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Infection and Immunity, March 2001, p. 1917-1921, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1917-1921.2001
Novel Genetic and Phenotypic Heterogeneity in
Bordetella bronchiseptica Pertactin
Karen B.
Register*
Respiratory Diseases of Livestock Research
Unit, National Animal Disease Center, USDA, Agricultural Research
Service, Ames, Iowa 50010
Received 28 August 2000/Returned for modification 10 October
2000/Accepted 24 November 2000
 |
ABSTRACT |
The Bordetella bronchiseptica outer membrane protein
pertactin is believed to function as an adhesin and is an important
protective immunogen. Previous sequence analysis of the pertactin gene
identified two regions predicted to encode amino acid repeat motifs.
Recent studies have documented DNA sequence heterogeneity in both
regions. The present study describes additional variants in these
regions, which form the basis for six novel pertactin types.
Immunoblotting demonstrated phenotypic heterogeneity in pertactin
consistent with the predicted combined sizes of the repeat regions. A
revised system for classifying B. bronchiseptica pertactin
variants is proposed.
| |
TEXT |
Bordetella bronchiseptica
causes respiratory disease in a wide range of domesticated and wild
animals, including atrophic rhinitis and pneumonia in pigs,
bronchopneumonia in dogs, and rhinitis in rabbits. Pertactin is an
outer membrane protein proposed to function as an adhesin for B. bronchiseptica, although this has yet to be proven. More clearly
defined is its role as a protective immunogen. Several studies have
demonstrated that pertactin-specific active or passive immunization
protects against mortality and disease in mice and pigs (6, 10,
14).
Previous sequence analysis of the B. bronchiseptica gene
encoding pertactin, prn, revealed two regions predicted to
encode either GGXXPn (region 1) or
PQPn (region 2) amino acid repeats
(8). A recent study first described heterogeneity in both
repeat regions of the B. bronchiseptica prn gene from a
limited number of swine field and vaccine isolates (K. B. Register, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. B187,
2000). Additional prn variants in both regions have
subsequently been reported based on an analysis of isolates of animal
and human origin (1). Since the sources of those isolates
were not further defined, the number of host species they represent is
not clear. Corresponding phenotypic evidence of pertactin variants
identified in these studies was not provided. The purpose of the
present study was to examine the prn repeat regions of
B. bronchiseptica isolates obtained from a variety of host
species to ascertain whether additional variants exist. Furthermore,
the pertactin proteins synthesized by strains possessing variations in
the prn sequence were evaluated by Western blotting to
determine whether accompanying phenotypic heterogeneity could be detected.
Genetic analysis of the repeat regions.
The 14 B. bronchiseptica isolates included in this study were chosen to
provide a diverse representation of host species, geographic origins,
and ribotypes (Table 1). All but one have been previously described (13, 16, 17). Isolate SO3287-99 was acquired during 1999 and has only recently been characterized and
ribotyped (C. Staveley, K. B. Register, S. Yang, S. L. Brockmeier, and M. Chechowitz, submitted for publication).
Detection of prn heterogeneity was initially based on a
comparison of the mobility in agarose gels of PCR amplicons
encompassing either region 1 or region 2, corresponding to bp 651 to
1211 or bp 1211 to 2063, respectively, relative to the published
sequence of strain CN7531 (8). Multiple variants differing
in mobility for each region were detected, some of which are shown in
Fig. 1. To more specifically define
regions of heterogeneity, fragments resulting from digestion of PCR
amplicons with restriction enzymes were evaluated. The previously
reported sequence predicts that digestion of region 1 amplicons with
Sau3A should generate three detectable fragments, with the
GGXXPn repeat contained in a fragment of 153 bp.
For some strains, the expected fragments were present and slight
differences in mobility could be detected in the
GGXXPn-containing fragment (Fig.
2A, lanes 1, 2, and 5). In other strains,
some Sau3A sites appeared to be absent or were located such
that fragments of an unexpected size were apparent (Fig. 2A, lanes 3 and 4). In these cases, the identity of the repeat-containing fragment
is not immediately obvious. Similar results were acquired when region 2 amplicons were digested with BstXI, which is predicted to
generate two detectable fragments under the conditions used here.
