Previous Article | Next Article 
Infection and Immunity, March 2001, p. 1671-1678, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1671-1678.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutagenesis and Functional Reconstitution of Chlamydial Major
Outer Membrane Proteins: VS4 Domains Are Not Required for Pore
Formation but Modify Channel Function
E. S.
Hughes,
K. M.
Shaw, and
R. H.
Ashley*
Department of Biomedical Sciences, University
of Edinburgh, Edinburgh EH8 9XD, United Kingdom
Received 26 July 2000/Returned for modification 23 October
2000/Accepted 14 December 2000
 |
ABSTRACT |
Chlamidial organisms are obligate intracellular pathogens
containing highly antigenic porin-like major outer membrane proteins (MOMPs). MOMP epitopes are of substantial medical interest, and they
cluster within four relatively short variable (VS) domains. If MOMPs
adopt a 
-barrel fold, like bacterial porins, the VS domains may form
extramembranous loops and the conserved regions of the protein may
correspond to predicted membrane-located -strands. However,
molecular studies on native MOMPs have been hampered by the need to
culture chlamydiae in eukaryotic host cells and purification and
reconstitution remain problematic. In addition, the organisms are
difficult to manipulate genetically, and it has also been difficult to
functionally reconstitute recombinant MOMPs. To help overcome these
problems and improve our understanding of MOMP structure and function,
we cloned and expressed C. trachomatis and C.
psittaci MOMPs and functionally reconstituted them at the single-channel level. We measured significant functional differences between the two proteins, and by removing and exchanging VS4, we
tested the hypothesis that the largest variable domain forms an
extramembranous loop that contributes to these differences. Proteins in
which VS4 was deleted continued to form functional ion channels,
consistent with the idea that the domain forms an extramembranous
protein loop and incompatible with models in which it contributes to
predicted membrane-located -strands. Additionally, the properties of
the chimeric proteins strongly suggested that the VS4 domain interacts
closely with other regions of the protein to form the channel entrance
or vestibule. Our approach can be used to probe structure-function
relationships in chlamydial MOMPs and may have implications for the
generation of effective antichlamydial vaccines.
| |
INTRODUCTION |
Chlamydiaceae
members are obligate intracellular pathogens responsible for a
broad spectrum of human and animal diseases (25, 26,
31). They include Chlamydia psittaci and
Chlamydia pecorum, which mainly infect animals, and the
human pathogens Chlamydia pneumoniae and Chlamydia
trachomatis. The long-term complications of C. trachomatis infection, including trachoma-related blindness, ectopic pregnancy, and female infertility, have a very substantial impact on human well-being (40). The respiratory tract
pathogen C. pneumoniae has also been linked to coronary
thrombosis (12, 37), although a recent epidemiological
study failed to confirm an association (39). Finally,
C. psittaci can also infect humans, leading to respiratory
disorders and abortion.
Previous studies have shown that chlamydial organisms carry
epitopes recognized by both T cells (for examples, see references 1, 22, 32, and 33) and B cells (for examples, see
references 2, 6, 9, 10, 29, 30, and 47), and following
extensive serotyping, they have been classified according to a range of partly overlapping serogroup- and species-specific epitopes
(2, 30, 45). These epitopes elicit both neutralizing
(20, 21) and nonneutralizing (18) antibodies,
some of which protect against infection (19, 23, 24, 46,
47). Many of the epitopes have been attributed to the major
outer membrane protein (MOMP), which comprises about 60% of the total
outer membrane protein of the infectious elementary body (EB)
(5). As a result, efforts have been focused on delineating
the role this protein has to play in the pathogenicity of
Chlamydia spp. and on determining whether it might form the
basis for useful antichlamydial vaccines. However, molecular studies
have been severely limited by technical problems. The organism is
difficult to grow in large amounts, and native MOMPs are particularly
difficult to purify and reconstitute. In addition, chlamydiae cannot
yet be routinely subjected to genetic manipulation, and the functional
reconstitution of heterologously expressed recombinant MOMPs has proven
to be problematic.
Despite these challenging technical problems, it has been discovered
that MOMPs have important structural and functional roles in the
complex chlamydial life cycle, in which noninfectious, metabolically
active reticulate bodies (RBs) alternate with the spore-like EB.
Functionally, MOMPs behave like bacterial porins (17, 21),
forming a pathway for the passage of ions and small solutes through the
outer membrane of the organism (4). However, unlike other
porins, MOMPs contain a large number of cysteine residues (up to nine
in C. trachomatis). These are relatively well conserved
throughout the genus, and the ability of MOMP to form intramolecular or
intermolecular disulfide bonds (or both) may contribute to the rigidity
of the free-living EB, which lacks the peptidoglycan layer found in
gram-negative bacteria (3).
Every known MOMP contains four domains of variable sequence (VS1 to
VS4) interspersed between five highly conserved regions or sequences
(CS1 to CS5) (29). As anticipated, the variable domains
have been shown to underpin much of the observed serological diversity
and represent major neutralization determinants that contain many
antigenic epitopes (2, 27, 47) and also serovar-specific T-cell epitopes (in VS1, VS2 and VS4 [1]). Proteolytic
digestion studies (2, 8, 20, 36) strongly support models
in which the highly immunogenic variable domains are surface exposed
and imply that the poorly immunogenic, conserved regions of MOMPs corresponding to CS1 to CS5 contain transmembrane domains. However, apart from studies such as these, we currently know very little about
the structure and function of chlamydial MOMPs at the molecular level,
because of the technical challenges mentioned previously.
We have speculated that chlamydial MOMPs assemble as -barrels, like
bacterial porins, and contain predicted transmembrane -strands
joined by protein turns and loops. In support of this, we purified
wild-type C. psittaci MOMP and showed by circular dichroism
that it had a porin-like secondary structure, with the high -sheet
and low 
-helix content typical of other porins (43). We
also confirmed that it formed poorly selective, high-conductance porin-like ion channels in planar lipid bilayers (43). The
reconstitution of functional channels extended the liposome swelling
studies of Bavoil et al. (4), who first suggested
that MOMP was a porin, and opened up a new route to structure-function
studies. The present study is the first investigation of
structure-function relationships in chlamydial MOMPs to combine cDNA
mutagenesis and functional reconstitution.
| |
MATERIALS AND METHODS |
Cloning of full-length C. trachomatis MOMP
cDNA.
