From the Departments of Pharmaceutical Chemistry and
Molecular Biosciences, University of Kansas,
Lawrence, Kansas 66045
Received for publication, August 16, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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Shigella flexneri causes a
self-limiting gastroenteritis in humans, characterized by severe
localized inflammation and ulceration of the colonic mucosa.
Shigellosis most often targets young children in underdeveloped
countries. Invasion plasmid antigen C (IpaC) has been identified as the
primary effector protein for Shigella invasion of
epithelial cells. Although an initial model of IpaC function has been
developed, no detailed structural information is available that could
assist in a better understanding of the molecular basis for its
interactions with the host cytoskeleton and phospholipid membrane. We
have therefore initiated structural studies of IpaC, IpaC I', (residues
101-363 deleted), and IpaC Shigella flexneri causes a self-limiting
gastroenteritis called shigellosis, which is characterized by severe
localized inflammation and ulceration of the colonic mucosa (1). An
estimated 360,000 Shigella cases occur in the United States
each year, although only 18,000 are reported (2). In developing
countries, the disease is present in most villages. It most commonly
strikes young children and is responsible for an estimated 600,000 deaths/year worldwide (all Shigella spp. combined) (1).
Shigellosis onset involves bacterial invasion of intestinal epithelial
cells by a process called "pathogen-induced phagocytosis" (2, 3) and requires the expression of the ipa operon. The invasion
plasmid antigens (or Ipa1
proteins) are effector proteins that are exported by a dedicated type
III secretion system (TTSS) at the host-pathogen interface. There they
directly interact with the host cell to promote actin cytoskeleton
rearrangements at the site of bacterial contact (2). These cytoskeletal
changes give rise to filopodia, which mature into membrane ruffles that
coalesce to trap the pathogen within a membrane-bound vacuole (2). The
vacuole is then quickly lysed to provide the bacterium with access to
the host cytoplasm, where it proliferates and is able to directly
invade neighboring cells (4).
IpaC has been identified as the primary effector protein for
Shigella invasion of epithelial cells (5-8).
Effector-related functions described for purified IpaC include:
(a) enhanced invasion of cultured cells by S. flexneri (5, 8, 9); (b) induced uptake of virulence
plasmid-cured S. flexneri (5); (c) interaction with phospholipid membranes (9-11); and (d) triggering of
cytoskeletal changes in cultured cells (7, 9, 12). Additional
activities associated with IpaC include oligomerization in solution
(8), reconstitution into complexes with IpaB in vitro (8),
in vivo formation of complexes containing IpaB that promote
the uptake of latex beads by cultured cells (6), and reconstitution
with IpaB and IpaD to form a complex that may allow entry of
noninvasive Escherichia coli into cultured cells (13).
It has been demonstrated that IpaC possesses a distinct functional
organization (Scheme I). The immediate N
terminus of IpaC is required for secretion, whereas a region near the N
terminus is responsible for association with IpaB, which is
probably needed for proper presentation of IpaC to the target cell
membrane. The central hydrophobic region of IpaC directs IpaC
penetration of phospholipid membranes and contributes to interactions
with IpaB (14).2 The ability
of IpaC to interact with phospholipid membranes does not require
secretion by the TTSS of S. flexneri (Ref. 9 and this work);
however, active insertion of IpaC into target cell membranes by the
TTSS greatly increases the efficiency of this process (15). The C
terminus of IpaC mediates IpaC-IpaC interactions and probably possesses
IpaC effector function (7, 14). This information, however, only
provides a very general picture of IpaC structure and function and its
role in the S. flexneri infection process. Unfortunately,
there exists no detailed structural information concerning IpaC that
could provide a molecular basis for its interactions with the host
cytoskeleton and phospholipid membranes.
H (residues 63-170 deleted). The
secondary and tertiary structure of the protein was examined as a
function of temperature, employing circular dichroism and high
resolution derivative absorbance techniques. ANS
(8-anilino-1-napthalene sulfonic acid) was used to probe the exposure
of the hydrophobic surfaces under different conditions. The interaction
of IpaC and these mutants with a liposome model (liposomes with
entrapped fluorescein) was also examined. Domain III (residues
261-363) was studied using linker-scanning mutagenesis. It was shown
that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein.
