Amyloid Formation Modulates the Biological Activity of a Bacterial Protein

Abstract

The aggregation of proteins into amyloid fibrils is the hallmark feature of a group of late-onset degenerative diseases including Alzheimer, Parkinson, and prion diseases. We report here that microcin E492, a peptide naturally produced by Klebsiella pneumoniae that kills bacteria by forming pores in the cytoplasmic membrane, assembles in vitro into amyloid-like fibrils. The fibrils have the same structural, morphological, tinctorial, and biochemical properties as the aggregates observed in the disease conditions. In addition, we found that amyloid formation also occurs in vivo where it is associated with a loss of toxicity of the protein. The finding that microcin E492 naturally exists both as functional toxic pores and as harmless fibrils suggests that protein aggregation into amyloid fibrils is an evolutionarily conserved property of proteins that can be successfully employed by bacteria to fulfill specific physiological needs. The aggregation of proteins into amyloid fibrils is the hallmark feature of a group of late-onset degenerative diseases including Alzheimer, Parkinson, and prion diseases. We report here that microcin E492, a peptide naturally produced by Klebsiella pneumoniae that kills bacteria by forming pores in the cytoplasmic membrane, assembles in vitro into amyloid-like fibrils. The fibrils have the same structural, morphological, tinctorial, and biochemical properties as the aggregates observed in the disease conditions. In addition, we found that amyloid formation also occurs in vivo where it is associated with a loss of toxicity of the protein. The finding that microcin E492 naturally exists both as functional toxic pores and as harmless fibrils suggests that protein aggregation into amyloid fibrils is an evolutionarily conserved property of proteins that can be successfully employed by bacteria to fulfill specific physiological needs. Bacteriocins form a vast and diverse group of antibacterial proteins that have been found in most lineages of Eubacteria and Archaebacteria. They kill or inhibit the growth of closely related species by binding to specific cell surface receptors and by forming ion channels in the cytoplasmic membrane, degrading DNA, blocking protein translation, or inhibiting peptidoglycan synthesis (1Riley M.A. Annu. Rev. Genet. 1998; 32: 255-278Crossref PubMed Scopus (199) Google Scholar). They have been proposed to be involved in maintaining bacterial diversity by mediating interactions at the population and community levels (2Riley M.A. Goldstone C.M. Wertz J.E. Gordon D. J. Evol. Biol. 2003; 16: 690-697Crossref PubMed Scopus (63) Google Scholar). Microcin E492 (Mcc) 1The abbreviations used are: Mcc, microcin E492; LB, Luria-Bertani; PIPES, 1,4-piperazinediethanesulfonic acid; CR, Congo red; ThT, thioflavin T; PK, proteinase K. is a low molecular weight bacteriocin naturally produced by Klebsiella pneumoniae RYC492 that is active against several strains of Enterobacteriaceae (3de Lorenzo V. Martinez J.L. Asensio C. J. Gen. Microbiol. 1984; 130: 391-400PubMed Google Scholar, 4de Lorenzo V. Arch. Microbiol. 1984; 139: 72-75Crossref PubMed Scopus (76) Google Scholar, 5Destoumieux-Garzon D. Thomas X. Santamaria M. Goulard C. Barthelemy M. Boscher B. Bessin Y. Molle G. Pons A.M. Letellier L. Peduzzi J. Rebuffat S. Mol. Microbiol. 2003; 49: 1031-1041Crossref PubMed Scopus (66) Google Scholar). Its mechanism of action has been shown to involve the formation of pores in the cytoplasmic membrane of target cells and subsequent lethal loss of membrane potential (5Destoumieux-Garzon D. Thomas X. Santamaria M. Goulard C. Barthelemy M. Boscher B. Bessin Y. Molle G. Pons A.M. Letellier L. Peduzzi J. Rebuffat S. Mol. Microbiol. 2003; 49: 1031-1041Crossref PubMed Scopus (66) Google Scholar, 6de Lorenzo V. Pugsley A.P. Antimicrob. Agents Chemother. 1985; 27: 666-669Crossref PubMed Scopus (66) Google Scholar, 7Lagos R. Wilkens M. Vergara C. Cecchi X. Monasterio O. FEBS Lett. 1993; 321: 145-148Crossref PubMed Scopus (61) Google Scholar). Whereas most known microcins are only produced when exponential growth ceases and the cells enter the stationary phase (8Baquero F. Moreno F. FEMS Microbiol. Lett. 1984; 23: 117-124Crossref Scopus (160) Google Scholar), Mcc activity has been reported to be highest during the exponential phase and to suddenly drop to virtually undetectable levels during the stationary phase (3de Lorenzo V. Martinez J.L. Asensio C. J. Gen. Microbiol. 1984; 130: 391-400PubMed Google Scholar, 9de Lorenzo V. J. Antibiot. 1985; 38: 340-345Crossref PubMed Scopus (16) Google Scholar). Surprisingly, Mcc was found to be similarly produced and accumulated both in exponential and stationary phases (10Corsini G. Baeza M. Monasterio O. Lagos R. Biochimie (Paris). 