The MOMP gene from C. trachomatis
was amplified by PCR from genomic DNA kindly provided by the
Chlamydia Genome Project. The primer pairs used were the
following: sense primer,
5'-GTCGACATGCTGCCTGTGGGGAATCCTGCTGAACC-3'; antisense primer,
5'-CCATGGTTTGCAAAAAAAACTGGACCCGACCG-3'. SalI and NcoI restriction sites (shown in
boldface type) were engineered into the sense and antisense primers,
respectively. The sense primer also contained an engineered methionine
start codon (underlined). Each reaction was carried out in a 50-µl
volume containing 100 ng of template, 200 µM deoxynucleoside
triphosphates, a 0.5 µM concentration of each primer, 200 mM
Tris-HCl (pH 8.8 at 25°C), 100 mM KCl, 100 mM
(NH4)2SO4,
20 mM MgSO4, 1% (vol/vol) Triton X-100, a
1-mg/ml concentration of nuclease-free bovine serum albumin, and 2.5 U
of Pfu DNA polymerase. Amplification of the target DNA was
accomplished by an initial denaturation step of 5 min at 94°C,
followed by a thermal cycle of 30 s at 94°C, 30 s at
65°C, and 1 min at 72°C with a final strand extension step of
72°C for 7 min. Twenty-five cycles of amplification were carried out.
In order to reduce the number of mutations introduced by PCR,
Pfu DNA polymerase (Promega) was used throughout. All PCR
products were purified using the QIAEX II gel extraction kit (Qiagen).
Cloning of full-length C. psittaci MOMP cDNA.
A truncated MOMP gene previously cloned from C. psittaci genomic DNA, which does not encode the first 16 amino
acids of the mature protein (44), was extended by PCR
using a three-step heminested procedure. The sense primers were the
following:
T1(ps), 5'-TTAATCCATGGTACTATGTGGGAAGGAGCTTCAGGAGATCC-3'; T2(ps),
5'-CCAGCTGAACCAAGTTTATTAATCGATGGCACTATGTGGGAAG-3'; and
T3(ps),
5'-CATATGTTGCCTGTGGGGAACCCAGCTGAACCAAGTTTATTAATCG-3'. We used the antisense primer T4(ps),
5'-CCATGGCAGAGATTCCTAGGTTCTGATAGCGGGACAA-3'. The
T3 sense and T4 antisense primers contained the engineered restriction
sites NdeI (to provide a methionine start codon) and NcoI, respectively, which are shown in boldface type.
Amplification of the target DNA was accomplished using the protocol
described above.
Amplification of recombinant cDNAs.
Both full-length inserts
were cloned into the EcoRV site of pSTBlue-1 (Novagen),
using the Perfectly Blunt cloning kit (Novagen). The nomenclature
adopted here is pSTB-ps and pSTB-tra for C. psittaci and
C. trachomatis constructs, respectively. These constructs were then transformed into Novablue cells (Novagen), which lack the
gene for T7 RNA polymerase, to amplify and recover DNA for examination
of the construct sequences. All subsequent mutations were carried out
in this nonexpressing vector prior to subcloning for protein
expression, and all the clones were sequenced prior to subcloning
(Oswell DNA). After verification, full-length constructs were subcloned
into the expression vector pET22b(+) (Novagen) using corresponding
restriction sites present in the multiple cloning site of pET22b(+) (to
give pET-ps and pET-tra). Ligations were carried out overnight at
16°C, and the plasmids were transformed into Novablue cells.
In vitro site-directed mutagenesis.
Site-directed
mutagenesis was carried out to introduce an AatII
restriction site into the 5' and 3' ends of the VS4-encoding domains of
the C. trachomatis and C. psittaci MOMP cDNAs.
The mutations were generated by the method described by Chen and
Przybyla (7), using two overlapping primers containing the
desired mutation. The primers used for C. psittaci MOMP cDNA
were the following: 5' sense,
5'-CGAGCAACTTTTGAC(T)GT(C)C(T)GACGCTATCCGCATC-3'; 5'antisense,
5'-GATGCGGATAGCGTCGACGTCAAAAGTTGCTCG-3'; 3'sense, 5'-AACAAATTCGCTGACG(T)TCTTGCAAATTGCTTCG-3';
and 3' antisense,
5'-CGAAGCAATTTGCAAGACGTCAGCGAATTTGTT-3'.
For C. trachomatis MOMP cDNA, the primers were the
following: 5' sense,
5'-CGAGCAAGCTTTGAC(T)GT(C)CGATACGATTCGTATAGC-3'; 5' antisense,
5'-GCTATACGAATCGTATCGACGTCAAAGCTTGCTCG-3'; 3'
sense,
5'-GGTCAGCTCGGAGACG(A)T(C)C(A)ATGCAAATCGTTTCC-3'; and 3' antisense,
5'-GGAAACGATTTGCATGACGTCTCCGAGCTGACC-3'. The
introduced restriction site is shown in bold, with original nucleotides in parentheses. Each PCR was carried out in a 50-µl volume containing 20 ng of plasmid, 200 µM deoxynucleoside
triphosphates, 125 ng of each primer, 200 mM Tris-HCl (pH 8.8 at
25°C), 100 mM KCl, 100 mM
(NH4)2SO4,
20 mM MgSO4, 1% (vol/vol) Triton X-100, a
1-mg/ml concentration of nuclease-free bovine serum albumin, and 2.5 U
of Pfu DNA polymerase. Amplification was accomplished by an
initial denaturation step of 1 min at 94°C followed by a thermal
cycle of 94°C for 30 s, 65°C for 1 min, 72°C for 8 min (i.e., 2 min/kb), and a final strand extension step at 72°C for 7 min. Each template was subjected to 18 cycles of amplification. Mutations were first introduced into the 5' end of the VS4-encoding region, and the DNA was then digested with DpnI (to remove
the methylated, template DNA) and transformed into DH5 cells to
repair the nicks in the recombinant DNA. 3' Mutations were then
introduced in the same way, using the first product as a template.
Final digestion with DpnI and passage through DH5 cells
produced constructs containing AatII restriction sites at
each end of the VS4 region [pSTB-ps(VS4) and pSTB-tra(VS4)].