In addition to these structural studies, investigation into the actin
nucleation properties of IpaC was conducted, and actin nucleation by
IpaC and some of the mutants was exhibited. Structure-function
relationships of IpaC are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Scheme 1.
Functional organization of IpaC. The N
terminus of IpaC harbors sequences for TTSS export and interaction with
IpgC and IpaB. The central hydrophobic region is involved in IpaB
binding, IpaC penetration of phospholipid membranes, and possibly
protein stabilization. The C terminus possesses essential
oligomerization and effector functions. Residues 101-363 are deleted
in IpaC I' ,and residues 63-170 are deleted in IpaC H.
In this work, we have initiated studies into the structure of IpaC by
employing a number of biophysical approaches. Truncated versions of
IpaC, specifically IpaC I' (residues 101-363 deleted) and IpaC H
(residues 63-170 deleted), were overexpressed in E. coli
and purified, permitting structural analysis and a more comprehensive investigation into the role of the individual domains described above.
The interaction of these mutants and full-length IpaC with a
liposome model was also examined. In addition to these
structural studies, an investigation into the actin nucleation
properties of IpaC and these deletion constructs was conducted.
Finally, domain III (residues 261-363), which has previously resisted
attempts at purification after recombinant expression, was studied
using an alternate technique, linker-scanning mutagenesis. From this, it is shown that domain III contains a sequence periodic
structure-activity relationship, which suggests that the activity may
be dependent on helical structure in this region. Interpretation of
IpaC structure with regard to the functional properties of the
wild-type protein is then discussed.
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EXPERIMENTAL PROCEDURES |
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Materials-- DOPC (dioleoylphosphatidylcholine) and DOPG (dioleoylphosphatidylglycerol) were purchased from Avanti. ANS (8-anilino-1-naphthalene sulfonic acid) was obtained from Across Organics, and fluorescein (5,6-carboxyfluorescein, high purity) was obtained from Molecular Probes. Dialysis materials were provided by Spectra. All other chemicals were of reagent grade and obtained from Sigma and Fisher.
Preparation of Affinity-purified Recombinant
Proteins--
Plasmids used to prepare recombinant IpaC and IpaC H
and sipC have been described previously (5, 16, 17). pWPI' was designed
to encode an IpaC peptide called region I', in which residues 101-363
are deleted. This plasmid was generated as pWPC15 except that the 3'
primer was designed to place a stop codon at amino acid 101. All new
plasmids were transformed into E. coli BL21(DE3) for high
level protein expression. Recombinant proteins were purified via the
N-terminal His6 tag by nickel chelation chromatography
under denaturing conditions as described in detail previously (5, 14,
17). Purified proteins were step-dialyzed against 10 mM
NaPO4, pH 7.2, 150 mM NaCl containing
0.5 mM dithiothreitol to remove the urea.
Sample Preparation--
Purified proteins in buffer were stored
at 70 °C for long term storage (IpaC only) or 4 °C for short
term use (<2 weeks). All protein samples were centrifuged at
13,400 × g for 5 min at 4 °C on the day of use to
remove aggregates formed during storage. Tween 20 (0.1%) was added to
the IpaC solutions to further stabilize them during freezing.
Unfortunately, neither centrifugation nor filtration was effective at
completely removing all aggregated material (observed as a small amount
of optical density above 300 nm in absorbance spectra). Concentrations
were therefore obtained employing derivative absorbance spectroscopy,
using N-acetyl-L-tyrosine-ethyl ester derivative
minima at 275 and 283 nm to establish a standard curve since IpaC
possesses no tryptophan residues. Biophysical studies were conducted in
10 mM NaPO4, 150 mM NaCl, pH 7.2, containing 1 mM dithiothreitol.
