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Infection and Immunity, May 2001, p. 3442-3446, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.3442-3446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular and Genomic Analysis of Genes Encoding
Surface-Anchored Proteins from Clostridium
difficile
Tuomo
Karjalainen,1
Anne-Judith
Waligora-Dupriet,1
Marina
Cerquetti,2
Patrizia
Spigaglia,2
Andrea
Maggioni,3
Pierluigi
Mauri,3 and
Paola
Mastrantonio2,*
Département de Microbiologie,
Faculté de Pharmacie, Université de Paris-Sud, 92296 Châtenay-Malabry Cedex, France,1 and
Laboratorio di Batteriologia e Micologia Medica, Istituto
Superiore di Sanità, Rome 00161,2 and
Istituto Tecnologie Biomediche Avanzate/CNR, 20090 Segrate-Milan,3 Italy
Received 27 November 2000/Returned for modification 30 January
2001/Accepted 8 February 2001
 |
ABSTRACT |
The gene slpA, encoding the S-layer precursor protein
in the virulent Clostridium difficile strains C253 and
79-685, was identified. The precursor protein carries a C-terminal
highly conserved anchoring domain, similar to the one found in the
Cwp66 adhesin (previously characterized in strain 79-685), an SLH
domain, and a variable N-terminal domain mediating cell adherence. The
genes encoding the S-layer precursor proteins and the Cwp66 adhesin are
present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include
surface-anchored proteins.
| |
TEXT |
Clostridium
difficile is responsible for many cases of
antibiotic-associated diarrhea and pseudomembranous colitis in
humans (6). It is considered a common microorganism in
nosocomial outbreaks (14). Few studies have focused
on the importance of virulence factors other than toxins A and B in the
pathogenesis of this disease. As in other bacteria, the role of surface
proteins in the first step of colonization, i.e., adherence to
enterocytes, could be of crucial importance.
Cerquetti et al. have previously described the presence of two
superimposed paracrystalline surface layers (S-layers) on different C. difficile strains (4). Each layer is
composed of a glycoprotein subunit which slightly varies in
molecular mass among the different strains examined. Two S-layer
proteins, called P36 and P47, extracted from the toxigenic and virulent
C. difficile strain C253, were purified by high-performance
liquid chromatography, and their N-terminal sequences were determined.
Moreover, properties of adhesiveness to Caco-2 cells were observed for
the P36 protein (3).
Karjalainen et al. (9) and Waligora et al.
(19) have shown that C. difficile strain
79-685, a toxigenic and virulent isolate, can adhere to various
culture cells in vitro and that certain stress factors can increase
adherence and expression of some surface proteins. The gene encoding
one of these surface proteins (Cwp66, a 66-kDa cell wall protein) has
been cloned and characterized (20) (GenBank accession
number AF194870). Cwp66 displays a two-domain structure, with an
N-terminal cell wall-anchoring domain and a C-terminal surface-exposed domain.
Since genetic manipulation within C. difficile is difficult,
thus preventing the construction of isogenic mutants, in the present
work we have identified and characterized the slpA gene, encoding the S-layer precursor protein of C. difficile C253
and C. difficile 79-685, by deriving peptide sequences
from purified proteins and by analyzing the genome sequence of C. difficile 630, now available (http://www.sanger.ac.uk). This is
the first evidence of two S-layer proteins derived from a common
precursor through posttranslational processing. A comparative analysis
of SlpA and Cwp66 as well as an analysis of the region containing the
genes on the genome of C. difficile strain 630 has also been performed.
(This report was presented in part as a poster at Microbial
Pathogenesis Session No. 137/B, abstract B-139, of the General Meeting
of the American Society for Microbiology, Los Angeles, Calif., 21 to 25 May 2000 [P. Mastrantonio, A.-J. Waligora, M. Cerquetti, A. Sebastianelli, P. Bourlioux, and T. Karjalainen, Abstr. 100th Gen.
Meet. Am. Soc. Microbiol., abstr. B-139, 2000].)
Characterization of the slpA gene.
In order to
search for sequence similarity to other proteins, the two S-layer
protein subunits of C. difficile strain C253, P36 and P47,
were digested with protease V8 (endoproteinase Glu-C; Sigma-Aldrich,
St. Louis, Mo.) and purified using a C18 high-performance liquid chromatography column (Vydac, Hesperia, Calif.). The purified peptides were analyzed by matrix-assisted laser desorption
ionization-time of flight (MALDI-TOF) to determine the molecular
weights and sequenced by means of Edman degradation. The sequences
obtained for five peptides are presented in Table
1.
