Clostridium difficile is the main cause of antibiotic-associated
diarrhea, leading to significant morbidity and mortality and putting
considerable economic pressure on healthcare systems. Current knowledge of the
molecular basis of pathogenesis is limited primarily to the activities and
regulation of two major toxins. In contrast, little is known of mechanisms used
in colonization of the enteric system. C. difficile expresses a
proteinaceous array on its cell surface known as the S-layer, consisting
primarily of the major S-layer protein SlpA and a family of SlpA homologues, the
cell wall protein (CWP) family. CwpV is the largest member of this family and is
expressed in a phase variable manner. Here we show CwpV promotes C.
difficile aggregation, mediated by the C-terminal repetitive
domain. This domain varies markedly between strains; five distinct repeat types
were identified and were shown to be antigenically distinct. Other aspects of
CwpV are, however, conserved. All CwpV types are expressed in a phase variable
manner. Using targeted gene knock-out, we show that a single site-specific
recombinase RecV is required for CwpV phase variation. CwpV is
post-translationally cleaved at a conserved site leading to formation of a
complex of cleavage products. The highly conserved N-terminus anchors the CwpV
complex to the cell surface. Therefore CwpV function, regulation and processing
are highly conserved across C. difficile strains, whilst the
functional domain exists in at least five antigenically distinct forms. This
hints at a complex evolutionary history for CwpV.
C. difficile is gram-positive spore forming anaerobe and a major
cause of antibiotic-associated diarrhea . C.
difficile infection (CDI) often occurs in the nosocomial environment
where infection management exerts significant economic pressure on healthcare
systems. The strong association of CDI with antibiotic usage reflects disruption of
the normal gut flora allowing effective colonization by C.
difficile. Strains causing disease produce one or two related toxins,
TcdA and TcdB, which modulate the activity of host cell Rho GTPases, destroying the
integrity of the epithelial cell barrier and inducing a variety of effects on
intestinal cells . Using isogenic mutants in a hamster model of infection,
recent studies have shown that toxin B plays an important role in pathogenesis but
the situation is less clear for TcdA , . C. difficile
strains causing CDI however are remarkably diverse. Despite a high prevalence of
certain strain types, for example NAP1/027 in North America and Europe around
2005–6, no particular type dominates and strain prevalences vary
geographically and temporally .
Factors expressed on the bacterial cell surface are likely to contribute to host
colonization via interactions with host tissue, the immune system and other
bacterial cells. In common with a large number of bacterial species , C.
difficile produces an S-layer, a paracrystalline proteinaceous array
that completely coats the bacterial cell wall. The C. difficile
S-layer is primarily composed of two proteins, the high molecular weight S-layer
protein (HMW SLP) and the low molecular weight (LMW) SLP. These proteins are derived
from the precursor SlpA  through cleavage mediated by the cysteine protease Cwp84
, . The LMW and HMW
SLPs form a stable non-covalently associated complex, which has been analysed by
small-angle X-ray scattering . The HMW SLP contains putative cell wall binding motifs
(Pfam 04122) which are thought to mediate attachment to the underlying cell wall.
The LMW SLP is highly immunogenic and variable between strains, with over 20
distinct sequences identified to date , , , .
C. difficile 630 contains 28 paralogs of the HMW SLP . Known as the
cell wall protein (CWP) family, all members contain two or three copies of the
putative cell wall binding motif in addition to a second unique domain that may
specify function. Transcriptomic and proteomic studies have shown several of these
proteins to be expressed in vitro, . Antibodies
against some of these proteins are found in serum of infected patients  implying at
least some of the CWPs are expressed in vivo during infection and
accessible to the host immune system.
In C. difficile 630 , CwpV is the largest member
of the CWP family and consists of three distinct domains: (1) an N-terminal region
with putative cell wall binding activity, (2) a region of unknown function
terminating in a serine-glycine-rich flexible linker; (3) nine repeats of 120 amino
acids each. We recently described the phase variable expression of CwpV and proposed
a mechanism of control involving a cwpV switch . Briefly,
between the cwpV promoter and open reading frame there is a 195 bp
invertible region flanked by imperfect 21 bp inverted repeats. In the OFF
orientation this sequence adopts a stem loop terminator, preventing formation of
full-length transcripts while in the other orientation (ON), the terminator is
absent so full length cwpV transcription proceeds and CwpV is
In this study we investigate the regulation, post-translational processing and
function of CwpV across a diverse panel of C. difficile strains. We
definitively identify the site-specific recombinase necessary for CwpV phase
variation, and show that the cwpV switch orientation is the primary
determinant of CwpV expression. We show that the CwpV N-terminal domain is
responsible for cell wall anchoring and is well conserved across strains. In
contrast, we find high sequence variation of the C-terminal repeat sequences across
C. difficile strains with five antigenically distinct repeat
types identified. Despite this variability, both post-translational processing and
function of CwpV appear to be conserved across C. difficile
strains. All types of CwpV exhibit an aggregation-promoting function, which is
conferred by the variable C-terminal domain. The implications of these findings
relating to the role of CwpV in C. difficile infection are
The recombinase RecV controls expression of CwpV in C.
