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Infection and Immunity, May 2000, p. 3056-3060, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
R1 Region of P97 Mediates Adherence of
Mycoplasma hyopneumoniae to Swine Cilia
F. Chris
Minion,*
Cary
Adams, and
Tsungda
Hsu
Department of Veterinary Microbiology and
Preventive Medicine, Veterinary Medical Research Institute, Iowa
State University, Ames, Iowa 50011
Received 23 November 1999/Returned for modification 11 January
2000/Accepted 27 January 2000
 |
ABSTRACT |
Adherence of Mycoplasma hyopneumoniae to the swine
respiratory tract is mediated by the membrane protein P97. This protein is located on the outer membrane surface, and its role in adherence has
been firmly established. The general region of P97 that mediates adherence to swine cilia is thought to be the R1 region near the carboxy terminus of the protein, but it was not clear if this region
could mediate adherence to swine cilia independently of other P97
sequences. To examine this in more detail, a series of R1 repeat
sequences containing different numbers of repeating units cloned in
frame with lacZ was used to produce R1-
-galactosidase fusion proteins. These proteins were then tested for adherence to swine
cilia and for reactivity to the adherence-blocking monoclonal antibody
F2G5 and convalescent-phase swine sera. In this way it was possible to
accurately define the cilium binding epitope of P97 and the minimal
epitope recognized by antibody. Our results indicate that eight R1
repeating units are required for cilium binding and that three
repeating units are needed for antibody recognition. These results
could lead to more effective therapeutic measures against this
important swine pathogen.
| |
TEXT |
Attachment to host tissue is
essential for colonization by most mucosal pathogens, including
mycoplasmas (1, 2). The best-defined mycoplasmal adhesins,
those of Mycoplasma pneumoniae and Mycoplasma
genitalium, are found in a specialized attachment organelle
(8). It is clear from numerous studies that their adhesins
work in concert with other membrane proteins and accessory factors in a
coordinated fashion to form the attachment organelle structure
(8). Thus, the process of adherence in these species is
multifactorial, requiring both membrane proteins and cytoskeletal elements (8). Most mycoplasmas, however, lack specialized
attachment organelles like M. pneumoniae, and consequently,
their adherence mechanisms differ. How most mycoplasma species adhere
to their respective host tissues is largely unknown, and the genetic
mechanisms of adherence are poorly defined.
As the etiological agent of porcine mycoplasma pneumonia and a major
component of the porcine respiratory disease complex, Mycoplasma
hyopneumoniae continues to present significant problems for the
swine industry. As is the case for many other mycoplasmal species, it
is host specific (for swine), it has no readily apparent external
structures or organelles that could be used for attachment, there are
no genetic systems for analyzing the adherence phenotype, and studies
of adherence have been largely qualitative rather than quantitative in
nature. For instance, M. hyopneumoniae closely adheres to
swine respiratory epithelium both in vivo and in vitro (3,
9). It has also been demonstrated that M. hyopneumoniae can attach to pig and human lung fibroblasts, to pig
kidney cells in culture (15), and to turkey red blood cells
(12).
Our interests in adherence mechanisms as the prelude to colonization
and disease led us to take advantage of the available adherence-blocking monoclonal antibodies (MAbs) (14) and a
cilium adherence assay (CAA) (13) to address adherence
mechanisms at the molecular level through a heterologous in vitro
system. The cloning and analysis of the P97 structural gene
(5) and analysis of nearby genes in the chromosome
(7) have been accomplished. DNA sequence analysis revealed
two repeat regions near the 3' end of the P97 gene, the R1 and R2
regions (5). It was clear from previous studies using
transposon mutagenesis and -galactosidase fusion proteins that the
R1 region participated in the adherence to swine cilia (6).
In these studies, the R1 region was cloned upstream of lacZ,
producing a recombinant R1--galactosidase fusion protein capable of
binding to swine cilia with same specificity as intact cells. These
studies demonstrated the potential of using fusion proteins to study
the functional regions of P97. The R1 region PCR fragment used in these
cloning experiments, however, contained an additional 78 bp upstream of
the R1 region because of PCR primer design limitations. Transposon
mutagenesis had completely ruled out the need for amino acids
downstream of the R1 region (6), but it was not clear
without more precise analysis if upstream sequences were important in
the structure of a functional binding epitope. These additional 26 amino acids in the translation product could have an important role in
the cilium binding epitope. Here we report studies using a different
approach that further defines the binding epitope of P97 to swine cilia.