Both constructs were sequenced prior to subcloning.
VS4 domain swapping and deletion.
The mutated clones
pSTB-ps(VS4) and pSTB-tra(VS4) were digested with AatII for
2 h at 37°C to yield four fragments: pSTB-ps minus VS4, ps(VS4),
pSTB-tra minus VS4, and tra(VS4). The fragments were
gel-purified using the QIAEX II gel extraction kit (Qiagen). ps(VS4)
was ligated into pSTB-tra minus (VS4), and tra(VS4) was ligated into
pSTB-ps minus (VS4). The ligation products were transformed into
Novablue cells to isolate mutated constructs pSTB-tra minus VS4 plus
ps(VS4) and pSTB-ps minus VS4 plus tra(VS4). Finally, these chimeric
constructs and the two deletion constructs, pSTB-ps minus VS4 and
pSTB-tr minus VS4, were subcloned into pET22b(+), as previously
described. Domain orientation in the chimeric constructs was verified
by PCR.
Protein expression and isolation of inclusion bodies (IBs).
Subcloned plasmid constructs isolated from Novablue cells were used to
transform BL21 (DE3) cells to express recombinant protein. Conditions
for expression were optimized in preliminary studies in each case. For
example, for C. trachomatis MOMP, a single colony was
inoculated into 5 ml of Luria broth containing ampicillin (100 µg/ml)
and the culture was incubated at 37°C with shaking (250 rpm)
overnight. This culture was diluted 1:100 into fresh Luria broth and
incubated at 37°C with shaking to an optical density of 0.6 at 590 nm. Isopropyl--D-thiogalactopyranoside was
then added to a final concentration of 1 mM to induce production of the
recombinant protein, and the cells were incubated at 37°C for a
further 3 h. Cells were harvested by centrifugation, and IBs were
prepared as previously described (44). The channel proteins were extracted and solubilized using
octylglucoside-dithiothreitol (octylglucoside-DTT), analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as
previously described (44), and reconstituted into planar
lipid bilayers either immediately or after storage at 
70°C for up
to 4 months.
Planar bilayer reconstitution.
Bilayers were formed by
drawing a dispersion of diphytanoyl phosphatidylcholine (Avanti) in
n-decane (Sigma) across a 0.3-mm hole in a polystyrene
partition separating two solution-filled chambers (35, 36,
38). The contents of both chambers were stirred (using stirbars)
as required, and all the bilayers used had a final capacitance of 200 to 300 pF and a conductance of 5 to 10 pS. Spontaneous thinning of the
lipid dispersion with bilayer formation was verified by monitoring the
increase in membrane capacitance until it stabilized at the expected
value (corresponding to a specific capacitance of 0.3 to 0.5 µF/cm2). Initially, both chambers contained 50 mM KCl, 20 mM Tris-HCl (pH 7.4), and 1 mM DTT, and the membrane was
voltage-clamped at 0 mV with an Axopatch 200B amplifier (Axon
Instruments). Potentials are always quoted as cis minus
trans, where the cis chamber corresponds to the
compartment to which protein was subsequently added. Channel incorporation was induced by adding MOMP to a final concentration of
1 ng/ml in the presence of 500 mM KCl in the cis chamber, with stirring. The membrane potential was repeatedly switched between
+60 and -60 mV, and following the appearance of unit
transmembrane currents, the cis chamber was extensively
perfused with 50 mM KCl-20 mM Tris-HCl (pH 7.4) containing 1 mM DTT to
limit the incorporation of additional channels. Thereafter, the salt
concentration in each chamber was changed by perfusion or by direct
addition of concentrated salt solutions, as required.
Single-channel recording and analysis.
Transmembrane
currents were low-pass filtered (2 kHz, 8-pole Bessel response) and
recorded. Voltage clamp protocols are described in detail in Results.
Recordings were postfiltered and analyzed using pClamp 8 software
(Axon) and PSI Plot (Polysoftware International). Positive (upgoing)
currents are defined as currents in which positive ions flow
cis to trans or (equivalently) negative ions flow
trans to cis. The results were analyzed by
nonpaired t testing, and levels of significance are given in
the appropriate sections in Results.
| |
RESULTS |
Cloning and expression of full-length MOMPs.
The
sequence of the full-length C. trachomatis MOMP cDNA
was confirmed to correspond to the published sequence of
serovar Da (accession number X62918.1), and that of full-length
C. psittaci MOMP corresponded to the full-length
version (accession number X51859, Chlamydophila
abortus) of the previously cloned ovine abortion strain
S36/3 (44). Schematic representations of the
full-length MOMPs, together with details of their VS4 regions, are
shown in Fig. 1a. Denaturing SDS-PAGE
(10% [wt/vol]) analysis of these and all the other recombinant
proteins used in this study showed protein bands of the predicted sizes
(40 kDa) (Fig. 1b).
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 1.
Expression of recombinant chlamydial MOMPs. (a)
Schematic representation of the approximate positions and sizes of
C. psittaci and C. trachomatis MOMP VS
domains. The amino acid sequences (single-letter code) include the
residues (underlined) that were mutagenized to valine after
AatII sites were introduced into the cDNAs to allow
protein engineering. The longer underlined sequence in C.
trachomatis VS4 is a species-specific conserved nonapeptide.
(b) A 10% (wt/vol) Coomassie blue-stained SDS-PAGE gel of
octylglucoside-dithiothreitol extracts containing the recombinant
proteins used in this study. Lanes 1 to 8 contain, respectively, 10 µg of full-length C. trachomatis MOMP, C.
trachomatis minus VS4, chimeric C.
trachomatis MOMP containing the VS4 domain from C.
psittaci MOMP, full-length C. psittaci MOMP,
C. psittaci MOMP minus, chimeric C.
psittaci MOMP containing the VS4 domain from C.
trachomatis, uninduced transformed cells, and nontransformed
cells. The position of a 42.5-kDa size marker is indicated.
|
|
Because of the high cysteine content of chlamydial MOMPs, we maintained
the recombinant proteins in reducing conditions (1
mM DTT) throughout
this study in order to prevent random inter-
or intramolecular
disulfide bond formation and protein aggregation
during extraction and
reconstitution.
Functional reconstitution.