Previously we identified the C. difficile recV gene as a strong
candidate for the site-specific recombinase responsible for inversion of the
cwpV DNA switch . To definitively determine
the role of recV in inversion of the cwpV
switch, we constructed a recV knock-out in
630Δerm using ClosTron technology . 630Δerm
(wild-type) and four erythromycin resistant recV mutants,
ΔrecV1-4, were confirmed by PCR (Figure 1). To establish the orientation of
the cwpV switch in these ΔrecV strains,
orientation-specific PCR was carried out  (Figure 1B). In wild-type clones, inversion of
the cwpV switch was occurring as expected, shown by products
with both primer pairs (Figure 1C
i). All four ΔrecV clones only amplified an OFF
product (Figure 1C ii) and
were therefore designated ΔrecV OFF.
A plasmid encoding recV (pRecV+) was introduced into
C. difficile ΔrecV OFF by conjugation.
The orientation of the cwpV switch in four
ΔrecV(pRecV+) transconjugants was tested by PCR.
In all four strains, cwpV switch inversion was observed
indicating successful complementation of the switching defect (Figure 1C iii). RecV is a
member of the phage integrase family of tyrosine recombinases and by alignment
tyrosine-176 was identified as a putative catalytic residue. To confirm the key
role of this residue, we constructed a Tyr-Phe mutant (pRecVY176F)
which was introduced into ΔrecV OFF. All four
ΔrecV(pRecVY176F) transconjugants only had
the cwpV switch in the OFF orientation (Figure 1C iv). Therefore RecVY176F
could not complement the ΔrecV OFF phenotype confirming the
key role of tyrosine-176 in RecV- mediated inversion of the
In order to isolate ΔrecV ON mutants,
ΔrecV(pRecV+) was serially subcultured without
thiamphenicol selection to allow loss of the pRecV+ plasmid.
Thiamphenicol-sensitive colonies were tested for cwpV switch
orientation and all were found to contain a single orientation, as the
pRecV+ plasmid had been lost (data not shown). Four clones, from which only
the ON orientation of the cwpV switch could be amplified, were
isolated and designated ΔrecV ON (Figure 1C v). The lack of inversion in these
strains confirmed the necessary role of RecV in cwpV switch
inversion, and suggests that this is the only recombinase responsible for
During the plasmid-curing process we noted two distinct colony morphologies
amongst the resulting thiamphenicol-sensitive colonies. Interestingly, these
were associated with the two orientations of the cwpV switch.
In Figure 1D it can be seen
that ΔrecV OFF colonies have the same profuse, ruffled
appearance as wild-type 630 colonies, whilst ΔrecV ON
colonies are smaller and smoother-edged. The hypothesis that CwpV expression in
a ΔrecV ON background causes a smaller colony phenotype was
tested (see below).
CwpV is the major CWP on ΔrecV ON cells
The expression of CwpV in S-layer extracts of 630Δerm (WT),
ΔrecV OFF and ΔrecV ON was
analyzed by SDS-PAGE (Figure
2A). In WT and
ΔrecV(pRecV+) extracts, the two
cleavage products of CwpV, the ∼42 kDa (N-terminal fragment) and ∼116
kDa (repeat domain) can be seen. However, as CwpV is only expressed by the
minority of ON cells, the overall level of expression in these cultures is low.
No expression of CwpV is seen in
ΔrecV OFF strains. Compared to WT cultures,
ΔrecV ON strains express a high amount of CwpV.
Densitometry analysis indicated that in ΔrecV ON strains,
CwpV constituted 13.3% of the protein present in the S-layer (data not
shown). These findings were confirmed by Western blot using anti-CwpVrptI
(detects the ∼116 kDa CwpV repeat domain) and anti-CwpVNter (detects the
∼42 kDa N-terminal fragment) antibodies (Figures 2C). In all cases, expression of CwpV
corresponded to cwpV switch orientation (Figure 2C).
In order to assess CwpV expression at the individual cell level, C.
difficile cultures were stained using CwpVrptI antibody and
analyzed by fluorescence microscopy (Figure 2D). In the WT culture, ∼5%
of cells are ON as previously reported . In
ΔrecV OFF, expression cannot be detected on any cell.
In ΔrecV(pRecV+) cultures, phase variable expression
of CwpV can be seen with approximately 15% of cells expressing CwpV. The
higher proportion of CwpV-positive cells is probably due to the altered level of
RecV expression from the multi-copy plasmid, pRecV+. In
ΔrecV OFF(pRecVY176F) no CwpV expression can
be detected, as expected due to a lack of switch inversion from OFF. In
ΔrecV ON, CwpV expression can be detected in all cells.
This suggests that cwpV switch orientation is the primary
determinant of CwpV expression.
The CwpV C-terminal repeat region is variable across C.
Given the clinical importance of C. difficile strain diversity
we sought to determine the level of conservation of cwpV across
a variety of strains. PCR analysis of a collection of C.
difficile strains using primers specific for the 5′ region of
cwpV revealed the presence of a common fragment in all
strains (Figure 3Ai).
