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Journal of Bacteriology, February 1999, p. 869-878, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Functional Analysis of a Rickettsial OmpA Homology
Domain of Shigella flexneri IcsA
Macarthur
Charles,1
Juana
Magdalena,1
Julie A.
Theriot,2 and
Marcia
B.
Goldberg1,*
Department of Microbiology and Immunology,
Albert Einstein College of Medicine, Bronx, New York
10461-1602,1 and
Department of
Biochemistry, Stanford University School of Medicine, Palo Alto,
California 94305-53072
Received 2 July 1998/Accepted 11 November 1998
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ABSTRACT |
Shigella flexneri is a gram-negative bacterium that
causes diarrhea and dysentery by invasion and spread through the
colonic epithelium. Bacteria spread by assembling actin and other
cytoskeletal proteins of the host into "actin tails" at the
bacterial pole; actin tail assembly provides the force required to move
bacteria through the cell cytoplasm and into adjacent cells. The
120-kDa S. flexneri outer membrane protein IcsA is
essential for actin assembly. IcsA is anchored in the outer membrane by
a carboxy-terminal domain (the 
domain), such that the
amino-terminal 706 amino acid residues (the 
domain) are exposed on
the exterior of the bacillus. The domain is therefore likely to
contain the domains that are important to interactions with host
factors. We identify and characterize a domain of IcsA within the domain that bears significant sequence similarity to two repeated
domains of rickettsial OmpA, which has been implicated in rickettsial
actin tail formation. Strains of S. flexneri and
Escherichia coli that carry derivatives of IcsA containing
deletions within this domain display loss of actin recruitment and
increased accessibility to IcsA-specific antibody on the surface of
intracytoplasmic bacteria. However, site-directed mutagenesis of
charged residues within this domain results in actin assembly that is
indistinguishable from that of the wild type, and in vitro competition
of a polypeptide of this domain fused to glutathione
S-transferase did not alter the motility of the wild-type
construct. Taken together, our data suggest that the rickettsial
homology domain of IcsA is required for the proper conformation of IcsA
and that its disruption leads to loss of interactions of other IcsA
domains within the amino terminus with host cytoskeletal proteins.
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INTRODUCTION |
The gram-negative bacterium
Shigella flexneri causes diarrhea and dysentery by directly
invading the colonic epithelium and eliciting a severe local
inflammatory response. After inducing its entry into the cell, S. flexneri rapidly lyses the phagocytic vacuole and is released into
the cytoplasm (6, 21, 27, 40). Within the cytoplasm,
bacteria assemble actin on one pole of the bacterial body into tails
that consist of bundled actin filaments. The continuous polymerization
of actin at the junction of the bacterial body with the tail and the
incorporation of the resulting actin filaments into the tail provide
the force to propel the microorganism through the cytoplasm at speeds
of 12 µm min
1. Then, in a process that also requires
actin assembly, bacteria induce cell surface protrusions, which enable
bacterial spread into adjacent cells (23, 35, 37, 41).
IcsA, a 120-kDa S. flexneri outer membrane protein, has been
shown to be essential and sufficient for actin tail formation (4,
17, 26, 30). A strain with a disruption of icsA
invades cells and lyses the phagocytic vacuole but fails to induce
polymerization of actin and remains confined to the cytoplasm
(9). In addition, introduction of icsA into
Escherichia coli confers upon this organism the ability to
form actin tails in cytoplasmic extracts (17, 26).
IcsA is an 1,102-amino-acid protein that is unusual in that it is
localized at one pole of S. flexneri (16). It is
anchored in the outer membrane via a domain within the carboxy-terminal 344 amino acid residues (the domain), thereby exposing its
amino-terminal 706 amino acid residues (the domain) on the exterior
of the bacillus (45). Once translocated across the outer
membrane, IcsA is slowly cleaved by IcsP (SopA) between
Arg758 and Arg759 to release the 95-kDa domain into the growth medium or the cytosol (12, 13, 42).
The specific domain(s) of IcsA that is required for actin assembly has
not been identified, although deletion of a large region encompassing
amino acids 320 to 507 leads to loss of actin recruitment on the
surface of the bacillus (46).
Several other intracellular microorganisms have been shown to assemble
actin tails that are similar to those assembled by Shigella:
the gram-positive bacterium Listeria monocytogenes (8, 32, 48), certain members of the Rickettsiaceae family
of small gram-negative bacteria (19), and vaccinia virus
(7). The specific protein that mediates actin assembly on
L. monocytogenes, ActA, has been extensively characterized,
while those that mediate actin assembly on rickettsiae and vaccinia
virus have not yet been definitively identified. ActA has been shown to
be the sole Listeria factor required for this process
(11, 25) and bears no significant sequence similarity to
IcsA. ActA has two domains that appear to have distinct roles in actin
tail formation: an amino-terminal domain (amino acid residues 128 to
151) that is required for actin recruitment and a domain containing a
series of four proline-rich repeats (amino acid residues 263 to 390) that directly binds the vasodilator-stimulated phosphoprotein (VASP), a
focal adhesion protein, which in turn recruits profilin-actin to the
surface of the bacterium (5, 24, 29, 36, 43). Deletion of
the actin recruitment domain leads to loss of actin association with
ActA (29, 36), whereas deletion of the proline-rich repeats
leads to slowing of the rate of actin assembly by approximately two-thirds (28, 33, 43). In addition, mounting evidence indicates that the Arp2/3 complex of seven proteins is also required for actin assembly on L. monocytogenes (49, 50).
