Modeling amyloids in bacteria
© Villar-Pique and Ventura; licensee BioMed Central Ltd. 2012
Received: 23 December 2012
Accepted: 23 December 2012
Published: 28 December 2012
An increasing number of proteins are being shown to assemble into amyloid structures, self-seeding fibrillar aggregates that may lead to pathological states or play essential biological functions in organisms. Bacterial cell factories have raised as privileged model systems to understand the mechanisms behind amyloid assembly and the cellular fitness cost associated to the formation of these aggregates. In the near future, these bacterial systems will allow implementing high-throughput screening approaches to identify effective modulators of amyloid aggregation.
The aggregation of proteins into amyloid structures is the triggering event on the onset of a growing number of human disorders, from neurodegenerative diseases as Alzheimer, Parkinson, Huntington or transmissible spongiform encephalopathies to non-neurodegenerative systemic and localized amyloidosis, as senile systemic amyloidosis or type II diabetes. In addition, it is now clear that different organisms, from virus to humans, exploit the special architecture of amyloids for functional purposes, such as cellular invasion or hormone storage. Therefore, protein aggregation has emerged from a neglected area of protein science to a central issue in biology and biomedicine.
Many biochemical pathways, from DNA replication to protein degradation, have been modeled first in bacteria. However, despite it has been long recognized that heterologous protein expression in bacterial cell factories results often in the formation of insoluble deposits composed essentially by the target protein[3, 4], only recently some groups have dared to exploit this well-characterized phenomena to model amyloid formation[5, 6]. This delay resulted mainly from the fact that these aggregates, known as inclusion bodies (IBs), were traditionally considered unstructured protein particles only useful to obtain denatured protein for refolding purposes. This old framework has changed into a new scenario where intracellular aggregation in bacteria is providing important clues on the molecular determinants of amyloid formation and its remediation[7, 8].
A first inflection in the field came along with early reports describing a selective molecular structure inside IBs. It has been found a substantial similitude between the properties of these bacterial aggregates and the pathological fibrils linked to amyloidosis. On one side, IBs generally bind thioflavin T and Congo Red, the typical amyloid dyes, and display seeding ability in the fibrillar assembly of homologous monomers[9, 10]. On the other side, low resolution techniques, such as infra-red spectroscopy, circular dichroism or X-ray difracction, denote the presence of signals corresponding to tightly packed intermolecular β-sheets, similar to those in amyloid fibrils[9–11]. Importantly, these findings come from independent studies using completely different protein models, not related in sequence or structure, thus suggesting that the amyloid signature might be a generic feature of bacterial aggregates. Furthermore, high resolution approaches, such as hydrogen/deuterium exchange by NMR or solid-state NMR have been used to study different bacterial IBs, defining their molecular structure at the residue level. These analysis prove that, at least for amyloidogenic proteins, bacterial IBs and fibrils share the same amyloid core. However, they also show that part of the polypeptide sequence or, alternatively, a fraction of the molecules remain disordered and/or in native-like conformations inside these aggregates[11, 12]. Contrary to the previous assumption that IBs were totally inactive, the presence of native-like structure endorse IBs with a certain degree of biological activity[13–17]. This observation has opened the door to the use of bacteria as small factories to produce promising functional materials and catalysts, boosting the investigation of the structural and functional properties of IBs[18–23].
It is now clear that they are the oligomeric assemblies populating the fibrillation pathway and not the mature fibrils that exert the main cytotoxic effect in conformational disorders. The structures of these oligomeric intermediates states have been the subject of debate for many years. The similarity between IBs and amyloids has arisen a critical question: do IBs exert a cytotoxic effect analogous to that recurrently observed for fibrils? The answer is: Yes, the addition of purified bacterial IBs to neuronal cultured cells produces a loss in cell viability equal to that promoted by the same concentration of amyloid material[24, 25]. Bacterial IBs contain small heat shock proteins (sHSPs), which are highly homologous to those found in the aggregates of the brains of patients suffering different neuronal pathologies. It has been proposed that in the brain these sHSPs might break down amyloid fibril structure, resulting in the accumulation of toxic oligomeric species. The observation that the neurotoxicity of IBs correlates with the amount of oligomeric assemblies and chaperones in these aggregates and the possibility to identify at the residue level the determinants of this effect are expected to provide new molecular insights on the structures of the deleterious species in amyloid assemblies. Thus, protein overexpression in bacterial cell factories, by mimicking the conditions in the cell under stress, will likely allow to address aspects of amyloid biology that are otherwise technically impossible to study in more complex contexts.