However, amplification using primers specific for the 630 repeat region
generated products only in a subset of strains (Figure 3A ii). We have previously shown that,
in these strains, the cwpV gene is highly similar to that of
630, but differs in the number of C-terminal repeats  (Figure 1A, panel vii). CDKK371 contains six
repeats and strains R8366 and Y each contain four repeats. The repeats, termed
type I, are highly similar to each other in DNA and predicted amino acid
sequence, both within any one strain and between strains. In each strain the
first repeat is more divergent from the remaining repeated sequences, although
it is still almost identical between strains.
We were unable to amplify the 3′ region of cwpV from the
remaining strains studied using primers designed against the 630 type I repeats
(Figure 3A ii),
consistent with a comparative genomic microarray study using DNA probes from
strain 630 which showed that, while the 5′ region of cwpV
is present in each of 75 strains studied, the 3′ region was only detected
in 17 strains . PCR amplification of the 3′ end of the
cwpV gene from two ribotype 027 strains, R20352 and R20291,
was achieved using primers found to be conserved in the 5′ end of the
R20352 cwpV gene and in CD0515, the gene located downstream of
cwpV in 630. The amino acid sequence of R20352 CwpV was
almost identical to 630 CwpV from the N-terminus to the serine/glycine-rich
region. However, instead of containing repeats of 120 amino acids, these 027
strains contained eight 79 amino acid repeat regions. PCR analysis with primers
specific for the R20352 CwpV repeats is shown (Figure 3A iii and vii). These repeats,
designated type II, were completely distinct from type I repeats. Several 027
strains were analyzed and the repeats were virtually identical in sequence to
one another with the exception of the first repeat, which was slightly divergent
from the consensus but conserved between strains.
In other C. difficile strains, where the 5′ region of the
gene was present but the 3′ region could not be amplified, genomic DNA was
digested with AseI and then re-ligated to form circular fragments. Following
inverse PCR, sequence analysis revealed cwpV from all strains
to be practically identical to 630 cwpV extending to the
serine/glycine-rich region. The 3′ region of cwpV from
strains CDKK167 and NF2029 encode six repeats of 94 amino acids distinct in
sequence from types I and II, and were designated type III. These repeats could
be amplified using repeat-specific primers (Figure 3A iv and vii). Two toxin A-B+
strains, CDKK17 and M9, were found to have a fourth type of repeat sequence. The
first repeat is very similar to the first type III repeat, whereas the remaining
seven repeats of 98–100 amino acids are distinct from all other repeats
and were designated type IV. Finally, non-toxic strains, AY1 and AY5, were found
to have a combination of type III repeats and a further novel 79 amino acid
repeat, designated type V. Primers designed against type IV and V repeats
reflected the sequence analysis, shown in Figure 3A v–vii.
It should be emphasized that the five types of CwpV repeat are completely
unrelated to one another. A diagrammatic representation of the eight unique CwpV
protein architectures discovered so far is shown in Figure 3B. A multiple sequence alignment of
the amino acid sequences of one repeat of each type is shown in Figure 3C, illustrating
visually how unrelated the sequences are as no meaningful alignment can be
Having characterized these five repeat types, a larger collection of strains were
analyzed by PCR to determine their repeat types (see Table 1). Our analysis of strains was not
exhaustive, and it seems possible that further CwpV repeat types may be present
amongst the considerable diversity of the C. difficile species.
However, we focused on the characterization of these five known CwpV repeat
types. For each type, a model strain was decided upon and used to represent that
type for the rest of the study. We chose; for type I 630, for type II R20352,
for type III CDKK167, for type IV M9 (which also contains one type III repeat)
and for type V AY1 (which also contains two type III repeats). A full multiple
sequence alignment of all the repeat sequences is shown in Figure
Different CwpV repeat types are antigenically distinct
Given the completely different amino acid sequences for each repeat type we
hypothesized that the repeats may be antigenically distinct. CwpV expression was
therefore investigated using antibodies raised against recombinant CwpV domains.
The CwpV domains used as antigens are indicated by black lines above the protein
cartoons in Figure 4A.
S-layer extracts were prepared for the representative strain of each type and
were visualized by SDS-PAGE (Figure
4B). The significant variation in SlpA across these strains can be
seen, as described previously . Analysis by Western blotting with anti-CwpVNter
antibody reveals a single band at ∼42 kDa in each strain (Figure 4C) as would be
expected by the high level of conservation of the CwpV N-terminal domain across
strains. This confirmed expression of CwpV and localization to the cell wall in
all these strains.
S-layer extracts were then analyzed using antibodies raised against the specific
repeat types I–V. Antibodies against repeat types I, II, IV and V
recognized only their cognate strains 630, R20352, M9 and AY1, respectively.
Anti-CwpVrptIII detects CwpV repeats in CDKK167, M9 and AY1 which all contain at
least one type III repeat. Detection of CwpV in strain M9 with anti-CwpVrptIII
is weak, this may be due to the presence of only one type III repeat in this
CwpV protein. These results demonstrate that, as expected based on distinct
amino acid sequence, the CwpV repeat types are antigenically distinct.
