Molecular Microbiology Unit, Women's and
Children's Hospital, North Adelaide, South Australia
5006,1 and Department of
Microbiology and Immunology, University of Adelaide, Adelaide,
South Australia 5005,2 Australia
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Streptococcus pneumoniae
(the pneumococcus) is an important cause of invasive disease in human
populations throughout the world, resulting in high morbidity and
mortality. Control of pneumococcal disease is being complicated by the
increasing prevalence of antibiotic-resistant strains and the
suboptimal clinical efficacy of existing vaccines. S. pneumoniae produces a polysaccharide capsule which is essential for virulence because it protects the pneumococcus from the nonspecific immune defenses of the host during an infection (2).
There are now 90 recognized serotypes of S. pneumoniae
(9), each of which produces a structurally distinct capsular
polysaccharide (CPS). Classical genetic studies carried out by Austrian
et al. (3) demonstrated that the S. pneumoniae
genes required for biosynthesis and expression of CPS are closely
linked on the pneumococcal chromosome. This fact enabled us to clone
and sequence the complete capsule locus from S. pneumoniae
type 19F (designated cps19f) (8, 17). Our studies
were concentrated on S. pneumoniae type 19F because it is
one of the commonest causes of invasive disease in children, and the
type 19F CPS is one of the poorest immunogens in this group
(6). Type 19F belongs to serogroup 19, which also
contains the immunologically cross-reactive types 19A, 19B, and
19C. S. pneumoniae type 19A is also an important
cause of disease, whereas types 19B and 19C are rare causes of disease (25).
We have previously examined the distribution of individual
cps19f genes among other pneumococcal serotypes, including
the other members of serogroup 19, by Southern hybridization analysis (17). Only homologues to cps19fA and
-B, the first two genes in the cps locus, were
present in all serotypes examined. Cps19fA is a putative
transcriptional attenuator, but the function of Cps19fB is unknown. The
next two genes in the cps locus, cps19fC and
-D, encode proteins which are predicted to be involved in chain length regulation and export of CPS (8, 17). Moreover, Cps19fC and -D are essential for CPS expression in S. pneumoniae type 19F, as in-frame deletion mutations in either
cps19fC or cps19fD result in the loss of CPS
production (16a). Thus, cps19fC and -D
homologues are probably essential for CPS production in all S. pneumoniae serotypes which are synthesized via lipid-linked repeat
unit intermediates in a fashion similar to type 19F CPS. To date, this
would include all pneumococcal serotypes which have been
characterized except type 3, which is synthesized by a processive transferase (1, 5, 21). Surprisingly, however, 10 of the 21 serotypes tested in previous hybridization studies, including type 19A,
did not contain high-stringency homologues of cps19fC and
-D.
The structures of the CPS for types 19F and 19A are almost identical,
consisting of the same rhamnose
N-acetyl
mannosamineglucose trisaccharide repeat units joined by
different glycosidic linkages (
[12] for 19F and [13]
for 19A) (10, 20). Thus the only predicted functional
difference between the protein products expressed by the
cps19f and cps19a loci would be that of the
polysaccharide polymerase. However, the type 19A cps locus
appears to be more divergent, with high-stringency homologues of only
eight of the cps19f genes present, compared to homologues of
13 out of the 15 cps19f genes present in types 19B and
19C (17). This study investigates the basis for this
apparent diversity.
Bacterial strains.
S. pneumoniae Rx1-19F has been
described previously (8). A clinical isolate of
S. pneumoniae type 19A, strain 1777/39, was obtained from
Jorgen Henrichsen, Statens Seruminstitut, Copenhagen, Denmark.
All other S. pneumoniae strains were clinical
isolates from the Women's and Children's Hospital, North Adelaide,
Australia. Pneumococci were routinely grown either in Todd-Hewitt broth
(Oxoid Limited, Basingstoke, England) supplemented with 0.5% (wt/vol) yeast extract (Difco Laboratories, Detroit, Mich.) or on blood agar
(Oxoid) and serotyped by the quellung reaction using sera obtained from
the Statens Seruminstitut.
Characterization of the 5' portion of cps19a.