Among the Rickettsiaceae, spotted-fever group rickettsiae
(Rickettsia rickettsii, R. conorii, and R. akari) form actin tails in the cytoplasm of infected cells,
whereas typhus-group rickettsiae do not. Since spotted-fever group
rickettsiae express the 190-kDa outer membrane protein OmpA and
typhus-group rickettsiae lack OmpA, it has been suggested that OmpA may
have a role in rickettsial actin tail formation (19, 20).
In this context, we were interested in defining the IcsA domain(s) that
is essential to its function in actin assembly. We identified a domain
of IcsA that bears significant sequence similarity to a repeated domain
of rickettsial OmpA. We therefore analyzed the role of this domain in
translocation of IcsA to the bacterial surface and in actin assembly.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and cell lines.
The bacterial
strains and plasmids used in this study are listed in Table
1. All strains carrying icsA
alleles are derived from S. flexneri serotype 5 wild-type
strain M90T (39) or E. coli MC1061
(31). Shigella strains were grown in tryptic soy broth, and E. coli strains were grown in Luria-Bertaini
broth. Where appropriate, antibiotics were used at the following
concentrations: ampicillin, 100 µg/ml; spectinomycin, 100 µg/ml;
kanamycin, 40 µg/ml. HeLa and L2 cells used for plaque assays and
Shigella intracellular motility assays were maintained in
minimal essential medium supplemented with 10% fetal bovine serum and
1% nonessential amino acids.
Construction of in-frame deletions within icsA.
Plasmid pMBG235 (icsA WT [wild-type]) contains the entire
coding sequence for icsA, as well as 523 bp of flanking DNA
upstream of the translational start site and 56 bp of flanking DNA
downstream of the translational stop codon. pMAC103 (icsA3),
pMAC216 (icsA16), and pMAC217 (icsA17) were
generated by reverse PCR on pMB6235 DNA, using oligonucleotide primer
pairs 5'-CTGTGGATCCTTTATCTGCACTTAG-3' and
5'-CTTACTGGATCCACTATTCTGGCA-3',
5'-ACCTGTGGATCCTTTATCTGCACTTA-3' and
5'-GCTATGGATCCGACCAGAATAAATTG-3', and
5'-CCAGACTGGATCCGTAGATCAATTA-3' and
5'-CTGAACTTACTGGATCCACTATTCT-3', respectively. To construct the three site-directed mutants (icsA10, icsA14,
and icsA15), reverse PCR was carried out with pMBG235 as a
template and three different oligonucleotide primer pairs. pMAC210
(icsA10) was made with
5'-CTGGAATGATACAGATGGCGCTAGCCATG-3' and
5'-TCCATGGCTAGCGCCAGCTGTAGCATTC-3', thus creating
NheI ends and resulting in the following three amino acid
substitutions: Asp487 to Ala, Asp489 to Ala,
and Asp491 to Ala. pMAC214 (icsA14) was made
with 5'-GACTATACAGCTAGCTATATCAGTGAC-3' and
5'-CTGATATAGCTAGCTGTATAGGCAATAG-3' to create NheI
ends and resulted in the following two amino acid substitutions:
Asp467 to Ala and Lys470 to Ala. pMAC215
(icsA15) was generated with
5'-GTGACCAGAACGCGTTGATCTACGGTTT-3' and
5'-CCGTAGATCAACGCGTTCTGGGCACTGA-3' to generate
MluI ends and resulted in the following two amino acid
substitutions: Asp475 to Ala and Lys477 to Ala.
The absence of the deleted segment or the presence of the point
mutations in each case was confirmed by DNA sequencing.
To construct pMAC107 (
icsA7), a region encompassing
R. rickettsii ompA bp 2671 to 2868 was amplified by PCR with
oligonucleotide
primers 5'-GGAGCGGTGATTAGATCTACTACG-3' and
5'-CTATATCCCCAGATCTTTGACTTAAC-3',
thereby generating
BglII ends. This fragment was inserted into
pMAC103 at the
BamHI site in
icsA3, thus resulting in insertion
of rOmpA amino acids 868 to 933 after IcsA amino acid 507 with
insertion of an Arg residue and a Ser residue. The presence of
an
insert was confirmed by restriction digestion and PCR analysis,
and the
orientation and nucleotide sequence were verified to be
correct by DNA
sequencing. pMAC108 (
icsA8) and pMAC109 (
icsA9)
were made similarly, except with oligonucleotide primers
5'-CGACTAAAATAACGAGATCTGTGCA-3'
and
5'-GACTTAAAGATCTATTTAAATTTAACACA-3'. To construct pMBG282
(
gst::icsA67), a fragment encompassing
icsA bp 1897 to 2105 was
amplified by PCR with
oligonucleotide primers 5'-GCAGATAAAAGATCTACAGGTTTCAG-3'
and
5'-GCCAGAATAGTGAATTCAGTAAGTTC-3', thus generating a
BglII
and an
EcoRI end. This fragment was ligated
into the cloning vector
pGEX-2T (Pharmacia) at its
BamHI and
EcoRI sites. The resulting
gene fusion codes for a 34-kDa
protein (GST::IcsA67) consisting
of glutathione
S-transferase (GST) and IcsA amino acids 447 to
505 with a
thrombin cleavage site (LVPRGS) between the two polypeptides
and six
additional amino acids at the carboxy terminus (EFIVTD).