The difficulty in cloning the region cleanly in the original studies
was due to the nature of the AT-rich mycoplasma genome and the lack of
good PCR primer binding sites. Thus, it was decided to construct the
region systematically, one repeat unit at a time, using the same
-galactosidase fusion strategy employed earlier (6). This
strategy had several advantages over the previous approach of direct
cloning. It would allow us to precisely identify the amino acids and
the number of repeat units needed for cilium binding. It would
unambiguously confirm the structure of the antigenic epitope recognized
by the adherence-blocking MAbs. It would also provide a way to perform
site-directed mutagenesis for a detailed in-depth analysis of the
binding epitope.
Bacterial strains and growth.
Escherichia coli strain
GM124 (CGSC 6498) [lacZ118(Oc) rpsL275 dam-4]
was used throughout this study as both the cloning and expression host.
This strain was Lac
to allow for scoring for in-frame
lacZ fusions during plasmid constructions. It was also
Dam so that plasmids isolated from this strain could be
digested with the dam methylation-sensitive restriction
enzyme BspEI.
Construction of R1--galactosidase gene fusions.
Our
overall strategy was to construct different numbers of R1 repeating
units fused to -galactosidase so that cilium binding experiments
could be performed with the fusion proteins. The approach needed to
construct the R1 region was not immediately apparent, however, because
of the amino acid sequence variability in the R1 region expressed as a
charged-noncharged alternating motif and the substitution of threonine
for alanine in the fourth repeat unit (Fig.
1). Thus, it was necessary that the
repeat region be constructed one unit at a time with the option of
altering the sequence within each new unit while maintaining the exact
amino acid sequence of the wild-type R1 region. Our approach combined construction of the two different repeat units found in the R1 region
with ligation of purified blunt-ended digestion fragments to construct
the R1 region systematically. Since it was necessary to allow for the
use of the repeat unit both as an additive element in further plasmid
constructions and as a final lacZ fusion, it was necessary
to construct two derivatives of each repeat region subset. One of these
contained an additional cytosine to bring the final gene fusion into
frame with the lacZ reading frame.
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FIG. 1.
Strategy for the construction of R1-lacZ
fusion plasmids. (A) Amino acid sequence of the R1 region of P97. The
underline indicates the substitution of threonine for alanine in the
fourth repeat unit. (B) Construction of plasmids pISM1301.1,
pISM1301.3, pISM1301.4, pISM1301.5, and pISM1301.6 using PCR-generated
fragments. These plasmids served as sources for the left and right
fragments for other plasmid constructions. Where appropriate, plasmid
designations are given as examples. In step 1, using pMLB1107 template
DNA, PCR was performed with different primer sets (Tables 1 and 2) to
obtain left and right fragments for cloning. Two different primers were
used at the R1-lacZ junction site; one primer (GGG)
maintained the reading frame within the repeating units. These plasmids
were used only in the construction of the R1 region with various
numbers of repeating units. During the addition of the last repeating
unit for final Lac+ constructs, the second primer (CGGG)
altered the final frame of the lacZ coding sequence to align
it with the upstream R1 region. In step 2, the fragments were digested
and purified. In step 3, the purified fragments were ligated to form
the starting plasmids for the R1-lacZ fusions. (C)
Construction of multiple repeating units fused to -galactosidase
(-gal). The plasmids were constructed consecutively, a repeat at a
time, in order to have the starting fragments for the next plasmid
construction. In step 4, for each step, the appropriate plasmids (Table
2) were digested, and the individual fragments were purified. In some
cases, a PCR step as described for panel B was performed for one of the
fragments, such as in the construction of pISM1302.1 (Table 2). In step
5, the fragments were blunt-end ligated to form the desired repeat.
This resulted in loss of the SmaI and SnaBI or
BstBI cloning sites at the cloning junction. In step 6, steps 4 and 5 were then repeated consecutively with the plasmids
described in Table 2 until the series of R1-lacZ fusions was
constructed.
|
|
The backbone for all plasmid constructions was plasmid pMLB1107, a
unique cloning vector that provided the
lacZ sequence for
the gene fusions, an isopropyl thio-
-
D-galactopyranoside
inducible
promoter, and
lacIq for controlling
gene expression (
6). The R1-
-galactosidase
fusions were
constructed with PCR products obtained using pMLB1107
template DNA. The
PCR primers used in the construction of these
plasmids are shown in
Table
1. These primers were designed to
accommodate the codon usage of
E. coli rather than that of
mycoplasmas.