Mobility variants of the 360-bp PQPn-encoding
fragment were obvious for some strains (Fig. 2B, lanes 1 to 4). Other
strains appeared to lack the BstXI site responsible for
generating these two fragments (Fig. 2B, lane 5). In some cases,
restriction digestion revealed heterogeneity in regions 1 or 2 that was
not apparent from the comparison of full-length amplicons.
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FIG. 1.
PCR amplicon mobility variants. Region 1 (A) or region
2 (B) PCR amplicons were resolved by agarose gel electrophoresis
and visualized by staining with ethidium bromide. (A) Lanes: 1, MBORD847; 2, PV6; 3, MBORD731; 4, MBORD831; 5, St. Louis. (B) Lanes: 1, MBORD901; 2, ATCC 19395; 3, MBORD591; 4, St. Louis; 5, MBORD731. Lanes
marked M contain molecular size markers, with their sizes indicated on
the left.
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FIG. 2.
PCR amplicon restriction fragment analysis. Fragments
resulting from digestion of region 1 amplicons with Sau3A
(A) or digestion of region 2 amplicons with BstXI (B) were
resolved by agarose gel electrophoresis and visualized by staining with
ethidium bromide. (A) Lanes: 1, MBORD731; 2, MBORD831; 3, MBORD901; 4, St. Louis; 5, 5107. (B) Lanes: 1, MBORD676; 2, MBORD606; 3, ATCC 19395;
4, MBORD626; 5, MBORD591. The locations of molecular size markers are
indicated on the left.
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A formal definition of the sequence encompassed by regions 1 and 2 has
not been previously proposed but would facilitate comparisons among
different studies. In the only published report describing B. bronchiseptica region 1 and 2 variants (1), they are
presented as including amino acids 254 to 299 and 559 to 610, respectively, relative to the published sequence of pertactin
(8). I propose maintaining these boundaries, even though
they include short stretches of sequence both upstream and downstream
of the repeats themselves. Substitutions, insertions, or deletions
immediately adjacent to the repeats may lead to local changes in
conformation, charge, or other properties that could directly affect
functions dependent upon GGXXPn or
PQPn sequences. Therefore, in accordance with
previous practice (1), novel variants in this study were
defined as those having any predicted amino acid substitution, insertion, or deletion in the areas specified as regions 1 and 2, as
compared to variants already identified.
The five region 1 amplicons exhibiting unique patterns in agarose gels
were purified and sequenced directly at the Iowa State University DNA
Sequencing and Synthesis Facility, Ames. The predicted amino acid
sequences of all five region 1 amplicons were found to constitute
unique variants of the GGXXPn region, based on
differences in the number of repeats as well as amino acid substitutions. Substitutions occurred both in the degenerate amino acid
positions and in sequence immediately adjacent to the repeats (Fig.
3A). Variant I-2 has been previously
described (1). None of the unique region 1 variants
identified here is identical to the six previously identified
Bordetella pertussis region 1 variants (9, 11,
12; H. F. L. M. van Oirschot, unpublished data [GenBank accession no. AJ132095]).
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FIG. 3.
Multiple alignments of the predicted amino acid
sequences for prn variants in region 1 (A) or region 2 (B).
Dots and dashes indicate identical amino acids and gaps in the
sequence, respectively. Unmarked positions indicate the location of
amino acid substitutions.
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Region 2 amplicons from the five strains with novel region 1 sequences,
as well as additional amplicons representing all unique patterns
observed in agarose gels, were analyzed similarly. Eight region 2 variants could be distinguished, based on the presence of six, seven,
or eight PQP repeats, as well as amino acid substitutions and/or
deletions in adjacent sequence (Fig. 3B). The predicted amino acid
sequences for four variants, II-1, II-2, II-4, and II-5, have been
previously reported (1). However, it is important to note
the sequences presented in that publication are not consistent with the
DNA sequence deposited in GenBank for the representative strains
indicated. Since the authors of that study did not include DNA
sequences in their report, it is not possible to determine the reason
for this discrepancy. Therefore, it is presently unclear whether
variants II-1, II-2, II-4, and II-5, as described here, have been
previously identified.