Genes
homologous to cps19fA, -B, -G, and
-H were predicted to be present in the cps19a
locus based on previous Southern hybridization data obtained with the
cps19f genes as probes (17). Thus, the 5' portion
of cps19a was amplified by long-range PCR using the Expand
Long Template PCR system (Boehringer, Mannheim, Germany), according to
the manufacturer's instructions, and was performed in a Hybaid
Touchdown Thermal Cycler. The two primers used to amplify this region
(CPS5' and J22) (Fig. 1A) were based on
regions of the cps19f sequence which are predicted to be
homologous to the cps19a locus. The resultant PCR product
was sequenced by using dye-terminator chemistry with specifically
designed primers on an Applied Biosystems model 373A automated DNA
sequencer. The sequences were analyzed with DNASIS software (version
7.0; Hitachi Software Engineering, South San Francisco, Calif.).
Analysis of the compiled DNA sequence revealed that the
cps19f and cps19a loci are indeed very closely
related. There are seven open reading frames (ORFs) in this portion of
the cps19a locus, designated cps19aA to
-G, which are organized in an order identical to those in
cps19f, with similarities to the cps19f genes
ranging from 70.1 to 90.9% identity. The sizes, G+C content, and
percent identity of the cps19a and cps19f protein
products are shown in Table 1, and the arrangement of
the cps19aA to -G genes compared to those from
cps19f is shown in Fig. 1A. The arrangement of the genes in
this region of the two loci are remarkably similar; even the intergenic
gaps between the cps19a genes and the cps19f
genes are similar in size. The most significant difference between the two loci is the start codon used for cps19G. Whereas the
start codon for both cps19aG and cps19aH is
TTG, only cps19fH has a TTG start codon in the
cps19f locus.
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FIG. 1.
The dexB-to-aliA region of the
S. pneumoniae type 19F chromosome. The regions
conserved among all pneumococcal serotypes are indicated in black, the
cps19G and -H genes are shaded grey to indicate a
higher degree of identity between cps19f and
cps19a in this region. (A) Arrangement of the
cps19a locus between the CPS5' and J22 primers. CPS5'
(5'-TGATGTTCAAGGTATAGGTGTTAATCA) is homologous to
nucleotides 146 to 169 of the cps19f sequence
(17), immediately preceding the cps19fA gene, and
J22 (5'-AATTGAATTCTTTTATAGATTTAACACAAG) is complementary to
nucleotides 6743 to 6772 of the cps19f sequence, in the 5'
region of cps19fH. (B) The portion of the cps
locus between cpsB and aliA from various
pneumococcal serotypes was amplified by using the two primers J39 and
J36. The positions of the two primers J39
(5'-TAGTTCATGTAGTTGCAAGTGACATGCACAA, homologous to
nucleotides 2190 to 2220 of the cps19f sequence, in the 3'
region of cps19fB) and J36
(5'-CAATAATGTCACGCCCGCAAGGGCAAGT, complementary to
nucleotides 16463 to 16490 of the cps19f sequence, located
just after the start of aliA) are indicated with half
arrows. Abbreviations for restriction sites are as follows: B,
BamHI; C, ClaI; E, EcoRI; H,
HindIII; K, KpnI; Nc, NcoI; Nr,
NruI; P, PstI; S, SphI.
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Interestingly, even though cps19aA and -B
sequences hybridize to cps19fA- and
cps19fB-specific probes (17), the overall identity between the genes is lower than expected (90.5 and 82%, respectively), with no clearly identifiable point from which downstream sequences diverge. Instead, the cps19aAB genes present a
mosaic pattern with small regions of varying degrees of identity to the cps19fAB genes, ranging from 76.6 to 100%, as shown in Fig.
2. This suggests that the
cps19a locus and the type 19A serotype may be the result of
several recombination events between the ancestral cps locus
and exogenous DNA. Some of these recombination events may have involved
small DNA fragments that did not affect the serotype, while others
resulted in the exchange of larger regions of the capsule locus, which
may have altered the structure and hence serotype of the expressed
CPS. A small region of cps19aB (nucleotides 3,221 to
3,374) has 100% identity to cps19fB. This region presumably
accounts for the high-stringency hybridization of the
cps19aB DNA to a cps19fB probe (17),
as there is only 76.7% identity between the remainder of the
cps19aB and cps19fB genes. The highly conserved
region either may encode a functionally important domain in the
cps19B gene product or may simply be the result of a
recombination event.
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FIG. 2.
Diagrammatic representation of the similarity of the
cps19aAB genes to the cps19fAB genes. There are
several possible recombination points in this region of the locus.
Increasing similarity is represented by progressively darker shades of
grey, and the percent identity is shown under the individual shaded
regions. The arrows indicate the points of divergence, and the number
below each arrow corresponds to the nucleotide number of the
cps19a sequence.
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Southern hybridization analysis.
