Báo cáo khoa học: Biological role of bacterial inclusion bodies: a model for amyloid aggregation potx

MINIREVIEW
Biological role of bacterial inclusion bodies: a model for
amyloid aggregation
Elena Garcı
´
a-Fruito
´
s
1–3,
*, Raimon Sabate
1,4,
*, Natalia S. de Groot
1,4
, Antonio Villaverde
1–3
and
Salvador Ventura
1,4
1 Institute for Biotechnology and Biomedicine, Universitat Auto
`
noma de Barcelona, Spain
2 Department of Genetics and Microbiology, Universitat Auto
`
noma de Barcelona, Spain
3 CIBER de Bioingenierı
´
a, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
4 Department of Biochemistry and Molecular Biology, Universitat Auto
`
noma de Barcelona, Spain
Biotechnology of bacterial inclusion

bodies; a historical view
Production of recombinant proteins in microorgan-
isms, powered in the late 1970s by the identification of
restriction enzymes, has provided much fewer products
than initially expected [1]. The ready-to-use concept of
recombinant DNA technologies has proved to be unre-
alistic and has faced severe obstacles associated with
the physiology of the host microorganism. This is
because the cellular protein factories are usually forced
to produce heterologous polypeptides, encoded in a
multicopy expression plasmid, over physiological rates
Keywords
aggregation; amyloid; FTIR; inclusion bodies;
protein folding; protein quality; recombinant
proteins
Correspondence
A. Villaverde, Institute for Biotechnology and
Biomedicine, Universitat Auto
`
noma de
Barcelona, Bellaterra, 08193 Barcelona,
Spain
Fax: +34 93 581 2011
Tel: +34 93 581 2148
E-mail:
S. Ventura, Institute for Biotechnology and
Biomedicine, Universitat Auto
`
noma de
Barcelona, Bellaterra, 08193 Barcelona,

Spain
Fax: +34 93 581 2011
Tel: +34 93 586 8956
E-mail:
*These authors contributed equally to this
work
(Received 28 January 2011, revised 18
March 2011, accepted 15 April 2011)
doi:10.1111/j.1742-4658.2011.08165.x
Inclusion bodies are insoluble protein aggregates usually found in recombi-
nant bacteria when they are forced to produce heterologous protein species.
These particles are formed by polypeptides that cross-interact through
sterospecific contacts and that are steadily deposited in either the cell’s
cytoplasm or the periplasm. An important fraction of eukaryotic proteins
form inclusion bodies in bacteria, which has posed major problems in the
development of the biotechnology industry. Over the last decade, the fine
dissection of the quality control system in bacteria and the recognition of
the amyloid-like architecture of inclusion bodies have provided dramatic
insights on the dynamic biology of these aggregates. We discuss here the
relevant aspects, in the interface between cell physiology and structural
biology, which make inclusion bodies unique models for the study of pro-
tein aggregation, amyloid formation and prion biology in a physiologically
relevant background.
Abbreviations
IB, inclusion body; PFD, prion forming domain.
FEBS Journal 278 (2011) 2419–2427 ª 2011 The Authors Journal compilation ª 2011 FEBS 2419
– a combination of facts that tend to saturate the pro-
tein synthesis machinery and activate the quality con-
trol system. Essentially, protein production processes
in bacteria (as well as in other microorganisms) suffer