Stop codons in
each of the three reading frames are located downstream
of the coding
sequence of the gene fusion. The fusion protein
was expressed in
E. coli BL21, and purification was carried out
according to
the manufacturer's recommendations. DNA isolations,
transformation,
and cloning were performed by standard methods
(
38).
Protein preparation and analysis.
Whole-cell and supernatant
extracts were prepared as described previously (1, 22).
Protein extracts, resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, were transferred to nitrocellulose and probed with the
monoclonal antibody VIF8, which recognizes an epitope within the domain of IcsA, or with affinity-purified IcsA antiserum
(16). The VIF8 monoclonal antibody was synthesized according
to standard procedures by using IcsA that had been purified as
previously described (16). Visualization was performed by
enhanced chemiluminescence (Amersham).
IcsA localization and cell infection assays.
Labeling for
IcsA on the surface of bacteria grown in vitro was performed
essentially as described previously (16). Labeling for IcsA
and actin on S. flexneri-infected HeLa or L2 cell monolayers was performed essentially as previously described (16, 18). Plaque assays were performed essentially as described previously (42) with the exception that, where appropriate, ampicillin was added to the agarose overlay at a final concentration of 250 µg/ml. Monolayers were photographed 48 h after infection. Speeds of motile intracytoplasmic bacteria were determined as previously described (42).
Motility assays in Xenopus laevis egg cytoplasmic
extracts.
Motility assays were performed as described previously
(17). For competition studies, 8 µl of
rhodamine-conjugated monomeric actin in G buffer (5 mM Tris [pH 8.0],
0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM dithiothreitol [DTT])
was mixed with 3.2 µl of Kron buffer (0.05 mg of catalase per ml,
0.10 mg of glucose oxidase per ml, 2.5 mg of glucose per ml, 0.5 mM
DTT). The following were then combined in a small Eppendorf tube: 5.5 µl of extracts; 0.7 µl of the rhodamine-conjugated actin mixture to
a concentration of 40 µg/ml; 0.5 µl of phosphate-buffered saline
(PBS), GST, or GST::IcsA67; and 0.5 µl of a suspension of
formaldehyde-fixed bacteria in XB buffer (100 mM KCl, 0.1 mM
CaCl2, 2 mM MgCl2, 5 mM EGTA, 10 mM K HEPES
[pH 7.7], 50 mM sucrose). Following incubation on ice for 1 h,
0.5 µl of the assay mixture was spotted on a glass slide, covered
with a 22- by 22-mm coverslip, sealed with nail polish, and examined by
phase and fluorescence microscopy on a Nikon Diaphot 200 microscope
with epifluorescence optics using standard rhodamine filters (Chroma,
Brattleboro, Vt.). Between 250 and 350 individual bacteria were
examined for each concentration of polypeptide per experiment and were
evaluated for an association with polymerized actin in the form of
clouds and tails.
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RESULTS |
Sequence similarity of IcsA amino acids 446 to 506 to rickettsial
OmpA repeats.
IcsA has been shown to be anchored in the outer
membrane by a domain within the carboxy-terminal 344 amino acids ( domain); as a result, the amino-terminal 706 amino acids of the mature protein ( domain) are exposed on the bacterial surface (45, 46) and thus constitute the region of the protein likely to be
involved in interactions with host proteins. To aid in defining functional domains of IcsA, we compared the domain of IcsA against the Swiss-Protein, EMBL, and GenBank databases. Sequence alignment reveals significant similarity of IcsA amino acids 446 to 506 to two
discontinuous domains of the rickettsial outer membrane protein OmpA
(Fig. 1B). Spotted-fever group
Rickettsiae (R. rickettsii, R. conorii, and R. akari), like S. flexneri,
form actin tails in the cytoplasm of cells, whereas typhus-group
Rickettsiae do not. Since spotted-fever group
Rickettsiae express OmpA and typhus-group Rickettsiae lack OmpA, it has been suggested that OmpA may
have a role in rickettsial actin tail formation (19, 20).
Significantly, the region that contains these two discontinuous domains
is repeated 13 times in R. rickettsii OmpA, 14 times in
R. conorii OmpA, and 11 times in R. akari OmpA
(2, 14, 15). Within IcsA, the domain is not repeated. Of
note, IcsA bears no sequence similarity to L. monocytogenes
ActA.
