiotics against infectiousdiseasesrdquo Phytomedicine vol 15 no 8 pp 639ndash652 2008

[40] S Hemaiswarya and M Doble ldquoSynergistic interaction ofphenylpropanoids with antibiotics against bacteriardquo Journal ofMedical Microbiology vol 59 part 12 pp 1469ndash1476 2010

[41] B M Kyaw S Arora and C S Lim ldquoBactericidal antibiotic-phytochemical combinations against methicillin resistantStaphylococcus aureusrdquo Brazilian Journal of Microbiology vol43 no 3 pp 938ndash945 2012

[42] J Jyot V Balloy G Jouvion et al ldquoType II secretion system ofpseudomonas aeruginosa in vivo evidence of a significant rolein death due to lung infectionrdquo Journal of Infectious Diseasesvol 203 no 10 pp 1369ndash1377 2011

[43] D S Toder M J Gambello and B H Iglewski ldquoPseudomonasaeruginosa LasA a second elastase under the transcriptionalcontrol of lasRrdquoMolecular Microbiology vol 5 no 8 pp 2003ndash2010 1991

[44] R Hoge A Pelzer F Rosenau and S Wilhelm ldquoWeapons of apathogen proteases and their role in virulence of Pseudomonasaeruginosardquo in Current Reseach Technology and EducationTopics in Applied Microbiology and Microbial Technology vol 1pp 383ndash395 2010

[45] J A Voynow B M Fischer and S Zheng ldquoProteases andcystic fibrosisrdquoThe International Journal of Biochemistry amp CellBiology vol 40 no 6-7 pp 1238ndash1245 2008

[46] LW Heck KMoriharaW BMcRae and E J Miller ldquoSpecificcleavage of human type III and IV collagens by Pseudomonasaeruginosa elastaserdquo Infection and Immunity vol 51 no 1 pp115ndash118 1986

[47] J W Costerton Z Lewandowski D E Caldwell D R Korberand HM Lappin-Scott ldquoMicrobial biofilmsrdquoAnnual Review ofMicrobiology vol 49 pp 711ndash745 1995

[48] D Davies ldquoUnderstanding biofilm resistance to antibacterialagentsrdquo Nature Reviews Drug Discovery vol 2 no 2 pp 114ndash122 2003

[49] G S Schulert H Feltman S D P Rabin et al ldquoSecretion of thetoxin ExoU is a marker for highly virulent Pseudomonas aerug-inosa isolates obtained from patients with hospital-acquiredpneumoniardquo Journal of Infectious Diseases vol 188 no 11 pp1695ndash1706 2003

[50] I A Holder A N Neely and D W Frank ldquoType IIIsecretionintoxication system important in virulence of Pseu-domonas aeruginosa infections in burnsrdquo Burns vol 27 no 2pp 129ndash130 2001


[51] J Moss M E Ehrmantraut B D Banwart D W Frank and JT Barbieri ldquoSera from adult patients with cystic fibrosis containantibodies to Pseudomonas aeruginosa type III apparatusrdquoInfection and Immunity vol 69 no 2 pp 1185ndash1188 2001

[52] M R Kaufman J Jia L Zeng U Ha M Chow and SJin ldquoPseudomonas aeruginosa mediated apoptosis requires theADP-ribosylating activity of ExoSrdquo Microbiology vol 146 no10 pp 2531ndash2541 2000

[53] C K Stover X Q Pham A L Erwin et al ldquoComplete genomesequence of Pseudomonas aeruginosa PAO1 an opportunisticpathogenrdquo Nature vol 406 no 6799 pp 959ndash964 2000

[54] I Kukavica-Ibrulj A BragonziM Paroni et al ldquoIn vivo growthof Pseudomonas aeruginosa strains PAO1 and PA14 and thehypervirulent strain LESB58 in a rat model of chronic lunginfectionrdquo Journal of Bacteriology vol 190 no 8 pp 2804ndash28132008

[55] S M J Fleiszig T S Zaidi M J Preston M Grout D J Evansand G B Pier ldquoRelationship between cytotoxicity and cornealepithelial cell invasion by clinical isolates of Pseudomonasaeruginosardquo Infection and Immunity vol 64 no 6 pp 2288ndash2294 1996

[56] G S Schulert H Feltman S D P Rabin et al ldquoSecretion of thetoxin ExoU is a marker for highly virulent Pseudomonas aerug-inosa isolates obtained from patients with hospital-acquiredpneumoniardquo The Journal of Infectious Diseases vol 188 no 11pp 1695ndash1706 2003

[57] H Mikkelsen R McMullan and A Filloux ldquoThe Pseudomonasaeruginosa reference strain PA14 displays increased virulencedue to a mutation in ladSrdquo PLoS ONE vol 6 no 12 Article IDe29113 2011

[58] S S Ricardo Obando M Jani V Aguiar-Pulido et al ldquoWhole-genome assembly of multi-resistant Pseudomonas aeruginosaisolaterdquo in Proceedings of the Pseudomonas Conference floridaInternational University Washington DC USA 2015