Circular Dichroism--
CD spectra were recorded with a Jasco
J-720 spectrophotometer (Tokyo, Japan) equipped with a Peltier
temperature controller. Far UV spectra (between 195 and 260 nm)
were collected using a 1-mm path length cuvette sealed with a
Teflon stopper. A resolution of 0.1 nm and a scanning speed of 20 nm/min with a 2-s response time were employed. Spectra presented are an
average of six consecutive spectra. Spectra were recorded at 5 °C
intervals employing a thermostated cuvette holder. An incubation time
of 3 min at each temperature interval (sufficient for equilibrium to be
obtained) and a temperature ramp rate of 20 °C/h were employed.
Protein concentrations of 8.3, 26.3, and 11.2 µM were
employed for IpaC, IpaC I', and IpaC H, respectively. Noise
reduction and data analysis were performed using Standard Analysis and
Temperature/Wavelength Analysis programs (Jasco) and MicroCal OriginTM
6.0 software. Secondary structure content was estimated using the
CONTIN (18), SELCON (19), and CDSSTR (20) analysis programs provided
with the CDPro software suite (21).
Derivative Absorbance Spectroscopy--
High-resolution
absorbance spectra were collected on a Hewlett-Packard 8453 UV-Visible
spectrophotometer (Agilent, Palo Alto, CA) fitted with a Peltier
temperature controller. Temperature perturbation studies were conducted
at protein concentrations of 12.7, 25.3, and 16.9 µM for
IpaC, IpaC I', and IpaC H, respectively. Spectra were collected for
25 s at 2.5 °C intervals with a 3-min equilibration time before
collection of each spectrum. Spectral analysis was conducted using
UV-Visible Chemstation software (Agilent) and Microcal OriginTM 6.0. Second derivative spectra were calculated employing a nine-point data
filter and fifth degree Savitzky-Golay polynomial and subsequently
fitted to a cubic function with 99 interpolated points/raw data point,
permitting 0.01-nm resolution (22). Peak positions were then determined
from the interpolated curves. Optical density data were simultaneously
monitored at 350 nm.
Lipid Interaction Studies-- Aliquots of DOPC and DOPG in chloroform were dried under nitrogen and vacuum to create thin films of either 100% DOPC or 50:50 [DOPC]:[DOPG]. Films were hydrated in a solution containing 100 mM 5,6-carboxyfluorescein at pH 7.0 in water for 10 min and then sonicated in a bath sonicator for 30 min prior to extrusion through a 100-nm pore size membrane 10 times at 45 °C. A final size of ~150 nm was determined with a Brookhaven ZetaPALS dynamic light scattering instrument (Holtsville, NY) equipped with a 25 mW 626-nm laser. Excess dye was separated from the bulk liposomes by size exclusion chromatography, employing a Sephadex G-25 column coupled to an AKTA FLPC (Amersham Biosciences). The run buffer was 10 mM NaPO4, 150 mM NaCl, pH 7.4. Peak fractions were collected and pooled, and lipid content was determined by a total phosphorous assay, as described (23). Solutions were stored at 4 °C and protected from light.
Time-based release studies were conducted with a PTI QuantaMaster spectrophotometer with a thermostated cuvette holder. Samples were excited at 492 nm, and the emission signal was monitored at 517 nm for 10 min. Excitation slits were set at 1 nm, and emission was set at 2 nm. Data points were collected at 0.2-s intervals. Data were collected as follows: buffer was incubated for 3 min at the desired temperature prior to adding liposome solutions. Baseline fluorescence was monitored with liposomes alone for 10 min to determine residual release of fluorescein from the liposomes. Maximum fluorescein release, as defined by release in the presence of 0.1% Triton, was determined by adding Triton to the liposome solution 3 min after the start of signal collection. Protein-induced fluorescein release was examined by monitoring the fluorescein signal at 512 nm after addition of protein to the liposome solution after 3 min of incubation. Both protein and Triton solutions were added during continuous signal collection through a syringe port in the sample compartment. The protein concentration employed was 1 µM, and the total lipid concentration was 100 µM. Release was monitored at 10, 20, 30, 35, 40, 45, 50, and 60 °C. Samples were measured in triplicate. The extent of release was calculated as a percentage of Triton-induced release at 600 s after background correction. Analysis was conducted using Felix (PTI) and MicroCal OriginTM software.
