Department of Bacteriology, Swedish Institute for Infectious Disease Control, S-17182, Solna, Sweden1
Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm, Sweden2
AstraZeneca Research Center India, PO Box 359 Malleswaram, 560 003 Bangalore, India3
Author for correspondence: Thomas kerlund. Tel: +46 8 4572467. Fax: +46 8 301797. e-mail: Thomas.Akerlund{at}smi.ki.se
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ABSTRACT |
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Keywords: extracellular proteins, outer membrane efflux proteins, PBSX prophage, S-layer
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INTRODUCTION |
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The tcdA (toxA) and the tcdB (toxB) genes are part of a 19·6 kb pathogenicity locus (PaLoc, toxigenic element), which comprises five ORFs (Braun et al., 1996 ; von Eichel-Streiber et al., 1992
; Hammond & Johnson, 1995
). Several mRNAs are transcribed from the toxigenic element, including a 17·5 kb polycistronic transcript (Hammond et al., 1997
; Hundsberger et al., 1997
). One of the genes, tcdD (txeR), encodes a 22 kDa protein necessary for transcription of the toxin genes (Moncrief et al., 1997
) and was recently shown to act as an alternative sigma factor (Mani & Dupuy, 2001
).
The toxin yield or activity may differ up to 106-fold between toxin-positive strains (Lyerly & Wilkins, 1986 ). The toxins are expressed mainly during the late exponential growth phase and the stationary phase (Kamiya et al., 1992
; Ketley et al., 1986
), and limiting nutrient levels (e.g. glucose, amino acids, biotin) lead to up-regulation of toxin expression (Dupuy & Sonenshein, 1998
; Haslam et al., 1986
; Karlsson et al., 1999
, 2000
; Yamakawa et al., 1994
, 1996
. Toxin export occurs by an unknown mechanism during the stationary phase (Ketley et al., 1984
), and may be affected by the oxidationreduction potential of the medium, heat shock, and certain antibiotics (Onderdonk et al., 1979
). The toxin export mechanism remains obscure, but due to their large size and lack of secretion signal peptides, one hypothesis is that the toxins are externalized by bacterial lysis. The aim of this study was to investigate the toxin export kinetics and to identify other extracellular proteins and putative virulence factors in the culture supernatant of C. difficile. Bacterial lysis could not explain release of toxins in the high-toxin-producing strain C. difficile VPI 10463. Five other extracellular proteins were identified and characterized at the protein and the genomic levels.
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METHODS |
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Sample preparation and membrane fractionation.
Culture samples were centrifuged at 16000 g for 3 min; the resulting supernatants were removed and stored at -20 °C for later analysis. The cell pellet was dissolved in 1 ml sterile water and sonicated on ice for 3x30 s at 100 W (Labsonic 1510, B. Braun). The cell extracts were centrifuged at 5000 g for 5 min, giving a low-speed pellet (LSP). The supernatant was removed and further centrifuged at 50000 g for 20 min, resulting in a high-speed pellet (HSP) and a soluble fraction. The LSP and the HSP were resuspended in PBS and all samples were stored at -20 °C. Protein was measured using a kit (Bio-Rad) and a BSA standard curve according to the manufacturers instructions. Proteins in culture supernatants were precipitated in 10% trichloroacetic acid.
SDS-PAGE and immunoprecipitation.
Proteins were analysed by SDS-PAGE (ExcelGel 818%, Amersham Biosciences) on a Multiphor II horizontal slab gel apparatus and stained with silver. The gels were digitized by scanning (Scanjet 3c/T, Hewlett Packard) and analysed using the Totallab software (Amersham Biosciences). Immunoprecipitation was performed in microtitre wells coated with antibodies against toxin A (PCG-4, r-Biopharm) or toxin B (2CV, r-Biopharm). Antibody (10 µg ml-1 in 40 mM Na2CO3, 60 mM NaHCO3, pH 9·6) was coated in microtitre wells by incubation for 1 h at 37 °C. The wells were washed four times with PBS containing 0·05% (v/v) Tween 20, pH 7·4. The wells were loaded with cell extract, culture supernatant medium or PBS (negative control), incubated for 1·5 h at 25 °C, and washed four times with PBS. Fifty microlitres of SDS sample buffer was added to each well and the samples were heated to 95 °C for 5 min prior to analysis by SDS-PAGE.