Diphytanoyl phosphatidylcholine
formed exceptionally stable bilayers that routinely withstood an
imposed membrane potential of ±100 mV for several minutes, and
potentials of up to ±150 mV for short periods (1 min), without
dielectric breakdown. The full-length proteins incorporated
spontaneously into bilayers in the presence of high salt concentrations
(500 mM) and their channel properties were consistent in over 50 independent experiments. This suggested that during membrane
incorporation, the proteins folded correctly and consistently into the
same structure. Full-length C. psittaci channels (Fig.
2) were similar in appearance to
those previously obtained from nondenatured native MOMP
(43) and from a recombinant protein containing an
N-terminal 16-residue truncation (44). As detailed
previously (44), we never observed channel activity
attributable to contaminating Escherichia coli porins.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 2.
Functional reconstitution of C. psittaci
MOMP. Typical ion channel activity obtained from recombinant
full-length C. psittaci MOMP incorporated into a planar
lipid bilayer. The recording was initiated by switching the membrane
holding potential from 0 to 150 mV, with 50 mM KCl on both sides of
the membrane. Note the successive closure of six initial unit
conductances over time, with little or no activity remaining after
32 s, and the brief closures illustrated in the magnified inset
(low-pass filtered at 0.2 kHz). The negative (downwards) currents are
consistent with K+ flowing trans to
cis and with Cl flowing cis
to trans (see Materials and Methods).
|
|
Characteristically, MOMP channels activated rapidly (within 1 ms) on
switching the membrane holding potential from 0 to
150
mV and
gradually inactivated over the course of
30 to 60 s. In
addition to the "staircase" effect produced by the shutting down
of
individual unit conductances (six in all in Fig.
2), open channels
also
displayed frequent short-lived closures (Fig.
2, inset).
Inactivated
channels could be reactivated by repolarizing or depolarizing
the
membrane, and similar activation was seen on switching to
positive
holding potentials (data not shown). The frequency of
short-lived
closures at negative potentials was increased compared
to that at
positive potentials, suggesting that the channels incorporated
in a
consistent
orientation.
Full-length
C. trachomatis MOMP channels behaved in a
broadly similar way to
C. psittaci MOMP (Fig.
3), except that they inactivated
more
rapidly at
150 mV (or at +150 mV [data not shown]). This
inactivation was slow or incomplete over the course of
30 s at
lower
holding potentials (e.g.,
100 mV) (Fig.
3). As previously
noted for
native
C. psittaci MOMP (
35), the number of
unit conductances
counted in independent experiments with both of the
recombinant,
full-length, wild-type proteins tended to occur in
groups of three,
six, or occasionally nine.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 3.
Functional reconstitution of C.
trachomatis MOMP. Typical ion channel activity obtained from
recombinant full-length C. trachomatis MOMP incorporated
into a planar lipid bilayer. The recording was initiated by switching
the membrane holding potential from 0 to 150 mV (upper trace) or to
100 mV (lower trace), with 50 mM KCl on both sides of the membrane
(filtered at 0.2 kHz). The baseline bilayer currents, with no channels
open, are indicated by dotted lines. The upper inset box magnifies the
period of 1 s immediately after switching the membrane holding
potential to 150 mV and illustrates three unit currents superimposed
on a typical exponentially decaying bilayer membrane capacitative
discharge current.
|
|
Channel conductances.
To enable a detailed analysis of the
properties of reconstituted channels under reproducible experimental
conditions, we used the bilayer voltage-clamp protocol summarized in
Fig. 4c. Briefly, we applied a holding
potential of 0 mV and then initiated repetitive sweeps consisting of
4 s at 100 mV, a voltage ramp over 32 s from 100 mV to
+100 mV, 4 s at +100 mV, and finally a step return to 100 mV.
This protocol was suitable for examining channel conductances over a
wide range of symmetric ion concentrations, from 50 to 2,000 mM on each
side of the bilayer. Repetitive sweeps (three to four) showed that the
measurements were highly reproducible for each experiment.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 4.
Measurement of channel conductances using voltage
ramps. C. trachomatis MOMP channels (in the same
experiment) were exposed to 1 M KCl (a) or 2 M KCl (b), present in each
case on both sides of the incorporated proteins. The membrane potential
was varied between 100 mV and +100 mV at a constant rate of 6.25 mV/s, using the voltage clamp protocol summarized in panel c. Each main
panel shows three unit conductances, and the dotted lines project
the "closed" levels of the top (third) conductance state.
Note that all the real and projected lines are linear and intersect at
the point of zero current and zero potential (the current and voltage
axes are indicated by fine horizontal and vertical lines,
respectively). The gradients of the channel traces (pA/V) correspond to
the appropriate multiple (1, 2, or 3) of the channel slope conductance
(pS).
|
|
Figure
4a and b illustrate experiments with reconstituted
C. trachomatis MOMP exposed to two different KCl concentrations,
1,000 and 2,000 mM, respectively. Up to three unit conductances
(single
channels) are open at
100 mV. As the membrane potential
is ramped
towards 0 mV, the slope of the line corresponds to the
total
conductance of all the open channels (measured in pS, where
1 pS = 1 pA per V). The crossover point (zero current level),
at 0 mV in this
case, represents the point at which there is no
chemical or electrical
driving force for ion flux (even though
all three channels are open).
Finally, as the potential is ramped
on towards +100 mV, the unit
conductances tend to close down sequentially.
It is important to note
that the slopes traced by the individual
unit conductances all project
to 0
mV.
We next carried out experiments to delineate specific functional
differences between
C. psittaci and
C. trachomatis MOMPs.
Figure
5
summarizes the KCl concentration dependence of the channel
conductances. Both curves are characteristic of porins, with
conductances
exceeding 1,000 pS at and above 1,000 mM KCl and no
evidence of
saturation even at 2,000 mM KCl. However, the conductances
clearly
differ between the two proteins, with that of
C. trachomatis MOMP
being consistently lower than that of
C. psittaci MOMP. The value
for
C. trachomatis MOMP is
20% less than that of
C. psittaci MOMP at the highest
salt concentration used (1,730 ± 165 pS versus
2,250 ± 125 pS [mean ± standard deviation {SD}, respectively,
n = 5). This difference is highly significant
(
P < 0.001).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 5.
