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

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

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

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

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

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
´

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

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
´
a-Fruito
´
s et al.
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,

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-

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

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

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
´

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