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Journal of Bacteriology, September 1999, p. 5838-5842, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The C-Terminal Domain of the Bordetella pertussis
Autotransporter BrkA Forms a Pore in Lipid Bilayer Membranes
Jennifer L.
Shannon and
Rachel C.
Fernandez*
Department of Microbiology and Immunology,
University of British Columbia, Vancouver, British Columbia, Canada
V6T 1Z3
Received 18 May 1999/Accepted 1 July 1999
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ABSTRACT |
BrkA is a 103-kDa outer membrane protein of Bordetella
pertussis that mediates resistance to antibody-dependent killing
by complement. It is proteolytically processed into a 73-kDa N-terminal domain and a 30-kDa C-terminal domain as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. BrkA is also a member of
the autotransporter family of proteins. Translocation of the N-terminal
domain of the protein across the outer membrane is hypothesized to
occur through a pore formed by the C-terminal domain. To test this
hypothesis, we performed black lipid bilayer experiments with purified
recombinant protein. The BrkA C-terminal protein showed an average
single-channel conductance of 3.0 nS in 1 M KCl. This result strongly
suggests that the C-terminal autotransporter domain of BrkA is indeed
capable of forming a pore.
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TEXT |
The autotransporters are a growing
family of extracellular proteins, found in many gram-negative bacteria,
that have many different functions but appear to have the same
mechanism of export (19, 20, 25). Members of this diverse
family include immunoglobulin A proteases from Neisseria
gonorrhoeae (21) and Haemophilus influenzae
(36); VacA (11), a vacuolating cytotoxin from
Helicobacter pylori; the AIDA-I adhesin (28, 43)
from Escherichia coli; IcsA (44) from
Shigella flexneri, which is involved in intracellular spread; the ring-forming protein (32) from
Helicobacter mustelae; Tsh (37), a
temperature-sensitive hemagglutinin from an avian E. coli
strain; EspP (8), an extracellular serine protease from
enterohemorrhagic E. coli; and tracheal colonization factor (17), the adhesin pertactin (10), and serum
resistance protein BrkA (15) from Bordetella
pertussis.
These proteins are grouped together by the following three
characteristics. (i) Most of the mature proteins are proteolytically processed into an approximately 30-kDa C-terminal domain and a much
larger N-terminal domain, (ii) the C-terminal domains are predicted to
form amphipathic 
-barrels in the outer membrane, and (iii) export
through the outer membrane does not require accessory proteins; hence,
the name autotransporters.
In the proposed model of autotransporter secretion (22), an
N-terminal signal sequence enables translocation across the cytoplasmic
membrane. Once the protein is in the periplasm, the signal sequence is
cleaved and the C-terminal domain then inserts itself into the outer
membrane. It presumably forms a pore through which the N-terminal
domain is exported by the formation of a hairpin loop. Cleavage of the
N-terminal domain is thought to occur after translocation through the
outer membrane, either autoproteolytically or by another protease
(12).
In this study, we investigated the putative pore-forming ability of the
C-terminal domain of the B. pertussis autotransporter BrkA
through black lipid bilayer analysis. We found that the purified recombinant BrkA C-terminal protein forms channels in lipid bilayers whereas the BrkA N-terminal protein and the protein from a vector-only clone do not form channels in lipid bilayers.
Construction of clones.
RF1065 and DO218 were subcloned from
RF1066 (16). To construct RF1065 (Fig.
1a), brkA from the
BamHI site to the HindIII site was ligated to
pRSETb (Invitrogen, Carlsbad, Calif.), which represents amino acids
(aa) 694 to 1010 of BrkA. This clone contains the C-terminal domain
plus 37 aa that are upstream of the C-terminal processing site, as well
as an N-terminal His tag. JS13 contains pRSETb (Invitrogen) without an
insert. For DO218 (Fig. 1a), brkA from the AflIII
site to the BamHI site was ligated to pET30b (Novagen, Madison, Wis.). This clone contains the first 693 aa of BrkA with N-
and C-terminal His tags. All constructs were transformed into E. coli BL21 (DE3) pLysS cells (Novagen). Cultures were grown at
37°C in Luria broth or on Luria agar supplemented with 100 µg of
ampicillin per ml and 34 µg of chloramphenicol per ml.
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FIG. 1.
