A fraction from Escherichia coli with anti-Aspergillus properties

A fraction from Escherichia coli with anti-Aspergillus properties
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  Journal of Medical Microbiology  (2005), 54 , 375–379 DOI 10.1099/jmm.0.45748-045748 & 2005 SGM Printed in Great Britain 375 Correspondence G. L. Received 18 May 2004 Accepted 2 December 2004 A fraction from Escherichia coli  with anti- Aspergillus  properties V. Yadav, 1 R. Mandhan, 2 Rajesh Dabur, 3 A. K. Chhillar, 1 J. Gupta 1 and G. L. Sharma 1,3 1 Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi, India 2 Department of Biotechnology, Kurukshetra University, Kurukshetra, India 3 Department of Biomedical Sciences, Bundelkhund University, Jhansi, India The products of various strains of Escherichia coli  (BL21, DH5 Æ , HB101 and XL Blue) wereinvestigated for antimycotic properties using pathogenic isolates of Aspergillus . Co-cultureexperimentsrevealedthat E.coli  strainsexhibitedvariableactivityagainst Aspergillusfumigatus .ThelysatespreparedfromDH5 Æ ,HB101andXLBluestrainsof E.coli  showedinhibitoryactivityagainst A.fumigatus intheproteinconcentrationrangeof62.50to250.00 ì  gml À 1 .Thehighestactivitywasseen in the lysate of BL21, which inhibited the growth of A. fumigatus and Aspergillus flavus completelyataconcentrationof31.25 ì  gproteinml À 1 .TheMICofBL21lysateagainst Aspergillusniger  was found to be 62.50 ì  g ml À 1 . The in vitro toxicity of BL21 lysate was evaluated using ahaemolyticassay.ABL21lysateproteinconcentrationof1250.00 ì  gml À 1 wasfoundtobenontoxicto human erythrocytes. The standard drug amphotericin B lysed 100% of erythrocytes at aconcentration of 37.50 ì  g ml À 1 . SDS-PAGE showed the presence of at least 15 major proteins inthe lysate of BL21. Ion-exchange chromatography resolved the BL21 lysate into five fractions andfraction III was found to be endowed with anti- Aspergillus properties. The MIC of this fraction wasfound to be 3.90 ì  g ml À 1 . Further work on the purification of the active molecule and itscharacterization is in progress. INTRODUCTION Infections due to opportunistic fungal pathogens such as  Aspergillus and Candida speciesareemergingasmajorcausesof morbidity and mortality in immunocompromised pa-tients despitewidespread use of several antifungal drugs (Walsh et al. , 1996;Richardson& Kokki, 1998). Theantifungal agents currently available for treatment of theseinfections include amphotericin B and its liposomal for-mulations,5-fluorocytosine,triazolesandallylamines,whichare used singly or in combination(Maertens et al. , 2002;Chiou et al. ,2000). These drugs not only have limitedefficacy, allof them induce severe toxicity or immuno-suppression(Georgopapadakou & Walsh, 1996). Further,resistance of fungi to most drugs has emerged, which hastriggered considerable interest inidentification of novelantifungal molecules(Viscoli & Castagnola, 1998).The recent past has witnessed the introduction of a few promising second-generation triazoles (voriconazole, ravu-conazole and posaconazole) but they are still undergoingclinical trials. These molecules are reported to show broadspectrum in vitro activity against clinical isolates of patho-genicfungalspecies(Graybill etal. ,1998).Studiescarriedouton voriconazole, ravuconazole and posaconazole usingimmunocompromised animal models(Sheehan et al. ,1999) and the published data from clinical trials havedemonstrated improved antifungal efficacy (Walsh et al. ,2002).However,theseriousproblemofcross-resistancemay still persist since these molecules have structural similaritieswithfirst-generationtriazoledrugsandusethesamepathway for exhibiting antifungal activity.Antifungal proteins such as thaumatin-like proteins (Ye et al. , 1999), glucanases(York  et al. , 2004), chitinases (Choi et al. , 2004), ribosome-inactivating proteins(Ng & Parkash,2002),cyclophillin-like proteins, miraculin-likeproteins (Ye etal. ,2000),cysteine-richpeptides,likethionins(Epple etal. ,1997), and lipid-transfer proteins(Regente & dela Canal,2000)havebeen reported fromavariety ofsources includingbacteria, mammals, insects and plants for treating fungalinfections(Selitrennikoff , 2001). Although these proteinshave been shown to inhibit the growth of pathogenic fungi,many of them were found to be highly toxic(Conlon et al. ,2003). However, less-toxic antif ungal proteins have beendescribed from bacterial sources(Woo et al. , 2002).  