Concentration dependence of MOMP conductances. The
conductances of full-length C. psittaci and
C. trachomatis MOMPs were measured under symmetric
ionic conditions over a range of KCl concentrations. Each point
represents the average value from at least three independent
experiments, and bars show ±1 SD calculated from five to eight
independent experiments.
|
|
Channel selectivity.
Channel selectivities for
Cl or K+ also differed to
a measurable extent. To quantify these differences, we measured
reversal (equilibrium) potentials with 250 mM KCl in the cis
chamber and 50 mM KCl in the trans chamber. Under these
conditions, the Nernst equation predicts a reversal potential of plus
or minus 38 mV for an ideally selective Cl or
K+ channel, respectively (correcting the ionic
concentrations for activities [13]).
We measured reversal potentials under asymmetric ionic conditions using
voltage ramps. Figure
6 shows single
examples of ramps
obtained from bilayers containing either
C. psittaci or
C. trachomatis MOMP channels. The reversal
potentials are indicated. As shown
here, current traces often showed a
clear break at this crossover
point. This represented the opening of an
additional channel as
the holding potential moved through the point at
which the current
reversed. The reversal potentials for
C. psittaci and
C. trachomatis MOMPs were
20.4 ± 1.5 mV (
n = 8) and
14.4 ± 0.8 mV
(
n = 8) for
n independent experiments,
respectively. Thus, both channels were
more selective for the cation
than for the anion, but their reversal
potentials (i.e., their relative
anion versus cation selectivities)
differed to a highly significant
extent (
P < 0.001).
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 6.
Measurement of channel selectivities using voltage
ramps. Recombinant MOMPs from C. psittaci (a) and
C. trachomatis (b) were subjected to voltage ramps
(lower panel) with 250 mM KCl in the cis bilayer chamber
and 50 mM KCl in the trans chamber. Positive, upgoing
currents at 0 mV (i.e., in the absence of an electrical driving force)
indicate a net flux of K+ flowing cis to
trans (i.e., channels from both species are more
selective for K+ than for Cl). The reversal
(equilibrium) potentials, where negative holding potentials (applied to
the cis chamber; see Materials and Methods) exactly
balance the chemical driving force, are indicated by arrows.
|
|
Domain deletion.
VS4 is the most extensive variable domain in
chlamydial MOMPs. It is known to be highly immunogenic and very likely
to be surface exposed. Interestingly, the VS4 domain of C. trachomatis MOMP also contains a conserved nonapeptide sequence
common to all serovars (see Fig. 1a). Do these VS4 domains form
extramembranous protein loops that contribute to the functional
differences between C. psittaci and C. trachomatis MOMPs? In order to answer this question, we first
inserted unique AatII sites into regions of the
MOMP cDNAs flanking the VS4 coding sequences, to facilitate manipulation of the VS4 domains. The resulting point mutations (A277V
and T318V for C. trachomatis MOMP and A303V and F343V for C. psittaci MOMP) had no significant effect on channel
selectivity or on channel conductances in symmetric 50 or 500 mM
KCl (Table 1).
MOMPs in which the entire VS4 domain was deleted remained capable of
forming functional ion channels (e.g., Fig.
7), although
both deletion constructs
gave rise to channels that were more
K
+ selective
than channels formed by the full-length recombinant
proteins. In each
case, reversal potentials measured in 250 versus
50 mM KCl
(Table
1) corresponded to a mean
K
+:Cl
selectivity ratio
(calculated as in references
14 and
43)
of
11:1 (correcting for
ionic activities). However, the conductances
of the deletion constructs
measured in symmetric 50 mM KCl were
not increased compared to those of
full-length proteins (Table
1). This suggested that the VS4 domain does
not form a partially
obstructing, pore-confined loop (
11,
41).
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 7.
Ion channels formed by C. trachomatis
MOMP lacking the VS4 domain. (a) With 250 mM KCl cis
versus 50 mM KCl trans, the reversal potential for
C. trachomatis MOMP lacking the VS4 domain is 30 mV
(arrow). (b) Longer recordings at membrane potentials of +100 mV and
100 mV (low-pass filtered at 0.2 kHz, with the zero-current baseline,
i.e., no open channels, indicated in each case by a dotted line). The
standard voltage-clamp protocol is also shown.
|
|
Domain swapping.
Does the VS4 domain influence channel
function by forming a nonessential but functionally significant part of
the MOMP channel entrance or vestibule? To test this idea, we used the
engineered restriction sites in our MOMP cDNAs to
interchange the VS4 domain in C. psittaci MOMP (see Fig. 1a)
with the VS4 domain of C. trachomatis MOMP. We then
reexamined the single-channel properties previously found to be
statistically different between the two full-length, recombinant
proteins (including proteins containing the point mutations introduced
by the restriction sites).
The ion channels formed from the chimeric
C. trachomatis
protein containing
C. psittaci VS4 (Fig.
8) tended to inactivate
within
20 s at
150 mV but remained active for much longer at
100 mV, similar to
the behavior of full-length
C. trachomatis MOMP. Strikingly,
the mean reversal potential of the chimera in
250 versus 50 mM KCl was
indistinguishable from that of full-length
C. psittaci MOMP,
or
C. psittaci MOMP containing point mutations,
but was
significantly different from that of the corresponding
C. trachomatis MOMPs (Table
1). Figure
8 also shows a subconductance
level (S) that was not detected in other recordings.
View larger version (32K):
[in this window]
[in a new window]
|
FIG. 8.
Functional reconstitution of C.
trachomatis MOMP containing C. psittaci
VS4. Typical ion channel activity obtained from recombinant C.
trachomatis MOMP containing the C. psittaci VS4
domain in place of C. trachomatis VS4 is shown. As in
Fig. 3, the recording was initiated by switching the membrane
holding potential from 0 to 150 mV (upper trace) or to 100 mV
(lower trace), with 50 mM KCl on both sides of the membrane. The
baseline bilayer currents, with no channels open, are indicated by the
dotted lines. The upper inset box details gating behavior on an
expanded time scale (filtered at 0.2 kHz), and S indicates a region of
prominent substate behavior.
|
|
The chimeric
C. psittaci protein containing
C. trachomatis VS4 also formed fully functional ion channels. These
tended to
behave like full-length
C. psittaci channels in
their activation
and conductance behavior, and their selectivity was
indistinguishable
from that of full-length
C. psittaci MOMP,
including
C. psittaci MOMP containing point mutations (Table
1). Finally, the conductances
of the two chimeras were not
significantly different in relatively
low concentrations of KCl
(symmetric 50 mM KCl), but like the
full-length proteins, their
conductances differed significantly
in symmetric 500 mM KCl (Table
1).
| |
DISCUSSION |
MOMPs as porins.