from protein degradation and lack of solubility and, to
a minor extent, toxicity exerted by the product on the
cells and consequent genetic instability (including plas-
mid loss). These events occur in the context of several
cell stress responses, which depending on the nature of
the host microorganism include triggering of oxidative
stress, the unfolded protein response, the heat shock
and the stringent response and the activation of the
DNA repair SOS system [2].
Traditionally, lack of solubility and the formation of
inclusion bodies (IBs), large insoluble clusters enriched
by misfolded versions of the recombinant protein spe-
cies [3], have been the main obstacle for the smooth
consecution of production processes, aimed at high
yields of soluble, biologically active species. Believed to
be irreversibly formed and containing inactive proteins,
how to minimize IB formation in midstream has been
a matter of extensive discussion. Essentially, reducing
the growth temperature, lowering the transcription rate
and co-producing folding modulators selected from the
quality control system have been thoroughly explored
strategies [4]. Also, at the downstream level, refolding
of IB proteins has also been approached [5]. Both mid-
stream- and downstream-focused approaches have
been successful for an important number of specific
proteins but they do not offer generic solutions to the
lack of solubility in protein production.
Despite the economical relevance of IB formation
for both catalysis and biotech industries, IBs have been
in general poorly characterized. Consequently, the dis-

covery of the reversibility of IB formation [6], the gen-
eral acceptance of IBs being formed by functional
proteins [7] and the recognition of the amyloid-like
architecture of IB proteins [8] have represented dra-
matic insights in the biology of these structures that
has favoured important advances in the comprehension
of their physiological and structural nature. For
instance, the conceptual unlinking between solubility
and functional quality [9], and the fact that enhanced
protein yields result in lower quality protein species
[10,11], has permitted IBs to be explored as powerful
biocatalysts (the embedded proteins acting as immobi-
lized enzymes) [12,13]. On the other hand, the fine
and timely analysis of the amyloid architecture of IB
proteins [14,15] has led to the use of these underesti-
mated bacterial aggregates as intriguing models for the
analysis of protein–protein interactions in the context
of amyloid and prion diseases.
Dynamics of IB formation and
biological activity
Intracellular electrodense proteinaceous granules had
been observed in classical experiments when bacteria
Soluble VP1LAC + (1/10) VP1LAC IBs
Soluble VP1LAC + (1/10) TSP IBs
Soluble VP1LAC + (1/10) SPC-PI3DT IBs
Soluble VP1LAC + (1/10) HIVP IBs
Soluble LACZ
Soluble LACZ + (1/10) VP1LAC IBs
B
A

C
Fig. 1. A nucleation ⁄ polymerization self-
assembly process drives the formation of
IBs in bacteria. (A) In vivo formation of IBs
in recombinant bacteria. Aggregation-prone
versions of the recombinant protein (green)
slowly form seeding nuclei by cross-molecu-
lar stereospecific interactions. These proto-
aggregates recruit further copies of the tar-
get protein, in a process compatible with
first-order kinetics. This promotes a fast
mass growth of IBs. Non-homologous cellu-
lar or recombinant proteins (red, black) are
excluded from these seeding events. (B)
Kinetics of aggregation monitored by time-
dependent increase of turbidity at 350 nm
using soluble VP1LAC and LACZ incubated
with different IBs (figure modified from [8]).
(C) Kinetics of Ab42 peptide seeding moni-
tored through thioflavin-T fluorescence emis-
sion (figure taken from [58]). All figures have
been reproduced with permission.
Biological role of bacterial inclusion bodies E. Garcı
´
a-Fruito
´
s et al.
2420 FEBS Journal 278 (2011) 2419–2427 ª 2011 The Authors Journal compilation ª 2011 FEBS
were cultured in the presence of non-natural amino
acids. This observation, which indicated the transient

nature of protein aggregates formed by conformation-
ally aberrant proteins, was more recently repeated with
bacterial IBs [6], so far believed to be irreversible pro-
tein clusters averse to in vivo protein refolding [16].
200 nm
50 nm
200 nm
500 nm
0.2
0.4
0.6
CR
free
CR +
CR +
CR +
HET-s (001–289)
HET-s (157–289)
Absorbance
HET-s (218–289)
375
475 575 675
0.0
Wavelength (nm)
0.3
CR +
CR +
HET-s (001–289)
0.8
1.0

1600162016401660
1680
1700
0.0
0.2
0.4
0.6
Absorbance
0.001
1628 cm
–1
Inter β-sheet band
1600
1620
1640
1660
1680
1700
–0.005
–0.003
–0.001
Wavenumber (cm
–1
)Wavenumber (cm
–1
)
Second derivative
50 nm
375
475