Two-dimensional (2-D) PAGE.
For 2-D PAGE, 40 µl aliquots of each protein sample (see above) were mixed with 160 µl buffer [9·9 M urea, 4% (v/v) Igepal CA630, 2·2% (v/v) Pharmalytes 310, 100 mM DTT, 2% (w/v) CHAPS]. Proteins were focused at 20 °C on 180 mm IPG Drystrip pH 47 (Amersham Biosciences). The second dimension was run on 12% SDS-PAGE and proteins were stained with silver. PDQuest (Bio-Rad) were used to quantify proteins. The chemicals were obtained from Sigma except for Pharmalytes (Amersham Biosciences). Proteins were transferred to PVDF membranes, stained with Coomassie brilliant blue, and spots of interest were excised and N-terminally sequenced.
PCR amplification and restriction cleavage of slpA.
The primers used in the detection of the surface layer gene slpA were 5'-TATAATGTTGGGAGGAATTTAAGA-3' and 5'-CAAATCCAAATTCACTATTTGTAC-3'. The PCR was performed using Expand Long Template PCR System from Boehringer Mannheim (as specified by the manufacturer). After an initial denaturation at 92 °C for 2 min, the PCR cycle conditions were 92 °C for 10 s, 40 °C for 30 s, and 68 °C for 2 min (30 cycles). The PCR products were cleaved with the restriction enzymes RsaI (Boehringer Mannheim) and SauIIIA (Amersham Pharmacia Biotech). PCR and digestion products were separated on 0·8% agarose gels and visualized by ethidium bromide.
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RESULTS |
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The PY-specific extracellular proteins 5 and 6 (39 and 30 kDa, respectively, Table 1) matched with two ORFs encoding proteins of 38 and 22 kDa, respectively, showing high similarity to FixB and FixA in Escherichia coli. The amounts of these were estimated to be <1% of those in the intracellular fraction (compare Fig. 2a
with 2d
, spots 5 and 6) and their presence in the extracellular fraction may thus represent a small fraction of the bacterial population being lysed. Importantly, <1% lysed cells cannot explain the efficient release of toxins from strain VPI 10463. The amounts of extracellular FixA and FixB were higher in strain 630 (Fig. 2e
), confirming that this strain was more prone to lyse.
Identification of the C. difficile surface layer proteins
The two most abundant extracellular proteins in strain VPI 10463 were present in both PY and PYG culture supernatants (Fig. 2a and 2b
, spot 1 [50 kDa] and 2 [36 kDa]) and these were highly abundant in the high-speed pellet fraction, which contained mainly membrane vesicles, cell wall fragments and large protein complexes (compare Fig. 2c
with Fig. 2a
). In strain 630, two abundant proteins with similar molecular masses (48 and 35 kDa) and different pI values than those of strain VPI 10463 were found in the extracellular (Fig. 2e
, spots 10 and 11) and particularly in the pellet fraction (Fig. 2f
).
The N-terminal sequence of the 50 kDa protein (spot 1) from strain VPI 10463 did not show homology to any protein in the C. difficile strain 630 genome database whereas the N-terminus of the 36 kDa protein (spot 2) showed partial similarity to the 72 kDa C. difficile S-layer protein SlpA (Fig. 3c, Table 1
). Spots 7 and 8 found in PYG cultures (Fig. 2b
) matched with ORF 2 from the same genomic fragment and spot 9 matched with SlpA (Table 1
). The N-terminal sequences of spots 10 and 11 from strain 630 both matched with the SlpA ORF (Table 1
). At least six other proteins less abundant than spots 1 and 2 were observed in both PY and PYG culture supernatants of strain VPI 10463 but were not further characterized. These results imply an extracellular or surface-located protelytic activity mainly involved in processing of the C. difficile S-layer protein.