The -sheet-rich secondary structure
and ion channel functions of chlamydial MOMPs (43, 44)
strongly resemble the properties of bacterial porins (11, 17,
41). Porin -barrels have turns and loops connecting
transmembrane -strands, and our general model for MOMP encompasses
the idea that some of its extramembranous loops and turns are formed by
the variable domains. In this paper, we combined channel engineering
and functional analysis by single-channel recording to test the
idea that VS4, the largest variable domain, contributes an
extramembranous loop. The VS4 domain of C. trachomatis is
also of particular interest because it contains a species-specific epitope comprising nine amino acids that is conserved between all the
known serovars (28, 34, 35).
When used in tandem with protein mutagenesis and engineering,
single-channel recording can provide a powerful assay to test
specific
structural hypotheses, even in the absence of a crystal
structure. A
particular advantage of single-molecule studies is
that they do not
rely on the availability of large amounts of
refolded proteins but can
proceed at the rate of just one refolded
molecule at a time, provided
the molecules fold consistently and
operate in the same way. The
full-length recombinant MOMPs reconstituted
in the present study
displayed highly reproducible functional
properties, similar
to purified
C. psittaci MOMP channels
(
43).
This strongly suggests that during reconstitution,
the proteins
fold consistently and adopt a native-like conformation. In
addition,
the channels appeared to insert into the bilayer in the same
orientation,
because they retained the same asymmetry in
voltage-dependent
gating.
The VS4 domain is not essential for pore formation.
Typical of general diffusion porins, the channels formed by full-length
C. psittaci and C. trachomatis MOMPs were of high conductance and showed little evidence of saturation, even at KCl
concentrations of 2,000 mM. However, we were able to detect significant
differences in conductance after increasing the salt concentration to
500 mM. Although both channels were poorly selective, we noted that
they were both more selective for K+ than for
Cl, similar to many bacterial porins and to
recombinant truncated MOMPs (44). We had previously noted
that purified native C. psittaci MOMP was slightly anion
selective (43), although it had a conductance similar to
that of the recombinant proteins (44). The native
preparation may have retained associated proteins or small molecules
that bind to the channel and modify its selectivity. By varying the
experimental conditions, we also found a difference in anion versus
cation selectivity between C. psittaci and C. trachomatis MOMPs, and this was statistically significant in 250 mM versus 50 mM KCl.
To investigate the role of the VS4 domain, we deleted the regions
encoding it from the respective genes and expressed and
functionally
reconstituted the corresponding channels. Both recombinant
MOMPs formed
functional ion channels. This provides very strong
support for the idea
that VS4 is not involved in the formation
of predicted transmembrane
-strands, because in that case proteins
lacking VS4 would be
very unlikely to be able to fold into a
-barrel
(i.e., a rolled-up
-sheet) (
16). In addition, the conductances
of the
channels in which VS4 was deleted were no higher than those
of either
the full-length or the chimeric proteins, suggesting
that VS4 does not
form a pore-confined "eyelet" loop (
11). In
other
porins, eyelet loops substantially limit the flow of ions,
and simple
geometric calculations suggest that porin
-barrels
in which such an
obstruction was removed would increase their
conductance by at least an
order of magnitude (
15).
The VS4 domain may contribute to the channel vestibule.
If VS4
domains do not form transmembrane -strands or pore-confined loops,
an alternative role would be to contribute to the formation of the
channel entrance or vestibule. In this context, the domain might exert
a particularly strong influence on ion selectivity. In particular, the
addition of fixed negative charges might increase the local activity of
cations and promote the permeation of K+ rather
than Cl. However, despite losing a substantial
net negative charge of 2 or 3 U, the MOMP channels in which the VS4
domain was removed became markedly more K+
selective, rather than more Cl selective.
In a simplistic model, the net loss of negative charge on
removing the VS4 domains would be expected to reduce the negative
surface potential at or near the pore entrance and reduce rather
than
enhance cation permeability. Also, transplanting the VS4
domain from
C. psittaci to
C. trachomatis MOMP made the
latter
more K
+ selective, not less, despite
the fact that this actually decreased
the net negative charge
(though only by one unit). In contrast
to their selectivities, the
conductances of the hybrid channels
continued to show a significant
difference (with slightly more
variability for the
C. psittaci protein containing the
C. trachomatis VS4
domain). These findings suggest that the VS4 domain influences
selectivity by complex interactions with other parts of the protein,
including other loops and turns in the channel vestibule, and
its role
cannot be considered in isolation from the rest of the
protein, with
which it must interact. However, it is clear that
the structure of this
loop is not important for proper assembly
of the putative
-barrel.
Conclusions.
The functional reconstitution of wild-type and
mutagenized chlamydial MOMPs in planar lipid bilayers provides a new
way to probe MOMP structure-function relationships. We have shown that the VS4 domain is not required for pore formation, strongly implying (for a -barrel model) that it does not contribute to predicted transmembrane -strands. It does not appear to be a pore-confined loop, and instead our results suggest that it may help to form the
channel vestibule, where it interacts closely with other protein loops
or turns. The conformations of these surface-exposed regions will be of
particular interest when the crystal structures of MOMPs from different
species become available.
Our findings may have implications for the design of anti-MOMP
vaccines. First, it appears to be likely that the utility of
subunit
vaccines based on the VS4 domain and, by implication,
other VS domains
will be limited by the dependence of protein
folding on the local
protein environment. With the possible exception
of further work based
on the highly conserved nonapeptide in the
C. trachomatis
VS4 domain, this suggests that only linear epitopes
(rather than
conformational epitopes) may be of value. On the
other hand, we have
shown that MOMPs can be substantially modified
yet still remain
functional. Organisms in which MOMPs are attenuated
could have a
valuable role in future vaccination
strategies.
| |
ACKNOWLEDGMENTS |
This work was supported by the Wellcome Trust.