(a) Diagram of E. coli BrkA clones used in
this study. Numbers below the boxes refer to amino acids. (b) SDS-PAGE
and Coomassie blue staining showing pooled fractions of BrkA C-terminal
protein obtained by denaturing Ni2+ chromatography after
dialysis. Dialysis was performed slowly at 4°C against decreasing
concentrations of urea and finally against 0.1% Triton X-100-10 mM
Tris (pH 8.0). M, low-molecular-weight markers (Pharmacia) (molecular
sizes are in kilodaltons). (c) Western immunoblot of BrkA C-terminal
protein (same as in panel b). Detection was performed with an anti-BrkA
C-terminal protein monoclonal antibody. Kaleidoscope Prestained
Standards (Bio-Rad) were used for molecular size determination
(molecular sizes are in kilodaltons).
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Purification of the BrkA C-terminal domain from RF1065.
The
RF1065 clone used as the source of our BrkA C-terminal protein is shown
in Fig. 1a. Protein was purified from RF1065 by denaturing
Ni2+-nitrilotriacetic acid purification using the Xpress
System Protein Purification protocol (Invitrogen). In order to renature
the protein, elution fractions containing the protein of interest were
pooled and then slowly dialyzed against decreasing concentrations of urea in phosphate-buffered saline (PBS). Essentially, 8 M urea was
diluted at a rate of 1 ml/min by PBS during the dialysis. The final
dialysis was done overnight against 0.1% Triton X-100-10 mM Tris (pH
8.0). All dialysis was performed at 4°C.
After dialysis, pooled elution fractions were run on a sodium dodecyl
sulfate (SDS)-11% polyacrylamide gel (
23) and the
proteins
were visualized following staining with Coomassie brilliant
blue. The
Low Molecular Weight Electrophoresis Calibration Kit
(Amersham
Pharmacia Biotech, Baie d'Urfé, Quebec, Canada) was
used to
determine the molecular weight. SDS-polyacrylamide gel
electrophoresis
(PAGE) (Fig.
1b) revealed a 37-kDa band which
corresponds in size to
the BrkA C-terminal domain (30 kDa) along
with the 37 aa upstream of
the processing site (Fig.
1a) and the
His
tag.
Western blot analysis was also performed to confirm the identity of the
protein. After electrophoresis was performed (
23),
the
proteins were transferred to an Immobilon-P membrane (Millipore,
Bedford, Mass.) at 100 V for 75 min by a wet transfer apparatus
(Trans-Blot Electrophoretic Transfer Cell; Bio-Rad, Hercules,
Calif.)
in accordance with the manufacturer's instructions. After
transfer,
the membrane was blocked with a 5% (wt/vol) skim milk
solution in PBS
for at least 1 h at room temperature. Washing
and antibody
incubation were carried out in a PBS solution containing
0.25% skim
milk and 0.5% Tween 20. Membranes were incubated with
a 1/30 dilution
of mouse anti-BrkA C-terminal protein monoclonal
antibody (a gift from
Roger Parton, University of Glasgow) for
2 h at 37°C and then
washed for 30 min. Secondary antibody incubation
with a 1/10,000
dilution of goat anti-mouse immunoglobulin G conjugated
to horseradish
peroxidase (Cappel, ICN Biomedicals, Costa Mesa,
Calif.) was carried
out for 1 h at room temperature and followed
by 30 min of washing.
Renaissance Western blot chemiluminescence
reagent (NEN Life Science
Products, Boston, Mass.) was used for
detection. Kaleidoscope
Prestained Standards (Bio-Rad) were used
for molecular weight
determination. The results of this Western
blot analysis (Fig.
1c)
confirmed that the BrkA C-terminal protein
had been
isolated.
Black lipid bilayer analysis of the BrkA C-terminal protein.
The pore-forming ability of the purified BrkA C-terminal protein was
assessed through black lipid bilayer experiments, which were performed
as previously described (3). Addition of the protein to a 1 M KCl solution bathing a membrane of 1.5% (wt/vol) oxidized
cholesterol in n-decane with an applied voltage of 50 mV
caused stepwise increases in conductance (Fig.
