1 Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway
2 School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK
Correspondence
Per Einar Granum
per.e.granum{at}veths.no
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ABSTRACT |
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Present address: Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
Present address: The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
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INTRODUCTION |
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The amino acid sequence of CytK is similar (about 30 % identity) to that of -haemolysin (
-toxin,
-HL), leucocidins and
-haemolysin of Staphylococcus aureus, and
-toxin of Clostridium perfringens, which all belong to the family of
-barrel pore-forming toxins (Song et al., 1996
; Prevost, 1999
; Steinthorsdottir et al., 2000
). The capacity to form pores in planar bilayers (Hardy et al., 2001
) is consistent with CytK being a member of this family of proteins with pore-forming ability.
Several proteins have been described as putative virulence factors in B. cereus. The extent to which they may belong to a family of pore-forming proteins is of interest, as many have been shown to possess haemolytic or cytotoxic activity. Beecher et al. (2000) partially characterized a novel haemolysin designated haemolysin IV. This toxin appeared to be one of the most rapidly acting or one of the most abundant haemolysins in crude culture supernatants from many B. cereus strains. Amino acid sequencing showed that 28 out of 30 amino acids in the N-terminal region were identical to those of CytK (Beecher et al., 2000
; Lund et al., 2000
).
A haemolysin of B. cereus designated haemolysin II (H-II) was first characterized by Coolbaugh & Williams (1978), although a similar haemolysin had been mentioned as early as 1963 (Fossum, 1963
). A similar haemolysin has also been isolated from Bacillus thuringiensis (Pendleton et al., 1973
). A genetic determinant of B. cereus, hly-II, supposedly encoding haemolysin II, has been cloned and sequenced (Sinev et al., 1993
; Baida et al., 1999
). hly-II was, however, more characteristic of B. thuringiensis than B. cereus (Budarina et al., 1994
). The deduced amino acid sequence of hly-II (mature protein 42·3 kDa) showed similarity to known
-barrel pore-forming toxins of S. aureus (Baida et al., 1999
).
CytK of B. cereus is probably an important virulence factor in many B. cereus strains. We have therefore studied the occurrence of this toxin in different strains, and have identified a new cytK variant, which we designate as cytK-2, referring to the original cytK from B. cereus 391-98 as cytK-1. The haemolytic and cytotoxic activity of the CytK-2 proteins, and their pore-forming ability, were determined and compared with those of CytK-1. The relationship and relative importance of CytK-1, CytK-2, and haemolysin II as virulence factors are discussed.
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METHODS |
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PCR.
PCR was carried out in an MJ Research Minicycler or an Eppendorf Mastercycler Gradient thermal cycler. The primers used are listed in Table 2. Depending on the primers, the PCR programme used was: 95 °C for 1 min, 30 cycles of 95 °C for 1 min, 4551 °C for 1 min and 72 °C for 1 min, and finally 72 °C for 7 min. PfuTurbo DNA Polymerase (Stratagene) was used for construction of pMS20 and cloning of cytK-1 and cytK-2 (from strain 1230-88) genes in pMS20, and DyNAzyme II DNA polymerase (Finnzymes) was used for all other PCRs.
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Sequence similarity search.
A database search for similar protein sequences was carried out using the BLAST algorithm (Altschul et al., 1997). The deduced amino acid sequences were compared with the nonredundant protein databases, including GenBank CDS translations, PDB, SWISS-PROT, SPupdate and PIR. The values for percentage identity of DNA and amino acid sequences were obtained using the SmithWaterman algorithm for local alignments (Smith & Waterman, 1981
).
Culture medium and growth conditions.
CGY culture medium containing 0·4 % glucose was used for production of enterotoxins in B. cereus (Beecher & Wong, 1994). The cultures were grown at 32 °C for 6 h; 1 mM EDTA was added at the time of harvest. Extracellular proteins were separated from the cells by centrifugation (10 000 g at 4 °C for 20 min). The supernatant proteins were precipitated with 70 % saturated (NH4)2SO4.
Purification of proteins.
CytK-2 proteins from B. cereus strains 23, FM-1 and 1230-88 were isolated essentially as described by Lund & Granum (1996), including chromatography on a DEAE-Sephacel column (Amersham Biosciences) with Bistris/HCl buffer at pH 5·9, and chromatography on a column of Gel HT hydroxylapatite (HA) (Bio-Rad) with sodium phosphate buffer at pH 6·8. The last purification step was chromatography on a 1 ml Resource Q (ReQ) column (Amersham Biosciences) with 20 mM triethanolamine buffer at pH 8·1 and a linear 40 ml NaCl gradient from 0 to 0·2 M. The concentration of purified proteins was calculated from A280 measurements (Beckman DU 650, path length 1 cm).
Protein sequencing.
Purified protein was sequenced from the N-terminus by Edman degradation using an Applied Biosystems 477 A automatic sequence analyser with an on-line 120 A phenylthiohydantoin amino acid analyser.
Electrophoresis.