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FIG. 1.
Schematic of IcsA and sequence alignment of IcsA with
the rickettsial homology domain. (A) Schematic of IcsA. SP and
vertically striped bar, signal peptide; open bar, domain;
diagonally striped bar, domain. (B) Sequence alignment of IcsA
amino acids 450 to 498 with R. rickettsii OmpA amino acids
686 to 789. Dashes represent gaps introduced in the sequence to
optimize alignment. (C) Sequence alignment of IcsA amino acids 446 to
506 with the three R. rickettsii OmpA domains exchanged for
it in this study. Amino acids in boldface type are conserved between
IcsA and the OmpA sequences. Amino acid designations for IcsA are those
in GenBank accession no. M22802, and those for OmpA are those in
GenBank accession no. M31227.
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As shown in Fig.
1A, two discontinuous regions within the rickettsial
OmpA repeat bear similarity to sequences within IcsA
amino acids 446 to
506. One, OmpA amino acids 686 to 705, shares
50% sequence identity
and 70% sequence similarity with IcsA amino
acids 450 to 469; the
other, OmpA amino acids 764 to 789, shares
34% sequence identity and
50% sequence similarity with IcsA amino
acids 473 to
498.
We constructed a series of in-frame deletions that encompass all or
portions of the coding region for the rickettsial homology
domain of
IcsA on plasmid pMBG235 (Fig.
2A).
icsA3 contains a
deletion that closely encompasses the
rickettsial homology domain,
while
icsA16 and
icsA17 contain deletions that roughly encompass
the coding
sequence for the amino-terminal and carboxy-terminal
halves of the
rickettsial homology domain, respectively. All
icsA alleles
constructed in this study contain the native
icsA promoter,
signal sequence, and coding region for the transmembrane domain.
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FIG. 2.
Schematic of IcsA constructs made in this study. (A)
Schema of internal in-frame deletions of IcsA. a, icsA WT,
which encodes wild-type IcsA; b, icsA3, which encodes a
deletion of amino acids 446 to 506, inclusive; c, icsA16,
which encodes a deletion of amino acids 446 to 473, inclusive; d,
icsA17, which encodes a deletion of amino acids 483 to 506, inclusive. Gaps represent deleted amino acids. Amino acid designations
are those in GenBank accession no. M22802. SP and vertically striped
bar, signal peptide; open bar, domain; diagonally striped bar, domain. (B) Alignment of IcsA amino acids 450 to 498 with the
site-directed mutations within this domain. a, icsA WT,
which encodes the wild-type amino acid sequence of IcsA (charged amino
acids are underlined); b, icsA10, which encodes the amino
acid substitutions Asp487 to Ala, Asp489 to
Ala, and Asp491 to Ala; c, icsA14, which encodes
the amino acid substitutions Asp467 to Ala and
Lys470 to Ala; d, icsA15, which encodes the
amino acid substitutions Asp475 to Ala and
Lys477 to Ala. Alanine residues indicated in boldface type
replace the charged residues underlined in line a.
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Expression of icsA constructs in S. flexneri and E. coli grown in vitro.
IcsA is
inserted into the outer membrane as a 120-kDa protein. Once in the
outer membrane, it is slowly cleaved by IcsP (SopA) between
Arg758 and Arg759, thereby releasing the 95-kDa
domain into the culture supernatant or host cell cytosol (10,
12, 13, 42). To determine whether the deletions within IcsA
altered either the amount of IcsA present or cleavage by IcsP, protein
samples prepared from approximately equivalent numbers of bacteria of
each construct in both S. flexneri SC560 and E. coli MBG263 were analyzed by Western blotting.
As expected,
S. flexneri strains M90T (wild type) and
MAC1000 (
icsA+ [i.e., the wild-type
icsA allele on pMBG235]) exhibited strong
bands
corresponding to the full-length 120-kDa protein in whole-cell
extracts
(Fig.
3A, lanes 1 and 3) and the cleaved
95-kDa polypeptide
in supernatant extracts (Fig.
3B, lanes 1 and 3).
MAC1016 (
icsA16)
gave bands in both whole-cell extracts
(Fig.
3A, lane 6) and supernatant
extracts (Fig.
3B, lane 6) that were
approximately as strong as
those seen for M90T and MAC1000. Each of the
other two constructs,
MAC1003 (
icsA3) and MAC1017
(
icsA17), produced less protein in
both whole-cell extracts
(Fig.
3A, lanes 4 and 7) and supernatant
extracts (Fig.
3B, lanes 4 and
7). For each construct, the apparent
molecular masses of the observed
bands correspond to the expected
sizes of the deletion constructs. The
relative faintness of the
bands for MAC1003 and MAC1017 suggest that
the mutant protein
produced by alleles
icsA3 and
icsA17 might be slightly less stable
than the native
protein. To verify that the relative faintness
of the signal was not
due to partial loss of the epitope by the
IcsA monoclonal antibody,
Western blot analysis was also performed
with a polyclonal antiserum
and gave similar results (data not
shown).
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FIG. 3.