Assay for Actin Nucleation-- The fluorescence of pyrene-labeled G-actin monomers increases following assembly into pyrene F-actin (24). For monitoring IpaC-mediated actin nucleation in vitro, pyrene G-actin was incubated at 4 °C in G-actin buffer (5 mM Tris-HCl, pH 8.0, 0.1 mM ATP, 0.2 mM CaCl2). Test protein (IpaC or an IpaC mutant) was then added to the sample, and pyrene fluorescence was monitored at 23 °C. A Spex FluoroMax instrument (Jobin Yvon Horiba, Edison, NJ) was used to measure pyrene fluorescence using a time-based acquisition mode with an excitation wavelength of 330 and an emission wavelength of 385 nm. After 15 min, 50× actin polymerization buffer (100 mM MgCl2, 50 mM ATP, 2.5 M KCl) was added, and the change in pyrene fluorescence was monitored as a function of time. Negative controls either contained no added protein or contained IpaD, which has no actin-nucleating activity. SipC from Salmonella typhimurium, which has been shown to nucleate actin in vitro, was used as a positive control (24).
Linker-scanning Mutagenesis-- Linker-scanning mutagenesis was used to introduce consecutive site-specific mutations into a predicted coiled-coil segment near the C terminus of IpaC. Either NheI cleavage sites encoding Ala-Ser pairs or XhoI sites encoding Leu-Glu pairs were generated throughout the length of the putative coiled-coil region (amino acids 309-344). Primers with a 5' NheI or XhoI site and the appropriate neighboring ipaC sequences running in either direction were used for inverse PCR. The product was digested with NheI or XhoI, respectively, and then ligated. The resulting plasmids were electroporated into the S. flexneri IpaC mutant strain SF621, and the ability to restore invasion and contact hemolysis functions was determined as described below.
S. flexneri Invasion of Cultured Cells and Contact-mediated
Hemolysis--
S. flexneri invasion of Henle 407 cells was
monitored using a standard gentamycin protection assay as described
(16). Semiconfluent monolayers of Henle 407 cells were seeded into
24-well plates and grown overnight. SF621 harboring the desired plasmid
was grown in trypticase soy broth containing 100 µg/ml ampicillin and
50 µg/ml kanamycin to an A600 of 0.4-0.6. The
bacteria were diluted with serum-free MEM containing 0.45% glucose
(MEM-glc), centrifuged onto the surface of semiconfluent Henle 407 monolayers, and incubated with the cells for 30 min at 37 °C. Free
bacteria were removed by aspiration, and the cells were washed with MEM
containing 5% calf serum and 50 µg/ml gentamycin. The cells were
incubated in the final gentamycin wash for 2 h (to kill adherent,
noninternalized bacteria) and rinsed with MEM-glc. The cells were lysed
by overlaying them with 250 µl of 0.5% agarose in water. The agarose
was then overlaid with 0.5% agar containing 2× LB medium. After
overnight incubation at 37 °C, internalized bacteria formed
subsurface colonies that were quantified using a ChemiImager 4400 system (Alpha Innotech Corp., San Leandro, CA).
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RESULTS |
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Statistics-- All errors are reported as standard error (S.E., n = 3) unless otherwise indicated.
Spectroscopic Analysis of Full-length IpaC, IpaC I', and IpaC
H--
To better understand the function of IpaC in the
S. flexneri invasion process, a series of
structural studies were conducted to characterize the secondary and
tertiary structure of full-length IpaC, IpaC I', and
H mutants and
their response to temperature. All three proteins are efficiently
secreted by the S. flexneri SF621 TTSS and are able to
interact with IpaB, indicating that biological functions associated
with the N terminus remain intact (14).