We thank the Chlamydia Genome Project for genomic DNA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biomedical Sciences, University of Edinburgh, George Square, Edinburgh EH8 9XD, United Kingdom. Phone: (0441) (0)31 650 3873. Fax: (0441) (0)31 650 3711. E-mail: Richard.Ashley{at}ed.ac.uk.
Editor:
D. L. Burns
| |
REFERENCES |
| 1.
|
Arno, J. N.,
C. Xie,
R. B. Jones, and B. van der Pol.
1998.
Identification of T cells that respond to serovar-specific regions of the Chlamydia trachomatis major outer membrane protein in persons with serovar E infection.
J. Infect. Dis.
178:1713-1718[CrossRef][Medline].
|
| 2.
|
Baehr, W.,
Y.-X. Zhang,
T. Joseph,
H. Su,
F. E. Nano,
K. D. E. Everett, and H. D. Caldwell.
1988.
Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes.
Proc. Natl. Acad. Sci. USA
85:4000-4004[Abstract/Free Full Text].
|
| 3.
|
Barbour, A. G.,
K.-I. Amano,
T. Hackstadt,
L. Perry, and H. D. Caldwell.
1982.
Chlamydia trachomatis has penicillin-binding proteins but not detectable muramic acid.
J. Bacteriol.
151:420-428[Abstract/Free Full Text].
|
| 4.
|
Bavoil, P.,
A. Ohlin, and J. Schachter.
1984.
Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis.
Infect. Immun.
44:479-485[Abstract/Free Full Text].
|
| 5.
|
Caldwell, H. D.,
J. Kromhout, and J. Schachter.
1981.
Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.
Infect. Immun.
31:1161-1176[Abstract/Free Full Text].
|
| 6.
|
Caldwell, H. D., and J. Schachter.
1982.
Antigenic analysis of the major outer membrane protein of Chlamydia spp.
Infect. Immun.
35:1024-1031[Abstract/Free Full Text].
|
| 7.
|
Chen, B., and A. E. Przybyla.
1994.
An efficient site-directed mutagenesis method based on PCR.
BioTechniques
17:567-659.
|
| 8.
|
Collet, R. A.,
W. J. Newhall,
R. A. Jersild, and R. B. Jones.
1989.
Detection of surface-exposed epitopes on Chlamydia trachomatis by immune electron microscopy.
J. Gen. Microbiol.
135:85-94[Medline].
|
| 9.
|
Conlan, J. W.,
I. N. Clarke, and M. E. Ward.
1988.
Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis.
Mol. Microbiol.
2:673-679[Medline].
|
| 10.
|
Conlan, J. W.,
M. Kajbaf,
I. N. Clarke,
S. Chantler, and M. E. Ward.
1989.
The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable Chlamydiae.
Mol. Microbiol.
3:311-318[CrossRef][Medline].
|
| 11.
|
Cowan, S. W.,
T. Schirmer,
G. Rummel,
M. Steiert,
R. Ghosh,
R. A. Pauptit,
J. N. Jansonius, and J. P. Rosenbusch.
1992.
Crystal structures explain functional properties of two E. coli porins.
Nature
358:727-733[CrossRef][Medline].
|
| 12.
|
Grayston, J. T.,
L. A. Campbell,
C.-C. Kuo,
C. H. Mordhorst,
P. Saikku,
D. H. Thom, and S.-P. Wang.
1990.
A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR.
J. Infect. Dis.
161:618-625[Medline].
|
| 13.
|
Harned, H. S., and B. B. Owen.
1957.
The physical chemistry of electrolyte solutions.
Reinhold, New York, N.Y.
|
| 14.
|
Hayman, K. A., and R. H. Ashley.
1993.
Structural features of a multisubstate cardiac mitoplast anion channel: inferences from single channel recording.
J. Membr. Biol.
136:191-197[CrossRef][Medline].
|
| 15.
|
Hille, B.
1994.
Ionic channels of excitable membranes, 2nd ed., p. 294-296.
Sinauer, Sunderland, Mass.
|
| 16.
|
Huang, H.,
D. Jeanteur,
F. Pattus, and R. E. W. Hancock.
1995.
Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD.
Mol. Microbiol.
16:931-941[CrossRef][Medline].
|
| 17.
|
Jap, B. K., and P. J. Walian.
1990.
Biophysics of the stucture and function of porins.
Q. Rev. Biophys.
23:367-403[Medline].
|
| 18.
|
Lampe, M. F.,
K. G. Wong,
L. M. Kuehl, and W. E. Stamm.
1997.
Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.
Infect. Immun.
65:317-319[Abstract].
|
| 19.
|
McCafferty, M. C.,
A. J. Herring,
A. A. Andersen, and G. E. Jones.
1995.
Electrophoretic analysis of the major outer membrane protein of Chlamydia psittaci reveals multimers which are recognized by protective monoclonal antibodies.
Infect. Immun.
63:2387-2389[Abstract].
|
| 20.
|
Newhall, W. J.
1988.
Antigenic structure of surface-exposed regions of the major outer membrane protein of Chlamydia trachomatis.
Rev. Infect. Dis.
10:S386-S390.
|
| 21.
|
Nikaido, H.
1992.
Porins and specific channels of bacterial outer membranes.
Mol. Microbiol.
6:435-442[CrossRef][Medline].
|
| 22.
|
Ortiz, L.,
M. Angevine,
S.-K. Kim,
D. Watkins, and R. DeMars.
2000.
T-cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein elicit serovar-specific immune responses in infected humans.
Infect. Immun.
68:1719-1723[Abstract/Free Full Text].
|
| 23.
|
Peeling, R.,
I. W. Maclean, and R. C. Brunham.
1984.
In vitro neutralization of Chlamydia trachomatis with monoclonal antibody to an epitope on the major outer membrane protein.
Infect. Immun.
46:484-488[Abstract/Free Full Text].
|
| 24.
|
Sa, S.,
A. Souriau,
F. Bernard,
J. Salinas, and A. Rodolakis.
1995.
An oligomer of the major outer membrane protein of Chlamydia psittaci is recognized by monoclonal antibodies which protect mice from abortion.
Infect. Immun.
63:4912-4916[Abstract].
|
| 25.
|
Schachter, J.
1988.
Overview of human diseases, p. 153-166.