575
675
–0.1
0.0
0.1
0.2
CR +
Wavelength (nm)
Differential absorbance
HET-s (157–289)
HET-s (218–289)
≈
540 nm
50 nm
15
190
200
210 220 230 240 250
–10
–5
0
5
10
Wavelength (nm)
θ (mdeg·cm
2
·dmol
–1
)
1.0

0
50 100
150
200
0.0
0.2
0.4
0.6
0.8
1.0
Without IBs
HET-s Full length IBs
HET-s (157–289) IBs
HET-s PFD IBs
[HET-s PFD] = 10 μM
Time (min)
Aggregated fraction
A
C
E
G
I
J
L
M
K
F
D
H
B

Fig. 2. Presence of amyloid-like structures in the IBs formed by the prion protein HET-s from P. anserina. (A) HET-s PFD IBs from E. coli
observed by cryo-electron microscopy in intact E. coli cells. (B) Transmission electron micrograph of negatively stained purified HET-s PFD
IBs. (C), (D) HET-s IB structure before (C) and after (D) 30 min of proteinase K digestion monitored by transmission electron microscopy,
showing the apparition of fibrillar structures. (E)–(H) Congo Red (CR) binding to different HET-s IBs monitored by UV ⁄ vis spectroscopy and
staining and birefringence under cross-polarized light using an optical microscope: (E), (F) CR spectral changes in the presence of different
HET-s IBs; (E) changes in k
max
and intensity in CR spectra in the presence of HET-s IBs; (F) difference absorbance spectra of CR in the pres-
ence and absence of IBs showing in all cases the characteristic amyloid band at  540 nm; (G) HET-s PFD IBs stained with CR and
observed at 40· magnification and (H) the same field observed between crossed polarizers displaying the green birefringence characteristic
of amyloid structures. (I)
13
C–
13
C solid-state NMR correlation spectrum (proton-driven spin-diffusion with a mixing time of 50 ms) of purified
HET-s PFD IBs (blue) compared with a spectrum of in vitro HET-s PFD amyloid fibrils (red) recorded under identical conditions. All the signals
assigned for the purified fibrils were also observed in the spectrum of the IBs. The insets demonstrate that no significant changes in the
chemical shifts appear and that the linewidths of the two samples are virtually identical. The individual spectra were recorded at a 1H fre-
quency of 600 MHz (static field B
0
= 14.9 T), 10 kHz magic angle spinning. (J)–(L) Secondary structure of HET-s PFD IBs: (J) CD spectra,
and (K), (L) FTIR absorbance and second derivative spectra in the amide I region of HET-s PFD spectra showing the characteristic spectral
bands of b-sheet conformations. (M) Seeding-dependent maturation of HET-s PFD amyloid growth. The aggregation reaction was seeded
with HET-s full length, HET-s (157–289), HET-s PFD, Ab40 or Ab42 IBs. The fibrillar fraction of HET-s PFD is represented as a function of
time. The formation of HET-s PFD amyloid fibrils is accelerated only in the presence of HET-s IBs. (A), (B) and (I) adapted, with permission,
from [60]; (C)–(H) and (J)–(M) adapted, with permission, from [15].
E. Garcı
´
a-Fruito
´