The CD spectrum of IpaC at 20 °C exhibits minima at 222 and 204 nm,
suggesting the presence of some helical structure (Fig. 1A). Secondary structure
estimates indicate that IpaC contains a mixture of -helical and
-sheet structure with significant turn and random structure also
present (Table I). In contrast, IpaC I'
and IpaC
H appear to be less structured, as indicated by a decrease
in CD intensity above 210 nm. The spectrum of IpaC I' exhibits
an increase in negative intensity near 200 nm, suggesting increased
random structure. Solution conditions prevented collection of data
below 200 nm, preventing secondary structure estimation for both
mutants. IpaC undergoes a significant change in secondary structure at
fairly moderate temperatures (Table I and Fig. 1). This transition
suggests a loss of helix content (Table I) and has a midpoint of
43.3 ± 0.3 °C (n = 3). In contrast, IpaC I' and IpaC
H display evidence of only weak transitions between 10 and
40 °C. Although limited aggregation of some samples was observed
above 30 °C, no red shifts or strong decreases in intensity indicative of absorption flattening were observed, indicating that the
spectral changes are not an artifact of aggregation-dependent phenomena.
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Because IpaC lacks tryptophan residues, derivative absorbance
spectroscopy was employed to monitor tertiary structure changes. This
method is especially useful in aggregating systems since it is not
sensitive to broad spectral components such as light scattering (25).
The spectra display two phenylalanine (Fig. 2, A and B, 253 and
260 nm) and three tyrosine (Fig. 2, C-E, 268, 276, and 285 nm) minima (22). Most plots of peak position versus temperature show similar linear increases with temperature, which is an
intrinsic property of the aromatic amino
acids.3
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However, deviations from these linear plots consistent with structural
alterations are observed in many cases. The IpaC Phe minimum at 253 nm
is shifted to longer wavelengths relative to the two mutants and shows
no temperature dependence at lower temperatures. Upon reaching the
temperature at which the protein begins aggregating, the noise in the
data begins to increase. The second Phe minimum shows a difference in
minimum position for each of the proteins with the IpaC peak once again
at the longest wavelength. An expanded version of the IpaC I' data
(Fig. 2F) shows that IpaC I' actually undergoes a small but
very reproducible transition between 30 and 55 °C. The 267 nm IpaC
I' tyrosine minimum is observed at longer wavelengths, disappearing
from the derivative spectrum at temperatures above 62 °C. Two strong
transitions are observed in the plot of the 276-nm tyrosine minimum of
IpaC with no similar transitions seen for either mutant. The 284-nm
IpaC I' and IpaC H tyrosine minima again manifest no evidence of
conformational change, whereas the IpaC absorption band shows evidence
of the two transitions and is again present at a longer wavelength
(Fig. 2E).
Trends in protein-associative behavior can be seen by monitoring the
turbidity (OD) at 350 nm as the temperature is increased (Fig.
3). Full-length IpaC shows a large change
in turbidity starting near 30 °C. Transitions are observed between
30 and 70 °C and above 70 °C. Although the changes are small, a
distinct transition is observed starting at 55 °C for IpaC I',
whereas IpaC H shows a small but steady increase in OD starting at
20 °C (Fig. 3, bottom panels). Both results are
consistent with the formation of soluble aggregates.
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Full-length IpaC but Not the IpaC Mutants Interacts with
Liposomes--
The effect of IpaC, IpaC I', and IpaC H upon
fluorescein-containing liposomes was examined as a function of the
temperature and lipid composition. Fluorescein can be sequestered in
liposomes at concentrations that produce self-quenching (26). Any
interaction of a protein with such loaded liposomes that is sufficient
to significantly perturb the bilayer should cause a leakage of the fluorescein, leading to its dilution and an increase in the
fluorescence intensity of the fluorescein dye. Using this approach, two
types of vesicles were employed: 100% DOPC, producing a neutral
surface charge, and 50:50 [DOPC]:[DOPG] to produce an overall
negatively charged surface.
The extent of dye release at 600 s at different temperatures is
shown in Fig. 4. No release is observed
for IpaC I' and IpaC H. IpaC shows little release at low
temperatures with DOPC liposomes, but a dramatic increase is observed
at 30 °C and higher. Release at lower temperatures is greater with
DOPC:DOPG liposomes. The rate of release is similar to that of the
Triton control (results not illustrated). The liposomes remained above
the phase transitions of the component lipids (
20 and
18 °C for
DOPC and DOPG, respectively (27)) under all conditions examined.