The C. difficile surface layer gene shows inter-strain diversity
As reported by Calabi et al. (2001) and Karjalainen et al. (2001)
we found that the gene segment containing the C. difficile S-layer gene slpA comprised secA and several additional genes with high similarity to slpA. The amino acid sequences of SlpA and the SlpA-like ORFs were characterized by a constant part showing significant homology to N-acetylmuramoyl-L-alanine amidase (CwlB/LytC) and amidase enhancer protein (LytB) from B. subtilis (Table 1
, Fig. 3c
). The N-terminus of all the N-acetylmuramoyl-L-alanine amidase-like ORFs contained a typical Sec-dependent signal peptide, and the predicted cleavage site in SlpA from strain 630 was identical to that found in the extracellular form. However, no typical secondary protease processing site was found that would allow the further cleavage of the expected 72 kDa SlpA protein to give the finally sizedS-layer proteins of apparent molecular masses 48 and 35 kDa in strain 630.
To test whether other C. difficile strains contained the slpA gene, a PCR with primers directed to the region upstream of slpA and to the 5' part of secA was applied to 18 serogroup type strains. All strains except those belonging to serogroups A, A5 and S4 yielded one major PCR product of approximately 2900 bp, although that of serogroup H was slightly larger (Fig. 5a). Thus the upstream region of slpA and secA and the size of the intervening DNA appeared to be conserved in most strains of C. difficile. Digestion of the PCR fragments using the restriction enzymes SauIIIA and RsaI revealed that each type strain yielded a distinct and unique banding pattern (Fig. 5b
, c
). The pattern of strain 630 and VPI 10463 was identical to that of the serogroup C and G type strains, respectively. In summary, these results showed that the C. difficile S-layer genes contain both constant and variable domains and the latter may be useful for grouping and typing of various C. difficile isolates.
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DISCUSSION |
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As compared to B. subtilis (Antelmann et al., 2001 ) the repertoire of extracellular proteins in C. difficile was small (about 15 vs 200 proteins), possibly reflecting the different ecological niches of the organisms. The 47 kDa extracellular C. difficile protein found during high toxin production (PY cultures), was identified as an ORF with weak similarity to outer-membrane efflux proteins (OEPs) of Gram-negative bacteria, e.g. TolC (Fig. 4a
). This ORF contained a putative signal peptide for export via the Sec-dependent pathway (Economou, 1999
; Izard & Kendall, 1994
). The extracellular form was truncated downstream of the predicted signal peptide cleavage site, suggesting that its release was a result of further proteolytic digestion. The two adjacent ORFs encoded proteins similar to cation/multidrug resistance efflux pumps of the AcrB/D/F family and periplasmic membrane fusion proteins of the HlyD family (Fig. 4a
). These proteins, including TolC, are thought to form a continuous channel that traverses both the inner and the outer membrane in Gram-negative bacteria, and enable direct transport of molecules to the extracellular compartment via proton-motive force (Paulsen et al., 1996
; Zgurskaya & Nikaido, 2000
). A similar architecture is found also in the type I secretion system which transports toxins in some Gram-negative bacteria, but the multidrug efflux pump protein is replaced by an ATPase. The three genes found in C. difficile may thus constitute a novel type of transporter system in Gram-positive bacteria that traverses both the cell membrane and the cell wall. The PY-specific XkdK-like protein neither contained a typical signal peptide, nor was processed at its N-terminus. Its gene was found in a segment on the C. difficile chromosome containing several genes similar to those encoded by the phage-like element PBSX in B. subtilis (Krogh et al., 1996
). B. subtilis also releases PBSX proteins including XkdK into the culture supernatant during growth in LB medium, most likely via phage-specific holins (Antelmann et al., 2001
). Interestingly, the tcdE gene located between the genes for toxin A and B in the pathogenicity locus of C. difficile encodes a putative holin (Tan et al., 2000
), suggesting that the toxins and the XkdK homologue may be secreted via similar mechanisms.
