This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule.. Our experiments indicate that EspA has the potentia
Trang 1component of the Escherichia coli O157:H7 type III
secretion system
Tomoaki Kato1,2, Daizo Hamada2, Takashi Fukui2, Makoto Hayashi1, Takeshi Honda3,
Yoshikatsu Murooka1and Itaru Yanagihara2
1 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
2 Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Japan
3 Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Japan
Enterohaemorrhagic and enteropathogenic Escherichia
coli (EHEC and EPEC, respectively) cause outbreaks
of serious diarrhoea These bacteria express type III
secretion systems [1], which consist of various protein
components encoded at the locus of enterocyte
efface-ment, LEE [2–5] To date, type III secretion systems
have been identified in more than 20 pathogenic
bac-terial species [6] The type III secretion system is a
fila-mentous multiprotein complex that assembles across
the bacterial and host cell surfaces For EHEC and
EPEC, such complex structures, which include the pro-teins, EspA, EspB, EspD [7,8], probably permit direct delivery of effector proteins, such as, Tir [9–11], EspF [12,13], EspG [14] and Orf19 [15], into the host cell [16]
EspA is a major component of this large, transiently expressed, filamentous surface organelle [17,18] EspA oligomerization may be mediated by interactions between coiled-coil regions of individual EspA mole-cules [19] in a manner similar to that of falgellin
Keywords
ANS binding; CD; FT-IR; partially unfolded;
sedimentation equilibrium
Correspondence
D Hamada, Department of Developmental
Infectious Diseases, Research Institute,
Osaka Medical Center for Maternal and
Child Health, 840 Murodo, Izumi,
Osaka 594-1011, Japan
Fax: +81 725 57 3021
Tel: +81 725 56 1220
E-mail: daizo@mch.pref.osaka.jp
(Received 21 September 2004, revised
1 March 2005, accepted 1 April 2005)
doi:10.1111/j.1742-4658.2005.04697.x
pH-Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluores-cence, and sedimentation equilibrium ultracentrifugation Far- and
near-UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures An increase in ANS fluorescence intensity (in the presence of EspA) was observed at aci-dic pH; whereas, no increased ANS fluorescence was observed at pH 7.0 These results suggest the presence of a partially unfolded state Interest-ingly, urea-induced unfolding transitions, monitored by far-UV CD spectro-scopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH Although increased ANS fluorescence was observed
at pH 3.0, the urea-induced unfolding curve is similar to that found at
pH 7.0 This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing a-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular b-sheets at
pH 2.0 Our experiments indicate that EspA has the potential to spontane-ously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonic acid; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; FT-IR, Fourier transform infrared; LB, Luria–Bertani.
Trang 2molecules, which assemble to form flagella filaments
[20] The EspA-containing filamentous apparatus may
form a conduit for translocation of bacterial proteins
into host cells [21] Recently, a model of EspA
fila-ments has been built based on negative-stain EspA
electron micrographs [18] Interestingly, the model is a
helical tube with a diameter of 120 A˚, enclosing a
cen-tral channel of 25 A˚ diameter, and has an axial rise of
4.6 A˚ per subunit EspA filaments may attach to host
cells via an EspB⁄ D pore-forming complex [22] and
the EspB⁄ D complex may also specifically interact with
the host-target protein, a-catenin [23] Such a
super-structure, formed by EspA⁄ B ⁄ D and a-catenin,
facili-tates the delivery of effector proteins into host cells
[16]
Although there is information available concerning
the roles and structural properties of EspA filaments,
to date, the conformation and the thermodynamic
properties of EspA have not been characterized
In this study, we characterized certain conformational
and thermodynamic properties of EspA in solution
using spectroscopic and physicochemical techniques
Far-UV CD shows that the protein has a substantial
amount of secondary structure throughout the pH
range of 2.0–7.0 However, an analysis of
8-anilino-2-naphthyl sulfonic acid (ANS) fluorescence (in the
presence of EspA) suggests that a conformational
transition occurs between pH 3.0 and 5.0, with
expo-sure of hydrophobic protein surfaces Consistent with
this observation, urea-induced EspA unfolding
transi-tions, as followed by far-UV CD, indicate that the
folded structure is less stable at pH 2.0 A
sedimenta-tion equilibrium study shows that EspA forms
oligo-mers at pH 7.0, indicating an ability by EspA to form
filamentous structures The data now reported suggest
that EspA, at near physiological conditions, assumes
short filamentous oligomers, but dissociates into
parti-ally unfolded species at acidic pH
Results
CD
The secondary structure prediction for EspA, based on
its amino acid sequence, suggests that the protein is
predominantly a-helical in conformation (Fig 1) The
secondary structures of the recombinant EspA
pre-pared here were analysed by far-UV CD spectra In
this study, the recombinant EspA protein was prepared
under either native or denaturing conditions To clarify
whether both preparations yielded protein with similar
propertiees, we first compared the CD spectra of EspA
prepared under the different conditions
Recombinant EspA prepared from soluble fractions without unfolding the protein, at pH 7.0 and 20 C, showed a CD spectrum typical of a protein with a significant amount of secondary structure (Fig 2A) Importantly, the spectrum for the sample prepared by urea-solubilized cells was almost completely super-imposable on the above spectrum This observation
Fig 1 Secondary structure prediction for EspA based on its amino acid sequence H and E refer to a-helical and b-strand propensities, respectively.