2002; 84: 539-544Crossref PubMed Scopus (24) Google Scholar), thereby ruling out an arrest of Mcc production and/or degradation as causes of this loss of activity. In addition, mass spectrometry studies revealed no difference in the molecular weight of Mcc purified from either growth phases, indicating that this effect was not related to any post-translational modifications of the protein under our experimental conditions. 2S. Bieler, R. Lagos, and C. Soto, unpublished data. This led us to hypothesize that the sudden disappearance of activity in the stationary phase could be because of a change in Mcc conformation and/or oligomerization state. We report here that purified active Mcc is able to be converted upon in vitro incubation from a soluble to an insoluble state consisting of amyloid-like fibrils. Electron microscopy, structural, biochemical, and kinetics studies indicate that these fibrils have properties identical to those previously described for the amyloid aggregates that are formed in the course of amyloid diseases. We also found that the assembly of Mcc into amyloid fibrils is accompanied by a loss of the antibacterial activity of the protein. Furthermore, amyloid fibrils are also formed in vivo, and this correlates with a sudden drop in Mcc activity in the culture. We therefore propose that amyloid formation modulates the activity of Mcc in vivo and is responsible for the previously reported loss of activity in the stationary phase. These data represent the first demonstration that amyloid formation in prokaryotes controls in vivo the biological activity of a protein and changes the phenotype of the bacteria. Thus far, formation of amyloid has been associated with several human diseases involving the misfolding, aggregation, and tissue deposition of proteins (11Soto C. FEBS Lett. 2001; 498: 204-207Crossref PubMed Scopus (290) Google Scholar, 12Dobson C.M. Trends Biochem. Sci. 1999; 24: 329-332Abstract Full Text Full Text PDF PubMed Scopus (1708) Google Scholar, 13Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar). Our findings suggest that the process of protein aggregation into amyloid fibrils is a conserved mechanism that may have an important role not only in triggering tissue damage and disease but also in modulating the biological activity of diverse proteins. Bacterial Strains and Plasmids—The Escherichia coli strains and plasmids used in this study are described in Table I.Table IBacterial strains and plasmidsStrain/plasmidGenotypeSource or referenceE. coli strainsVCS257DP50 supF [supE44 supF58 hsdS3(rB-mB-) dapD8 lacY1 glnV44 Δ(gal-uvrB)47 tyrT58 gyrA29 tonA53 Δ(thyA57)]StratageneBL21F- ompT hsdS(rB-, mB-) gal dcmAmersham BiosciencesBL21(DE3)BL21 λ(DE3)NovagenPlasmidspJEM15Mcc determinants28Wilkens M. Villanueva J.E. Cofre J. Chnaiderman J. Lagos R. J. Bacteriol. 1997; 179: 4789-4794Crossref PubMed Google Scholarnp108Mcc determinants mceG::Tn527Lagos R. Baeza M. Corsini G. Hetz C. Strahsburger E. Castillo J.A. Vergara C. Monasterio O. Mol. Microbiol. 2001; 42: 229-243Crossref PubMed Scopus (66) Google Scholarp11α2Ampr, fragment of tubulin cloned in pT7-728Wilkens M. Villanueva J.E. Cofre J. Chnaiderman J. Lagos R. J. Bacteriol. 1997; 179: 4789-4794Crossref PubMed Google Scholar Open table in a new tab Growth Conditions—Bacteria were grown at 37 °C with shaking in LB (Luria-Bertani) Miller medium (14Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1972: 431-435Google Scholar) that was filtered through a 0.22-μm cellulose acetate filter system (Corning). When specified, the medium was buffered with 100 mm PIPES-NaOH at pH 6.5. Antibiotics were used at the following concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml. Cultures were started using 1:1000 dilutions of fresh overnight cultures to minimize the amount of amyloid-like Mcc from the inocula. Purification of Mcc—E. coli VCS257pJEM15 was grown at 37 °C with shaking to an absorbance at 600 nm of 1.5 in M9 minimal medium containing 0.2% glucose, 0.2% sodium citrate, 1 g/liter casamino acids, 1 mg/liter thiamine, and 100 mg/liter ampicillin. 200 ml of culture supernatant were passed through a Sep-Pak C18 cartridge (Waters). The cartridge was washed with 3 ml of 65% methanol and 3 ml of 25% acetonitrile. Mcc was eluted with 3 ml of 50% acetonitrile and concentrated by a CentriVap Concentrator (Labconco). Samples were brought to pH 8.0 by adding NaOH and incubated at 37 °C for 15 min to ensure full solubilization of aggregates formed during the concentration step. Finally, samples were centrifuged at 75,000 × g for 1 h at 4 °C to remove any remaining aggregates, and supernatants were stored at –20 °C. Purity was calculated to be >90% by amino acid composition analysis (420/H Amino Acid Analysis System; Applied Biosystems). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed in a Voyager DE STR biospectrometry work station (Applied Biosystems) revealed one single peak at 7887.5 Da corresponding to the previously reported molecular mass of mature Mcc without post-translational modifications (5Destoumieux-Garzon D. Thomas X. Santamaria M. Goulard C. Barthelemy M. Boscher B. Bessin Y. Molle G. Pons A.M. Letellier L. Peduzzi J. Rebuffat S. Mol. Microbiol. 2003; 49: 1031-1041Crossref PubMed Scopus (66) Google Scholar, 15Pons A.M. Zorn N. Vignon D. Delalande F. Van Dorsselaer A. Cottenceau G. Antimicrob. Agents Chemother. 2002; 46: 229-230Crossref PubMed Scopus (29) Google Scholar). Electron Microscopy—Samples of purified Mcc or bacterial culture were placed onto 300-square mesh nickel grids (Maxtaform), negatively stained with 2% uranyl acetate for 90 s, and examined in a Philips EM410 electron microscope. Circular Dichroism Analysis—CD spectra were obtained using an AVIV CD spectrometer model 215 with a 0.1-cm path length cell. Five consecutive readings at 1-nm bandwidth, 0.25-s response time, and 1-nm resolution were taken over the wavelength range 190–260 nm at 25 °C, averaged, buffer subtracted, and noise reduced. Mean residue molar ellipticities of Mcc were calculated using 7886.5 as molecular weight and 84 as the number of residues. Samples were to analysis to ensure Congo Mcc was obtained by the protein at in aggregation buffer mm pH at 37 °C with for 15 Samples were and incubated with Congo for 15 min at 37 °C. was from to nm with a resolution of 1 nm using a The of was obtained by the of Mcc from the of Mcc in the of The of was obtained by the of the buffer from the of in buffer in the of amyloid formation by Mcc samples were incubated with for 15 min at 37 °C and centrifuged at × g for min at The absorbance of supernatants was at nm using an The of was obtained by with the absorbance of a of protein. Mcc was in the aggregation buffer to a concentration of 3 and thioflavin min of incubation at were out a The wavelength was at from to and were mm for and amyloid formation by was in the of at The wavelength was at and were mm for and is as were incubated for min at 37 °C under with the of proteinase Applied The was by the samples at °C for min in buffer the of soluble and Mcc to of PK, purified Mcc was at 15 in culture medium 100 mm pH and either or incubated for 15 h at 37 °C with to was in aggregation buffer at a concentration of and incubated at 37 °C with The aggregation of the protein was by the absorbance at nm using a and of Mcc of Mcc at in aggregation buffer were incubated at 37 °C with for the and centrifuged at × g for min at concentration in supernatants was using a protein The amount of protein was obtained by this from the concentration of Mcc used to aggregation was obtained by the protein at in aggregation buffer at 37 °C with for 15 Mcc was obtained by the protein to for 3 at using a Mcc by the to be were in 50% In the of cells were previously by at × g for min at of samples were onto LB previously with 3 ml of LB containing × E. coli 15 h of incubation at 37 °C the activity was by the and in A. in New York1972: Scholar). Mcc by of samples to be were to of E. coli cells grown overnight in LB min of incubation at 37 °C with cells were by dilutions onto LB The activity was as a as a the activity obtained using a Mcc that was not were in buffer at °C for and were to incubated with a against a fragment of Mcc incubated with a and with Mcc into in electron of samples of soluble purified Mcc incubated in LB 100 mm PIPES, pH for 15 h at 37 °C revealed amyloid-like fibrils of and no fibrils could be in samples of the purified protein that not been incubated from those obtained in culture medium were observed upon incubation in 100 mm pH Circular studies that soluble Mcc a conformation in buffer or a one in methanol Mcc is in in both and These are with the that has been proposed for amyloid fibrils M. C. 1997; PubMed Google Scholar). from a soluble conformation into aggregates is the hallmark and the triggering in protein diseases (11Soto C. FEBS Lett. 2001; 498: 204-207Crossref PubMed Scopus (290) Google Scholar, C. Rev. 2003; PubMed Scopus Google Scholar). feature of amyloid fibrils that is used to from of aggregates is the specific binding and with as and N. J. PubMed Scopus Google Scholar, J. Biol. 130: PubMed Scopus Google Scholar). previously described 1999; PubMed Scopus Google Scholar), Mcc a change in the absorbance of with a difference and at nm The of Mcc to a of in a at nm is identical to the by amyloid proteins 1999; PubMed Scopus Google Scholar). feature of amyloid aggregates is to is the for the in from the in the disease PubMed Scopus Google Scholar, C. J. J. L. J. Biol. Full Text PDF PubMed Google Scholar). soluble Mcc was by low of PK, Mcc to this at and protein were employed to amyloid formation by Mcc over as described under The obtained are of previously described amyloid formation kinetics Annu. Rev. Biochem. 