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Linker-scanning Mutagenesis Indicates That the IpaC C Terminus Is
Important for Directing Uptake by Cultured Cells--
The region
between amino acids 309 and 344 of IpaC is predicted to possess a
coiled-coil trimerization domain based on primary structure analysis
(28). Attempts to purify a recombinant form of this region have been
unsuccessful, so a structural analysis of this region was conducted
using linker-scanning mutagenesis. Initially, XhoI linkers
were used to substitute Leu-Glu amino acid pairs for existing amino
acids in this region of IpaC. Sequence periodicity in the generation of
inactivating mutations with respect to restoring invasiveness to
S. flexneri SF621 was consistent with the presence of a
coiled -helix in this region (Fig.
5A). Although linker-scanning
mutations near the C-terminal end of the putative coiled-coil show
periodicity in reducing invasiveness, they do not eliminate IpaC
activity completely. In contrast, when NheI linkers
(encoding Ala-Ser) were introduced into the same region, substitution
for the Leu335-Ile336 pair did completely
eliminate IpaC invasion function, as did substitution for
Leu339-Leu340 (Fig. 5B). This
difference in the effect of amino acid substitutions at this location
could be explained by the fact that the Leu-Glu led to the replacement
of a nonpolar pair of amino acids by a nonpolar/polar pair. On the
other hand, NheI scanning results in the replacement of
nonpolar residues by a polar pair. In total, these data are consistent
with the presence of an
-helix within this portion of IpaC.
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Because a single amino acid change at Ile336 and
Leu340 in the putative IpaC coiled region only partially
reduced the ability of IpaC to restore invasiveness, other single amino
acid changes were introduced, using Pro as the substituted amino acid
because of its incompatibility with -helix formation. When Pro was
used to replace Ser314 at the N terminus of the putative
IpaC coil, invasion function was not affected. Replacement of
Lys326 reduced IpaC invasion function to less than half
that of native IpaC (Table II). When
Ile336 was replaced with Pro, however, IpaC invasion
function was reduced more than 80% (Table II). During linker-scanning
analysis, the equivalent of a single amino acid change at this position
(Ile336
Glu336) resulted in only a 48%
reduction. Replacement of Leu340 with Pro completely
eliminated IpaC invasion function, whereas replacement with Glu only
reduced invasion function 64% (Table II). The large negative effect of
Pro replacement of amino acids near the C terminus of the putative IpaC
coiled region, when compared with Glu substitution, is consistent with
this region existing as a functionally important
-helix.
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IpaC Nucleates Actin in Vitro--
It has been reported that the
IpaC homologue from S. typhimurium (SipC) nucleates actin
in vitro (24). We find that IpaC also nucleates actin
in vitro with an efficiency that is at least equal to that
of SipC (Fig. 6). Deletion of the IpaC N
terminus does not affect this activity, whereas deletion of the C
terminus (data not shown) or hydrophobic region does eliminate the
ability of IpaC to nucleate actin (Fig. 6). Furthermore, the C-terminal addition of a 15-residue tag that eliminates IpaC effector function in vivo also eliminates the ability of IpaC to direct actin
nucleation (data not shown), indicating that the C terminus of IpaC is
required for actin nucleation.
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DISCUSSION |
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IpaC is an essential participant in the invasion process of
S. flexneri (3). It is involved in actin
rearrangements, pathogen entry into phagosomal vacuoles, and the
subsequent lysis of those vacuoles (2, 4). Although the broad
functional organization of IpaC has been described (14, 29), little is
known of the structure of the protein. We find that under physiological
conditions, recombinant IpaC is composed of an -helix (<10%), a
-sheet, and significant random structure (Table I). Thus, this
protein may be considered a member of that recently recognized class of proteins whose native state contains a significant amount of disordered structure (30, 31).