Previous Southern
hybridization data have shown that high stringency homologues of
cps19fA and -B are present in all serotypes tested, whereas cps19fF and -G are serogroup
specific. However, high-stringency cps19fC,
-D, and -E homologues were present in some
serotypes tested but not others (17). The presence of
homologues to the divergent cps19aC, cps19aD, and
cps19aE genes in the cps loci of various
S. pneumoniae serotypes was therefore
examined by Southern hybridization. Digoxigenin (DIG)-labelled
DNA fragments corresponding to the cps19aC,
cps19aD, and cps19aE genes were used
to probe, at high stringency, ClaI-restricted
chromosomal DNA from representative pneumococci belonging to serotypes
2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 12, 14, 16, 17, 18C, 19F, 19B, 19C, 20, 22, 23F, and 24. The hybridization data for the type 19A
cps19aC, cps19aD, and cps19aE gene
probes and previous data obtained for the type 19F cps19fC,
cps19fD, and cps19fE gene probes (17) are compared in Table 2.
The most remarkable feature seen in Table 2 is that all the serotypes
tested contained high-stringency homologues of either cps19fC to -E or cps19aC to
-E, except types 3 and 4, which do not have a
high-stringency homologue of either cps19fE or
cps19aE (the gene encoding the glucose-1-phosphate
transferase which catalyzes the addition of glucose-1-phosphate to the
lipid carrier, a common first step in biosynthesis of the lipid-linked
repeat unit [12, 17]). The absence of a
cpsE homologue in types 3 and 4 is not surprising, because
the type 4 CPS does not contain glucose, and the mode of type 3 CPS
biosynthesis is atypical, occurring via a processive transferase
(1, 5). Type 4 also contains a hybrid cpsC gene,
hybridizing to both the cps19fC and the cps19aC probes, as described below. Thus, these Southern hybridization data
suggest that S. pneumoniae cps loci can be
divided into two distinct classes, designated class I and class
II, where class I loci contain high-stringency cps19fC
to -E homologues and class II loci contain high-stringency
cps19aC to -E homologues.
Amplification of capsule loci by long-range PCR.
In order to
directly characterize the two classes of cpsC gene,
long-range PCR was used to amplify the portion of the cps loci between cpsB and aliA (Fig. 1B) from several
S. pneumoniae serotypes so that DNA sequencing could be
undertaken. DNAs prepared from serotypes or groups 2, 4, 6A, 6B, 7F, 8, 9N, 9V, 12, 14, 16, 17, 18C, 19F, 19A, 19B, 19C, 20, 22, 23F, and 24 were used as templates for long-range PCR. PCR products were obtained
from at least one pneumococcal isolate of types 2, 4, 6A, 6B, 8, 9N, 14, 18C, 19F, 19A, 19B, 20, and 23F but not from types 7F, 9V, 12, 16, 17, 19C, 22, and 24. Analysis of the DNA fragments reveals that the PCR
products ranged in size from 15 to 20 kb, as shown in Fig.
3A. The PCR products were digested
with the restriction endonuclease ClaI and
electrophoresed on a 1% agarose gel in a Tris-borate-EDTA
(TBE) buffer system as described by Maniatis et al.
(16) (Fig. 3B). Identical restriction patterns were obtained for three different isolates of serotypes 4 and 23F. However, a
restriction site polymorphism was observed in two of the five PCR products from different type 19F strains (Fig. 3B).
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FIG. 3.
Long-range PCR products. PCR products, not digested (A)
or digested with ClaI (B), were electrophoresed on a 1%
agarose gel in the presence of ethidium bromide.
ClaI-restricted PCR product was subjected to Southern
hybridization analysis using DIG-labelled probes specific for
cps19fC (C) or cps19fL (D). The probes specific
for cps19fC and cps19fL correspond to nucleotides
2380 to 2998 and 11539 to 12493 of the cps19f sequence
(17). The molecular size standards are shown on the
right-hand side of the figure and correspond to
HindIII-digested  phage DNA.
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Southern hybridization analysis of long-range PCR
products.
In order to confirm that they contained
cps-related sequences, the long-range PCR products from the
various S. pneumoniae serotypes were restricted with
ClaI and subjected to Southern hybridization analysis using
probes specific for two different type 19F gene probes,
cps19fC (located in the 5' region of the cps19f
locus) and cps19fL (located in the 3' region of the
cps19f locus) (Fig. 3C and D).