Fig 2 CD spectra of EspA at various pH values (A) Far- and (B) near-UV CD spectra at pH 2.0 (broken line), pH 3.0 (dotted line), and pH 7.0 (continuous line) Circles indicate the spectrum at
pH 7.0 for recombinant EspA prepared from soluble fraction of cell lysate (C) Plot of ellipticity at 222 nm vs pH.
Trang 3suggests that our preparation of recombinant EspA
using urea, which included unfolding and refolding
steps was successful, and that this protein reversibly
unfolds and refolds, at least under the controlled
con-ditions used here
The ellipticity at 222 nm for EspA at pH 7.0 was
)13 600 degẳcm2ẳdmol)1 This ellipticity value yields an
a-helical content of 37.2% when used in the equation:
fHỬ đơh222ợ 2340ỡ=30300
where fH and [h]222 are the a-helical fraction and the
ellipticity at 222 nm, respectively [24] This value,
derived from the pH 7.0 CD spectrum, is smaller than
that estimated from the secondary structure prediction
using the amino acid sequence (62.0% and 8.3% for
a-helices and b-sheets, respectively, Table 1) The
sec-ondary structure contents, estimated by the program
CDPro, are 39.6% and 13.6% for a-helices and
b-sheets, respectively This a-helical value is also
smal-ler than that predicted using the amino acid sequence
(62.0% and 8.3% for a-helices and b-sheets,
respect-ively, Table 1)
Although the intensity is significantly low, the
near-UV CD spectrum of EspA at pH 7.0 and 20C,
showed a minimum and a maximum around 280 and
290 nm As in the case of far-UV CD, the near-UV
CD spectrum at pH 2.0 was similar to the spectrum at
pH 7.0, although the intensity of each peak bacome
slightly smaller It is in the near-UV region that
aro-matic residues display optical activity EspA contains
five tyrosines at positions 22, 51, 53, 110 and 182, and
no tryptophans Therefore, the shape of the near-UV
CD spectra of EspA suggests the presence of some
ter-tiary contacts around at least one of the tyrosines both
at pH 7.0 and 2.0 (Fig 2B)
To gain further insight into the conformational
properties of EspA, we recorded far-UV CD spectra
for solutions with the pH adjusted between 2.0 and
7.0 Interestingly, when the solution pH was between 3.0 and 7.0, the spectra are almost identical and the derived secondary structure estimates are similar to each other (Fig 2C and Table 1) The pH 2.0 spec-trum also indicates a significant amount of secondary structure, although the spectral intensity is smaller than those obtained at higher pH This observation suggests that at pH 2.0 EspA is less ordered than at
pH 3.0Ờ7.0
ANS binding ANS binds to solvent-accessible hydrophobic surfaces and when bound its fluorescence intensity at 500 nm increases This property of ANS is often used to detect partially unfolded protein intermediates [25], e.g mol-ten globules, which are compact intermediates with significant amounts of native-like secondary structure, but with disordered tertiary contacts and solvent-exposed hydrophobic clusters [26Ờ34] To determine if partially unfolded EspA species are present as a result
of solution conditions, we recorded ANS fluorescence spectra, with EspA present at various pH conditions Between pH 6.0 and 9.0 the ANS fluorescence was insignificant (Fig 3), suggesting that negligible amounts of hydrophobic surfaces are solvent-accessible However, ANS fluorescence increased when the pH decreased from 6.0 to 2.0 (Fig 3) This observation suggests that hydrophobic surfaces become exposed upon decreasing the pH Since the protein maintains a significant amount of secondary structure (as estimated
Table 1 EspA Secondary structure composition at various pH
val-ues as estimated using the far-UV CD spectral data Valval-ues were
calculated using CDPro [44,45].
Conditions a-Helix (%) b-Sheet (%) Turn (%) Others (%)
pH 2.0 32.6 ổ 3.0 16.1 ổ 2.4 21.9 ổ 0.9 29.9 ổ 0.4
pH 3.0 40.9 ổ 6.9 12.0 ổ 4.1 19.6 ổ 2.1 29.1 ổ 2.0
pH 5.0 39.8 ổ 8.1 13.0 ổ 1.1 19.0 ổ 2.0 28.5 ổ 1.0
pH 7.0 39.6 ổ 7.1 13.6 ổ 5.2 19.7 ổ 1.7 27.6 ổ 0.4
Predicted values a 62.0 8.3 29.7 b
a Estimated from the secondary structure prediction (Fig 1) using
the PHDSEC algorithm available at the PREDICTPROTEIN server [46Ờ48].
b The value is for non-a-helical and non-b-strand regions.