[59] B Siedle A J Garcıa-Pineres R Murillo et al ldquoQuantita-tive structure-activity relationship of sesquiterpene lactones asinhibitors of the transcription factor NF-kappaBrdquo Journal ofMedicinal Chemistry vol 47 no 24 pp 6042ndash6054 2004

[60] E D Brutinel C A Vakulskas K M Brady and T L YahrldquoCharacterization of ExsA and of ExsA-dependent promotersrequired for expression of the Pseudomonas aeruginosa type IIIsecretion systemrdquo Molecular Microbiology vol 68 no 3 pp657ndash671 2008

[61] A Mathee Y E R von Schirnding J Levin A Ismail RHuntley and A Cantrell ldquoA survey of blood lead levels amongyoung Johannesburg school childrenrdquo Environmental Researchvol 90 no 3 pp 181ndash184 2002

[62] A E Clatworthy E Pierson and D T Hung ldquoTargetingvirulence a new paradigm for antimicrobial therapyrdquo NatureChemical Biology vol 3 no 9 pp 541ndash548 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


to currently available drugs Since the pathogenicity of Paeruginosa is regulated by several secretory-systemsmediatedcell-to-cell communications inhibition of this system cancause attenuation of virulence and protect against infection[14 15]

Artemisia the largest diverse genera of Asteraceae familypossesses medicinally valuable essential oils and secondarymetabolites [16 17] Many studies indicate antimicrobialactivity in Artemisia (A) spp [18ndash20] A douglasiana is welldocumented as preventive folk medicine in Argentina [21]Dehydroleucodine (DhL) a sesquiterpene lactone of the gua-ianolide group is one of the principal active secondary met-abolites in A douglasiana [21] DhL first isolated from Lid-beckia pectinata [22] possesses cytoprotective activity [23] aswell as antimicrobial activity against several pathogens [24ndash27]

In this study we tested the antibacterial activity of DhLand against P aeruginosa virulence At first we evaluated theminimum inhibitory concentration (MIC) and its synergisticeffect on other commercial antibiotics and thenwe examinedthe antipseudomonal properties of this phytochemical interms of arresting growth and attenuating virulent factorssuch as biofilm formation and secretion of Type III effectorselastase A and elastase B We also assessed the capacity ofthis phytocompound to inhibitP aeruginosa induced host celltoxicity

2 Material and Methods

21 Reagents Dehydroleucodine was extracted from A dou-glasiana (Voucher specimen 2012-1018-F Fairchild TropicalBotanical Garden Miami FL) as previously documented[21 28] DH-DhL was prepared as described previously [21]All chemicals and reagents were purchased from Sigma-Aldrich (St Louis MO) unless otherwise indicated Primaryand secondary antibodies used in immunoblotting werepurchased from Cell Signaling Technology Inc (DanversMA) Culture supplies were purchased from Invitrogen LifeTechnologies (Carlsbad CA) Prototypic P aeruginosa strainsused in this study are PAO1 clinical isolates PA103 and PA14(kindly provided by Dr Dara Frank University ofWisconsinMadison) and P aeruginosa strain PA103ΔUΔT expressingpUCP plasmid-encoded ExoS tagged with a hemagglutininepitope (ExoS-HA) (kindly provided by Dr Joan C OlsonWest Virginia University) In addition Staphylococcus aureus(ATCC 12600) was used in the LasA assay Cells weremaintained in Luria Broth (LB) (Life Technologies CarlsbadCA) or Agrobacterium (AB) minimum medium (Bio-WorldVisalia CA) to which glucose and casein amino acids [20wtvol] were added

22 Determination of Minimum Inhibitory Concentration(MIC) andMinimumBactericidal Concentration (MBC) Theminimum inhibitory concentration of DhL was determinedby micro dilution broth assay [12] for PA01 PA103 PA14 andCDN118 strains of P aeruginosa Serial doubling dilution ofDhL was made ranging from 224mgmL to 0224120583gmLTwenty (20) 120583L of standard inoculums (05 McFarland) of all

three P aeruginosa strains was introduced to desired volumeof growth medium and incubated at 37∘C for 18 hr Controlswere set up as follows (1) sterility control LB broth only (2)growth control (negative) LB broth + bacteria (3) positivecontrol LB broth with Gentamycin (4) vehicle control LBbroth + bacteria + DMSO MIC was interpreted as the leastconcentration with no observable turbidity Optical densityreadings (120582 = 600 nm) were taken using microplate readerat 0 and 18 hr MIC

90and MIC

50were determined as the

concentration of DhL that inhibited bacterial growth by 90and 50 respectively Results were reported as the MICMIC50 and MIC

90for growth at 18 hr after inoculation

Minimum bactericidal concentration (MBC) wasrecorded as a lowest extract concentration killing 999of thebacterial inocula after 24 h incubation at 37∘C Each experi-ment was repeated at least three times MBC values weredetermined by removing 100 120583L of bacterial suspension fromsubculture demonstrating no visible growth and inoculatingnutrient agar plates Plates were incubated at 37∘C for a totalperiod of 24 h The MBC is determined with the wells whosethe concentrations are ge MIC [29] According to the ratioMBCMIC we appreciated antibacterial activity If the ratioMBCMIC is 1 or 2 the effect was considered bactericidalbut if the ratio MBCMIC is 4 or 16 the effect was defined asbacteriostatic [30]