Initially, plasmids with one to four repeating units,
i.e., plasmids
pISM1301.1, pISM1302.1, pISM1303.1, pISM1304.1, and
pISM1304.2,
were constructed by generating left and right plasmid
halves by
PCR which were then ligated. Plasmids pISM1301.3, pISM1301.4,
pISM1301.5, and pISM1301.6 were also constructed in this fashion,
but
they included additional restriction sites to facilitate the
second
round of cloning. This second set of four plasmids included
the valine
and glutamic acid codon-containing repeat units and
corresponding
constructs with an additional cytosine at the R1-
lacZ junction to bring
lacZ into the proper reading frame.
The PCR conditions used to generate the fragments were as follows. The
primer pairs TH148-TH150 and TH154-TH157 were used
to generate
fragments representing the left and right halves,
respectively, of the
plasmid pISM1301.3 (Fig.
1B; Tables
1 and
2). The primer mixture for the left
fragment contained 25 pmol
of each phosphorylated primer, 2.5 mM
MgCl
2, 2.5 U of
Pfu polymerase
(Stratagene), 1×
Stratagene
Pfu polymerase buffer, 0.2 mM deoxynucleoside
triphosphates, and 200 pg of template DNA in a total volume of
100 µl. The reaction conditions were denaturation at 94°C for
3 min; 35 cycles of denaturation at 94°C for 0.5 min, renaturation
at 56°C
for 0.5 min, and extension at 72°C for 8 min; and a final
14-min
extension step at 72°C. The primer mixture for the right
fragment was
the same except that no additional MgCl
2 was added.
The
reaction conditions for the right fragment were denaturation
for at
94°C 3 min; 5 cycles of denaturation at 94°C for 0.5 min,
renaturation at 58°C for 0.45 min, and extension at 72°C for 7.5
min; 30 cycles of denaturation at 94°C for 0.5 min and extension
at
68°C for 8 min; and a final extension at 72°C for 14 min. The
fragment from each PCR was digested with restriction enzymes prior
to
gel purification, ligation, and transformation. For left fragments,
the
fragments were digested with
SmaI and
BspEI; for
right fragments,
the enzymes
SnaBI (for plasmids with
regions containing an initial
valine codon) or
BstBI (for
plasmids containing an initial glutamic
acid codon) and
BspEI were used.
The second set of four plasmids was used in other plasmid constructions
by ligating left and right fragments from these and
subsequent plasmid
constructions to form functional R1-
lacZ fusions
with
differing numbers of repeating units (Table
2). To streamline
the
cloning procedure, most of the plasmids required
SnaBI
digestion
of the PCR fragment to generate a blunt end (Table
2). The
BstBI
digestion, filling in, and mung bean digestion were
used only
in construction of plasmids 1301.5 and 1301.6. Because of the
sensitivity of restriction enzyme
BspEI to
dam
methylation, the
ligation mixtures were transformed into
E. coli GM124, which is
phenotypically Lac
Dam
. In this way, plasmids could be isolated from this
background
and used directly for the next round of cloning or scored
for
the Lac
+ phenotype on X-Gal
(5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside)-containing
media. The left and right fragments were obtained by restriction
digestion of each plasmid shown in Table
2 followed by purification
of
the appropriate fragment. The variation in the fourth repeat
unit in
pISM1304.1 and pISM1304.2 was introduced by producing
the right
fragment by PCR. Cloning of the repeat regions required
joining of
blunt ends within the repeat region and a
BstBI site
within
the vector region of the plasmid (Fig.
1C). The addition
of each repeat
unit was monitored by the size of the
HindIII-
EcoRV
digestion product, and each
final construct was sequenced through
the repeating region to ensure
that no errors had been introduced
during the cloning
process.
CAA.
The CAA has been described previously (6, 13).
Lysates from induced E. coli cultures containing different
R1--galactosidase fusions were prepared, and the -galactosidase
activity and protein content were measured. The assay was modified to
normalize the units of -galactosidase activity added to each well
(20 units per well) as determined by the method of Miller
(10). In this way the assay was more reproducible than it
would have been by adding a standard amount of protein as in previous
studies (6) (data not shown). Each lysate was tested in
triplicate, and the experiment was repeated twice. Figure
2 shows the results of two cilium binding
experiments using the series of R1-lacZ fusion plasmids.
-Galactosidase had no affinity for swine cilia, as demonstrated by
its lack of binding in the Lac+ control. This was also
observed in previous studies (6). There was also little
cilium binding of the R1--galactosidase fusions with seven
repeating units or fewer. Binding was not significant until there were
at least eight repeating units. This was reproducible between the two
experiments shown and in other experiments not shown.
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FIG. 2.