Fig 3 ANS fluorescence at 460 nm as a function of pH Circles indicate the raw data The line is drawn only to assist the reader and has no theoretical relevance The approximated baselines for
NIIand NI(see text in detail) are shown by dotted and broken lines, respectively.
Trang 4from far-UV CD spectra between pH 2.0 and 5.0,
Fig 2), the results of the ANS study suggest formation
of a partially unfolded state, possibly similar to the
a-lactalbumin molten globule characterized at acidic
pH [34]
Sedimentation equilibrium
Under physiological conditions, during EHEC or
EPEC infection, EspA is associated with filamentous
structures We therefore tested, using sedimentation
equilibrium ultracentrifugation, whether recombinant
EspA has the potential to form oligomers
Figure 4 shows the results for the EspA
sedimenta-tion equilibrium experiments at pH 7.0 and 20C If a
protein solution contains only a single molecular
weight species, then a plot of the natural logarithm of
the protein absorption at 280 nm [ln(A280)] vs the
square of the radial distance (r2) shows a linear
correlation between ln(A280) and r2 However, the data (Fig 4A) indicate that ln(A280) exponentially increases with increased r2 This sedimentation equilibrium pro-file indicates either the presence of large protein oligo-mers or a contribution to the plot by nonideal solution behaviour Probably, the solution behaves as an ideal system under the experimental conditions, i.e., 10 mm sodium phosphate, pH 7.0, 100 mm NaCl Thus, it is unlikely that the curvature shown in Fig 4A is caused
by nonideal behaviour
Figure 4B shows that Mapp increases with an increase in the concentration of EspA The data of Fig 4B suggest that the size distribution of EspA ran-ges from that of the monomer to approximately that
of a 30-mer when the protein concentration is
1 mgÆmL)1, i.e 44 lm
We also attempted to analyse the sedimentation pro-file of the partially unfolded state that exists at pH 2.0 However, it was extremely difficult to obtain the exact size of protein at pH 2.0 probably due to the forma-tion of irreversible aggregates during the long period
of centrifugation Although no visible precipitates were found at the beginning, protein absorption started to decrease after about 24 h, and become almost unde-tectable after 48 h This may be caused by the require-ment of high protein concentration (> 1 mgÆmL)1) due
to the lack of tryptophan residues in EspA for reliable detection as well as the need for a long equilibration period (> 24 h) essential for the sedimentation equilib-rium study The result is, however, consistent with the idea that the protein is partially unfolded at pH 2.0, because partially unfolded species are generally prone
to form nonspecific aggregates (see below) Thus, com-pared with other simple spectroscopic measurements such as CD, ultracentrifugation may generally not be suitable for the analysis of partially folded proteins which are prone to aggregate
FT-IR spectroscopy The previous sedimentation equilibrium study indica-ted that after a long incubation at pH 2.0 the EspA solution contains aggregates, although these are invis-ible just after preparation of the sample FT-IR spectro-scopy also confirmed the presence of molecular species containing intermolecular b-strands typical for nonspecific aggregates
As indicated by CD, the soluble EspA at pH 7.0 shows an IR spectrum suggestive of the formation of a-helical structures with a peak around 1650 cm)1 (Fig 5) However, the spectrum taken for the solution
at pH 2.0 has an additional maximum peak around
1620 cm)1 which is characteristic for the
intermole-Fig 4 Sedimentation equilibrium (A) Plot of the logarithm of the
absorbance at 280 nm, A280, as a function of the square of the
radial distance, r2 Data were collected at pH 7.0 with 1.0 mgÆmL)1
EspA (B) Plot of Mappvs protein concentration.