s et al. Biological role of bacterial inclusion bodies
FEBS Journal 278 (2011) 2419–2427 ª 2011 The Authors Journal compilation ª 2011 FEBS 2421
This is indicative of cellular activities acting on these
protein aggregates, including release to the soluble cell
fraction but also proteolytic events [6,17,18] that might
promote degradation of IB proteins in situ [19]. In
addition, for protein species that are found in both sol-
uble and insoluble cell fractions, the conformational
quality and biological activity of IB embedded proteins
evolve in parallel with those of the soluble counter-
parts, under different environmental conditions affect-
ing folding, such as temperature and chaperone
availability [20,21]. Therefore, IB proteins appear not
to be excluded from quality control [22], in which a
complex network of chaperones and proteases survey
the folding status of cellular proteins [23], soluble but
also insoluble.
In agreement with this concept, the main Escherichia
coli chaperone DnaK (a holding agent and a foldase
and disaggregase), is almost exclusively found, in IB-
producing bacteria, attached at the IB surface, while
the foldase GroEL is present within the IB core [24].
DnaK, which participates in the in vivo refolding of
bacterial thermal aggregates [25,26], appears to be
highly active on bacterial IBs [20,27,28]. In fact, we
have recently shown that the chaperone DnaK pro-
motes protein extraction from bacterial IBs but that
this event is intimately associated with proteolysis
[10,11]. This explains the reduction of protein yield
eventually observed during co-production of this chap-

erone and others [10], as a side effect of this strategy
[29] addressed to improve the solubility of recombinant
proteins. Interestingly, the specific dependence of the
DnaK-mediated stimulation on bacterial chaperones
makes this chaperone very useful for co-production in
eukaryotic systems [30].
The simultaneous surveillance of soluble and IB pro-
tein species by bacterial chaperones and proteases indi-
cates the occurrence of similar targets in both protein
versions and strongly suggests a highly dynamic transi-
tion between the two forms. In fact, aggregation and
disaggregation seem to be simultaneous events in
actively producing recombinant bacteria [16], while dis-
aggregation will be highly favoured in the absence of
protein synthesis [6]. Such a bidirectional protein tran-
sit between the cells’ virtual fractions (soluble and
insoluble [22]) accounts for the unexpected and
recently determined abundance of soluble aggregates in
recombinant cells [31]. These particles, either globular
or fibrillar, might be intermediates in the in ⁄ out IB
protein transition, or just members of the conforma-
tional spectrum that recombinant proteins can adopt
in host bacteria, irrespective of whether they are found
in soluble or insoluble cell fractions. Interestingly,
increasing evidence supports the presence of biologi-
cally active proteins embedded in IBs, indicating
that both folded and misfolded polypeptides coexist
in these proteinaceous aggregates [32]. Regarding
the presence of functional protein in such aggregates,
different enzyme-based IBs have been successfully

tested as catalysts of different bioprocesses [33]. Galac-
tosidases [7,34], reductases [7], oxidases [35], kinases
[36], phosphorylases [37] and aldolases [38] are just
some examples of the enzymes used in aggregated form
to catalyse specific reactions, opening a promising mar-
ket in the biotechnological industry [33]. In this con-
text, other authors have also described the use of IBs
for the intracellular capture of a co-synthesized target
enzyme, obtaining IB particles with the enzyme of
interest immobilized in their surface [39].
Stereospecific interactions in protein
aggregation
Chiti and coworkers pointed out that the intrinsic
physicochemical properties of an amino acid sequence,
such as hydrophobicity, secondary structure propensity
and charge, can determine the aggregation behaviour
of a given polypeptide [40,41]. Many examples support
the correlation between protein aggregation tendency
and amino acid sequence, and it is also possible to
identify the aggregation-prone regions of polypeptides
using software such as aggrescan [42] or tango [43].
Protein aggregation can be understood as an anoma-
lous type of protein–protein interaction. As for native
interactions, the attainment of ordered aggregated
structures requires the establishment of stereospecific
intermolecular contacts. Accordingly, it has been
observed that both bacterial [8] and mammalian pro-
tein aggregates are formed through a conserved, selec-
tive and sequence-specific process. Specificity during
protein aggregation is best exemplified by the nucle-