Prion proteins are a particularly interesting and dangerous type of amyloids, since their aggregated states have self-perpetuating ability and thus become infectious. Het-s, from the fungus Podospora anserina, was the first prion protein whose bacterial IBs were shown to display amyloid-like properties[12, 26]. The differential trait of these aggregates emerged when they were transfected into prion-free fungal strains, as they promoted prionic conversion at levels comparable to those induced by homologous amyloid fibrils. This result has been later corroborated in the case of the yeast prion Sup35. The IBs of this protein have been used to induce the prion phenotype in prion-free yeast strains, with the novel evidence that the infectivity rate can be easily modulated by tuning the environmental conditions during the formation of IBs. When instead of being expressed intracellularly, this protein is directed to the secretory pathway, the aggregates are formed in the cell surface of bacteria, but they are also able to template the conformational prionic change. These recent observations provide perhaps the best confirmation that the IBs molecular structure highly resembles to the fine architecture of amyloid fibrils, in such a way that even the infectious properties of amyloids, which depend on very specific conformational properties, are conserved in the two type of aggregates. This evidence bears important implications for the use of bacteria to model amyloids, since prion-like behaviour is currently receiving preferential attention in the field, due to the growing realisation that protein-based infection may be behind frequently occurring neurodegenerative disorders such Alzheimer’s and Parkinson’s diseases.
The increasing medical and economic impact of aggregation-linked diseases in our society has fueled the development of methods to identify chemical compounds that can interfere with amyloidogenic pathways, having thus therapeutic potential to treat or prevent these disorders. Generally, these assays, used by many biopharma companies, are cumbersome, lack reproducibility, use expensive synthetic peptides and are performed in physiologically non-relevant contexts. Several labs are focusing their efforts towards bacterial systems to overcome these limitations. In this context, fluorescent tag reporters of aggregation have been employed in bacteria to measure in a straightforward manner the amyloid assembly rate, as the final fluorescence of the aggregate is the result of a kinetic competition between folding and aggregation[15, 30, 31]. Any compound that enhances or inhibits one of these two competing reactions can be easily detected by spectrofluorometry. This property has been exploited recently both in living bacteria and in vitro, using purified IBs, to implement high-throughput, 96-well-plate based, assays able to identify and characterize novel amyloid modulators in large compound libraries[32, 33]. As stated above, the amyloid nature of bacterial aggregates can be assessed using dyes such as Thioflavin-S (Th-S), whose spectroscopic properties change upon binding to amyloid structures. This characteristic, together with the ability of the dye to enter intact cells can be used to detect in vivo the formation of amyloid structures inside bacteria. The application of flow cytometry to detect (Th-S) fluorescence has been shown to be a fast, robust, quantitative, non-invasive method to screen for the presence of in vivo intracellular amyloid-like aggregates in bacteria as well as for monitoring the effect of amyloid inhibitors in intact cells, skipping the need for a genetically encoded reporter. Although still in an early stage, it is clear that, apart from its academic interest, modeling amyloid formation in bacteria might render important economic revenues. The above examples illustrate how bacterial cell factories can be easily adapted to develop screening tools for amyloid aggregation inhibitors that will outperform the conventional screening procedures used by the industry.
Work in our lab is supported by grants BFU2010-14901 from Ministerio de Ciencia e Innovación (Spain) and 2009-SGR 760 from AGAUR (Generalitat de Catalunya). SV has been granted an ICREA ACADEMIA award (ICREA).