Trang 5cular b-sheets usually formed in the nonspecific
aggre-gates
Importantly, precipitates are also formed at pH 7.0
in the presence of EspA > 1 mgÆmL)1 The IR
spec-trum for these aggregates, however, is significantly
similar to the spectrum taken for the soluble EspA
(Fig 5) This observation suggests that EspA has an
intrinsic potential to self-associate into oligomeric
structures, which consist of a-helical secondary
struc-tures
Urea-induced unfolding
The stability of EspA at various pH values was
ana-lysed using far-UV CD spectroscopy By plotting the
ellipticity at 222 nm as a function of urea
concentra-tion, cooperative unfolding transitions were obtained
at all pH values (Fig 6) Between pH 3.0 and 7.0, the
transitions occurred between 3.0 and 6.0 m urea
How-ever, the transition region shifted towards lower urea
concentrations of about 0.0–3.0 m at pH 2.0 This
observation is qualitatively consistent with the ANS
binding results, which show that the protein, at
pH 2.0, assumes a partially unfolded conformation
with exposed hydrophobic surfaces Interestingly, the
stability of the protein at pH 3.0 seems comparable to
that at pH 7.0 This observation would seem to be
inconsistent with the pH 3.0 ANS binding experiment
as that experiment indicates a degree of unfolding
resulting in hydrophobic surface solvent-exposure
Therefore, between pH 3 and 5, a partially unfolded
state with highly ordered native-like tertiary contacts,
but also with fluctuating regions, may exist This
conformational state is clearly distinguishable from the typical molten globule formed by EspA at pH 2.0
Discussion
pH-dependence of EspA conformations The present analysis suggests that the amount of EspA secondary structure, at various pH conditions, is highly conserved, even at pH 2.0 However, ANS bind-ing experiments indicate that a conformational change occurred upon decreasing pH The characteristics of this conformational change are consistent with the for-mation of a partially unfolded species, probably sim-ilar to a molten globule Molten globules are compact denatured states with significant amounts of native-like secondary structure, but with disrupted tertiary inter-actions [26–34] Although peak intensity is slightly dif-ferent, the near-UV CD spectrum at pH 7.0 is closely similar to that at pH 2.0 (Fig 2B) This is apparently inconsistent with the idea that the conformational spe-cies of EspA at pH 2.0 is in a typical molten globule state In this sense, the partially unfolded structure at
pH 2.0, which exposes hydrophobic clusters to the sol-vent, may contain rather rigid tertiary conformation compared with the classical molten globule state It should be noted that the urea-induced denaturation data indicated a decreased stability and cooperativity against urea-induced unfolding for EspA at pH 2.0 compared with that at pH 7.0 This suggests that some conformational transitions may occur around
Fig 6 Urea-induced EspA unfolding transitions at various pH val-ues, 20 C The transition curves are obtained from far-UV CD spectra at pH 2.0 (triangles), pH 3.0 (squares) and pH 7.0 (circles) The approximated baselines for folded (N I or N II ) and unfolded states are drawn by dotted and broken lines, respectively, The ideal ellipticity for 50% of folded or unfolded species is shown by a thin line.
Fig 5 FT-IR spectroscopy of EspA The spectra at pH 2.0 (broken
line), aggregates formed at pH 7.0 (dotted line), and soluble fraction
at pH 7.0 (continuous line).
Trang 6pH 2.0–3.0 Importantly, at pH 2.0, the ellipticity at
222 nm decreased compared with the value at pH 3.0–
7.0 Thus, some of the a-helical structure formed at
pH 3.0–7.0 may be disrupted at pH 2.0, whereas
ter-tiary contacts, at least, around one of the tyrosine
resi-dues are conserved
Recently, the three-dimensional structure of EspA
complexed with its chaperone, CesA, has been solved
by X-ray crystallography [35] In this model, only the
N-terminal 29 and C-terminal 43 residues (amino acid
positions at 31–59 and 148–190) of EspA
correspond-ing to the bindcorrespond-ing interface of CesA could be clearly
solved The other regions corresponding to the amino
acid positions between 60 and 147 could not be solved,
possibly due to the conformational disorder or
mul-tiple conformations If the unsolved regions in the
EspA–CesA complex structure are disordered, the
a-helical content of EspA should be 37.5% This value
is highly consistent with the a-helical content estimated
here from far-UV CD spectra of EspA at pH 7.0
(39.6%) It is generally considered that the native-like
secondary structures are present in the partially folded
state of a protein Thus, it would be natural to assume
that the two a-helices of EspA shown in the EspA–
CesA complex structures may be also formed in the
partially folded state of EspA at pH 2.0 According to
the EspA–CesA complex structure, only Y53 forms
tertiary contacts with the C-terminal a-helix of EspA
and CesA, and other tyrosines located in these
a-heli-ces are exposed to the solvent Therefore, the near-UV
CD signals observed at pH 7.0 and 2.0 in Fig 2B
might be responsible for the formation of tertiary
con-tacts around Y53 The formation of nonspecific
aggre-gates which occurred at pH 2.0 in the presence of high
concentration of EspA indicate that the oligomeric
EspA at pH 7.0 can tend to dissociate into monomers
at pH 2.0 since the oligomerization into native
struc-ture should prevent the formation of nonspecific
aggre-gates In this sense, the near-UV CD signal observed
at pH 2.0 can be responsible for the intramolecular
tertiary contacts around Y53, whereas the signal at
pH 7.0 might reflect the intermolecular tertiary
con-tacts However, the information on three-dimensional
structure of EspA at different pH, particularly around
the amino acids between 60 and 147, which could not
be resolved by X-ray crystallography of EspA–CesA
complex, is critical to evaluate such a possibility
Importantly, the pH 3.0, urea-induced unfolding
transition is almost superimposable onto the pH 7.0
transition curve This observation suggests that the
protein, at pH 3.0, is as stable as that at pH 7.0
How-ever, the ANS binding data indicate exposure of
hydrophobic surfaces at pH 3.0, probably due to
partial unfolding One possible explanation, reconciling this discrepancy, is that, unlike the traditional molten globule, EspA maintains a well-ordered native-like domain, but also has less structured regions with exposed hydrophobic patches at pH 3.0 We designate this conformational state, NII, the native structure at acidic pH, which has a distinctive character compared with the native conformation at neutral pH (NI) Thus, the conformational change of EspA, associated with changing pH, can be schematically represented as in Scheme 1:
IA … NII … NI The evidence suggests that the partially folded state at
pH 2.0 may have native-like tertiary contacts but a lower a-helical structures content compared with NI and NII It is now designated as IA, i.e acid-induced intermediate structure
In an attempt to understand how pH and urea centration affect the conformations of EspA, we con-structed an EspA pseudo phase diagram with urea concentration as a function of pH (Fig 7), according
to Scheme 1 For ANS binding (Fig 3), the ANS transition midpoint can be considered to be the appar-ent NI to NII transition midpoint, assuming that the maximum ANS intensity in Fig 3 corresponds to the ANS fluorescence for NI The urea-induced unfolding transitions, between pH 3.0 and 5.0 (Fig 6), provide apparent midpoints for the transitions from either NII
or NIto the unfolded state (U); whereas, the transition
Fig 7 Pseudo phase diagram for EspA: urea concentration vs pH
at 20 C The boundaries are defined by the ANS binding and the urea-induced unfolding curves shown in Figs 3 and 5 U, Unfolded state; I A , acid-induced intermediate state; N I , native state at neutral pH; NII, native state at acidic pH The transition midpoints for NI(or
NII) to U (circles), IAto U (squares) and NI to NII (triangles) are shown by lines.