2a). This indicates that channels were
being formed in the membrane. The distribution of these conductance
measurements is shown in Fig. 2b. The average single-channel
conductance of the BrkA C-terminal protein in 1 M KCl was found to be
3.0 nS. This average was calculated from 127 conductance increases
obtained from two separate experiments. Similar-size channels were also
observed when BrkA C-terminal protein from a second purification was
used.
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FIG. 2.
Black lipid bilayer analysis. (a) Single-channel
conductance measurements after addition of the BrkA C-terminal protein
from RF1065 to a 1 M KCl solution bathing a membrane of 1.5% oxidized
cholesterol in n-decane. There was an applied voltage of 50 mV. (b) Histogram of single-channel conductance measurements showing
pore size distribution.
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To help rule out the possibility of contaminants in our protein
preparation, we performed black lipid bilayer experiments
with BrkA
C-terminal protein that had undergone an additional
gel purification
step. In this case, the BrkA C-terminal protein
was electrophoresed on
an 11% polyacrylamide gel (
23) and then
a portion of the
gel was stained with Coomassie brilliant blue
so that it could be used
as a guide for cutting of the protein
out of the unstained segment of
the gel. The protein was eluted
from the gel overnight at room
temperature with 0.1% Triton X-100-10
mM Tris (pH 8.0). Addition of
this protein caused increases in
conductance (Fig.
3) similar in size to those seen
previously.
This indicates that the channels seen were formed by the
BrkA
C-terminal protein and not by possible contaminants.
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FIG. 3.
Black lipid bilayer analysis. Single-channel conductance
measurements after addition of BrkA C-terminal protein that had been
further purified by elution from an SDS-PAGE gel. Protein was added to
a 1 M KCl solution bathing a membrane of 1.5% oxidized cholesterol in
n-decane. There was an applied voltage of 50 mV. The arrows
indicate stepwise increases in conductance.
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Black lipid bilayer analysis of the BrkA N-terminal protein and
protein from a vector-only clone.
RF1065 overexpresses the BrkA
C-terminal protein in the form of inclusion bodies, which necessitated
purification under denaturing conditions. Porin proteins may
contaminate preparations when proteins are produced in inclusion bodies
(9a). In order to ensure that our results were not due to
these possible contaminants, we used the BrkA N-terminal protein from
DO218 (Fig. 1a) in black lipid bilayer experiments. This protein is
also expressed in inclusion bodies, and we purified it in a way similar
to that used for the C-terminal protein, except that the final dialysis
was against PBS. Addition of the N-terminal protein diluted in 0.1%
Triton X-100-10 mM Tris (pH 8.0) caused no increases in conductance
(Fig. 4), even when large amounts of the
protein were added. Upon addition of the BrkA C-terminal protein to the
system (Fig. 4), channels were again observed.
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FIG. 4.
Black lipid bilayer analysis. Single-channel conductance
measurements were done after addition of first the BrkA N-terminal
protein (DO218) and then the BrkA C-terminal protein (RF1065). Protein
was added to a 1 M KCl solution bathing a membrane of 1.5% oxidized
cholesterol in n-decane. There was an applied voltage of 50 mV. Protein from DO218 was purified in a way similar to that used for
the protein from RF1065. The N-terminal protein was diluted in 0.1%
Triton X-100-10 mM Tris (pH 8.0) before addition.
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As another control for our protein purification method, we performed a
black lipid bilayer experiment with protein that had
been purified from
a vector-only clone (JS13; does not contain
a
brkA insert)
in the same manner as the BrkA C-terminal protein.
Increases in
conductance were not observed upon addition of protein
from JS13 (Fig.
5). Addition of the BrkA C-terminal
protein again
caused the appearance of channels (Fig.
5). Both of these
results
help to rule out contaminants as the source of the channels
seen
with the BrkA C-terminal protein.
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FIG. 5.
Black lipid bilayer analysis. Single-channel conductance
measurements were done after addition of first protein from a clone
containing the vector alone (JS13) and then the BrkA C-terminal protein
(RF1065). Protein was added to a 1 M KCl solution bathing a membrane of
1.5% oxidized cholesterol in n-decane. There was an applied
voltage of 50 mV. The protein from JS13 was purified in the same way as
the protein from RF1065.
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Discussion.