Expression of all CwpV types is phase variable
Inversion of the cwpV switch has been detected in a wide variety
of strains, but phase variable expression of CwpV has only been reported in
strain 630 . It therefore seemed possible that CwpV would be
expressed in a phase variable manner in strains expressing all types of CwpV.
Cultures of representative strains for each type were stained using the
appropriate anti-CwpVrpt antibody for analysis by immunofluorescence microscopy
(Figure 5). All strains
expressed CwpV in a subset of cells, ranging from 0.1–10% of total
cells, indicative of phase variable expression. The factors determining the
proportion of CwpV-positive cells are yet to be determined, but it is clear that
under standard laboratory culture conditions, a minority of cells express
A conserved post-translational cleavage mechanism for all CwpV
The five different CwpV proteins from 630, R20352, CDKK167, M9 and AY1 were
modified to include a C-terminal Strep-Tag II and expressed in C.
difficile 630ΔcwpV using a constitutive
promoter. S-layer extracts were prepared from these strains, designated
ΔcwpV(pOE I-V), and analyzed by SDS-PAGE (Figure 6A). A high level of
CwpV expression can be seen for each CwpV protein, with CwpV constituting a
significant proportion of the total S-layer extract. All five proteins are
cleaved into the ∼42 kDa N-terminal fragment and C- terminal fragment of
between 90 and 120 kDa, depending on their CwpV type. Western blotting using
antibodies against the N-terminal domain detected the 42 kDa fragment in each
extract and those against the Strep-Tag II detected the 90–120 kDa
C-terminal CwpV fragment (Figure
6B). In order to determine the cleavage site for each CwpV protein,
the N-terminal sequence of each strep-tagged C-terminal fragment was obtained.
The first 5 amino acids of every C-terminal CwpV fragment was found to be TFVNY,
unambiguously locating the cleavage site in the domain of CwpV between the
N-terminal domain and the serine/glycine rich region, as depicted in Figure 6D. This domain is
well-conserved between CwpV proteins of different types, and indicates that the
CwpV post-translational cleavage mechanism is conserved across CwpV types. The
protease responsible for SlpA cleavage has been identified as the surface
expressed cysteine protease Cwp84 ,  however it has been shown that
Cwp84 is not required for CwpV cleavage . We have analyzed expression
of CwpV in strains containing knock-outs of either cwp84 or its
paralog cwp13, and found that neither protease is required for
cleavage of CwpV (data not shown).
All types of CwpV form a complex of the N-terminal and C-terminal
Post-translational cleavage of CwpV into two fragments is reminiscent of
post-translational cleavage of SlpA into the HMW and LMW SLP fragments. In the
case of SlpA these two fragments form a complex allowing the HMW SLP, containing
the putative cell wall binding motifs, to anchor the LMW SLP to the underlying
cell wall .
We therefore reasoned that CwpV cleavage may also be followed by complex
formation to allow the N-terminal domain, containing putative cell wall binding
motifs, to anchor the repetitive C-terminal fragment to the cell surface. To
test this hypothesis, the C-terminal repeat region from strain
ΔcwpV(pOEI) was purified by affinity chromatography
using a StrepTactin resin. As shown in Figure 7A, the 42 kDa and 116 kDa fragments
of CwpV were co-eluted despite the presence of the Strep-Tag II only in the
larger fragment. This experiment was then repeated with S-layer extracts from
ΔcwpV strains over-expressing CwpV types II–V.
The complete S-layer extracts (S) and first elutions (E) are visualized by
SDS-PAGE (Figure 7B). In all
cases, both CwpV fragments co-eluted, clearly demonstrating complex formation.
As this interaction is independent of repeat type, this strongly suggests that,
as in SlpA, it is the highly conserved regions adjacent to the cleavage site
that mediate complex formation.
The CwpV N-terminal fragment is responsible for cell wall anchoring
The role of the N-terminal fragment of CwpV containing putative cell binding
motifs was then investigated. A truncated form of the 630 cwpV
gene which lacks the whole repetitive sequence downstream of the serine/glycine
region was created as depicted in Figure 7C. The plasmid encoding this protein, pOE Nter, was
transferred to 630ΔcwpV to create
ΔcwpV(pOENter). S-layer extracts from wild-type,
ΔcwpV, ΔcwpV(pOE I) and
ΔcwpV(pOENter) cultures were analysed by SDS-PAGE
(Figure 7D). The ∼42
kDa N-terminal CwpV fragment is seen in all extracts other than
ΔcwpV and its identity was confirmed by Western blot
with anti-CwpVNter (Figure
7E). This suggests that the N-terminal fragment of CwpV alone is
sufficient for stable association with the cell wall. The same amount of the
N-terminal CwpV fragment is seen in the ΔcwpV(pOEI) and
ΔcwpV(pOENter) S-layer extracts, showing that the
truncated version of CwpV is equally well expressed on the cell surface as the
full-length CwpV protein. We therefore named the N-terminal fragment of CwpV as
the cell wall anchoring (CWA) fragment. This is the first reported experimental
evidence that PF04122 motifs are responsible for cell wall anchoring.