Trang 7midpoint for IA to U is found using the pH 2.0
urea-induced unfolding data Importantly, since we have no
clear information on the transition between NIIand NI
by the addition of urea due to the spectral similarity
between these species, the boundary between NIand U
shown around neutral pH may actually correspond to
the boundary between NIIand U Also, unfortunately,
the experiments reported herein do not provide the
boundary between NIIand IA Additional
experimenta-tion using, for example, NMR or calorimetry is needed
to construct a more complete EspA phase diagram
Although the phase diagram of Fig 7 is incomplete, it
contains sufficient information such that, for a given
set of solution conditions, the existing conformational
state(s) can probably be identified
The C-terminal regions (Val138 to Gln181) of two
EspA molecules may associate to form coiled-coil
structures [19] These coiled-coils may then associate
further, forming oligomers Based on our data, we
pro-pose that the oligomeric native state, found at neutral
pH, dissociates at pH 3 into a monomeric native-like
state with an ordered N-terminal domain and less
structured hydrophobic C-terminal tail
The dissociation of oligomers into monomers upon
decreasing pH was previously observed for Salmonella
strain SJ25 flagellin [36] In that case, the protein, at
acidic pH, assumes a conformation with an associated
ellipticity at 222 nm of )3800 deg CÆm-2Ædmol)1
Thus, some residual conformation may be present in
monomeric flagellin at acidic pH It is possible that the
structural properties of monomeric flagellin, at acidic
pH, are similar to those of molten globules
Oligomerization
The EspA filamentous superstructure has been
ana-lysed by electron microscopy [17,18] It was suggested
that other factors, such as molecular chaperones, are
required to form an ordered EspA filamentous
assem-bly [18] However, based on our sedimentation
equili-brium data, we suggest that recombinant EspA
spontaneously forms oligomers For flagellin, several
additives, e.g salts or polyethylenglycoles, are required
to induce formation of long, ordered filaments [37–41]
Unfortunately, we were unable to produce long EspA
filaments even when such additives were present (data
not shown) The results of the sedimentation
equili-brium experiment indicate that the largest oligomer
formed by the recombinant protein is approximately a
30-mer According to the model derived from electron
microscopy, an axial rise for one filament is 4.6 A˚ per
subunit [18] Thus, a 30-mer, formed by recombinant
EspA, corresponds to a filament with a length of
approximately 14 nm This is significantly shorter than the length of EspA filaments formed on EHEC and EPEC cell surfaces Therefore, the assistance of addi-tional factors, such as molecular chaperones, may be needed to form longer EspA filaments, or the addi-tional residues present at the N-terminal region of our recombinant protein can destabilize the filaments Alternatively, time scales longer than those used in our experiments may be necessary for the formation of suf-ficiently long filaments
In summary, we provide, herein, the first study con-cerning the properties of the secondary structure of EspA EspA is shown to spontaneously associate into oligomeric structures at neutral pH However, two dis-tinctive partially unfolded species occur at lower pH Based on these results, a phase diagram, illustrating potential EspA conformational transitions, was con-structed Additional studies are necessary to character-ize the EspA filamentous structure at the atomic level and to elucidate the thermodynamic requirements for filament formation Such information should clarify the role of EspA during host cell infection by EPEC and EHEC
Experimental procedures
Expression and purification of recombinant EspA
The espA gene was amplified from an E coli O157:H7 cos-mid library (RIMD 0509890, Sakai strain) [42,43] by PCR and PCR product was cloned into pT7 vector (Novagen, Madison, WI, USA) The650 bp NdeI–SacI fragment con-taining the espA gene was then inserted into the expression vector, pET28a (Novagen) The recombinant EspA has
MGSSHHHHHHSSGLVPRGSH on the N-terminal side
of the native sequence The plasmid pET28a-EspA was transformed into E coli BL21 (DE3)
kanamycin, was inoculated with E coli BL21 colonies and
the overnight culture was diluted 100-fold into fresh LB
expression was induced by addition of IPTG (at concentra-tions up to 1 mm) when the cultures reached an optical density of 0.5 at 600 nm