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TABLE 1.
Amino acid sequences of peptides obtained by digesting
C. difficile C253 S-layer proteins with V8 protease
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When the peptide sequences were submitted to the
C. difficile strain 630 genome database using TBLASTN, all peptides
could
be mapped to the same contig. Analysis of the sequence of this
region revealed a unique 2,160-bp gene capable of coding for a
protein of 73.4 kDa. To identify the corresponding gene from strain
C253, oligonucleotides based on the sequence of
C. difficile
630
were synthesized and used for PCR amplification assays (Table
2). The PCR conditions consisted of an
initial denaturation at
94°C followed by 30 cycles of 1 min at
94°C, 1 min of annealing
at 54°C, and 1 min of extension at 72°C.
At the end of the cycling,
samples were held at 72°C for 5 min.
Amplification was carried
out in a final volume of 100 µl with a
reaction mixture containing
a buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM
MgCl
2), a 200 µM concentration
of each deoxynucleoside
triphosphate, 100 pmol of each primer,
2.5 U of Takara Ex Taq/TM
(Takara Shuzo Co., Ltd., Kyoto, Japan),
and 100 ng of template DNA. The
complete sequence was obtained
by sequencing the overlapping PCR
products. The purified PCR products,
together with the appropriate
oligonucleotide primers, were used
as templates for sequencing
reactions in a Perkin-Elmer ABI 373A
DNA sequencer and an ABI prism dye
terminator cycle sequencing
ready reaction kit (PE Applied Biosystems,
Monza, Italy). All
PCR products were sequenced on both strands.
Analysis of the sequence revealed the presence of the 2,160-bp gene
also in
C. difficile C253 (EMBL accession number
AJ291709),
exhibiting 100% identity with that found in
C. difficile 630 (the
sequencing of the gene from strain 630 revealed
nucleotide change
A
T at position 314 compared to the sequence
available on the
Internet). This gene was referred to as
slpA according to the
nomenclature used for other bacteria.
The encoded protein shows
a characteristic procaryotic signal sequence
at the N terminus
and a two-domain structure as described previously
for the cell
wall-anchored adhesin Cwp66 isolated from strain
79-685 (
20)
(Fig.
1A). The
C-terminal domain carries three imperfect intramolecular
repeated
sequences, a common feature of bacterial surface-exposed
proteins
(Fig.
1B). The fact that structural analysis of the S-layer
of strain
C253 revealed the presence of two proteins, P36 and
P47, suggests that
a specific cleavage of the precursor protein
occurs during the
maturation process. This cleavage has also been
observed for Cwp66; in
the cell wall this adhesin is always found
in two or even three
fragments (
20).
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FIG. 1.
(A) Diagram of the domain structure of the protein SlpA:
a potential leader peptide (black box) is followed by the SLH domain
and two larger domains named N and C. The numbers refer to amino acid
positions in the protein. The regions corresponding to the P36 and P47
S-layer proteins of C. difficile strain C253 are indicated,
as are the positions of repeat sequences (arrows). (B) Alignment of the
repeats of the SlpA protein in the C-terminal domain. The length of the
C-terminal repeats is between 85 and 111 amino acids. The amino acid
positions in the SlpA protein are indicated. * and +, two and three
identical amino acids, respectively;  and !, two and three
functionally identical amino acids (A, S, and T; D and E; N and Q; R
and K; I, L, M, and V; F, Y, and W), respectively.
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|
The availability of the N-terminal sequence of the two proteins
permitted us to determine that P47 corresponds to the C-terminal
part
and P36 corresponds to the N-terminal part of the precursor
protein
(Fig.
1). The molecular masses of the peptides determined
by liquid
chromatography-mass spectrometry and MALDI-TOF analyses
corresponded to
those obtained by theoretical digestion of the
virtual protein from the
contig. Moreover, the molecular masses
of the virtual proteins are
34,244 and 39,522 Da for P36 and P47,
respectively. These data are in
good agreement with the previous
experimental results obtained by
electrospray ionization-mass
spectrometry and MALDI-TOF mass
spectrometry (
13).
Sequence similarity to other proteins.
Homology searches were
conducted with Fasta3 (16) or Blast 2.0 (1).