Proposed modifications to nomenclature.
The results presented
identify four novel repeat variants in region 1 of the prn
gene of B. bronchiseptica, as well as four in region 2. Together with data already reported (1), a total of seven
region 1 variants and 13 region 2 variants have been described. New
variants identified here were initially assigned names consistent with
the classification system previously used (1). However,
given the large number of variants now identified and the existence of
additional variants (K. B. Register, unpublished data), I propose a
modified nomenclature that may be more informative and less cumbersome
than the existing one. This system, unlike the current one, will
clearly convey the number of GGXXP or PQP repeats present without
having to refer to some other source and will also indicate which
variants differ only in amino acids that do not affect the total number
of repeats.
All studies describing pertactin variants (9, 11, 12),
except for the most recent one (1), designate the use of Arabic numerals in referring to the repeat regions. Therefore, it is
suggested that the originally established designations for regions 1 and 2 not be modified. The remainder of the proposed epithet consists
of an additional Arabic numeral, coinciding with the predicted number
of amino acid repeats, followed by a lowercase letter to distinguish
variants sharing the same number of amino acid repeats (e.g., 1-1a).
The first variant reported having a particular number of repeats would
be assigned the letter "a," and subsequently identified variants
would assume the next available letter, proceeding alphabetically.
Region 1 variants differing only in the degenerate positions of the
repeats would be assigned the numeral indicative of the total number of
repeats, followed by the next available lowercase letter. To provide an
example, the region 1 variants in this report previously referred to as I-5 and I-6 each contain eight GGXXP repeats and are distinguished only
by a single amino acid substitution in the second X position of repeat
4 (Fig. 3A). Under the proposed system, these variants would be
designated 1-8a and 1-8b, respectively. As another example, the
previously described GGXXP3 variant I-2 would be renamed
1-3a. The designation 1-3b would be reserved for the next
GGXXP3 variant to be identified. Table
2 lists the previous and proposed
designations for each of the currently known GGXXP and PQP variants.
For the sake of clarity, the proposed nomenclature will be adopted for the remainder of this report.
Pertactin types predicted by DNA sequence.
Based on the
combined region 1 and region 2 sequences observed in this study, eight
pertactin types could be distinguished (Table
3). Two have been previously described
(1), assuming the region 2 sequences in that report are
not in error. However, it should also be noted that the prn
gene from strain CN7531, previously sequenced by Li et al.
(8), codes for pertactin type 1-3a/2-7a, not type
1-3a/2-7b (or I-2/II-4 as it originally appears) as reported
(1). That study, together with the present one, identifies
16 pertactin types from 56 isolates of B. bronchiseptica. This degree of heterogeneity is quite remarkable, considering that only
six pertactin types have been identified from several hundred strains
of B. pertussis (9, 11, 12; van Oirschot, unpublished data [GenBank accession no. AJ132095]). Recent evidence
suggests that the emergence of B. pertussis prn variants may
have been driven by immune selection in response to vaccination (9, 11, 12). Studies addressing this possibility with
respect to B. bronchiseptica have not been undertaken. While
many factors undoubtedly contribute to the multiplicity of B. bronchiseptica prn variants, the extensive host range of this
species and the use of many different vaccine strains (which likely
represent multiple pertactin variants) may have led to the
simultaneous, but independent, evolution of pertactin in multiple host
species.
Boursaux-Eude and Guiso (1) indicated that host
specificity was not observed with respect to the pertactin types
described, although information was not provided concerning the number
or exact identity of hosts represented. While nearly all novel
pertactin types reported here were found in isolates from different
hosts, only one or a few isolates were examined from each species
represented. Furthermore, isolates chosen for sequencing were those
from which region 1 or 2 PCR amplicons, or restriction fragments
derived from them, displayed unique patterns on agarose gels.
Consequently, the pertactin types identified may not be representative
of those most frequently occurring in a given host. Analysis of
additional strains is necessary before conclusions can be reached
regarding the distribution and frequency of pertactin types with
respect to host species, geographic location, or other criteria.
Phenotypic heterogeneity detected by immunoblotting.