Temperature perturbation studies show that IpaC undergoes a distinct structural transition involving both secondary and tertiary structure between 30 and 60 °C (Figs. 1 and 2). This does not involve complete unfolding since the majority of secondary structure is retained post-transition (Table I). Additionally, shifts in the derivative absorbance minima to shorter wavelengths, which would indicate exposure of buried aromatic resides to bulk solvent, were not observed. This conformational change is presumably related to temperature-induced aggregation of IpaC, as detected by turbidity measurements (Fig, 3, top). Two transitions are observed, the first between 30 and 70 °C and the second above 70 °C. This is consistent with the initial formation of soluble aggregates followed by more extensive aggregation and with previous observations that IpaC forms oligomers in solution (8). IpaC homotypic interaction involving the central hydrophobic region have also been detected using yeast two-hybrid analysis (32). The biologically relevant oligomerization state of IpaC is currently under investigation.
Examination of the N-terminal region of IpaC (IpaC I') shows reduced structural content in comparison with the wild-type protein. This is accompanied by a reduced effect of temperature on the protein. IpaC I' exhibits a small change in secondary structure above 30 °C (Fig. 1C), as well as a small tertiary structure transition between 30 and 60 °C (Fig. 2F). Differences in the environment of the aromatic residues of IpaC I', detected as shifts in derivative absorbance wavelength when compared with that of IpaC, are also observed. These shifts may also reflect differences in aromatic amino acid content. IpaC contains three Phe residues, whereas only one is present in IpaC I'. Some limited oligomerization is also observed above 50-55 °C in this form of the protein (Fig. 3).
IpaC H (residues 63-170 deleted) possesses significantly less
ordered structure than IpaC, verging on a completely disordered conformation as shown by the shift of the CD minima from ~204 to 200 nm (Fig. 1A). No major temperature-dependent
structural changes are observed for IpaC
H, although slight
aggregation is apparent at higher temperatures. Thus, much of the
disordered structure seen in the complete protein may be reflected in
the behavior of this mutant.
The C terminus region of IpaC appears to contain the coiled-coil region postulated to contribute to IpaC self-interaction and effector function. Linker-scanning analysis provides an indirect picture of this region. A periodicity in loss of invasion activity is observed with different amino acid substitutions, which is consistent with functionally significant helical structure in this region (Fig. 5). Additional substitutions of proline residues, known helix breakers, also reduce invasion activity, further supporting this hypothesis.
During IpaC-mediated Shigella invasion of epithelial cells, IpaC is inserted into the host cytoplasmic membrane and, following uptake, mediates vacuolar escape (16), possibly by disrupting the phospholipid membrane following penetration (11). Previous studies have indicated that the ability to penetrate and disrupt liposomes occurs optimally when the lipid bilayer possesses a net negative charge (11). We find, however, that although interactions are enhanced in the presence of negatively charged liposomes, IpaC also causes low level disruption of neutral liposomes. The mechanism of disruption does not appear to be purely a detergent effect since at low temperatures and in the presence of neutral liposomes, the rate and extent of fluorescein release is much less than that observed by Triton disruption of liposomes. A strong temperature dependence is observed for the IpaC-liposome interaction for both lipid compositions. With negatively charged liposomes, the extent of release increases until the temperature reaches ~30 °C, at which point IpaC is nearly as effective in dye release as Triton. In contrast, neutral liposomes exhibit a sigmoidal temperature dependence in the release process. This may reflect the structural change occurring between 30 and 60 °C in IpaC, which may produce a more "penetration-efficient" form of IpaC. Alternatively, the concurrent aggregation process occurring in this temperature range may reflect the ability of oligomerized protein to disrupt the bilayer.
Neither IpaC I' nor IpaC H was able to disrupt liposomes. This is
consistent with previous findings that the IpaC hydrophobic region is
needed for association with Langmuir phospholipid monolayers (9).
Coupled with the potential importance of the central hydrophobic region
of IpaC for its overall stabilization, it is possible that this region
contributes to overall tertiary structure stabilization, but when
partially exposed, it directs penetration of phospholipid membranes.
This is consistent with the results of ANS binding experiments in which
titrations of IpaC with ANS at 40 °C demonstrate interaction of the
dye with IpaC, indicating that hydrophobic regions of the protein
become exposed under membrane-interactive conditions (not illustrated).