- Fernandez-Busquets X, de Groot NS, Fernandez D, Ventura S: Recent structural and computational insights into conformational diseases. Curr Med Chem. 2008, 15: 1336-1349., 10.2174/092986708784534938View ArticleGoogle Scholar
- Invernizzi G, Papaleo E, Sabate R, Ventura S: Protein aggregation: mechanisms and functional consequences. Int J Biochem Cell Biol. 2012, 44: 1541-1554., 10.1016/j.biocel.2012.05.023View ArticleGoogle Scholar
- Sorensen HP, Mortensen KK: Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact. 2005, 4: 1-, 10.1186/1475-2859-4-1View ArticleGoogle Scholar
- Jurgen B, Breitenstein A, Urlacher V, Buttner K, Lin H, Hecker M, Schweder T, Neubauer P: Quality control of inclusion bodies in Escherichia coli. Microb Cell Fact. 2010, 9: 41-, 10.1186/1475-2859-9-41View ArticleGoogle Scholar
- Ventura S: Sequence determinants of protein aggregation: tools to increase protein solubility. Microb Cell Fact. 2005, 4: 11-, 10.1186/1475-2859-4-11View ArticleGoogle Scholar
- Ventura S, Villaverde A: Protein quality in bacterial inclusion bodies. Trends Biotechnol. 2006, 24: 179-185., 10.1016/j.tibtech.2006.02.007View ArticleGoogle Scholar
- de Groot NS, Sabate R, Ventura S: Amyloids in bacterial inclusion bodies. Trends Biochem Sci. 2009, 34: 408-416., 10.1016/j.tibs.2009.03.009View ArticleGoogle Scholar
- Garcia-Fruitos E, Sabate R, de Groot NS, Villaverde A, Ventura S: Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J. 2011, 278: 2419-2427., 10.1111/j.1742-4658.2011.08165.xView ArticleGoogle Scholar
- Carrio M, Gonzalez-Montalban N, Vera A, Villaverde A, Ventura S: Amyloid-like properties of bacterial inclusion bodies. J Mol Biol. 2005, 347: 1025-1037., 10.1016/j.jmb.2005.02.030View ArticleGoogle Scholar
- Morell M, Bravo R, Espargaro A, Sisquella X, Aviles FX, Fernandez-Busquets X, Ventura S: Inclusion bodies: specificity in their aggregation process and amyloid-like structure. Biochim Biophys Acta. 2008, 1783: 1815-1825., 10.1016/j.bbamcr.2008.06.007View ArticleGoogle Scholar
- Wang L, Maji SK, Sawaya MR, Eisenberg D, Riek R: Bacterial inclusion bodies contain amyloid-like structure. PLoS Biol. 2008, 6: e195-, 10.1371/journal.pbio.0060195View ArticleGoogle Scholar
- Wasmer C, Benkemoun L, Sabate R, Steinmetz MO, Coulary-Salin B, Wang L, Riek R, Saupe SJ, Meier BH: Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218–289) are amyloids. Angew Chem Int Ed Engl. 2009, 48: 4858-4860., 10.1002/anie.200806100View ArticleGoogle Scholar
- Garcia-Fruitos E, Gonzalez-Montalban N, Morell M, Vera A, Ferraz RM, Aris A, Ventura S, Villaverde A: Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact. 2005, 4: 27-, 10.1186/1475-2859-4-27View ArticleGoogle Scholar
- Garcia-Fruitos E, Aris A, Villaverde A: Localization of functional polypeptides in bacterial inclusion bodies. Appl Environ Microbiol. 2007, 73: 289-294., 10.1128/AEM.01952-06View ArticleGoogle Scholar
- de Groot NS, Ventura S: Protein activity in bacterial inclusion bodies correlates with predicted aggregation rates. J Biotechnol. 2006, 125: 110-113., 10.1016/j.jbiotec.2006.02.026View ArticleGoogle Scholar
- Peternel S, Grdadolnik J, Gaberc-Porekar V, Komel R: Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb Cell Fact. 2008, 7: 34-, 10.1186/1475-2859-7-34View ArticleGoogle Scholar
- Wu W, Xing L, Zhou B, Lin Z: Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli. Microb Cell Fact. 2011, 10: 9-, 10.1186/1475-2859-10-9View ArticleGoogle Scholar
- Garcia-Fruitos E: Inclusion bodies: a new concept. Microb Cell Fact. 2010, 9: 80-, 10.1186/1475-2859-9-80View ArticleGoogle Scholar