ation-driven polymerization of proteins into amyloid
aggregates [44], a mechanism reminiscent of that
occurring in crystallization processes [45]. Mature amy-
loid fibrils possess the faculty to accelerate the forma-
tion of new fibrils by acting as a nucleus that seeds the
growth of fibrillar structures [46]. However, molecular
recognition between aggregated and soluble proteins
only occurs when they share a high sequence similar-
ity. The requirement for stereospecific interactions
during protein aggregation would explain why disease-
linked amyloid deposits are composed almost exclu-
sively of the pathogenic protein [47] and bacterial IBs
are highly enriched in the target recombinant protein
[22]. The distribution of side chains in the sequence,
such as occurs in protein folding, plays a pivotal role
in determining the conformational properties of the
Biological role of bacterial inclusion bodies E. Garcı
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2422 FEBS Journal 278 (2011) 2419–2427 ª 2011 The Authors Journal compilation ª 2011 FEBS
aggregated state and the way in which this supramolec-
ular ensemble is reached from the initial soluble state.
This control is so exquisite that a protein and its
backward version (a protein with exactly the same
succession of side chains but with a reverted backbone)
do not cross seed each other and form aggregates dis-
playing different conformational and functional prop-
erties [48]. However, apart from the primary sequence,

the particular structural and thermodynamic properties
of proteins modulate their deposition in physiologically
relevant conditions, making it difficult to predict the
effective aggregation propensities of polypeptides in
cellular environments.
Protein aggregation into amyloid
structures
Protein aggregation can occur from multiple structural
conformations such as intrinsically disordered polypep-
tides, oligomeric species or globular proteins [47,49].
The macromolecular assemblies formed by these pro-
teins are all sustained by intermolecular interactions
but their arrangement and specificity define the degree
of order in the structure of the final aggregate. The
energy landscape of protein aggregation is rough and
complex, comprising both highly energetic amorphous
deposits and well-ordered amyloid fibrils of lower
energy than the native structure of the protein [50,51].
Amorphous aggregates can be formed rapidly by sim-
ple precipitation of the protein, whereas ordered fibril-
lation requires specific intermolecular contacts, the
formation of which is strongly influenced by the pro-
tein local environment [47,52].
The number of identified amyloid-forming proteins
increases each year. These fibrillar structures were
initially discovered in human tissues of patients
suffering from amyloidoses such as Alzheimer’s or
Parkinson’s diseases. The study of these deposits has
shown that mature fibrils can be less cytotoxic than
the intermediary forms in the aggregation pathway

suggesting that the amyloid structure might play in
fact a protective function [47,53]. Importantly, amy-
loid conformations are not only associated with path-
ological conditions but are also exploited by Nature
to execute important regulatory, structural and
genetic functions [54,55]. In fact, the ability to form
amyloid assemblies has been suggested to be a gen-
eric protein property [47,56] and, as we shall see in
the next sections, a conformation accessible to struc-
turally and sequentially unrelated proteins upon
recombinant expression [51].
Despite their diverse origin, all amyloid structures
share common morphological characteristics: straight
unbranched fibrils 7–12 nm in diameter made up of
two to six protofilaments 2–5 nm in diameter with a
cross-b-sheet spine [47,57] in which each polypeptide
chain is structured into b-strands and each b-strand is
arranged perpendicular to the long axis of the fibril.
This arrangement allows a tightly packed quaternary
structure sustained mainly by generic hydrogen bonds
and hydrophobic contacts [58], explaining why, in spite
of the high sequential specificity driving amyloid for-
mation pathways, any sequence able to be accommo-
dated in a b-sheet conformation can, potentially, reach
the amyloid state [51,56].
Amyloid-like properties of bacterial IBs
The architecture and mechanisms of IB formation in
bacteria have remained unexplored for years. However,
important insights in this field have lately emerged.
Although IBs were conventionally described as disor-