and the pellet was placed on ice for 15 min Most
However, some EspA are also present in the soluble frac-tion Therefore, we prepared the recombinant EspA from total cell solubilized by urea or from only soluble frac-tions
Trang 8For preparation of EspA from urea-solubilized total
cells, the cells were resuspended in 100 mm sodium
sonication The solution was centrifuged at 10 000 g for
The soluble fraction was diluted drop-wise 100-fold into
50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 10 mm
agarose (Qiagen, Valencia, CA, USA) and eluted using a
0–0.5-m imidazole gradient The eluted EspA was dialysed
against 50 mm sodium phosphate pH 8.0, 300 mm NaCl,
10 mm imidazole and rechromatographed over Ni–NTA
agarose Eluted EspA was concentrated by ultrafiltration
using a YM10 filter (Millipore, Billerica, MA, USA) and
then dialysed against 10 mm sodium phosphate pH 7.0
For purification from the soluble fraction, cells collected
by centrifugation were resuspended in 50 mm sodium
phosphate pH 8.0, 300 mm NaCl, 10 mm imidazole
concentra-tions, respectively) were then added Incubation was
The supernatant was applied to Ni–NTA agarose
equili-brated with 50 mm sodium phosphate pH 8.0, 300 mm
NaCl, 20 mm imidazole, and washed with the same buffer
The recombinant EspA was eluted with 50 mm sodium
phosphate pH 8.0, 300 mm NaCl, 250 mm imidazole The
eluted protein was dialysed against, 50 mm sodium
phos-phate pH 8.0, 300 mm NaCl, 10 mm imidazole, and
puri-fied again by Ni–NTA agarose
The purity of the recombinant protein was checked by
mole-cular weight of 20 kDa, a value consistent with calculated
molecular weight of recombinant EspA About 1 mg of
EspA were purified from 1 L culture by urea-solubilization
procedure, whereas only 0.1 mg of protein could purified
without solubilization by urea
CD spectroscopy
CD spectra were recorded using a J-600 spectropolarimeter
(Jasco, Tokyo, Japan) The temperature was adjusted to
con-nected to a circulating water bath For far- and
near-UV CD spectra, cells of 1 mm and 1 cm path length were
used, respectively Protein concentrations were 0.1 and
respectively The samples were prepared about 12 h before
the measurements and the measurements were completed
within 24 h after preparation of samples The sample pH
was checked by pH electrode, Horiba compact pH meter,
B-212 (Horiba, Kyoto, Japan) after each measurement The
data were expressed as mean residue ellipticity, [h], where [h] is defined as [h]¼ 100 hobs(c· l))1, hobsis the observed intensity, c is the concentration in residue moles per litre, and l is the path length in cm The secondary structure composition of EspA was estimated using the program package CDPro [44,45] Reported values are the average of the results obtained from three independent programs:
250 nm with an interval of 0.2 nm taken at different pH were directly used for input data
The urea-induced unfolding curves were obtained by plotting the ellipticity at 222 nm against urea concentration
To estimate the urea concentration of midpoint of the
unfol-ded species are approximated from the plateau regions of pre- and post-transition, respectively The data were ana-lysed according to the assumption of two-state transition between a native and an unfolded state However, we should stress here that this analysis should be incorrect because various oligomeric forms are present among native conformers However, without any data about the propor-tion of each native oligmer, this treatment is the only the
values without any bias The details in the analysis and the parameters for unfolding are available as supplementary material in Table S1
Fluorescence spectrum
ANS fluorescence spectra were recorded using a FP-777 fluorimeter (Jasco) The excitation wavelength was 350 nm and fluorescence emission spectra were recorded between
400 and 650 nm The protein concentration was 0.1
thermostatically controlled cell holder
Sedimentation equilibrium
Sedimentation equilibrium experiments were performed using a Beckman Optima XL-I analytical ultracentrifuge
Various amounts of protein were dissolved in 20 mm sodium phosphate pH 7.0, 100 mm NaCl Using the pro-gram, AA comp (RASMB web site: http://www.rasmb bbri.org/rasmb/mac/aa_comp-stafford), in conjunction with the EspA amino acid composition, the partial specific vol-ume of EspA was calculated as 0.731 The apparent
fol-lowing equation:
ð1 tqÞx2
d lnðCÞ
Trang 9where R is the gas constant, T is the absolute temperature,
x is the angular velocity, q is the solvent density and c is