A model for post-translational CwpV processing
The determination of the CwpV cleavage site, evidence of complex formation by the
two CwpV cleavage products, and role of the N-terminal fragment in cell wall
anchoring leads to an overall model for post-translational CwpV processing. The
three steps involved are depicted in Figure 7F, and are compared to the steps
known to occur for SlpA processing. The first step is export of the protein
across the cell membrane and cleavage of the signal peptide. Once on the surface
the protein is cleaved (step 2) by Cwp84 in the case of SlpA, or by an unknown
mechanism in the case of CwpV. The third step is complex formation of the
cleavage products, allowing the HMW SLP or the CwpV CWA fragment to anchor the
complex to the cell surface. All three steps appear to be conserved amongst
different CwpV types.
CwpV is an aggregation-promoting factor
As noted previously, ΔrecV ON strains showed a smaller,
smoother-edged colony morphology than ΔrecV OFF or
wild-type strains (see Figure
1D). We therefore investigated this phenomenon in greater detail. As
shown in Figure 8A,
wild-type and ΔcwpV strains exhibit the same disperse,
ruffled colony morphology. Over-expression of the CwpV N-terminal CWA alone in
ΔcwpV did not alter this colony morphology. However,
over-expression of any of the five types of full-length CwpV caused the same
smaller, smoother-edged colony morphology seen in ΔrecV ON
mutants (Figures 1D and
8A). These results
strongly suggest that it is the repetitive C-terminal domain of CwpV that
mediates this phenotype. In order to investigate more closely the basis of this
difference, the edges of colonies grown between glass and agar were visualized
microscopically. Detail of the colony morphology at the cellular level can be
seen in the 63× magnification images shown alongside the 1× images
in Figure 8A. Wild-type,
ΔcwpV and ΔcwpV(pOENter) colonies
were seen to have diffuse edges, with directional protrusions of bacterial
growth common at the colony edge. However, ΔcwpV(pOEI-V)
exhibited denser, straighter colony boundaries, with rare if any directional
protrusions. The observed macroscopic differences in colony morphology are
therefore due to altered cellular organization within bacterial colonies.
This altered cellular organization may result from CwpV-mediated
auto-aggregation. Therefore the propensity of C. difficile to
aggregate from suspension was assessed across the panel of strains.
Over-expression of all CwpV types, except type IV, induced aggregation of
C. difficile from a liquid suspension with a starting
OD600 nm of 10 (Figure 8B). Aggregation was followed in real time, and could be
observed in tubes where aggregation was occurring from as soon as 1 hour after
suspension of bacteria (data not shown). It was found that only 630 CwpV (type
I) induced aggregation of C. difficile from suspensions with an
OD600 nm of 3, while 630 CwpV and R20352 CwpV (type II) induced
aggregation from an OD600 nm of 6 (data not shown). An OD600
nm as high as 40 did not lead to type IV-mediated aggregation, nor
aggregation of wild-type, ΔcwpV or
ΔcwpV(pOENter) (data not shown). Therefore it appears
that the ability to induce aggregation is highest for CwpV from 630, followed by
R20352, then CDKK167 and AY1, with no observed propensity of M9 CwpV or the CwpV
CWA fragment to induce aggregation of C. difficile from
We previously described the phase-variable expression of CwpV, employing a novel
transcriptional termination mechanism . Here we show that CwpV can
constitute a major cell wall protein and that it exhibits an auto-aggregation
promoting function. Surprisingly, this function is mediated by the highly variable
C-terminal repeat domain which we find in five entirely distinct sequence types.
Auto-aggregation was analyzed at both the cellular level on solid media and within
liquid culture. In vitro colonies are organized populations of
bacteria and differences in colony morphology reflect the cellular organization of a
that can translate to important differences affecting natural growth dynamics.
Differences in the cellular organization of colonies was clearly demonstrated by
microscopy (Figure 8A) with CwpV
expression in all cells causing a denser, more randomly packed cellular organization
than the diffuse, directional patterns of cellular packing seen at the edge of
wild-type colonies. The CwpV-mediated aggregation of C. difficile
cells from suspension strongly suggests that CwpV has a direct aggregation-promoting
function rather than a direct effect on motility. In fact the motility of strains in
soft agar was found to be similar, regardless of whether or not they expressed CwpV
S2 and Text S1).