Variation
in the number of predicted amino acids in regions 1 and 2 for each of
the pertactin types identified here suggests that differences in
mobility should be detectable by Western blotting for some strains,
provided that compensatory insertions or deletions do not occur in
other regions. The monoclonal antibody BPE3 (2) was used
for detection of pertactin in immunoblots prepared with bacterial
extracts enriched in outer membrane proteins, as described (15). Alterations in the size of pertactin that are at
least partially consistent with the total number of amino acids
predicted in regions 1 and 2 were detectable in three strains.
Pertactin from strain MBORD901, predicted to contain a total of 122 amino acids in regions 1 and 2, displayed the highest apparent
molecular mass of all strains assessed. Pertactin from strains St.
Louis and PV6, predicted to contain a total of 119 and 106 amino acids in the repeat regions, respectively, also demonstrated an increased molecular mass compared to the remaining strains. Other pertactin types
are predicted to contain a total of 98 to 102 amino acids in regions 1 and 2. Reproducible differences in the mobility of pertactin from these
strains were not observed under the conditions of this study. However,
pertactin size heterogeneity may also be partially due to DNA
polymorphisms outside regions 1 and/or 2. Compared to strain PV6,
pertactin from the St. Louis strain is predicted to contain an
additional 13 amino acids, yet the mobility of pertactin from these
isolates is indistinguishable. Comparison of the sequences for the
entire prn gene is required to determine the basis for this observation.
A vital question not addressed by this study is the functional
significance of alterations in pertactin amino acid sequence. Investigations with B. pertussis suggest that region 1 amino
acids may play a role in adherence (5, 7). Although
analogous studies have not been carried out with B. bronchiseptica, it could be hypothesized that alterations in this
region affect the specificity of binding to host cells, with certain
pertactin types preferentially adherent to the tissues of one, or a
related group, of host species. At least some B. bronchiseptica isolates do show reduced ability to infect
heterologous hosts (4, 18). The presence of an immunodominant protective epitope in region 2 (3) suggests that polymorphisms in this region may permit escape from immune surveillance through the emergence of novel antigenic epitopes. Future
studies are clearly needed to clarify the implications of pertactin
heterogeneity in B. bronchiseptica.
| |
ACKNOWLEDGMENTS |
I acknowledge the excellent technical assistance of Pamala Beery
and the graphical design skills of Mary Sue Brown, as well as helpful
comments from John Neill.
| |
FOOTNOTES |
*
Mailing address: Swine Respiratory Diseases Project,
USDA/ARS/National Animal Disease Center, P.O. Box 70, 2300 Dayton Rd., Ames, IA 50010. Phone: (515) 663-7700. Fax: (515) 663-7458. E-mail: kregiste{at}nadc.ars.usda.gov.
Editor:
J. T. Barbieri
| |
REFERENCES |
| 1.
|
Boursaux-Eude, C., and N. Guiso.
2000.
Polymorphism of repeated regions of pertactin in Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.
Infect. Immun.
68:4815-4817[Abstract/Free Full Text].
|
| 2.
|
Brennan, M. J.,
Z. M. Li,
J. L. Cowell,
M. E. Bisher,
A. C. Steven,
P. Novotny, and C. R. Manclark.
1988.
Identification of a 69-kilodalton nonfimbrial protein as an agglutinogen of Bordetella pertussis.
Infect. Immun.
56:3189-3195[Abstract/Free Full Text].
|
| 3.
|
Charles, I. G.,
J. L. Li,
M. Roberts,
K. Beesley,
M. Romanos,
D. J. Pickard,
M. Francis,
D. Campbell,
G. Dougan,
M. J. Brennan,
C. R. Manclark,
M. A. Jensen,
I. Heron,
A. Chubb,
P. Novotny, and N. F. Fairweather.
1991.
Identification and characterization of a protective immunodominant B cell epitope of pertactin (P. 69) from Bordetella pertussis.
Eur. J. Immunol.
21:1147-1153[Medline].
|
| 4.
|
Collings, L. A., and J. M. Rutter.
1985.
Virulence of Bordetella bronchiseptica in the porcine respiratory tract.
J. Med. Microbiol.
19:247-255[Medline].
|
| 5.
|
Emsley, P.,
I. G. Charles,
N. F. Fairweather, and N. W. Isaacs.
1996.