the protein concentration at the radial distance r
FT-IR
Infrared spectra were recorded using Avatar 370 (Thermo
Nicolet Co., Madison, WI, USA) under continuous purge
with dry nitrogen gas Normal spectral resolution used
Happ–Genzel apodization function was applied before
Fourier transformation The samples were transferred to
separated by a 15-lm spacer FT-IR measurements were
carried out at room temperature Recombinant protein
(5 mg) dissolved in 5 mL 10 mm sodium phosphate was
lyophilized and resuspended in 200 lL 10 mm sodium
phosphate⁄2
visible precipitates were found in the solution The spectra
of soluble and insoluble fractions were individually taken
absorp-tion spectrum At pH 2.0, no visible precipitates were
found Thus, the concentration of EspA is considered to
species was obvious from the FT-IR spectrum as discussed
in the text
Acknowledgements
We thank Prof Yuji Goto for the use of the CD
spec-trometer and Miyo Sakai for performing the
ultracen-trifugation experiments This work was supported in
part by grants-in-aid for scientific research from the
Japan Ministry of Education, Culture, Sports, Science
and Technology (MEXT)
References
1 Galan JE & Collmer A (1999) Type III secretion
machines: bacterial devices for protein delivery into host
cells Science 284, 1322–1328
2 McDaniel TK, Jarvis KG, Donnenberg MS & Kaper
JB (1995) A genetic locus of enterocyte effacement
con-served among diverse enterobactrial pathogens Proc
Natl Acad Sci USA 92, 1664–1668
3 McDaniel TK & Kaper JB (1997) A cloned
pathogeni-city island from enteropathogenic Escherichia coli
con-fers the attaching and effacing phenotype on K-12
E coli Mol Microbiol 23, 399–407
4 Perna NT, Mayhew GF, Po´sfai G, Eliott SJ,
Donnen-berg MS, Kaper JB & Blattner FR (1998) Molecular
evolution of a pathogenicity island from
enterohemor-rhagic Escherichia coli O157: H7 Infect Immun 66, 3810–3817
5 Zhu C, Agin TS, Elliott SJ, Johnson LA, Thate TE, Kaper JB & Boedeker EC (2001) Complete nucleotide sequence and analysis of the Locus of Enterocyte efface-mant from rabbit diarrheagenic Escherichia coli
RDEC-1 Infect Immun 69, 2107–2115
6 Shuch R & Maurelli AT (2000) The type III secretion pathway Dictating the outcome of bacterial–host inter-actions In Virulence Mechanisms of Bacterial Pathogens (Brogden KA, Roth JA, Stanton TB, Bolin CA, Minion
FC & Wannemuehler MJ, eds), 3rd edn ASM Press, American Society for Microbiology, Washington, DC
7 Clarke SC, Haigh RD, Freestone PP & Williams PH (2003) Virulence of enteropathogenic Escherichia coli, a global pathogen Clin Microbiol Rev 16, 65–78
8 Roe AJ, Hoey DE & Gally DL (2003) Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157 Biochem Soc Trans 31, 98–103
9 Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey
EA & Finlay BB (1997) Enteropathogenic E coli (EPEC) transfers its receptor for intimate adherence into mammalian cells Cell 91, 511–520
10 Vlademir VC, Takahashi A, Yanagihara I, Akeda Y, Imura K, Kodama T, Kono G, Sato Y & Honda T (2001) Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropatho-genic Escherichia coli, and is essential for pedestal for-mation Cell Microbiol 3, 745–751
11 Vlademir VC, Takahashi A, Yanagihara I, Akeda Y, Imura K, Kodama T, Kono G, Sato Y, Iida T & Honda
T (2002) Cortactin is necessary for F-actin accumulation
in pedestal structure induced by enteropathogenic
12 Crane JK, McNamara BP & Donnenberg MS (2001) Role of EspF in host cell death induced by
enteropathogenic Escherichia coli Cell Microbiol 3, 197–211
13 McNamara BP & Donnenberg MS (1998) A novel pro-line-rich protein, EspF, is secreted from enteropatho-genic Escherichia coli via the type III export pathway FEMS Microbiol Lett 166, 71–78
14 Elliott SJ, Krejany EO, Mellies JL, Robins-Browne
RM, Sasakawa C & Kaper JB (2001) EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coliwith similarities to VirA of Shigella flex-neri Infect Immun 69, 4027–4033
15 Kenny B & Jepson M (2000) Targeting of an entero-pathogenic Escherichia coli (EPEC) effector protein to host mitochondria Cell Microbiol 2, 579–590
16 Nougayre`de J-P, Fernandes PJ & Donnenberg MS (2003) Adhesion of enteropathogenic Escherichia coli
to host cells Cell Microbiol 5, 359–372
Trang 1017 Sekiya K, Ohishi M, Ogino T, Tamano K, Sasakawa
C & Abe A (2001) Supermolecular structure of the
enteropathogenic Escherichia coli type III secretion
system and its direct interaction with the
EspA-sheath-like structure Proc Natl Acad Sci USA 98, 11638–
11643