- Garcia-Fruitos E, Vazquez E, Diez-Gil C, Corchero JL, Seras-Franzoso J, Ratera I, Veciana J, Villaverde A: Bacterial inclusion bodies: making gold from waste. Trends Biotechnol. 2012, 30: 65-70., 10.1016/j.tibtech.2011.09.003View ArticleGoogle Scholar
- Peternel S, Komel R: Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells. Microb Cell Fact. 2010, 9: 66-, 10.1186/1475-2859-9-66View ArticleGoogle Scholar
- Rodriguez-Carmona E, Cano-Garrido O, Seras-Franzoso J, Villaverde A, Garcia-Fruitos E: Isolation of cell-free bacterial inclusion bodies. Microb Cell Fact. 2010, 9: 71-, 10.1186/1475-2859-9-71View ArticleGoogle Scholar
- Vazquez E, Corchero JL, Burgueno JF, Seras-Franzoso J, Kosoy A, Bosser R, Mendoza R, Martinez-Lainez JM, Rinas U, Fernandez E, et al: Functional inclusion bodies produced in bacteria as naturally occurring nanopills for advanced cell therapies. Adv Mater. 2012, 24: 1742-1747., 10.1002/adma.201104330View ArticleGoogle Scholar
- Villaverde A, Garcia-Fruitos E, Rinas U, Seras-Franzoso J, Kosoy A, Corchero JL, Vazquez E: Packaging protein drugs as bacterial inclusion bodies for therapeutic applications. Microb Cell Fact. 2012, 11: 76-View ArticleGoogle Scholar
- Gonzalez-Montalban N, Villaverde A, Aris A: Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies. Biochem Biophys Res Commun. 2007, 355: 637-642., 10.1016/j.bbrc.2007.01.192View ArticleGoogle Scholar
- Dasari M, Espargaro A, Sabate R, del Amo JM L, Fink U, Grelle G, Bieschke J, Ventura S, Reif B: Bacterial inclusion bodies of Alzheimer's disease beta-amyloid peptides can be employed to study native-like aggregation intermediate states. Chembiochem. 2011, 12: 407-423., 10.1002/cbic.201000602View ArticleGoogle Scholar
- Sabate R, Espargaro A, Saupe SJ, Ventura S: Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion. Microb Cell Fact. 2009, 8: 56-, 10.1186/1475-2859-8-56View ArticleGoogle Scholar
- Espargaro A, Villar-Pique A, Sabate R, Ventura S: Yeast prions form infectious amyloid inclusion bodies in bacteria. Microb Cell Fact. 2012, 11: 89-, 10.1186/1475-2859-11-89View ArticleGoogle Scholar
- Sivanathan V, Hochschild A: Generating extracellular amyloid aggregates using E. coli cells. Genes Dev. 2012, 26: 2659-2667., 10.1101/gad.205310.112View ArticleGoogle Scholar
- Aguzzi A, Calella AM: Prions: protein aggregation and infectious diseases. Physiol Rev. 2009, 89: 1105-1152., 10.1152/physrev.00006.2009View ArticleGoogle Scholar
- Waldo GS, Standish BM, Berendzen J, Terwilliger TC: Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol. 1999, 17: 691-695., 10.1038/10904View ArticleGoogle Scholar
- Villar-Pique A, de Groot NS, Sabate R, Acebron SP, Celaya G, Fernandez-Busquets X, Muga A, Ventura S: The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity. J Mol Biol. 2012, 421: 270-281., 10.1016/j.jmb.2011.12.014View ArticleGoogle Scholar
- Kim W, Kim Y, Min J, Kim DJ, Chang YT, Hecht MH: A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide. ACS Chem Biol. 2006, 1: 461-469., 10.1021/cb600135wView ArticleGoogle Scholar
- Villar-Pique A, Espargaro A, Sabate R, de Groot NS, Ventura S: Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors. Microb Cell Fact. 2012, 11: 55-, 10.1186/1475-2859-11-55View ArticleGoogle Scholar
- Espargaro A, Sabate R, Ventura S: Thioflavin-S staining coupled to flow cytometry. A screening tool to detect in vivo protein aggregation. Mol Biosyst. 2012, 8: 2839-2844., 10.1039/c2mb25214gView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.