Detection of adherence of R1--galactosidase fusion
proteins to swine cilia. R1--galactosidase fusion proteins in
E. coli lysates were examined for adherence to purified
swine cilia using the microtiter plate CAA as described in the text.
Data are represented as the means plus standard deviations of the
optical densities at 412 nm of triplicate wells. White and gray bars
represent separate experiments on different days. C ( ),
Lac control; C (+), Lac+ control.
|
|
Immunoblot analysis.
In addition to the need to define the
repeat structure necessary for swine cilium adherence, it was also
useful to determine the size of the R1 repeat sequence required to form
the antigenic epitope recognized by adherence-blocking MAbs. This was
accomplished by developing immunoblots of the E. coli
lysates containing the different R1--galactosidase fusion proteins
with MAb F2G5 (14) and with anti--galactosidase
antibodies. The results of the immunoblot analysis are shown in Fig.
3. F2G5-immunoreactive bands were
observed with three, four, and six repeating units but not with one or two repeating units (Fig. 3A). To confirm that -galactosidase fusion
protein was present in each lane, an identical blot was reacted with
anti--galactosidase antibodies (Fig. 3A). In this experiment, 10 µg of protein was loaded per lane, except for lane 1, which contained
20 µg of protein to control for nonspecific binding of antibodies to
the fusion protein. Thus, it appeared that three repeating units formed
an antigenic epitope recognized by the MAb. Convalescent-phase swine
sera also recognized the R1 repeat structure (Fig. 3B).
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FIG. 3.
Immunoblot analysis of R1--galactosidase fusion
proteins. Induced E. coli lysates were resolved by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto
nitrocellulose membranes, and developed as follows. Each well contained
10 µg of protein, except for lane 1, which contained 20 µg of
protein. (A) -gal, blot developed with antibodies against E. coli -galactosidase (Sigma); F2G5, blot developed with MAb F2G5
(14). (B) Blot developed with convalescent-phase swine serum
S27 (11). Lane numbers indicate the number of repeating
units in the -galactosidase fusion protein. Lane +; Lac+
E. coli control containing pMLB1107 with no R1 repeat
unit.
|
|
Our results indicate that the cilium binding domain of P97 is found
exclusively within the R1 region. The amino acids upstream
of the R1
region are not necessary for cilium binding. The functional
site
required a minimum of eight repeating units, supporting our
previous
transposon mutagenesis study (
6). This gives a good
idea of
the size of the functional binding domain but leaves a
number of
unresolved issues. For instance, we could not determine
with our
existing fusions if the first eight repeating units,
including the
modified fourth unit, are required for binding or
if any of the
repeating units would suffice as long as eight units
were involved. It
was also not possible to determine the role,
if any, of the
charged-noncharged motif of the R1 region in cilium
binding. There was
also the possibility that cellular proteases
could be degrading the
fusion product by cleaving the product
at the fusion junction.
Immunoblot analysis of
E. coli lysates
with
anti-
-galactosidase antibodies and the F2G5 MAb with a large-format
gel system gave no evidence of product degradation (data not shown).
Since these lysates could be stored for at least 24 h at 4°C
without
loss of binding activity, and since the data fit nicely with
previous
studies (
6), it seemed unlikely that degradation of
the fusion
product was
occurring.
Finally, the sequence of the antigenic epitope of P97 is now better
understood. It is clear from these studies that three
repeat units (15 amino acids) are needed for the proper antigenic
structure. It seems
logical that the epitope is conformational
in nature because of its
size. Interestingly, this epitope appears
to be the major P97 epitope
in mice, because attempts to produce
MAbs against a non-R1 epitope of
P97 have all failed (R. F. Ross
and T. F. Young, unpublished
data). Likewise in swine, serum antibodies
are produced against this
epitope (Fig.
3B), but it is not known
if antibodies of this
specificity are found in significant levels
in the respiratory tract
during disease. Since the role of this
region in adherence is now
known, it is possible that development
of mucosal vaccines using four
or more R1 repeating units might
prove effective in preventing disease
or possibly even colonization.
If high anti-R1 antibody levels
could be produced at the respiratory
surface, perhaps by fusion with
the cholera toxin A2/B subunit
as described by Hajishengallis et al.
(
4), protection against
colonization might be
possible.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Iowa State
University, Veterinary Medical Research Institute, 1802 Elwood Dr.,
Ames, IA 50011. Phone: (515) 294-6347. Fax: (515) 294-1401. E-mail: fcminion{at}iastate.edu.
Present address: Department of Microbiology and Immunology, Albert
Einstein College of Medicine, Bronx, NY 10461.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, May 2000, p. 3056-3060, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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