Propensities of different CwpV types to cause aggregation varied, as observed by the
different starting densities of suspensions required for aggregation, apart from
type IV, which did not induce aggregation. This is likely to reflect different
affinities of the different CwpV proteins for their ligand(s). The lack of an
observable aggregation phenotype due to type IV repeats may be explained by a weaker
affinity between this repeat type and its ligand, however the observed colony
morphology phenotype does suggest that it carries out the same general function as
the other types. Functional interactions between CwpV and motility factors, such as
type IV pili and flagella, are worthy of further study. Indeed, the diffuse
directional organization of wild-type colony edges seen in this study is reminiscent
of twitching motility mediated by type IV pili in Clostridium
difficile does have type IV pili clusters, which are conserved across
Type IV pili may therefore be involved in the cellular organization of wild-type
colonies grown on solid surfaces observed here. Auto-aggregative proteins have been
shown to play roles in biofilm formation , , . No studies on C.
difficile biofilms have been reported, and in our experience C.
difficile does not exhibit significant biofilm formation on abiotic
surfaces (data not shown). However in infected mice large aggregates of C.
difficile cells, described as exaggerated mats, were reported to be
associated with regions of severe inflammation . This description is indicative
of a biofilm-like growth of C. difficile during infection.
Multispecies biofilms in the human GI tract have been observed  and C.
difficile is likely to require factors that promote appropriate intra-
and inter-species interactions to colonize the gut. CwpV may play important roles in
Aside from its aggregation-promoting function CwpV may have other functions. We
investigated bacterial adhesion to Caco-2 cells, but no significant difference in
adhesion was caused by CwpV expression (data not shown). Further work is required to
assess whether CwpV expression affects colonization levels in vivo,
and if so whether this is directly due to its aggregation-promoting activity.
Understanding the roles of CwpV during in vivo infection is a
priority and will be addressed using the isogenic mutants described in this study.
Established animal models of infection focus more on the acute stage of C.
difficile infection, rather than colonization . However, recent developments of
animal models better suited to studying the roles of C. difficile
genes likely to be involved in colonization, such as CwpV, have been described ,  that will
facilitate our future investigation. The phase-variable expression of CwpV may be
highly relevant to any in vivo role; its expression in a sub-set of
cells as a contingency may be beneficial to the survival of the population as a
The variation of CwpV repeat sequences uncovered in this study is extensive, with
strains expressing one or two of five antigenically distinct types encoded by
unrelated sequences. The lack of sequence homology between repeat types suggests
acquisition by horizontal gene transfer. Any known mechanism of chromosomal sequence
acquisition; homologous recombination, illegitimate recombination or additive
integration could account for introduction of these sequences to the C.
difficile chromosome . However, without knowing the origin of these sequences,
the mechanisms of transfer to C. difficile and incorporation into
the genome are unclear. Human microbiome sequencing may shed light on the origins of
CwpV sequences. It would seem that CwpV type exchange is occurring at a low
frequency, as all strains tested from within one ribotype contain the same CwpV
type. Current understanding of the phylogenetics and diversity of C.
difficile strains is limited , . However current data suggests a
single clade is associated with one CwpV type; the HY (027) clade associates with
type II CwpV, the A-B+ clade with type IV CwpV, the HA2 (078) and HA1 clades
with type I CwpV. The locations of strains expressing type III and V CwpV within
this phylogenetic tree of C. difficile are not yet known. As more
C. difficile strains are sequenced and our understanding of
evolutionary relationships between strains improves, it will become clearer as to
when different CwpV types were acquired, and whether single or multiple independent
acquisition events for each type have occurred. Host immune pressure is one possible
selection that could promote CwpV variability, as one key difference between CwpV
types is their antigenicity. Antigenic variability is also seen in the C.
difficile surface proteins SlpA and Cwp66 . Variation in surface proteins
may also be involved in competition between different C. difficile
strains or in resistance to bacteriophages.
Apart from the marked diversity in CwpV repeat sequences, other aspects of CwpV are
remarkably conserved. Expression of all types of CwpV is phase variable suggesting
that this complex regulatory mechanism, involving the conserved
cwpV switch and the RecV recombinase, is important for optimal
function of CwpV. Phase variation of bacterial surface proteins has been reported to
be involved in numerous processes including immune evasion and colonization . Given the
sequence variability and aggregation-promoting function of CwpV, several
explanations behind its phase variable regulation can be suggested. However,
investigation into CwpV expression during in vivo infection is
likely to shed most light on the importance of CwpV phase variability.
Conserved post-translational processing of CwpV and formation of a CWA-repeat domain
complex again suggests that these processes are important for optimal CwpV
expression and function. To date we only observe complex formation in cell wall
proteins CwpV and SlpA, with all other CWPs studied (e.g. Cwp84, Cwp66, Cwp2) being
presented on the surface without post-translational cleavage between the CWA and
functional domains. An unidentified protease could mediate cleavage of CwpV, or it
could undergo non-enzymatic auto-processing as reported for a number of protein
Uncleaved SlpA is poorly tolerated on the cell wall, and is found in large
quantities in the culture supernatant of a Δcwp84 mutant . SlpA monomers
once cleaved must interact on the cell surface to form a two-dimensional array.