Expression of IcsA constructs in S. flexneri
SC560 (A and B) and E. coli MBG263 (C). Western blot
analysis of whole-cell extracts (A and C) and supernatant extracts (B)
was performed with monoclonal antibody to IcsA. For each panel,
approximately equivalent amounts of protein preparations were loaded in
each lane. Apparent molecular masses are indicated, in kilodaltons.
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When expressed in
E. coli, the level of protein expression
from each of the mutant alleles was no different than that from
the
wild-type allele (Fig.
3C, lanes 3, 5, and 6 versus lane 1),
suggesting
that polypeptides expressed from
icsA3 and
icsA17
might
be more stable in
E. coli than in
S. flexneri. The apparent molecular
mass of each of the mutant
constructs was appropriate. Since IcsA
is not cleaved from the surface
of
E. coli MBG263, we did not
examine supernatant protein
preparations from the constructs in
these
strains.
Localization of IcsA on the surface of S. flexneri and
E. coli is not altered by deletions within the rickettsial
homology domain of IcsA.
IcsA is distributed unipolarly on the
surface of S. flexneri (16) and circumferentially
on E. coli MBG263 (17). To assess whether the
deletions involving the rickettsial homology domain affected surface
localization of IcsA, indirect immunofluorescence was performed on each
strain. IcsA is present on the surface of S. flexneri SC560
carrying the alleles icsA3, icsA16, and
icsA17 and is in the same unipolar distribution that it is
on wild-type S. flexneri (Fig.
4 and data not shown). For the strains
carrying the icsA3, icsA16, and icsA17
alleles, up to 60 to 75% of the bacteria did not have detectable IcsA
on the surface. Individual bacteria that had IcsA on the surface
appeared qualitatively to have less surface IcsA than did individual
wild-type organisms, as indicated by a less intense signal on indirect
immunofluorescence (data not shown).
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FIG. 4.
Localization of IcsA on the surface of S. flexneri cells expressing IcsA constructs. Indirect
immunofluorescence (A, C, and E) and phase-contrast (B, D, and F)
micrographs are shown. (A and B) M90T (WT). (C and D) MAC1000
(icsA WT). (E and F) MAC1003 (icsA3). Arrows
indicate unipolar localization of IcsA on bacteria. MAC1016
(icsA16) and MAC1017 (icsA17) each had a labeling
pattern that was indistinguishable from that of MAC1003 (data not
shown).
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The expression of the
icsA alleles on the surface of
E. coli MBG263 is shown in Fig.
5. As for
E. coli carrying the
wild-type
allele of
icsA (MAC2000) (Fig.
5A and B),
E. coli carrying each
of the mutant alleles displayed IcsA
distributed circumferentially
on the bacterial surface (Fig.
5 C to F
and data not shown). In
E. coli MBG263, more than 90% of
the bacteria observed for each
strain displayed IcsA on the surface. In
addition, the intensity
of IcsA on the surface of MAC2003
(
icsA3), MAC2016 (
icsA16), and
MAC2017
(
icsA17) was approximately the same as that on MAC2000
(
icsA WT).
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FIG. 5.
Localization of IcsA on the surface of E. coli cells expressing IcsA alleles. Indirect immunofluorescence
(A, C, and E) and phase-contrast (B, D, and F) micrographs are shown.
(A and B) MAC2000 (icsA WT). (C and D) MAC2003
(icsA3). (E and F) MAC2009 (icsA9). MAC2016
(icsA16) and MAC2017 (icsA17) each had a labeling
pattern that was indistinguishable from that of MAC2000 (data not
shown).
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Deletion of the rickettsial homology domain of IcsA results in loss
of plaque formation in HeLa and L2 cell monolayers.
The ability of
a Shigella strain to enter and spread between mammalian
cells can be assessed by its ability to form plaques on cell monolayers
(34). Plaques result when S. flexneri
successfully enter cells, multiply within them, and spread by
actin-based motility into cells that are immediately adjacent and,
subsequently, into the next layer of adjacent cells. SC560
(icsA), while capable of entering cells and dividing within
them, is unable to recruit actin and does not form plaques
(10). To evaluate whether MAC1003 (icsA3) could
form plaques, HeLa or L2 cell monolayers were infected with this
strain. Unlike MAC1000 (icsA WT) (Fig.
6A, panel c, and 6B, panel c) and
wild-type strain M90T (Fig. 6A, panel a, and 6B, panel a), MAC1003
(icsA3) (Fig. 6A, panel d, and 6B, panel d) did not form
plaques on HeLa or L2 cells, indicating that it was either unable to
enter cells or, once within the cell, unable to spread into adjacent
cells. Of note, the plaques formed by wild-type strain M90T are larger
than those formed by MAC1000 (icsA WT) (Fig. 6A and B,
panels a versus panels c).
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FIG. 6.