The S-layer of C. difficile is composed of two proteins with apparent molecular masses of 3236 and 4547 kDa and has been characterized previously at the genomic, protein and structural levels (Calabi et al., 2001 ; Cerquetti et al., 2000
; Karjalainen et al., 2001
; Mauri et al., 1999
; Takeoka et al., 1991
; Waligora et al., 2001
). The two highly abundant extracellular and membrane fraction proteins in strain 630 were identical to the S-layer protein SlpA-630/C253 (Karjalainen et al., 2001
). The differences in size and particularly pI between the S-layer proteins of strains 630 and VPI 10643 as shown by 2D-PAGE indicate significant sequence variation of the S-layer protein among different strains. A significant variation in pI of these proteins was also observed for other serogroup type strains (unpublished). The restriction cleavage pattern of PCR products from the slpAsecA region of various serogroup reference strains verified this sequence variability and also that strains 630 and VPI 10463 showed patterns identical to reference strains representing those of serogroup C and G, respectively. Using PCR ribotyping (Stubbs et al., 1999
), we found strain 630 to be of PCR ribotype 12, whose reference strain again belongs to serogroup C. Strain 630 has however been assigned to serogroup X (cf. http://www.sanger.ac.uk/Projects/C_difficile/), a result that is not in agreement with these data. This indicates that serotyping may give ambiguous results (or that strain 630 has been assigned to the wrong serogroup), or that the strain has switched its slpA gene. Strain VPI 10463 clustered by PCR ribotyping close to a group belonging to serogroup G (unpublished). The PCR ribotype of VPI 10463 is not determined according to the nomenclature of Stubbs et al. (1999)
but the N-terminal sequences of its S-layer proteins were identical to those of a strain belonging to PCR ribotype 1 (Calabi et al., 2001
), whose reference strain belongs to serogroup G. These data show that S-layer genomic typing methods should be developed for improved molecular typing of C. difficile strains.
The C-terminal part of SlpA showed significant homology to N-acetylmuramoyl-L-alanine amidase (CwlB/LytC) and amidase enhancer protein (LytB) from B. subtilis (Lazarevic et al., 1992 ), which confirmed recent findings (Calabi et al., 2001
; Karjalainen et al., 2001
). This motif may thus have cell wall peptidoglycan binding properties. Karjalainen et al. (2001)
suggested that the N-terminal part of C. difficile SlpA has homology to the SLH domain present in many S-layer proteins (cf. Lupas et al., 1994
), but like Calabi et al. (2001)
we did not find such similarity at the sequence level. It is possible, however, that this part of the C. difficile S-layer protein shows weak similarities to that of the SLH domain.
Considering the highly competitive situation of closely related organisms in their natural habitats, it is obvious that the S-layer has to contribute to diversification rather than to conservation. With respect to this, the importance of S-layer variation during different stress conditions such as those imposed by the immune system of a host in response to an S-layered pathogen or drastic changes in the growth and environmental conditions for non-pathogens is conceivable (Dworkin & Blaser, 1997 ; Luckevich & Beveridge, 1989
; Sara et al., 1996
). A protein named Cwp66 encoded in the same chromosomal segment as SlpA was shown to have putative adhesive properties to eukaryotic cells (Waligora et al., 2001
). Moreover, it was shown by RT-PCR that most ORFs in the S-layer gene segment were simultaneously transcribed (Calabi et al., 2001
), although the protein yield (as found here) may differ significantly between the ORFs. Whether the different S-layer ORFs in this chromosomal segment have similar or different functions and are under common or different regulatory control is yet to be verified. An interesting question is whether these genes are important for disease development and whether changes of organization or expression of the S-layer genes occur in vivo.
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ACKNOWLEDGEMENTS |
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Received 11 March 2002;
accepted 8 April 2002.