SlpA carries in its N terminus (Fig. 1A and
2A) a 78-amino-acid sequence showing
homology to the SLH (S-layer-homologous) domain, the presence of which
has been detected in numerous S-layer proteins (12). This
sequence is characterized by several highly conserved residues which
are present in SlpA (Fig. 2A) and a particular predicted secondary
structure, i.e., two 
-helices flanking a 
-strand (7)
followed by a coiled-coiled domain (11), which has been
found in other S-layer proteins (12). Only one of these domains is present in SlpA; in other proteins, several domains can be
present, suggesting duplication of a gene fragment. The SLH domains,
usually found in proteins that are anchored to the cell wall, were
originally proposed to be involved in anchoring the proteins to the
peptidoglycan, but more recently, secondary cell wall polymers have
been identified as anchoring structures for the SLH domain (5, 8,
15).
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FIG. 2.
(A) Amino acid sequence alignment of the N-terminal
domains of SlpA from three C. difficile strains, C253, 630, and 79-685. Note the pronounced variability. The SLH domain is shaded
in gray; the conserved residues of this domain (12) are in
boldface letters. Asterisks or colons denote amino acids that are
identical or similar, respectively. The positions of the two peptides
of C. difficile C253 (Table 1) are marked above the
sequence. The N-terminal amino acids of the P36 and P47 proteins are
indicated with arrows. (B) Amino acid sequence alignment of the
putative cell wall-anchoring domains of SlpA and Cwp66 from C. difficile strains 630 and C253 and strain 79-685. Asterisks or
colons denote amino acids that are identical or similar, respectively,
in the SlpA proteins only. Conserved residues in the four proteins are
in boldface.
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The 339-amino-acid C-terminal domain of SlpA (residues 380 to 719)
shows significant homology (29% identity and 51% similarity)
to a
domain found in
N-acetylmuramoyl-
L-alanine
amidase CwlB of
Bacillus subtilis (GenBank accession number
Q02114). CwlB
is known to be released from the bacteria and reattached
to the
cell wall peptidoglycan (
10) through the
domain displaying homology
to SlpA (corresponding to the P47 protein).
These data suggest
that both P36 and P47 carry a motif that could
anchor the S-layer
proteins to the cell wall, although in the case of
P36 the attachment
could be indirect through a secondary protein. A
similar domain
was also found in Cwp66 (
20).
Antibodies raised against P47 of
C. difficile C253 recognize
in other
C. difficile strains proteins with molecular masses
close to 47 kDa (
4). This suggests that this likely
cell-anchoring
domain is conserved among
C. difficile
strains. To verify whether
this is the case in strain 79-685, the
slpA gene was amplified
from the strain by PCR as described
above using primers listed
in Table
2 and its nucleotide sequence was
determined. The encoded
precursor protein has the same two-domain
structure as in strain
C253 (Fig.
2). Interestingly, alignments
performed with the ClustalW
program (
18) revealed that one
of the domains is well conserved
(C-terminal domain) among the three
strains studied (77% identity
between strains C253 and 630 and strain
79-685) and displays sequence
conservation with the cell
wall-anchoring N-terminal domain of
Cwp66 (Fig.
2B). The second
domain is highly variable: only 33%
of the residues are identical
between strains C253 and 630 and
strain 79-685 (Fig.
2A).
P36 (N-terminal domain) shows remote homology (<25% identity) with
the SlaP S-layer protein of
Bacillus stearothermophilus (
P35825), glycosyltransferase-
S of
Streptococcus
mutans (
D89979),
and various flagellins of
Salmonella (e.g.,
U06197 and
U06227).
However,
glycosyltransferases have been found to mediate adherence
in other
bacteria (for a review, see reference
17). Even if
the
N-terminal domain does not show significant homology to known
adhesins, preliminary experiments using the Caco-2 cell line and
specific antisera have shown it to be involved in the adhesion
process (
3). Antiserum against whole cells of
C. difficile C253 and monospecific antiserum against P36
were raised in adult
New Zealand rabbits by following procedures
previously described
(
2). The P36 antiserum, prepared
using the antigen obtained
by electroelution of the P36 band in sodium
dodecyl sulfate-polyacrylamide
gel electrophoresis, recognized only P36
when assayed by immunoblot
analysis against EDTA- or urea-extracted
surface proteins of
C. difficile C253 (
2,
4).