Plaque formation of S. flexneri cells
expressing IcsA constructs. (A) HeLa cell monolayers. a, M90T (WT); b,
SC560 (icsA); c, MAC1000 (icsA WT); d, MAC1003
(icsA3). (B) L2 cell monolayers. a to d, same as in panel A;
e, MAC1010 (icsA10). Plaques formed by MAC1014
(icsA14) and MAC1015 (icsA15) were not
significantly different from those formed by MAC1010
(icsA10). Petri dishes were photographed 48 h after
infection. Bar, 2 mm.
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Deletions within the rickettsial homology domain of IcsA abrogate
actin tail formation and actin-based motility in S. flexneri.
To determine whether MAC1003 (icsA3) did not
form plaques because of impairment in actin-based motility, HeLa cells
were infected with this strain and the association of intracellular
bacteria with actin was characterized. As expected, both the wild-type strain M90T (Fig. 7A and B) and MAC1000
(icsA WT) elaborated actin tails (Fig. 7C and D) and formed
cell surface protrusions (data not shown). In contrast, MAC1003
(icsA3) gained access to the cytoplasm, but was not
associated with actin and did not form tails (Fig. 7E and F) or cell
surface protrusions (data not shown). In addition, while both MAC1016
(icsA16) and MAC1017 (icsA17) also gained access
to the cytoplasm, neither was associated with actin or formed actin
tails in the cytoplasm of HeLa cells (data not shown).
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FIG. 7.
Actin tail formation of S. flexneri
expressing IcsA constructs. Rhodamine-phalloidin signal (A, C, and E)
and phase-contrast (B, D, and F) micrographs are shown. (A and B)
S. flexneri M90T (WT). (C and D) MAC1000 (icsA
WT). (E and F) MAC1003 (icsA3). Arrows indicate bacteria
with tails; arrowheads indicate bacteria lacking tails.
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One possible explanation for the inability of MAC1003
(
icsA3), MAC1016 (
icsA16), and MAC1017
(
icsA17) to associate with actin
could be that the bacteria
observed in the cytoplasm lacked surface
IcsA. To ascertain whether the
inability of MAC1003 (
icsA3), MAC1016
(
icsA16),
and MAC1017 (
icsA17) to recruit actin was attributable
to
reduced levels of surface IcsA, we evaluated the ability of
the mutant
constructs to recruit actin in the
E. coli MBG263
background,
since more than 90% of MBG263 cells carrying these alleles
had
levels of IcsA on their surfaces comparable to those on
E. coli carrying the wild-type allele (
icsA WT) (Fig.
5).
We have previously
shown that
X. laevis egg cytoplasmic
extracts support actin-based
motility of IcsA-expressing
E. coli MBG263 (
15). Unlike MAC2000
(
icsA WT),
which moved at an average rate of 12.9 ± 7.8 µm/min
in
Xenopus egg cytoplasmic extracts (Fig.
8B), MAC2003 (
icsA3),
MAC1016
(
icsA16), and MAC1017 (
icsA17) did not associate
with
actin or move (Fig.
8A and data not shown).
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|
FIG. 8.
Motility assay of E. coli MBG263 carrying
IcsA constructs. Phase-contrast time-lapse images in Xenopus
egg cytoplasmic extracts of MAC2003 (icsA3) (A) and MAC2000
(icsA WT) (B) are shown. The actin tail is seen as seen as a
phase-lucent streak behind moving MAC2000. Sequential frames are
separated by 30-s intervals. Images of MAC2016 (icsA16) and
MAC2017 (icsA17) were indistinguishable from those of
MAC2003.
|
|
Deletion of the rickettsial homology domain of IcsA leads to
relative unmasking of IcsA within the host cell cytosol.
As
mentioned above, a possible explanation for the inability of
MAC1003 (icsA3), MAC1016 (icsA16), and
MAC1017 (icsA17) to associate with actin in the
cytosol would be a lack of IcsA on the surface of these strains in this
setting. We therefore labeled MAC1003-infected cells for IcsA by using
affinity-purified polyclonal antiserum. Approximately 70 to 75% of the
observed intracellular bacteria displayed fluorescent label.
Surprisingly, the signal was consistently more intense on MAC1003
(icsA3) (Fig. 9G and H)
than on wild-type M90T (Fig. 9A and B) or MAC1000 (icsA WT) (Fig. 9E and F). This suggests that the mutant IcsA on MAC1003 was more
accessible to the antibody than native IcsA on M90T or MAC1000
(icsA WT). Consistent with this, the signal from
IcsA+ extracellular bacteria labeled with the same
antiserum is significantly more intense than that from intracellular
bacteria of any of the strains examined (data not shown).
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|
FIG. 9.
Immunolabeling of IcsA on intracellular S. flexneri strains carrying IcsA alleles. Indirect
immunofluorescence micrographs (A, C, E, and G), with affinity-purified
polyclonal antiserum to IcsA, and phase-contrast micrographs (B, D, F,
and H) are shown. (A and B) M90T (WT). (C and D) SC560
(icsA). (E and F) MAC100 (icsA WT). (G and H)
MAC1003 (icsA3). The apparent output of the
immunofluorescence signal was normalized for panels A, C, E, and G by
setting the background nonspecific signals to equivalent levels. Arrows
indicate intense signal from IcsA on MAC1003; arrowheads indicate
significantly weaker signal from IcsA on M90T and MAC1000.
|
|
Replacement of IcsA amino acids 446 to 506 with the rickettsial
repeat domain does not restore actin tail assembly.