For each assay 012mgmL (MIC50) of DhL was added

to P aeruginosa culture at early log phase unless otherwisestated The effect of DhL on bacterial cell proliferation wasdetermined by monitoring the growth curve of P aeruginosastrain PAO1 Briefly an overnight culture (in LB medium) ofPAO1was diluted toOD

600005 in LBmedium (control) or LB

and DhL (012mgmL) and incubated at 37∘C while shakingThe OD

600was monitored at 30min intervals until OD

600

of approximately 17 was obtained (approximately 8 hr) AllOD600

measurements were verified at a 110 dilution forgreater accuracy

23 Determination of Antibiotic Synergy with DhL by theE-Test Strip Bacterial suspensions (PAO1) homogenized insterile saline were prepared from overnight fresh culturesto a McFarland standard of 05 and were spread with asterile cotton swab on 150mm Mueller-Hinton agar controland containing DhL (MIC

50) plates The agar plates were

allowed to stand for 15ndash20min at room temperature toallow any excess surface moisture to be absorbed beforeplacement of MIC test strips Antibiotic gradient strips (E-test Liofilchem Italy) containingGentamycin (concentrationrange 0016ndash256mgL) Ciprofloxacin (concentration range0002ndash32mgL) or Chloramphenicol (concentration range0016ndash256mgL) were then used the MHA plates with theover layered strips were incubated at 37∘C or 24 hr and thegrowth inhibition zones were measured Differences in MICvalues between the control and test plates were recorded(end points were determined according to themanufacturerrsquosinstructions)

The Muller Hinton agar (MHA) dilution method wasused to evaluate the Fractional Inhibitory ConcentrationIndex (FICI) of DhL with Chloramphenicol on PAO1 being




595) due to lysis










495of






546











3 Results




90)lowast


90MIC50














60017 that





90failed to




H O

O

O

(a)

PAO1


lowast

lowast

lowast

lowastlowast

lowast

PAO1 + DhL

30 60 90 120 150 180 210 240 270 300 3300

Time (min)

0

02

04

06

08

10

12

14

OD600

(log

scal

e)

(b)




038 038 0064MIC50 120 (120583gmL)

MIC (120583gmL)

(a)

10

(b)

038

(c)


50concentration














4 Discussion



PAO1


PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)


595as described in



540of 35ndash






lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















595) due to lysis










495of






546











3 Results




90)lowast


90MIC50














60017 that





90failed to




H O

O

O

(a)

PAO1


lowast

lowast

lowast

lowastlowast

lowast

PAO1 + DhL

30 60 90 120 150 180 210 240 270 300 3300

Time (min)

0

02

04

06

08

10

12

14

OD600

(log

scal

e)

(b)




038 038 0064MIC50 120 (120583gmL)

MIC (120583gmL)

(a)

10

(b)

038

(c)


50concentration














4 Discussion



PAO1


PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)


595as described in



540of 35ndash






lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results




90)lowast


90MIC50














60017 that





90failed to




H O

O

O

(a)

PAO1


lowast

lowast

lowast

lowastlowast

lowast

PAO1 + DhL

30 60 90 120 150 180 210 240 270 300 3300

Time (min)

0

02

04

06

08

10

12

14

OD600

(log

scal

e)

(b)




038 038 0064MIC50 120 (120583gmL)

MIC (120583gmL)

(a)

10

(b)

038

(c)


50concentration














4 Discussion



PAO1


PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)


595as described in



540of 35ndash






lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H O

O

O

(a)

PAO1


lowast

lowast

lowast

lowastlowast

lowast

PAO1 + DhL

30 60 90 120 150 180 210 240 270 300 3300

Time (min)

0

02

04

06

08

10

12

14

OD600

(log

scal

e)

(b)




038 038 0064MIC50 120 (120583gmL)

MIC (120583gmL)

(a)

10

(b)

038

(c)


50concentration














4 Discussion



PAO1


PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)


595as described in



540of 35ndash






lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























4 Discussion



PAO1


PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)


595as described in



540of 35ndash






lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PAO1


PAO1 + DhL

0

02

04

06

08

10

12

LasA

pro

teas

e con

cent

ratio

n

5 10 20 30 40 50 600

Time (min)

LB + S aureus

( o

f con

trol)

(a)

lowast

PAO1 PAO1 + DhL0

005

01

015

02

025

(OD495)

Elas

toly

tic ac

tivity

(b)PAO1

lowast

PAO1 + DhLminus8

minus6

minus4

minus2

0

2

4

6

8

10

12

Rela

tive u

nits

(fold

chan

ge)

(c)


595as described in



540of 35ndash






lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

PAO1 PAO1 + DhL0

02

04

06

08

10(O

D546)

Crys

tal v

iole

t ass

ay


546of





PAO1

Anti-HA(IB)

250

175150

100

50

35

25

DhL

ExoS

ExoS

+ +minus minus

PA103ΔUT





0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Apo

ptos

is (

of c

ontro

l)

lowast








Acknowledgments


References







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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