Expression of CwpV does not appear to disrupt the integrity of the S-layer and, by
analogy, it seems likely that cleavage of CwpV is important for its stable
incorporation into the S-layer. Given the high proportion of the total S-layer
accounted for by CwpV in ΔrecV ON strains, it seems likely that
CwpV interacts positively with other S-layer proteins to maintain the integrity of
Our finding that the CwpV CWA domain is responsible for anchoring of the CwpV
C-terminal domain to the underlying cell wall is the first direct evidence of the
function of Pf04122 domains. These domains are also present in the HMW SLP of the
C. difficile S-layer where the presumably mediate a similar
role. They are also found in a large variety of Gram-positive bacteria and some
Archaea, where they presumably fulfill a similar function of anchoring cell wall
proteins to the underlying structure. This then adds to the diversity of mechanisms
used by Gram-positive bacteria to anchor and display cell wall proteins , .
Phylogenetic studies  show that outbreak-causing strains of C.
difficile have emerged independently from multiple lineages, indicating
that virulent strains can emerge from across the diversity of the species, rather
than being confined to a specific pathogenic lineage. This suggests that certain
genetic elements common to all C. difficile strains underlie
virulence. It is likely that the incidences of different lineages causing disease
are dynamic, and we cannot predict which lineages may cause future outbreaks.
Therefore understanding the common features of C. difficile as an
ancient species is important, as in order for intervention strategies to be
successful in the long-term they must target the full diversity of C.
difficile strains, rather than specific lineages. CwpV appears to be a
core component of C. difficile, including its phase variable
regulation controlled by RecV, post-translational processing mechanism, and
aggregation-promoting function. This highlights the importance of CwpV to C.
difficile survival throughout evolution of this diverse species.
Despite this conservation CwpV appears to have been under positive selection for
antigenic variability. Further investigation into the roles of CwpV in the
C. difficile life cycle may teach us a lot about what makes
C. difficile a successful pathogen, and provide opportunities
for new intervention strategies.
Materials and Methods
Bacterial strains and growth conditions
The C. difficile strains characterised for CwpV type in this
study are described in Table
1. C. difficile laboratory-generated mutant and
recombinant-plasmid containing strains used in this study are described in Table S3.
C. difficile was routinely cultured on blood agar base II
(Oxoid) supplemented with 7% horse blood (TCS Biosciences), brain-heart
infusion (BHI) agar (Oxoid) or in BHI broth (Oxoid). Cultures were grown in an
anaerobic cabinet (Don Whitley Scientific) at 37°C in an atmosphere of
10% CO2, 10% H2 and 80%
N2. C. difficile strains containing recombinant
plasmids were grown under thiamphenicol selection. Commercial chemically
competent TOP10 E. coli cells (Invitrogen) were used for
cloning and recombinant plasmid maintenance. Recombinant expression of CwpV
fragments was carried out in Rosetta E. coli cells (Novagen).
E. coli strains were routinely grown at 37°C on LB-agar
plates or in LB liquid culture in the presence of selective antibiotics where
appropriate to any transformed plasmid.
DNA manipulations were carried out according to standard techniques. C.
difficile genomic DNA for use in cloning and PCR analysis was
prepared as described previously . Polymerase chain
reactions (PCR) used KOD (Novagen), Expand Long-Template Polymerase (Roche
Diagnostics) or Taq polymerase (Sigma) in accordance with the
manufacturers' protocols using primers detailed in Table
Production of recombinant plasmids
In order to produce a C. difficile 630Δerm
knock out of recV, a retargeted ClosTron vector was designed
using the intron design tool www.clostron.com. The
top-scored target site in recV was selected (424/425s) and the
intron targeting region required was commercially synthesized and cloned into
pMTL007C-E2 (DNA 2.0) to generate a plasmid pLRP028. For complementation of the
ΔrecV mutant, the recV gene from
C. difficile 630 was amplified using primers NF1357 and
NF1358 and cloned using SacI and BamHI into a
C. difficile plasmid expression vector, based on the
pMTL960 replicon ,  to generate pCBR113 (pRecV+). Inverse PCR with
primers NF1411 and NF1412, using pCBR113 as the template, was carried out to
mutate the recV tyrosine176 to phenylalanine, producing pCBR115
(pRecVY176F+). To express CwpV fragments for use as antigens
cwpV repeats from strains R20352 (type II), CDKK167 (type
III), M9 (type IV) and AY1 (type V) were amplified by PCR using KOD polymerase
(Novagen/Merck Chemicals Ltd) and primers described in Table S1.
These fragments were directionally cloned using NcoI and
XhoI into pET28a E. coli expression vector
then transformed into TOP10 E. coli (Invitrogen) to generate
plasmids pCBR069-072 described in Table S2. To clone the cwpV
genes from strains of unknown genomic sequence, genomic DNA was digested
overnight at 37°C with AseI (NEB) and then ligated
overnight at 16°C using T4 ligase (NEB). Inverse PCR using primers NF401 and
NF654 was carried out and amplification products were cloned into pCR4-TOPO
(Invitrogen). pCBR044, containing the full-length cwpV gene
from 630 under the control of the cwp2 promoter within the
pMTL960 backbone , was modified to contain a 5′ XhoI site and
strep-tag II (WSHPQFEK) encoding sequence by inverse PCR, generating the
C. difficile strep-tagged 630 CwpV expression plasmid
pCBR080 (pOEI). The cwpV genes from R20352, CDKK167, M9 and AY1
were amplified with the primers described in Table S1
and cloned in place of the 630 gene in pCBR080 to create plasmids pCBR105-107
and 109 (pOEII-V) (Table S2). Successful construction of
recombinant plasmids was always confirmed by DNA sequencing (GATC Biotech).