dered aggregates being formed by non-specific interac-
tions of exposed hydrophobic surfaces, an increasing
amount of evidence is showing that in fact IBs are highly
ordered protein deposits formed through a process simi-
lar to that observed during amyloid deposition [8,14].
Just as occurs for amyloids, IB formation is driven by
intermolecular interactions occurring through homolo-
gous protein patches in a nucleation-dependent manner
(Figure 1) [8]. On the one hand, a study published by
Carrio
´
and coworkers demonstrates that target recombi-
nant protein aggregation in vitro is a tightly regulated
phenomenon, and recombinant proteins preferentially
associate with themselves rather than with other pro-
teins in the environment in a dose-dependent way [8].
On the other hand, an in vivo study performed using
fluorescence resonance energy transfer shows that, when
co-producing two different recombinant proteins in the
complex bacterial cytoplasmic environment, the distri-
bution of the two proteins in the formed IBs is also
tightly regulated through specific contacts, each protein
being specifically localized in a different region of the
aggregate depending on its sequence. Therefore, it is not
surprising that, in spite of the IBs’ amorphous macro-
scopic appearance, recently different groups have con-
verged to demonstrate unequivocally the effective
existence of amyloid-like structures inside bacterial
aggregates [14,59]. Accordingly, relative to the native
conformation, proteins embedded in IBs appear to be

enriched in b-sheet secondary structure elements dis-
playing the minimum at 217 nm characteristic of this
conformation in the far-UV circular dichroism spectra
(which can be displaced slightly to higher wavelengths
due to the stacking of aromatic residues) as well as a
band at 1620–1630 cm
)1
in the infrared spectra, typical
E. Garcı
´
a-Fruito
´
s et al. Biological role of bacterial inclusion bodies
FEBS Journal 278 (2011) 2419–2427 ª 2011 The Authors Journal compilation ª 2011 FEBS 2423
of the tightly bound intermolecular b-strands in amyloid
structures [8,15,59–61], and X-ray diffraction patterns
with meridional (4.8 A
˚
) and equatorial (10–11 A
˚
) reflec-
tions compatible with the presence of a cross- b structure
[59]. In addition, amyloid-specific dyes like Congo Red
or thioflavin-T and S bind to bacterial IBs with similar
affinity to the affinity they exhibit for amyloid structures
[8,15,59–61], confirming a high degree of conforma-
tional similarity between the two types of aggregates. As
in amyloid fibrils, IBs display regions with high resis-
tance against proteolytic attack, probably correspond-
ing to a preferentially protected b-sheet core. The

presence of fibrillar structures with amyloid-like mor-
phology in IBs has been observed directly or after con-
trolled proteolytic digestion by transmission electronic
microscopy, cryo-electron microscopy [59,62] and
atomic force microscopy [15,60,61]. In addition, IBs
formed by amyloid proteins display the capacity to seed
and accelerate in a highly specific manner the formation
of amyloid structures by their soluble and monomeric
forms [15,60–62] (see Figure 2).
Aggregated structures are non-crystalline and insolu-
ble and are therefore not amenable to X-ray crystallog-
raphy and solution NMR, the classical tools of
structural biology, making it difficult to characterize
the fine structure of these assemblies at the residue
level, even when they display a high degree of internal
order [63]. Quenched hydrogen ⁄ deuterium exchange
with solution NMR allows the identification of sol-
vent-protected backbone amide protons involved in
hydrogen bonds. Interestingly enough, three recent
studies using this approach to study the IBs formed by
different protein models convincingly demonstrate
the presence of sequence-specific motifs displaying
protection compatible with a cross-b conformation
[59,61,62]. High resolution information on the confor-
mation of proteins in the aggregated state can be
obtained by solid-state NMR, a technique that has
allowed amyloid fibrils to be modelled at atomic reso-
lution [64]. Two recent works have exploited solid-state
NMR to address the fine structure of the IBs formed
by two amyloidogenic proteins, the HET-s prion form-