To examine
whether the rickettsial repeat domain that bears sequence similarity to
IcsA amino acids 446 to 506 could functionally complement the similar
sequence of IcsA, the coding sequence of each of three distinct repeat
sequences from R. rickettsii OmpA was exchanged for that for
amino acids 446 to 506 of IcsA, creating alleles icsA7,
icsA8, and icsA9 (Fig. 1C).
The rickettsial repeats exchanged for IcsA amino acids 446 to 506 differ in length (Fig.
1C);
icsA7,
icsA8, and
icsA9 are
predicted to encode fusion proteins of 122, 126, and 120 kDa,
respectively. On Western blot analysis, the
S. flexneri strains
carrying these alleles, strains MAC1007
(
icsA7), MAC1008 (
icsA8),
and MAC1009
(
icsA9), exhibited bands corresponding to the predicted
masses of the fusion proteins that were fainter than the bands
corresponding to wild-type IcsA (Fig.
3A and B, lane 5, and data
not
shown versus Fig.
3A and B, lanes 1 and 3). For each of these
constructs in
S. flexneri, a significantly lower percentage
of
bacteria expressed IcsA on the surface than for MAC1000
(
icsA WT). None of these constructs in
S. flexneri formed plaques on
HeLa or L2 cell monolayers or
associated with actin in the cytoplasm
of infected HeLa cells (data not
shown).
The
E. coli MBG263 strains carrying these alleles, MAC2007
(
icsA7), MAC2008 (
icsA8), and MAC2009
(
icsA9), exhibited bands
corresponding to the predicted
masses of the fusion proteins that
were of the same approximate
intensity as the bands corresponding
to wild-type IcsA (Fig.
3C, lane
4, and data not shown versus
Fig.
3C, lanes 1 and 3). Each of these
strains expressed IcsA
on the bacterial surface to the same extent and
in the same circumferential
distribution as did MAC2000
(
icsA WT). However, none either associated
with actin or
moved in
Xenopus egg cytoplasmic extracts (data
not
shown).
To evaluate potentially critical contact residues within IcsA amino
acids 446 to 506, clusters of charged residues, which
are likely to be
exposed on the surface of the protein and therefore
to be involved in
protein-protein interactions, were identified
and subjected to
site-directed mutagenesis. This strategy has
been successfully applied
in a number of cases (
3,
51). Three
sets of mutations were
introduced in the region (Fig.
2B): the
first replaced amino acids
Asp
487, Asp
489, and Asp
491 with
alanines (giving
icsA10); the second replaced
Asp
467 and Lys
470 with alanines
(
icsA14); and the third replaced Asp
475 and
Lys
478 with alanines (
icsA15).
S. flexneri strains carrying the resultant
icsA alleles
were no different from
S. flexneri carrying the wild-type
allele (MAC1000) in any of the assays performed: they expressed
protein
at wild-type levels on Western blot analysis, expressed
IcsA on the
surface of approximately the same percentage of bacteria
and at
approximately the same intensity, formed plaques on HeLa
and L2 cell
monolayers that were not significantly different from
MAC100 (Fig.
6B,
panel c versus panel e), formed actin tails in
HeLa cells, and moved in
the cytoplasm of HeLa cells at speeds
comparable to MAC1000
(
icsA WT) (data not
shown).
Competition of the rickettsial homology domain polypeptide in actin
recruitment assays.
To further examine the role of IcsA amino
acids 446 to 506 in actin-based motility, a polypeptide that consists
of GST fused to amino acid residues 447 to 505 of IcsA
(GST::IcsA67) was used in competition assays with MAC2000
(icsA WT) in the Xenopus egg cytoplasmic
extracts. The polypeptide is greater than 90% pure (Fig.
10). As shown in Table
2, the fusion protein had no significant effect on either (i) the percentage of bacteria associated with actin
clouds, which are accumulations of polymerized actin that is not
organized into a bundle, or actin tails or (ii) the percentage of
bacteria assembling actin tails. Further, the IcsA-derived peptide
alone (after removal of GST) had no effect on actin polymerization in
these assays (unpublished data).
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|
FIG. 10.
Purified IcsA rickettsial homology domain used in
competition assays. A Coomassie-stained sodium dodecyl
sulfate-polyacrylamide gel of GST (lane 1) and GST::IcsA67
(lane 2) is shown. Apparent molecular masses are indicated, in
kilodaltons.
|
|
| |
DISCUSSION |
Shigella spp., like L. monocytogenes,
spotted-fever group Rickettsiae, and vaccinia virus, are
able to recruit host cell monomeric actin into a tail that consists of
filamentous actin bundled in a parallel array, with the barbed end of
the filaments uniformly directed towards the bacterium. Monomeric actin
is incorporated into the filaments at the junction of the bacterium
with the tail (47). The mechanism of actin tail formation
can therefore be presumed to involve recruitment of monomeric actin to
the bacterial pole, nucleation of this actin, orientation of the nuclei
such that the barbed end faces the bacterium, polymerization from the barbed end while protecting it from barbed end capping proteins, and
bundling of the filament into a parallel array. We have previously shown that the S. flexneri outer membrane protein IcsA is
located unipolarly on the bacterium at the site of the forming tail
(16), and we and others have shown that IcsA expressed in
E. coli is sufficient to permit actin tail formation in
cytoplasmic extracts (17, 26). Thus, IcsA is able to induce
the specific series of steps outlined above.