18 Daniell SJ, Kocsis E, Morris E, Knutton S, Booy FP &
Frankel G (2003) 3D structure of EspA filaments from
enteropathogenic Escherichia coli Mol Microbiol 49,
301–308
19 Delahay RM, Knutton S, Shaw RK, Hartland EL,
Pallen MJ & Frankel G (1999) The coiled-coil domain
of EspA is essential for the assembly of the type III
secretion translocon on the surface of enteropathogenic
Escherichia coli J Biol Chem 274, 35969–35974
20 Hyman HC & Trachtenberg S (1991) Point mutations
that lock Salmonella typhimurium flagellar filaments in
the straight right-handed and left-handed forms and
their relation to filament superhelicity J Mol Biol 220,
79–88
21 Knutton S, Rosenshine I, Pallen MJ, Nisan I, Neves
BC, Bain C, Wolff C, Dougan G & Frankel G (1998) A
novel EspA-associated surface organelle of
enteropatho-genic Escherichia coli involved in protein translocation
into epithelial cells EMBO J 17, 2166–2176
22 Ide T, Laarmann S, Greune L, Schillers H, Oberleithner
H & Schmidt MA (2001) Characterization of
transloca-tion pores inserted into plasma membranes by type
III-secreted Esp proteins of enteropathogenic Escherichia
coli Cell Microbiol 3, 669–679
23 Kodama T, Akeda Y, Kono G, Takahashi A, Imura K,
Iida T & Honda T (2002) The EspB protein of
entero-haemorrhagic Escherichia coli interacts directly with
a-catenin Cell Microbiol 4, 213–222
24 Chen Y-H, Yang JT & Martinez HM (1972)
Determina-tion of the secondary structures of proteins by circular
dichroism and optical rotatory dispersion Biochemistry
11, 4120–4131
25 Semisotnov GV, Rodionova NA, Kutyshenko VP,
Ebert B, Blanck J & Ptitsyn OB (1987) Sequential
mechanism of refolding of carbonic anhydrase B FEBS
Lett 224, 9–13
26 Ohgushi M & Wada A (1983) ‘Molten-globule state’: a
compact form of globular proteins with mobile
side-chains FEBS Lett 164, 21–24
27 Ohgushi M & Wada A (1984) Liquid-like state of side
chains at the intermediate stage of protein denaturation
Adv Biophys 18, 75–90
28 Ptitsyn OB (1995) How the molten globule became
Trends Biochem Sci 20, 376–379
29 Ptitsyn OB (1995) Molten globule and protein folding
Adv Protein Chem 47, 83–229
30 Arai M & Kuwajima K (2000) Role of the molten
globule state in protein folding Adv Protein Chem 53,
209–282
31 Creighton TE (1997) How important is the molten glob-ule for correct protein folding? Trends Biochem Sci 22, 6–10
32 Kuwajima K (1989) The molten globule state as a clue for understanding the folding and cooperativity of glob-ular-protein structure Proteins 6, 87–103
33 Kuwajima K (1992) Protein folding in vitro Curr Opin Biotechnol 3, 462–467
34 Kuwajima K (1996) The molten globule state of a-lact-albumin FASEB J 10, 102–109
35 Yip CK, Finlay BB & Strynadka NCJ (2005) Structural characterization of a type III secretion system filament protein in complex with its chaperone Nature Struct Mol Biol 12, 75–81
36 Uratani Y, Asakura S & Imahori K (1972) A circular dichroism study of Salmonella flagellin: evidence for conformational change on polymerization J Mol Biol
67, 85–98
37 Novikov VV, Metlina AL & Poglazov BF (1994) A study on the mechanism of polymerisation of Bacillus
38 Abram D & Koffler M (1964) In vitro formation of flagella-like filaments and other structures from flagellin
J Mol Biol 116, 168–185
39 Asakura S, Eguchi G & Iino T (1966) Salmonella flagella: in vitro reconstruction and over-all shapes of flagellar filaments J Mol Biol 16, 302–316
40 Asakura S, Eguchi G & Iino T (1964) Reconstitution of bacterial flagella in vitro J Mol Biol 10, 42–56
41 Wakabayashi K, Hotani H & Asakura S (1969) Poly-merization of Salmonella flagellin in the presence of high concentrations of salts Biochim Biophys Acta 175, 195– 203
42 Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii
K, Yokoyama K, Han CG, Ohtsubo E, Nakayama K, Murata T, Tanaka M, Tobe T, Iida T, Takami H, Honda T, Sasakawa C, Ogasawara N, Yasunaga T, Kuhara S, Shiba T, Hattori M & Shinagawa H (2001) Complete genome sequence of enterohemorrhagic
a laboratory strain K-12 DNA Res 8, 11–22
43 Perna, NT, Plunkett G, 3rd Burland V, Mau B, Glas-ner JD, Rose DJ, Mayhew GF, Evans PS, Gregor J, Kirkpatrick HA, Posfai G, Hackett J, Klink S, Boutin
A, Shao Y, Miller L, Grotbeck EJ, Davis NW, Lim
A, Dimalanta ET, Potamousis KD, Apodaca J, Anan-tharaman TS, Lin J, Yen G, Schwartz DC, Welch
RA & Blattner FR (2001) Genome sequence of enterohaemorrhagic Escherichia coli O157: H7 Nature
409, 529–533
44 Sreerama N & Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set Anal Biochem
287, 252–260