Structure of Bordetella pertussis virulence factor P. 69 pertactin.
Nature
381:90-92[CrossRef][Medline].
|
| 6.
|
Kobisch, M., and P. Novotny.
1990.
Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets.
Infect. Immun.
58:352-357[Abstract/Free Full Text].
|
| 7.
|
Leininger, E.,
C. A. Ewanowich,
A. Bhargava,
M. S. Peppler,
J. G. Kenimer, and M. J. Brennan.
1992.
Comparative roles of the Arg-Gly-Asp sequence present in the Bordetella pertussis adhesins pertactin and filamentous hemagglutinin.
Infect. Immun.
60:2380-2385[Abstract/Free Full Text].
|
| 8.
|
Li, J.,
N. F. Fairweather,
P. Novotny,
G. Dougan, and I. G. Charles.
1992.
Cloning, nucleotide sequence and heterologous expression of the protective outer-membrane protein P.68 pertactin from Bordetella bronchiseptica.
J. Gen. Microbiol.
138:1697-1705.
|
| 9.
|
Mastrantonio, P.,
P. Spigaglia,
H. van Oirschot,
H. G. van der Heide,
K. Heuvelman,
P. Stefanelli, and F. R. Mooi.
1999.
Antigenic variants in Bordetella pertussis strains isolated from vaccinated and unvaccinated children.
Microbiology
145:2069-2075[Medline].
|
| 10.
|
Montaraz, J. A.,
P. Novotny, and J. Ivanyi.
1985.
Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica.
Infect. Immun.
47:744-751[Abstract/Free Full Text].
|
| 11.
|
Mooi, F. R.,
H. van Oirschot,
K. Heuvelman,
H. G. van der Heide,
W. Gaastra, and R. J. Willems.
1998.
Polymorphism in the Bordetella pertussis virulence factors P. 69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution.
Infect. Immun.
66:670-675[Abstract/Free Full Text].
|
| 12.
|
Mooi, F. R.,
Q. He,
H. van Oirschot, and J. Mertsola.
1999.
Variation in the Bordetella pertussis virulence factors pertussis toxin and pertactin in vaccine strains and clinical isolates in Finland.
Infect. Immun.
67:3133-3134[Abstract/Free Full Text].
|
| 13.
|
Musser, J. M.,
D. A. Bemis,
H. Ishikawa, and R. K. Selander.
1987.
Clonal diversity and host distribution in Bordetella bronchiseptica.
J. Bacteriol.
169:2793-2803[Abstract/Free Full Text].
|
| 14.
|
Novotny, P.,
M. Kobisch,
K. Cownley,
A. P. Chubb, and J. A. Montaraz.
1985.
Evaluation of Bordetella bronchiseptica vaccines in specific-pathogen-free piglets with bacterial cell surface antigens in enzyme-linked immunosorbent assay.
Infect. Immun.
50:190-198[Abstract/Free Full Text].
|
| 15.
|
Register, K. B., and M. R. Ackermann.
1997.
A highly adherent phenotype associated with virulent Bvg+-phase swine isolates of Bordetella bronchiseptica grown under modulating conditions.
Infect. Immun.
65:5295-5300[Abstract].
|
| 16.
|
Register, K. B.,
A. Boisvert, and M. R. Ackermann.
1997.
Use of ribotyping to distinguish Bordetella bronchiseptica isolates.
Int. J. Syst. Bacteriol.
47:678-683[Abstract/Free Full Text].
|
| 17.
|
Register, K. B., and T. Magyar.
1999.
Optimized ribotyping protocol applied to Hungarian Bordetella bronchiseptica isolates: identification of two novel ribotypes.
Vet. Microbiol.
69:277-285[CrossRef][Medline].
|
| 18.
|
Ross, R. F.,
W. P. Switzer, and J. R. Duncan.
1967.
Comparison of pathogenicity of various isolates of Bordetella bronchiseptica in young pigs.
Can. J. Comp. Med. Vet. Sci.
31:53-57[Medline].
|
Infection and Immunity, March 2001, p. 1917-1921, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1917-1921.2001
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