In this work, we have identified a region of IcsA that is necessary for
the interaction of IcsA with host cytoskeletal factors involved in
actin assembly on S. flexneri. Deletion of the domain of
amino acid residues 446 to 506 totally eliminates actin-based motility
of either S. flexneri or E. coli strains that
express the IcsA deletion construct. The mutant Shigella
strain does not form actin tails or cell surface protrusions, does not
recruit actin into a cloud at the bacterial surface, and does not form plaques on cell monolayers, indicating an inability to spread from cell
to cell. Most striking, however, is the relative unmasking of the IcsA
on the surface of S. flexneri strains expressing the mutant
allele when in the cytoplasm of infected cells (Fig. 9). This suggests
that native IcsA is made relatively inaccessible to antibody by its
interactions with host cell factors, while the mutant allele is
accessible by virtue of few or no such interactions.
These observations suggest that the deletion of amino acids 446 to 506 of IcsA either (i) leads to loss of a domain that directly interacts
with key host cell factors or (ii) disrupts the native conformation of
IcsA so as to impair the interaction of the IcsA domain that binds key
host cell factors with those factors. On the basis of data presented
here, we favor the latter. First, with the exception of the IcsA
construct encoded by icsA16, all of the IcsA deletion
constructs examined in this study are less stably expressed in S. flexneri and presented in lower amounts of the bacterial surface
(Fig. 3 to 5). Second, alanine mutagenesis of clusters of charged
residues within the region, which would be predicted to be on the
surface of the protein in this region and therefore likely to interact
with outer proteins, resulted in maintenance of the wild-type
phenotype. Third, each of the small nonoverlapping deletions within
amino acid residues 446 to 506 encoded by icsA16 and
icsA17 has the phenotype of the larger deletion, which
suggests that the intact rickettsial homology domain may be required
for proper spacing or proximity of other domains. And finally, in vitro
competition of a polypeptide of the region fused to GST did not alter
the motility of the wild-type construct (Table 2). These data provide
further refinement on the large region, encompassing amino acids 320 to
507, that was reported by Suzuki et al. to be required for actin
recruitment (46).
IcsA is thought to cross the bacterial cytoplasmic membrane via the Sec
secretion machinery, and subsequent translocation of the domain of
IcsA to the bacterial surface has been proposed to occur through a pore
formed by 13 passages of the domain through the outer membrane
(45). We observed a moderate decrease in the surface
presentation of most of the mutant deletion constructs expressed in
S. flexneri. Notably, when expressed in E. coli, the amount of IcsA on the bacterial surface was not detectably decreased from those for native IcsA: IcsA was nevertheless unable to
induce actin-based motility. The differences in levels of expression on
the bacterial surface between the S. flexneri background and the E. coli background suggest that the mechanism of
translocation of IcsA to the bacterial surface may differ in S. flexneri and E. coli.
Despite the sequence similarity between IcsA amino acids 446 to 506 and
the repetitive domains of the rickettsial OmpA proteins, exchange of
these domains did not yield a functional form of IcsA. One possible
explanation for this is that the exchange of the rickettsial domain did
not reconstitute the protein to its native conformation, as suggested
by the observation that the levels of expression on the bacterial
surface and the level of protein in whole-cell preparations did not
return to those seen with wild-type IcsA. The presence of homologous
domains in rickettsial OmpA suggests that the function served by these
domains may be generalizable among a group of outer membrane proteins.
How the function of the multiple copies of the domain that are present
in rickettsial OmpA would differ from the function of the single copy
that is present in IcsA is unclear.
| |
ACKNOWLEDGMENTS |
We thank T. Hackstadt for providing R. rickettsii
genomic DNA, T. R. Evans for providing X. laevis eggs,
J. Condeelis for helpful discussions and providing rhodamine-conjugated
monomeric actin, S. Almo for helpful discussions and critical review of the manuscript, and the Analytical Imaging Facility and the DNA Sequencing Facility at the Albert Einstein College of Medicine for
technical assistance.
This work was supported by NIH grants GM16654 (M.C.), AI35817 (M.B.G.),
and AI36929 (J.A.T.); the Pew Scholars Program in the Biomedical
Sciences (M.B.G.); and Established Investigator and Grant-in-Aid awards
from the American Heart Association (M.B.G.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461-1602. Phone: (718) 430-2118. Fax:
(718) 430-8711. E-mail: mgoldber{at}aecom.yu.edu.
| |
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Journal of Bacteriology, February 1999, p. 869-878, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