In an inhibition-of-adhesion assay,
3-day-old monolayers of Caco-2
cells infected with
C. difficile C253 bacteria preincubated
with nonimmune serum, anti-C253 whole-cell
serum, and anti-P36 serum
showed mean numbers of adherent bacteria
per cell ± standard
deviations of 43.4 ± 16.42, 0.16 ± 0.20 (
P = 0.01), and 3.66 ± 3.23 (
P < 0.05),
respectively (values were
determined from three separate experiments;
statistical analysis
was performed using Student's
t test).
Adherence of
C. difficile C253 bacteria was specifically
blocked by the homologous antiserum
and significantly reduced by the
P36 antiserum compared to the
adherence of bacteria pretreated with a
nonimmune
serum.
Sequence analysis of SlpA from
C. difficile strains 79-685
and C253 (Fig.
2A) revealed pronounced variations in the N-terminal
domains suggesting that SlpA is surface exposed and thereby subject
to
antigenic drift. The surface localization of P36, previously
demonstrated by immunofluorescence studies using the P36 antiserum
(
2), and the above-mentioned data on its role as adhesin
may
support this
hypothesis.
Analysis of a gene cluster carrying slpA and
cwp66.
Examination of a 37-kb DNA fragment flanking
cwp66 and slpA of the genome sequence of C. difficile strain 630 revealed 17 open reading frames (ORFs) in the
same orientation. Eleven of these ORFs encode a domain, present in
either the N- or C-terminal part of these putative secreted proteins,
showing homology to the cell wall-anchoring domain of the Cwlp
autolysin of B. subtilis (Fig.
3). This conserved domain is
characterized by the presence of three PII/LL motifs, although the
third repeat is less conserved than the first two. The anchoring domain
is associated in all cases with a putative peptide signal and with
another domain (except for Orf14), whose function appears variable: in
Cwp66 and SlpA, this domain carries adhesive function; Cwp84 carries in
this domain a cysteine protease active site and has proteolytic
activity (our unpublished data); and Orf12 carries an active site found
in N-muramyl-L-alanine amidases. The variable
domains encoded by the other ORFs display remote homology (<20%) to
various bacterial outer membrane proteins. The variable domains of
Cwp66 and Orf10 and of Orf5, Orf6, and Orf2 are up to 40% homologous.
We suspect that all these proteins could be cell wall anchored through
their CwlB-like domain. The presence in multiple genes of sequences
encoding the two domains suggests modular evolution and subsequent
gene duplication from a common ancestor for B. subtilis cwlB
and C. difficile. During evolution, this domain was placed
in either the N or C terminus and became associated with another domain
as in Cwp66 or many autolysins. Interestingly, the cluster also carries
a large gene (orf3) capable of encoding a 220-kDa protein
that does not exhibit the two-domain structure. It displays remote
homology to various membrane-associated proteins from Plasmodium
falciparum.
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FIG. 3.
Genetic organization of a 37-kb DNA fragment carrying
the slpA and cwp66 genes from the genome of
C. difficile 630. The proteins encoded by these genes and
their sizes, molecular masses, and domain structures are indicated.
Orf7 exhibits significant homology with the SecA protein from B. subtilis, Orf9 with dehydrogenases, Orf12 with
N-acetyl-L-alanine-muramyl amidase, and Orf13
with glycosyltransferases. A.A., amino acids.
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|
In conclusion, we report here on the characterization of the
slpA gene in
C. difficile C253 and 79-685. This
gene in
C. difficile 630 is located in a cluster
including genes encoding proteins
with adhesive properties. The
isolation of this cluster is an
important step in the characterization
of the process of colonization
by
C. difficile: analogously
to many other bacteria,
C. difficile possesses multiple
surface-exposed proteins acting as adhesins.
Furthermore, surface
proteins are attractive targets for the development
of a vaccine
against this troublesome
pathogen.
Nucleotide sequence accession number.
The DNA sequence of the
slpA gene from C. difficile strains C253 and
79-685 have been deposited in the EMBL database under accession number
AJ291709 and in the GenBank database under accession number AY004256.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratorio di
Batteriologia e Micologia Medica, Istituto Superiore di Sanità,
Viale Regina Elena 299, Rome 00161, Italy. Phone: 39-06 49902335. Fax: 39-06 49387112. E-mail: pmastran{at}iss.it.
Editor:
V. J. DiRita
| |
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Infection and Immunity, May 2001, p. 3442-3446, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.3442-3446.2001
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