In this study, we demonstrated that the C-terminal
autotransporter domain of BrkA is capable of forming a pore. Black
lipid bilayer analysis showed the formation of channels upon addition of the BrkA C-terminal protein but not upon addition of the BrkA N-terminal protein or protein from a vector-only clone, all of which
had been purified similarly. As well, the BrkA C-terminal protein that
had been further purified by being cut out of an SDS-PAGE gel still
formed channels.
As evidenced by our results, black lipid bilayer analysis can be used
to determine the channel-forming capabilities not only
of typical
trimeric porins but also of a wide variety of proteins,
including those
involved in protein export (
5) and those from
mycobacterial
(
46,
47) and gram-positive (
38) cell walls.
Some
examples of pore-forming proteins and their pore sizes are
listed in
Table
1.
As can be seen in Table
1, the 3.0-nS pore size of the BrkA C-terminal
protein is larger than those reported for the typical
E. coli porins OmpF and OmpC, as well as many other proteins,
but is
not without precedent. OprF from
Pseudomonas aeruginosa and
the 53-kDa outer sheath protein of
Treponema denticola were
found to have single-channel conductances of 5.6 nS (
3) and
10.9 nS (
13), respectively. As well, a 59-kDa cell wall
protein
from
Mycobacterium chelonae was found to have a
channel size of
2.7 nS (
47).
A larger pore size for the BrkA C-terminal domain would be expected
based on its protein-exporting function. PapC, an outer
membrane usher
through which the subunits of P pili are exported
in uropathogenic
E. coli, has a pore diameter of at least 2 nm
(
45). This is large enough to allow the passage of
unravelled
pilus subunits. It appears that a similar requirement
for unfolded
passenger proteins also applies to
autotransporters.
In order for the BrkA C-terminal protein to have consistently formed
channels, one would expect it to be properly folded but
the denaturing
purification procedure that we used brings up the
question of whether
or not the protein should be capable of assuming
its native
conformation. In order to renature the protein, we
slowly removed the
urea and used a detergent. Examples of the
renaturation of outer
membrane proteins into a native conformation
using related procedures
have been shown previously. Outer membrane
proteins extracted from
inclusion bodies and renatured were found
to have the same pore-forming
characteristics as those obtained
from the outer membrane (
40,
41). As well, crystals of monomeric
outer membrane protein A
(OmpA) were obtained from inclusion bodies
(
35) and then
used for structure determination by X-ray diffraction
analysis
(
34).
The proposed native conformation of the autotransporter C-terminal
domains, as appears to be the case for all outer membrane
proteins, is
a multistranded
-barrel. Even though all of the
outer membrane
proteins examined to date contain this same basic
structure
(
34), they do not all form pores, which is one reason
why
the pore-forming ability of the BrkA C-terminal domain needed
to be
tested. The computer-predicted models of these proteins
give a general
idea of their structure, but as evidenced by the
recently published
structures of OmpA (
34) and FepA (
9),
they can be
wrong. The main structural features of autotransporters
that need to be
elucidated are the exact number of strands, whether
the extreme
N-terminal strand of the barrel faces in or out, and
whether or not the
pore is blocked after export of the N-terminal
domain of the protein.
We are currently addressing these questions
by mapping the topology of
the BrkA C-terminal
domain.
In summary, we have shown that the BrkA C-terminal domain is capable of
forming a pore, which supports the proposed model
of autotransporter
export. To our knowledge, this is the first
time that pore-forming
ability of the C-terminal domain has ever
been demonstrated for a
member of the autotransporter
family.
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ACKNOWLEDGMENTS |
This work was supported by Natural Sciences and
Engineering Research Council grant OGP0194599. J.L.S. was a recipient
of an NSERC PGS A scholarship.
We thank the R. E. W. Hancock laboratory for assistance with
the black lipid bilayer experiments and helpful discussions of the data
and, in particular, Fiona Brinkman for critical reading of the
manuscript. We thank Roger Parton for the gift of the anti-BrkA C-terminal protein monoclonal antibody and Dave Oliver and Carrie Mathewson for the DO218 protein.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, #300-6174 University Blvd., Vancouver,
B.C., Canada V6T 1Z3. Phone: (604) 822-6824. Fax: (604) 822-6041. E-mail: rachelf{at}interchange.ubc.ca.
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Journal of Bacteriology, September 1999, p. 5838-5842, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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