As expected, little binding of ANS to the two mutants is seen over a
wide temperature range.
IpaC H and IpaC I' fail to nucleate actin in vitro,
whereas IpaC and IpaC
I nucleate actin efficiently (Fig. 6). This
suggests that actin nucleation requires sequences at the IpaC C
terminus; however, it is possible that conformational changes induced
by mutations at the C terminus are responsible for this loss of
activity. The IpaC C terminus contains a sequence predicted to form a
coiled-coil that may be required for effector function. From these
observations and linker-scanning studies that reinforce the coiled-coil
hypothesis, it seems reasonable to conclude that this helical
conformation is important for some aspect of actin nucleation. This is
supported by preliminary data that the purified linker-scanning mutant
AS339, which cannot direct S. flexneri invasion of cultured
cells, is also unable to nucleate actin in vitro (not illustrated).
An analogous ability to nucleate actin was described for SipC of S. typhimurium (24); however, SipC has not been shown to act as a "direct effector" in Salmonella invasion. It was recently reported that SspC (SipC) from S. typhimurium co-purifies with HeLa cell actin in immunoprecipitation assays, but this relationship may be mediated by interactions with keratin 8 (33). Deletions introduced at the SspC C terminus and point mutations near its C terminus (most notably a Leu to Pro mutation) eliminate its ability to restore invasiveness to a Salmonella SspC mutant (33), suggesting similar structure-function relationships for the C termini of IpaC and SspC(SipC). How such mutations affect the ability for SspC/SipC to nucleate actin are not known, but this warrants further study. In the same report, mutations at the C terminus of SspC caused the protein to no longer associate with HeLa cell membranes following incubation with S. typhimurium (33). How similar mutations influence the ability of IpaC to disrupt liposomes has yet to be determined.
As with Salmonella, the role of actin nucleation in invasion by S. flexneri is not immediately obvious. It is possible that IpaC-mediated actin nucleation: 1) contributes to the localization of actin polymerization in host cells (34, 35) or 2) permits the quick burst of actin polymerization needed for rapid entry. It is also possible that actin nucleation by IpaC has a more direct role in Shigella invasion. A recent report demonstrates that a cdc42 knockout fibroblast-like cell line is still invaded by S. flexneri, albeit only at 15% of the levels seen in wild-type cells (36). Whatever its precise role, the IpaC protein is a key element in the invasion process of S. flexneri and may provide an important therapeutic target.
In conclusion, this work presents the first structural analysis of
IpaC, based on the functional organization scheme presented previously
(14). IpaC appears to be partially disordered with conformational
lability increasing under physiological temperature. The existence of a
helical domain at the C terminus of the protein, deemed necessary for
effector function in the invasion process, is probed, and the presence
of such a structure is strongly supported. Additionally, the
interaction of IpaC with phospholipid bilayers is further
characterized, and distinct domains of the protein are identified as
necessary for this interaction. Finally, in vitro actin
nucleation is demonstrated for IpaC, and this activity is preliminarily
localized to the C terminus.
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FOOTNOTES |
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* This work was supported by an NIGMS, National Institutes of Health Biotechnology Training Grant to the University of Kansas, a Bristol Myers Squibb tuition fellowship (to L. A. K.), and NIAID, National Institutes of Health funding (Grant AI34428) to (W. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Present address: University of Colorado Health Sciences Center, School of Pharmacy, 4200 E. 9th Ave., Denver, CO, 80262.
** Present address: Dept. of Molecular Microbiology, University of Texas at San Antonio, Health Science Center, San Antonio, TX 78229.
To whom correspondence should be addressed. Tel.: 785-864-3299;
Fax: 785-864-5294; E-mail picking@ku.edu.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208383200
2 A. Harrington and W. Picking, manuscript in preparation.
3 L. A. Kueltzo and C. R. Middaugh, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Ipa, invasion plasmid antigens; IpaC, invasion plasmid antigen C; IpaD, invasion plasmid antigen D; ANS, 8-anilino-1-napthalene sulfonic acid; TTSS, type III secretion system; MEM, minimum Eagle's medium; SipC, Salmonella invasion protein C.
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