ing domain (PFD) of the fungus Podospora anserina
and the Alzheimer’s amyloid b peptide (Ab). The com-
parison between the signals of the in vitro formed amy-
loid fibrils and the corresponding IBs indicates the
existence of regions with highly similar structural dis-
position in these aggregates, in particular in the case of
HET-s PFD where the NMR signals of the two types
of aggregates overlap significantly [61,62,65]. Overall,
it appears that the formation of amyloid-like assem-
blies is an omnipresent process in both eukaryotic and
prokaryotic cells.
Infectious conformations in bacterial
IBs
Prions represent a particular subclass of amyloids in
which the aggregation process becomes self-perpetuat-
ing in vivo and thus infectious [14]. The possibility that
the bacterial IBs formed by recombinant prion pro-
teins could display infectious properties has important
implications. On the one hand, bacteria might become
a simple and tunable in vivo system to study the deter-
minants of prion formation. On the other hand, bacte-
rial IBs would be an ideal system for the production
of significant amounts of infectious proteins ready to
use for cell biology studies, without the requirement of
the highly inefficient in vitro unfolding ⁄ refolding and
controlled aggregation procedures necessary to obtain
proteins in transmissible conformations. Therefore, the
infectious capacity of prion proteins deposited in bac-
teria during recombinant production is receiving
increasing attention. Meier and co-workers have tested

the ability of HET-s PFD IBs purified from E. coli to
infect strains of its natural host, P. anserina, using dif-
ferent protein transfection methods [62]. Strains trans-
fected with HET-s PFD IBs acquired the [Het-s] prion
phenotype at a frequency comparable with that
obtained with HET-s PFD infectious fibrils assembled
in vitro, confirming that bacterial HET-s PFD IBs dis-
play a high prion infectivity [62]. In contrast, the IBs
of a heterologous amyloid protein were not infectious.
The yeast prion protein Sup35 has also been shown
recently to access an infectious structure when pro-
duced in E. coli cells [66]. These two independent
observations confirm that the content of the bacterial
cytoplasm can support the formation of infectious con-
formations and suggest that bacterial aggregation
might become a generic model system to understand
prion biology.
Bacteria as model systems to study
protein aggregation
In addition to being the default protein production cel-
lular factories, bacterial cells are valuable systems to
understand the integration of metabolic, regulatory
and structural features in living cells. The similarities
between bacterial aggregates and the deposits formed
in higher organisms in pathological processes like amy-
loid fibrils, nuclear inclusions and aggresomes [67,68]
provide a unique opportunity to dissect the molecular
pathways triggering these disorders in a simple, yet
physiologically relevant, organism. Accordingly, E. coli
has been used to study the link between protein aggre-

gation and ageing [69], the role of the highly conserved
Biological role of bacterial inclusion bodies E. Garcı
´
a-Fruito
´
s et al.
2424 FEBS Journal 278 (2011) 2419–2427 ª 2011 The Authors Journal compilation ª 2011 FEBS
protein quality machinery on the conformational prop-
erties of aggregated states [20,67], the effect of the pro-
tein sequence on in vivo aggregation kinetics [41], the
influence of extrinsic factors like temperature on pro-
tein aggregation properties [21,70] or the control of
polypeptide solubility in biological environments by
the thermodynamic [71] and kinetic stability of pro-
teins [72]. In addition, the possibility of labelling
aggregation-prone proteins with natural [41] or artifi-
cial fluorophores [73] allows in vivo deposition path-
ways to be tracked in real time and compounds able
to block the self-assembly process to be identified [74].
Finally, bacteria provide a means to trap and study
the highly toxic, unstable and transient intermediates
in the fibrillation reaction, illuminating one of the
more obscure but crucial steps in amyloid fibril forma-
tion [61].
Acknowledgements
We appreciate the financial support from MICINN
(BFU2010-17450 and BFU2010-14901), AGAUR
(2009SGR-00108 and 2009SGR-00760) and CIBER de
Bioingenierı
´

a, Biomateriales y Nanomedicina (CIBER-
BBN, Spain), an initiative funded by the VI National
R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Con-
solider Program, CIBER Actions and financed by the
Instituto de Salud Carlos III with assistance from the
European Regional Development Fund. A.V. and S.V.
have been distinguished with an ICREA Academia
award.
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