Escherichia coli is a common commensal and an importantcomponentofgutflora.Itservesusefulfunctionsinthebody bysuppressingthegrowthofharmfulbacterialspeciesandby synthesizing appreciable amounts of vitamins. It is reportedto be useful in treating severe pseudomembranous colitis(Matricardi et al. , 2003)and is also found to be associatedwith anti-allergic effects(Georg & Schlorer, 1998).However,it is not known if  E. coli interacts with mycotic infections toexert any antimycotic effect. In view of the importance of  E.coli in human health, the present study was undertaken toidentify, isolate and characterize the potential antifungalfractions of  E. coli . METHODS Bacterial strains. E. coli BL21 (MTCCB1678), DH5 Æ (MTCCB1652)and HB101 (MTCCB82) were obtained from the Institute of MicrobialTechnology,Chandigarh,India,whereastheXLBluestrainof  E.coli waspurchased from Banglore Genei. Pathogens. Aspergillus fumigatus isolates ITCC4517 (IARI, Delhi),ITCC1634 (IARI, Delhi) and 190/96 (VPCI, Delhi), Aspergillus flavus isolates ITCC5192 (IARI, Delhi), ITCC5076 (IARI, Delhi) and 223/96(VPCI, Delhi), and Aspergillus niger  isolates ITCC5405 (IARI, Delhi)and 56/96 (VPCI, Delhi) were employed in the current study. Thesepathogenic species of  Aspergillus were cultured in the laboratory onSabouraud dextrose agar plates. Preparationof lysate of E. coli  strains. The 72 h exponential phaseculturesof  E.coli strainswerecentrifugedat5000r.p.m.for30 min.Thepellet was suspended in sonication buffer (50 mM Tris/HCl, 50 mMEDTA,5 mMDTT,1 mMPMSF)andsonicatedfor20sburstsat200Wand 10 s cool period using a sonicator (Misonix, Sonicator 3000). Thesonicate was centrifuged at 15000 r.p.m. for 30 min using a Sorvall RC5C centrifuge. The supernatant was used as lysate. The lysate wasdialysed against water at 4 8 C for 24 h and lyophilized. The proteinconcentrationof bacterial lysatewas determined by the bicinchoninicacid method of Smith et al  . (1985). Antimycotic activity. The antifungal activity of bacterial componentswas analysed by microbroth-dilution, disc-diffusion and spore-germi-nation-inhibition assays(Rajesh & Sharma, 2002). These assays wererepeated at least three times. Microbroth-dilution assay. The spores (1 3 10 6 ) of  Aspergillus wereharvested from 96 h cultures and treated with different concentrationsof bacterial products in a 96-well culture plate. The plates wereincubated at 37 8 C and examined macroscopically after 48 h for thegrowth of  Aspergillus mycelia. Disc-diffusion assay. This test was performed in radiation-sterilizedPetri plates of 10.0 cm diameter (Tarsons). Sterilized discs (5.0 mm of Whatman paper) impregnated with different concentrations of thebacterial products were placed on the surface of agar plates already inoculated with Aspergillus spores (1 3 10 6 ). The plates were incubatedat 37 8 C and examined at 48 h for the zone of inhibition, if any, aroundthe discs. Spore-germination-inhibitionassay. Various concentrations of thetest samples in 90 ì  l of culture medium were prepared in 96-well flat-bottom micro-culture plates (Nunc Nunclon) by double-dilutionmethod. The wells were prepared in duplicate for each concentration.The wells of the culture plates were inoculated with 10 ì  l of sporesuspension containing 100 Æ 5 spores. The plates were incubated at37 8 C for 16 h and then examined for spore germination under aninverted microscope. The number of germinated and non-germinatedspores was counted.Based on the results of co-culture antimycotic activity experiments, theBL21 strain of  E. coli was further investigated to identify its activefraction(s). Fractionation of E. coli  (BL21) lysate. The lysate prepared from theBL21 strain of  E. coli was subjected to fractionation by ion-exchangechromatography. A 15.00 mg amount of lysate protein was dissolved inTris/HCl, pH 7.4, and loaded onto a DEAE-cellulose column(5.00 3 