The cps19fC probe hybridized at high stringency with a
0.9-kb DNA fragment in types 4, 9N, 14, 18C, 19F, 19B, and 20. Both the hybridization pattern and the size of the DNA fragment which hybridized with the cps19fC probe are consistent with the
Southern hybridization data obtained when probing
ClaI-restricted chromosomal DNA with the cps19fC
probe (data not shown).
The cps19fL probe hybridized with DNA fragments ranging in
size from 4 to 10 kb in the ClaI-restricted PCR products
from types 2, 6A, 6B, 18C, 19F, 19B, and 23F. Hybridization was
consistent with that obtained from Southern hybridization with
ClaI-restricted chromosomal DNA from these isolates,
although the sizes of the restriction fragments differ (data not
shown). The size of this ClaI fragment is affected because
there is no ClaI site between cps19fL and the end
of the PCR product in type 19F.
DNA sequencing of the 5' portion of cpsC in the
long-range PCR products.
The long-range PCR products were
subjected to one round of sequence analysis with the J39 primer
(located at the 5' end of the PCR product) in order to determine the
presence of a cpsC homologue. No sequence data were obtained
from the type 18C PCR template, presumably due to the low yield of the
PCR product obtained. Analysis of the sequence data obtained from all
the other templates and that available for types 1, 3, and 14 (1,
13, 18) showed that, indeed, there were two distinct
cpsC genes in these loci. Types 1, 3, 9N, 14, 19F, 19B, and
20 have class I cpsC genes which exhibit >95% nucleotide
sequence identity to cps19fC, whereas types 2, 6A, 6B, 8, 19A, and 23F have class II cpsC genes which exhibit 72 to
74% identity to cps19fC and >95% identity to
cps19aC (Fig. 4). The
sequences obtained from the PCR products also included the last 75 nucleotides of cpsB; this region can also be separated into
the same two classes as described above (Fig. 4).
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FIG. 4.
Comparison of class I and class II cps
sequences. The first 500 nucleotides of the sequence obtained are shown
(100 nucleotides per line). Dots indicate nucleotides which are
identical to that for cps19f. The stop codon of the
cpsB gene is indicated with an asterisk. The start codon
of cpsC is underlined. (T) denotes an extra
nucleotide, and - denotes the absence of a nucleotide in the
cps14 DNA sequence. The vertical arrows indicate the region
where the crossover between class I and class II sequences has occurred
in cps4.
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An interesting exception is found in type 4, the cps4C gene
of which is a hybrid consisting of a class II 5' region and a class I
3' region, with a distinct crossover point in the vicinity of
nucleotide 345 of the cps4 sequence (Fig. 4). Comparison of the type 4 cps sequence data (available from the TIGR
microbial database) with the cps19f sequence showed another
point of divergence within the cpsB gene. The
cps4B gene is almost identical (except for the first 42 nucleotides) to the cps19aB gene and shows the same point of
sequence divergence from the cps19fB gene (nucleotide 3374 in Fig. 2). Thus, in the cps4 locus a region of 852 nucleotides, including most of cps4B and part of
cps4C, has approximately 74% identity to cps19f,
whereas the remainder of the cps4A to -D
region exhibits >95% identity to cps19fA to -D.
This may have arisen as a consequence of recombination between a class
I cps locus and a DNA fragment (approximately 852 nucleotides long) from a class II cps locus, resulting in a
mosaic cpsB-cpsC region. Analysis of the available type 23F
sequence data (4, 22) indicated that the class II
cps23f locus also diverges from the class I cps19f locus within the cpsB gene, but 98 nucleotides further downstream from the point of divergence for
cps19a and cps4. This suggests that the point of
sequence divergence from class I to class II within cpsB may
vary between different serotypes.
Phylogenetic analysis.
To further confirm the presence of two
distinct classes of cpsC sequences, their phylogenetic
relationship was investigated. An alignment of the partial
cpsC sequences was generated by using CLUSTAL W
(24) (data not shown), and this alignment was used to
generate a phylogenetic tree by the neighbor-joining method and the
distance measure of Tamura and Nei (23), as implemented in
the program MEGA (15). The tree in Fig.
5 shows two highly significant clusters
of cpsC sequences (based on a bootstrapping test with 500 replications) and confirms the observations initially made on the basis
of sequence homology that the cpsC genes are divided into
two classes. The cps4C sequence forms a third cluster; as
described above, this gene is a hybrid of the two cpsC
classes and has a recombination crossover point at or near nucleotide 345 (as shown in Fig. 4) within the cpsC gene. The
cps19bC gene is also separated from the other class I
cpsC sequences; cps19bC also appears to have a
mosaic structure with a small region of class II sequence (nucleotides
409 to 444 in Fig. 4), which is presumably the result of a
recombination event.