1997; PubMed Scopus Google Scholar). a phase where virtually no amyloid-like could be was by a sudden and in or protein kinetics was observed when Mcc aggregation was by the in of the not The course aggregation data suggest that Mcc, similarly to amyloid assembles into fibrils following a This a phase in the to form an that is to Annu. Rev. Biochem. 1997; PubMed Scopus Google Scholar). a model that the phase could be by adding or this soluble Mcc was incubated in the of previously Mcc, and protein aggregation was by the phase was by with the incubation of the soluble protein these of mature fibrils that are not as as a was performed using fibrils of of fibrils in a of the phase indicating that was in the fibrils and the number of for as we described previously for the of the prion protein B. C. 2001; PubMed Scopus Google Scholar). In addition, this amyloid formation model that the concentration of soluble Mcc to be over a to as concentration to This concentration also to the concentration of protein that in when amyloid formation is and the system is at Annu. Rev. Biochem. 1997; PubMed Scopus Google Scholar). that only Mcc can be from a of incubated that under these the concentration is or this concentration is a is for to the formation of a that protein In of Mcc into by a of Mcc activity and amyloid formation was by following over the of soluble or Mcc to kill The activity of Mcc and h of incubation was by the revealed a loss of of the activity upon aggregation In addition, the activity was by the number of cells to Mcc incubated for The that the activity was during a of was by a sudden and loss of activity The kinetics of the change in activity closely the kinetics of amyloid formation as shown in that the loss of activity was related to protein out the that the loss of activity was because of protein we the amount of protein and h of shown in and no difference was observed in the amount of Mcc and The of the of Mcc upon incubation was also Mcc to for h and to this correlates with the loss of activity shown in formation of amyloid is by of we from these that Mcc toxic activity upon The of Mcc in with culture of bacteria coli was by at 600 nm and the Mcc activity in the medium The activity was found to during growth to highest at the of the exponential phase of growth and to drop and at virtually undetectable levels during the stationary phase. This is not to our experimental involving a E. coli an identical of activity was previously reported in the K. pneumoniae RYC492 where both active and of Mcc were observed in the exponential and stationary phases of (3de Lorenzo V. Martinez J.L. Asensio C. J. Gen. Microbiol. 1984; 130: 391-400PubMed Google Scholar, 9de Lorenzo V. J. Antibiot. 1985; 38: 340-345Crossref PubMed Scopus (16) Google Scholar). an for this been amyloid was related to this sudden loss of Mcc the in vivo formation of amyloid was by and electron an of culture samples or not with the amount of Mcc was found to during cell no Mcc could be to h of culture. a was observed h and was in subsequent with the reported loss of activity. In with our in vitro data a Mcc activity and to these in vivo suggest that the formation of amyloid also occurs in a bacterial culture and that it the activity of the protein. study the Mcc aggregation and we performed culture samples to the amount of soluble and protein at time, as described under The that most Mcc to be soluble to virtually no soluble protein could be at h and These are in with was observed in where Mcc was found to insoluble several of incubation This finding is in with the that amyloid formation is responsible for the reported loss of Mcc activity. Finally, to that amyloid-like fibrils were formed in vivo, samples of E. coli culture grown for 15 h were by electron fibrils those obtained in vitro were observed and no could be in samples of E. coli that not R. Baeza M. Corsini G. Hetz C. Strahsburger E. Castillo J.A. Vergara C. Monasterio O. Mol. Microbiol. 