8.00 cm) pre-equilibriated with the same buffer. The non-adsorbed proteins were eluted with Tris/HCl, pH 7.4. The elution of adsorbed proteinswascarriedoutusinga200.0 mllinear gradientfrom0 to 1.00 M of NaCl in Tris/HCl, pH 7.4. The flow rate was adjusted to1.00 ml min À 1 and 2.00 ml fractions were collected. The OD 280 of thefractions was measured. The OD 280 values of various fractions wereplotted against the fraction number. The peaks were pooled andanalysed for antifungal activity. The proteins recovered in peaksshowing antimycotic activity were subjected to SDS-PAGE using12.5% gel. SDS-PAGE was carried out according to the method of Laemmli & Favre (1973). Haemolyticassay. ThebasicmethodofLatoud etal  .(1986)withslightmodifications was employed to determine the haemolytic effect of bacterial proteins with antifungal activity. Human erythrocytes, col-lected from apparently healthy individuals, were washed three timeswith PBSbycentrifugationat1500r.p.m. for10 min.A2%erythrocytesuspension was incubated at 37 8 C for 1 h with different concentrationsof lysate ranging from 5000.00 to 1.22 ì  g protein ml À 1 . After incuba-tion, cells were pelleted at 5000 r.p.m. for 10 min. The supernatant wascollected and the A 450 was determined using a spectrophotometer (UVVis Spect Lambda Bio 20, Perkin Elmer). In negative control sets, only buffer was used for background lysis, whereas in positive controls, lysisbuffer was used for completely lysing the erythrocytes. For each samplethe percentage of maximum haemolytic activity was determined. RESULTS AND DISCUSSION Antifungal activity of E. coli  lysates TheantifungalactivityoflysatespreparedfromBL21,DH5 Æ ,HB101 and XL Blue strains of  E. coli was examined by microbroth-dilution assay. These strains exhibited mild tomoderate activity (MICs in ì  g ml À 1 : DH5 Æ , 62.50; HB101,125.00; XL Blue, 250.00). The lysate of the BL21 strain of  E.coli showed potent and broad-spectrum antifungal activity against Aspergillus species. It was observed that a proteinconcentration of 31.25 ì  g ml À 1 of BL21 lysate inhibited thegrowth of  A. fumigatus and A. flavus in microbroth-dilutionand spore-germination-inhibition assays (Fig.1). A higherconcentration of bacterial lysate of BL21 was required toinhibit the growth of  A. niger  . In the disc-diffusion assay, theMIC of BL21 lysate against A. fumigatus and A.flavus wasfoundtobe6.25 ì  gdisc À 1 (Table1).Magnusson et al  .(2003)screened 1200 isolates of lactic acid bacteria for anti-  Aspergillus activity and observed strong inhibitory activity against A. fumigatus but several isolates showed reducedantifungal activity after storage and handling. Lactobacilluscoryniformis subsp. coryniformis strain Si3 was also found tobe a producer of broad-spectrum proteinaceous antifungalcompound(Magnusson & Schnurer, 2001). V. Yadav and others376 Journal of Medical Microbiology  54  Fractionation of E. coli  BL21 lysate The results of SDS-PAGE show ed various protein bands inthe lysate of  E. coli BL21 (Fig.2). It was, therefore, pertinenttofractionatetheseproteinsandidentifytheactiveantifungalfraction(s). The lysate of  E. coli BL21 was fractionated by DEAE cellulose column. The proteins were resolved into fivefractions (FI to FV). The elution profile of proteins of  E. coli (BL21) obtainedafter ion-exchange chromatography isshown in Fig.3.Different fractions were collected andexamined for antifungal activity as described. Antifungalactivityoffractionsof E. coli  BL21lysate It was observed that activity resided mainly in FIII as its MICwas found to be 3.90 ì  g ml À 1 against A. fumigatus usingmicrobroth-dilution and spore-germination-inhibition as-says. In disc-diffusionassay, the MIC of FIII was found to be1.25 ì  g disc À 1 (Fig.4). The SDS-PAGE of FIII on 12.5% geldemonstrated two major bands in the molecular mass rangeof 29 to 43 kDa (Fig.2).Dahot (1998)studied the antifungal properties of proteinfractions of  Moringa oleifera leaves and found that one of thechromatographicfractionsinhibitedthegrowthof   A.niger  ataminimumconcentrationof75.0 ì  gml À 1 .Thisfractionwasnot found tobe effective against A. flavus and A. fumigatus .Dahot (1999)also fractionated the leaf extract of  Indigoferaoblongifolia using Sephadex G-25 column to obtain fourfractions. Their chromatographic fraction AP4 was activeagainst A. fumigatus , A. flavus and A. niger  at the concentra-tion of 50.0, 15.0 and 25.0 ì  g ml À 1 , respectively. But AP3inhibited the growth of  A. fumigatus and A. flav us at theconcentrationof50.0and10.0 ì  gml À 1 ,respectively .Bottone&Peluso(2003)identifiedthe Bacilluspumilus (MSH)whichhad antifungal activity against Mucoraceae and Aspergillus species,but it was nottested against A. niger  . No data weregiven by Bottone & Peluso (2003) regarding the toxicity of the antifungal compound. The results of the present inves-tigation showed that lysate of  E. coli (BL21) inhibited the Fig. 1. Microwellsshowinggrowthof A.fumigatus  sporesat16 h.(a)Treated with 31.25 ì  g BL21 lysate ml À 1 ; (b) untreated control.Magnification 3 60. Table 1. Activity of BL21 lysate against Aspergillus species by microbroth-dilution and disc-diffusionassays +, Activity, À , no activity. Concn of BL21lysate ( ì  g ml À 1 )Microbroth-dilution assay Disc-diffusion assay zone of inhibition(mean diameter in mm)  A. fumigatus A. flavus A. niger  A. fumigatus A. flavus A. niger  125.00 + + + 11.0 11.0 10.562.50 + + + 10.5 10.5 6.531.25 + + À 6.0 7.0 À 15.60 À À À À À À 7.825 À À À À À À TP FIIIkDa97·466·043·029·021·014·0 Fig. 2. Protein profile of a BL21 lysate fraction on 12.5% gel. FIII,fraction III; TP, total bacterial protein. 81716151413121111 00·20·40·60·81·01·21·41·6FIFII FIIIFIVFVFraction number    O   D    2   8   0 Fig. 3. Elution profile of BL21 lysate on DEAE-cellulose column.Antifungal fraction from E. coli 377  growth of  A. fumigatus at a concentration of 31.25 ì  g ml À 1 and the activity mainly resided in ion-exchange chromato-graphic fraction FIII, the MIC being 3.90 ì  g ml À 1 . Cytotoxicity The results of toxicity experiments revealed that the totalprotein lysate of  E. coli investigated in the present study wasnontoxic up to 1250.0 ì  g ml À 1 to human erythrocytes. Thehigher doses exerted insignificant toxicity and only marginalhaemolysis (5.8%) w as detected at the concentrations up to5000 ì  g ml À 1 (Fig.5). The amphotericin B lysed allerythro- cytes at a concentration of 37.5 ì  g ml À 1 . Similarly,Hong et al  . (1999) found that a novel antimicrobial peptide did notshow haemolytic activity up to the concentration of 500 ì  gml À 1 .Woo et al  . (2002) also found antifungal SAP protein,isolated from Streptomyces , nontoxic up to 250.0 ì  g ml À 1 tohuman dermal fibroblasts, but it was f ound to be toxic at allhigher doses. However,Sorensen et al  . (1996) found 100%haemolysis by syringomycins at the concentration of 20.0 ì  gml À 1 and found it to be more toxic than amphotericin B toerythrocytes. Thionins and defensins have been reported tobeeffectiveantifungalproteinsagainsthumanpathogensbutthey exerted nonspecific cytotoxic activity against a widerange of normal and malignant targets, including cellsresistant to TNF (tumour necrosis factor)-alpha and NK(natural killer)-cytolytic factor. They appear to kill mamma-liantargetcellsandmicro-organismsbyacommonmechan-ism(Lehrer et al. ,1993). The antifungal peptides purifiedfrom Bacillus cereus were also reported to be highly toxic tohuman erythrocytes(Latoud et al. ,1986).The toxicity of most reported antifungal proteins to mam-malian cells, therefore, has become a major limitation inusing them as lead molecules for developing new antifungaldrugs and using them for further formulations of betterdrugs. The much lower toxicity of the potent antifungalfraction (FIII) identified in the current study thus empha-sizesitsusefulnessinnewtherapeutics.Itmayalsobepossibleto develop specialized probiotics using E. coli strains fortreating Aspergillus- induced disorders. Conclusion TheobservationsofthepresentstudyindicatedthattheBL21strain of  E. coli synthesizes potent anti-  Aspergillus proteinswith extremely low toxicity to human cells. Such prepara-tions may be important leads for developing new therapiesfor treating fungal infections. 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