In
A. L. Barron (ed.), Microbiology of Chlamydia. CRC Press, Boca Raton, Fla.
|
| 26.
|
Schachter, J.
1988.
The intracellular life of Chlamydia.
Curr. Top. Microbiol. Immunol.
138:109-139[Medline].
|
| 27.
|
Stephens, S. P.,
E. A. Wagar, and G. K. Schoolnik.
1988.
High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis.
J. Exp. Med.
167:817-831[Abstract/Free Full Text].
|
| 28.
|
Stephens, R. S.,
F. S. Fawaz,
K. A. Kennedy,
K. Koshiyama,
B. Nichols,
C. Ooij, and J. N. Engel.
2000.
Eukaryotic cell uptake of heparin-coated microspheres: a model of host cell invasion by Chlamydia trachomatis.
Infect. Immun.
68:1080-1085[Abstract/Free Full Text].
|
| 29.
|
Stephens, R. S.,
R. Sanchez-Pescador,
E. A. Wagar,
C. Inouye, and M. S. Urdea.
1987.
Diversity of Chlamydia trachomatis major outer membrane protein genes.
J. Bacteriol.
169:3879-3885[Abstract/Free Full Text].
|
| 30.
|
Stephens, R. S.,
M. R. Tam,
C. C. Kuo, and R. C. Nowinski.
1982.
Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization.
J. Immunol.
128:1083-1089[Abstract].
|
| 31.
|
Storz, J.
1988.
Overview of chlamydial diseases, p. 167-192.
In
A. L. Barron (ed.), Microbiology of Chlamydia. CRC Press, Boca Raton, Fla.
|
| 32.
|
Su, H., and H. D. Caldwell.
1992.
Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein.
J. Exp. Med.
175:227-235[Abstract/Free Full Text].
|
| 33.
|
Su, H., and H. D. Caldwell.
1993.
Immunogenicity of a synthetic oligopeptide corresponding to antigenically common T-helper and B cell neutralizing epitopes of the major outer membrane protein of Chlamydia trachomatis.
Vaccine
11:1159-1166[CrossRef][Medline].
|
| 34.
|
Su, H.,
L. Raymond,
D. D. Rockey,
E. Fischer,
T. Hackstadt, and H. D. Caldwell.
1996.
A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.
Proc. Natl. Acad. Sci. USA
93:11143-11148[Abstract/Free Full Text].
|
| 35.
|
Su, H.,
N. G. Watkins,
Y.-X. Zhang, and H. D. Caldwell.
1990.
Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.
Infect. Immun.
58:1017-1025[Abstract/Free Full Text].
|
| 36.
|
Su, H., and Y.-X. Zhang.
1988.
Differential effect of trypsin on infectivity of Chlamydia trachomatis: loss of infectivity requires cleavage of major outer membrane protein variable domains II and IV.
Infect. Immun.
56:2094-2100[Abstract/Free Full Text].
|
| 37.
|
Valtonen, V. V.
1991.
Infection as a risk factor for infarction and atherosclerosis.
Ann. Med.
23:539-543[Medline].
|
| 38.
|
Villeneuve, A.,
L. Brossay,
G. Paradis, and J. Hebert.
1994.
Determination of neutralizing epitopes in variable domains I and IV of the major outer membrane protein from Chlamydia trachomatis serovar K.
Microbiology
140:2481-2487[Abstract/Free Full Text].
|
| 39.
|
Wald, N. J.,
M. R. Law,
J. K. Morris,
X. Zhou,
Y. Wong, and M. E. Ward.
2000.
Chlamydia pneumoniae infection and mortality from ischaemic heart disease: large prospective study.
Br. Med. J.
321:204-207[Abstract/Free Full Text].
|
| 40.
|
Ward, M. E.
1992.
Chlamydial vaccines future trends.
J. Infect.
25:S11-S26.
|
| 41.
|
Weiss, M. S.,
A. Kreusch,
E. Schiltz,
V. Nestel,
W. Wette,
J. Weckesser, and G. E. Schultz.
1991.
The structure of porin from Rhodobacter capsulatus at 1.8 A resolution.
FEBS Lett.
280:379-382[CrossRef][Medline].
|
| 42.
|
Williams, A. J.
1995.
The measurement of the function of ion channels reconstituted into artificial membranes, p. 43-67.
In
R. H. Ashley (ed.), Ion channels, a practical approach. IRL/OUP, Oxford, United Kingdom.
|
| 43.
|
Wyllie, S.,
R. A. Ashley,
D. Longbottom, and A. J. Herring.
1998.
The major outer membrane protein in Chlamydia psittaci functions as a porin-like ion channel.
Infect. Immun.
66:5202-5207[Abstract/Free Full Text].
|
| 44.
|
Wyllie, S.,
D. Longbottom,
A. J. Herring, and R. H. Ashley.
1999.
Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.
FEBS Lett.
445:192-196[CrossRef][Medline].
|
| 45.
|
Zhang, Y.-U.,
S. J. Stewart, and H. D. Caldwell.
1989.
Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants.
Infect. Immun.
57:636-638[Abstract/Free Full Text].
|
| 46.
|
Zhang, Y.-X.,
S. Stewart,
T. Joseph,
H. R. Taylor, and H. D. Caldwell.
1987.
Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis.
J. Immunol.
138:575-581[Abstract].
|
| 47.
|
Zhong, G.,
R. E. Reid, and R. C. Brunham.
1990.
Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides.
Infect. Immun.
58:1450-1455[Abstract/Free Full Text].
|
Infection and Immunity, March 2001, p. 1671-1678, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1671-1678.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Nunes, A., Borrego, M. J., Nunes, B., Florindo, C., Gomes, J. P.
(2009). Evolutionary Dynamics of ompA, the Gene Encoding the Chlamydia trachomatis Key Antigen. J. Bacteriol.
191: 7182-7192
[Abstract]
[Full Text]
-
Lister, N. A, Tabrizi, S. N., Fairley, C. K., Smith, A., Janssen, P. H., Garland, S.
(2004). Variability of the Chlamydia trachomatis omp1 Gene Detected in Samples from Men Tested in Male-Only Saunas in Melbourne, Australia. J. Clin. Microbiol.
42: 2596-2601
[Abstract]
[Full Text]
Top
Abstract
Introduction