Conjugation into C. difficile
Plasmids were transformed into E. coli CA434 and then conjugated
into C. difficile as described previously  using thiamphenicol (30 mg
ml−1) to select for pMTL960-based plasmids and cycloserine
(250 mg ml−1) to counter-select for E.
Purification of recombinant CwpV protein and generation of antibodies
Plasmids pCBR069-072 expressing repeat types II–V were transformed into
E. coli Rosetta (Novagen/Merck Chemicals Ltd) and grown
overnight at 37°C in Overnight Express media (Novagen/Merck Chemicals Ltd).
Bacteria were collected, lysed using BugBuster (Novagen/Merck Chemicals Ltd) and
the His-tagged fusion proteins purified by affinity chromatography using Ni-NTA
agarose (Qiagen). Following purification, proteins were dialysed into 10 mM
HEPES 150 mM NaCl. Antisera to the proteins were generated in rabbits using a
commercial service (Covalab, Cambridge, UK). Anti-CwpVNter and anti-CwpVrptI
were described previously .
S-layer extraction, SDS-PAGE and western immunoblotting
C. difficile cultures were grown overnight, harvested by
centrifugation and S-layer extracts were prepared using low pH glycine
incubation as described previously . Proteins in the S-layer
extracts were subjected to SDS-PAGE and western blotting according to standard
protocols. All rabbit primary anti-CwpV antibodies (anti-CwpVNter,
anti-CwpVrptI, anti-CwpVrptII, anti-CwpVrptIII, anti-CwpVrptIV and anti-
CwpVrptV) were used at 1/5000 dilution, followed by anti-rabbit-HRP (Dako
Cytomation) at 1/2000 dilution. Signal was detected using SuperSignal® West
Pico Chemiluminescent Substrate (Pierce).
C. difficile cells from liquid culture were washed with PBS then
fixed in 8% formaldehyde, which was quenched with 20 mM NH4Cl
for 15 min prior to staining. Washed cell suspensions were re-suspended in 40
µl 1% BSA in PBS containing 1/20 dilutions of rabbit primary
antibodies (anti-CwpVNter, anti-CwpVrptI, anti-CwpVrptII, anti-CwpVrptIII,
anti-CwpVrptIV and anti- CwpVrptV) for 45 min. Bacteria were then washed and
incubated with 1/40 anti-rabbit-rhodamine red-X (Jackson ImmunoResearch) in the
dark for 45 min. Bacteria were washed with PBS and dH20 then allowed
to air dry onto a microscope slide. Coverslips were mounted using ProLong gold
anti-fade mounting reagent (Invitrogen) and allowed to set overnight. Bacteria
were visualised using a Nikon Eclipse E600 microscope fitted with a Nikon
S-layer extracts were separated on 10% SDS-PAGE gels and transferred to a
PVDF membrane. The membrane was washed in water for 10 min, stained in
0.1% Coomassie Blue R in 50% MeOH for 5 min. After destaining in
50% MeOH, 10% acetic acid (3×5 min with rocking) and washing
in water (3×5 min with rocking) the membrane was allowed to dry
thoroughly. N-terminal sequencing by Edman degradation was then carried (PNAC
Facility, Department of Biochemistry, University of Cambridge).
C. difficile S-layer extract streptactin binding/elution
StrepTactin resin (Novagen) was used according to
manufacturers' instructions. Briefly, resin was washed with
dH2O, equilibrated with wash buffer (150 mM NaCl, 100 mM Tris-HCl, 1
mM EDTA, pH 8.0) and mixed with S-layer extract. The mixture was incubated
overnight at 4°C with rotation. The next day the resin was washed with wash
buffer to remove non-specifically bound proteins. Elution buffer (150 mM NaCl,
100 mM Tris-HCl, 1 mM EDTA, 2.5 mM desthiobiotin, pH 8.0) was then used to elute
strep-tagged proteins. All fractions were collected for analysis using SDS-PAGE
and Coomassie staining.
Visualization of C. difficile colony morphology
For macroscopic visualization of C. difficile colony morphology
strains were grown on BHI agar (Oxoid) for 5 days from an appropriate C.
difficile inoculum such that isolated colonies grew on the plate.
Plates were then scanned using a flat-bed scanner to record images of the
colonies. For microscopic visualization of C. difficile colony
morphology, cultures taken from a 24-hour old colony were inoculated directly on
to glass-bottomed dishes (MatTek corporation). Anaerobic BHI 0.7% agar at
40°C was then carefully poured over the glass to cover the dish and allowed
to set. C. difficile was grown between the glass and the agar
for 3 days. Dishes were removed and images taken using a Zeiss Axiovert 200
widefield microscope at FILM, Imperial College London. Microscope images were
analysed using Volocity 5.3.2 software (Improvision).