2001; 42: 229-243Crossref PubMed Scopus (66) Google Scholar) culture grown for 15 h and In addition, no fibrils could be observed in samples of E. coli culture grown for h only not is with the finding that Mcc is soluble and to at this The misfolding, aggregation, and tissue of proteins into amyloid is a hallmark process in a of and protein diseases (11Soto C. FEBS Lett. 2001; 498: 204-207Crossref PubMed Scopus (290) Google Scholar, 12Dobson C.M. Trends Biochem. Sci. 1999; 24: 329-332Abstract Full Text Full Text PDF PubMed Scopus (1708) Google Scholar, 13Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar, C. Rev. 2003; PubMed Scopus Google Scholar). that the formation of and is the triggering in these including Alzheimer, Parkinson, and prion diseases as as These findings have led to the that amyloid formation is an process in in we have shown that amyloid formation also occurs in prokaryotes and is involved in modulating the biological activity of the biochemical, structural, morphological, and properties of the Mcc aggregates are identical to those observed in the disease protein In addition, the mechanism of amyloid formation following a process and the formation of are also hallmark properties of protein aggregation in the disease conditions. be as a process associated with diseases in but be as a conserved mechanism to the biological of diverse proteins. The of action of Mcc has been shown to involve the formation of pores in the cytoplasmic membrane of target bacteria. purified Mcc was found to and the E. coli cytoplasmic membrane (5Destoumieux-Garzon D. Thomas X. Santamaria M. Goulard C. Barthelemy M. Boscher B. Bessin Y. Molle G. Pons A.M. Letellier L. Peduzzi J. Rebuffat S. Mol. Microbiol. 2003; 49: 1031-1041Crossref PubMed Scopus (66) Google Scholar, 6de Lorenzo V. Pugsley A.P. Antimicrob. Agents Chemother. 1985; 27: 666-669Crossref PubMed Scopus (66) Google Scholar) and to form ion channels in in vitro R. Wilkens M. Vergara C. Cecchi X. Monasterio O. FEBS Lett. 1993; 321: 145-148Crossref PubMed Scopus (61) Google Scholar). Mcc is not only active against bacteria but was also found to and in cell C. Lagos R. Sci. S. A. 2002; PubMed Scopus Google Scholar). The finding that Mcc is able to cell and into amyloid fibrils is and could a model by to study the mechanism of of protein the in protein diseases is that the toxic species be that may cell damage by B. Annu. Rev. 2003; PubMed Scopus Google Scholar). pores have been proposed to be responsible for cell in and diseases J. M. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, D. 2002; PubMed Scopus Google Scholar). has been proposed to be involved in as amyloid was found to by a mechanism M. 2002; PubMed Scopus Google Scholar). and were reported to the from bacteria as D. 2002; PubMed Scopus Google Scholar, A.P. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), that a could be responsible for toxic membrane the formation of amyloid-like fibrils of bacterial not been reported and this was only a The oligomerization of the soluble Mcc and of the toxic that are formed in target as as with the that are during the amyloid formation are under in our The finding that Mcc aggregation into amyloid fibrils is associated with a loss of toxicity against target bacteria the that mature amyloid fibrils in may not be toxic but may be as or as a mechanism to the toxic B. Annu. Rev. 2003; PubMed Scopus Google Scholar). Mcc amyloid formation in vivo is a process involving post-translational modifications or interactions with proteins is an that to be Mcc activity has been reported to be under growth by the post-translational to of a to a of X. D. Peduzzi J. C. A. N. Goulard C. L. R. Rebuffat S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the mechanism by Mcc is active one be that this toxic and/or the formation of harmless mature amyloid fibrils. it has been reported that a of post-translational modifications could the of amyloid proteins to into amyloid fibrils M.A. G. 2002; 23: PubMed Scopus Google Scholar). proteins and have also been shown to or inhibit amyloid formation by proteins C. Mol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). a that is naturally produced by has been reported to inhibit the activity of Mcc C. Lagos R. FEMS Microbiol. Lett. PubMed Google Scholar). this effect is related or not with amyloid formation to be Our findings with Mcc that protein aggregation into amyloid fibrils be a property of proteins not associated to a may be by of specific of aggregates by the of the proteins involved M. E. F. F. L. N. G. C.M. M. 2002; PubMed Scopus Google Scholar). in with where the protein aggregates in an in the of Mcc, bacteria these properties to kill target cells as of a physiological process that formation was previously reported in bacteria when produced by E. coli were shown to of amyloid fibrils R. J. M. S. 2002; PubMed Scopus Google Scholar). Mcc, are not involved in toxicity but to have related to the of and The of amyloid in the biological activity of a bacterial protein suggests that formation of these aggregates is an evolutionarily conserved property of proteins that is not only in but also be used for specific physiological We and Monasterio for and