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FIG. 5.
Phylogenetic tree of cpsC sequences. The
cpsC gene sequences were aligned by using CLUSTAL W
(24), and the phylogenetic tree was generated by using MEGA
(15), as described in the text. The numbers associated with
the branches are bootstrapping confidence limits, resulting from 500 replications, as defined in MEGA. The scale represents the number of
nucleotide substitutions per site.
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Conclusions.
The 5' portion of the cps locus from
S. pneumoniae type 19A is similar to cps19f,
in that it has the same number of genes arranged in the same order.
However, many of these genes demonstrate only 70 to 80% nucleotide
sequence identity with their cps19f counterpart, suggesting
either that the two loci diverged a long time ago or that portions of
these loci have separate origins. Some regions within the
cps19aA, -B, and -G genes do have
>90% identity to those in cps19f, which may be a
consequence of recombination between the two loci or perhaps is due to
a requirement for a higher degree of conservation in regions encoding
functionally important domains.
Southern hybridization analysis identified two classes of
cpsC, cpsD, and cpsE genes in
S. pneumoniae cps loci, which were designated as either
class I or class II. Class I pneumococcal cps loci contain
high-stringency cps19fC to -E homologues, whereas class II loci contain high-stringency cps19aC to
-E homologues. Direct sequencing of the long-range PCR
products obtained confirmed the presence of two classes of
cpsC gene. Phylogenetic analysis of the sequence data
also confirmed that the pneumococcal cpsC gene is divided
into two closely related classes. The presence of the cpsC
and cpsD genes in all cps loci examined is
consistent with the important role of CpsC and CpsD in pneumococcal CPS
production. Both are predicted to be involved in chain length
regulation and export of the CPS (8, 17). At this stage, it
is not possible to determine whether the differences between class I
and class II cpsC and cpsD gene products are
functionally significant. Translation of the genes indicates a similar
degree of amino acid sequence divergence between class I and class II
CpsC proteins (approximately 70% identity). Interestingly, even
small differences between the functionally homologous Rol (Wzz)
proteins of Shigella species has previously been shown to
affect the modal chain length of the lipopolysaccharide O antigen
(11).
The cpsE gene was also present in all S. pneumoniae serotypes tested which contain glucose in their CPS,
except type 3, which has a different mode of CPS biosynthesis (1,
5). This gene is also separated into either class I or class II,
along with the preceding cpsC and cpsD genes.
However, the two different classes do not appear to affect the function
of CpsE, which is a glucose-1-phosphate transferase. Kolkman et al.
(14) demonstrated glucose-1-phosphate transferase activity
in several pneumococcal serotypes now known to contain either class I
or class II cpsE genes. In all S. pneumoniae
cps loci sequenced to date, the gene which follows cpsE
is serotype or serogroup specific (21).
All S. pneumoniae cps loci examined contain highly
conserved cpsA and cpsB genes, indicating that
they probably evolved from a common ancestor. However, their
cpsC, cpsD, and cpsE genes can be
separated into either class I or class II sequences, suggesting that
recombination between the original S. pneumoniae
ancestor (either class I or class II) and exogenous DNA resulted in the formation of two distinct clonal S. pneumoniae strains
from which all subsequent serotypes have evolved. The presence of DNA
homologous to cps19fA to -D, even though these
genes are not functional in type 3 pneumococci (7), probably
reflects the common origin between type 3 and other class I pneumococci.
The type 4 and 19B cpsC sequences both show evidence of
recombination within the cps loci. Two recent studies have
demonstrated that natural recombination events involving exchange of
entire cps loci (or major portions thereof) have resulted in
switching of capsule type (e.g., from 23F to 19F) by multiply
drug-resistant pneumococcal clones on numerous occasions (4,
19). The current study indicates that recombination events
involving small fragments within pneumococcal cps loci may
also be common in nature and may represent a mechanism whereby
additional serotype diversity is generated.
Nucleotide sequence accession numbers.
The cps19a
sequence has been deposited with GenBank under accession no.
AF094575. The sequences for the 5' region of cpsC from
serotypes 2, 6A, 6B, 8, 9N, 19B, and 20 are available under GenBank
accession no. AF106132, AF106133, AF106134, AF106135, AF106136,
AF106137, and AF106138, respectively.
This work was supported by a grant from the National Health and
Medical Research Council of Australia.
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