SDS-PAGE was carried out using a Bio-Rad Mini-Protean II Dual Slab Cell. The gels (12 % acrylamide) were silver stained according to Blum et al. (1987) and the molecular mass of the CytK-2 protein was estimated using the Bio-Rad Low Range SDS-PAGE standard.
Haemolysis assay.
The CytK toxins were twofold serially diluted in PBS (pH 7·4), containing 1 mg BSA ml1. The diluted toxin was added to an equal volume of 2 % bovine red blood cells diluted in PBS, pH 7·4, and incubated for 1 h at room temperature. At the end of the incubation, the samples were centrifuged and the A545 of the supernatants was recorded (Beckman DU 650, path length 1 cm) to measure the release of haemoglobin. PBS was used as the blank.
Caco-2 cell and Vero cell assays.
The human colon cancer cell line Caco-2 (Pinto et al., 1983) was cultivated in RPMI 1640 plus fetal calf serum, gentamicin and L-glutamine. Vero cells (derived from monkey kidney) were cultivated in MEM plus fetal calf serum, penicillin and streptomycin. The cells were grown in 24-well plates until confluence. Toxicity was determined using the inhibition of protein synthesis according to Sandvig & Olsnes (1982)
.
Expression of cytK-1 and cytK-2 in E. coli.
The kanamycin-resistance gene from pUC4K (Amersham Biosciences) was cloned into the SacI/NcoI site of pGK12 (Kok et al., 1984). The strong, constitutive promoter p32 from plasmid pMG36e (van de Guchte et al., 1989
) was cloned into the KpnI/MluI site of the resulting plasmid, and the final construct was designated pMS20. Primers with MluI restriction sites incorporated in their 5' termini (Table 2
) were used to PCR-amplify cytK-1 from B. cereus 391-98 (primers 391F and 391R) and cytK-2 from B. cereus 1230-88 (primers 1230F and 1230R). The PCR fragments were cloned into the MluI site of pMS20. The inserts were confirmed to be correct by DNA sequencing.
Preparation of concentrated supernatants and periplasmic extracts.
Overnight cultures of E. coli XL10-Gold (Stratagene) containing the vector constructs pMS20-cytK-1, pMS20-cytK-2 and pMS20 were diluted 1 : 100 in 400 ml BHI (brain heart infusion) medium containing 50 µg kanamycin ml1, and cultured at 200 r.p.m. and 37 °C until an OD600 of 0·60·7 was reached (Shimadzu UV-160A, path length 1 cm). Then 10 mM Tris/HCl (pH 7·3) and 10 mM NaCl was added to the cultures, followed by incubation for a further 10 min. The cultures were centrifuged at 6000 g for 20 min. The supernatants were concentrated by precipitation with 70 % saturated (NH4)2SO4, followed by centrifugation at 10 000 g and 4 °C for 20 min and dialysis against 25 mM Bistris (pH 5·9) with 1 mM EDTA at 4 °C overnight. Periplasmic extracts were prepared from the bacterial cell pellets, using the following procedure. The pellets were resuspended in 10 ml 33 mM Tris/HCl pH 7·3; 10 ml freshly prepared 33 mM Tris/HCl (pH 7·3) with 40 % sucrose and 4 mM EDTA was added. The cells were gently mixed for 10 min at room temperature, followed by centrifugation at 9000 g for 15 min. The pellets were gently resuspended in 2 ml ice-cold distilled H2O. Ice-cold MgSO4 was added to a concentration of 5 mM, followed by incubation on ice for 10 min. The samples were centrifuged at 16 000 g for 15 min, and the supernatants (periplasmic extracts) were collected and stored on ice.
Planar lipid bilayer recording.
Planar lipid bilayer recordings were carried out using a system described by Williams (1995). Briefly, lipid bilayers were formed from a dispersion of 15 mg palmitoyloleoylphosphatidylethanolamine ml1 and 15 mg palmitoyloleoylphosphatidylserine ml1 in n-decane, which was drawn across a 0·4 mm diameter hole in a polystyrene cup separating two solution-filled chambers, designated cis and trans. The cis chamber (to which the toxin was added) was held at ground (0 mV), and the trans chamber was clamped to a range of potentials (GeneClamp 500 patch-clamp amplifier, Axon Instruments). The sign of the membrane potential refers to the trans chamber, and currents are defined as positive when cations flow from trans to cis. Transmembrane currents were low-pass filtered at 0·51 kHz (8 pole Bessel) digitized at 5 kHz. Membrane capacitance was measured by differentiating a triangle wave input of 0·2 kHz. Only bilayers that had a conductance of less than 10 pS and initial capacitance of at least 300 pF were used. Unless otherwise stated, lipid bilayers were bathed in 250 mM NaCl containing 5 mM HEPES/NaOH (pH 7·0), and all recordings were made at room temperature (1922 °C). The recordings were analysed off-line using 30180 s recordings carried out for each holding potential and analysed using PAT and win EDR software (Strathclyde Electrophysiology Software). Current steps yielding conductances over twice the size of CytK-1 (i.e. >220 pS) were excluded as simultaneous openings of more than one channel.
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RESULTS |
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Sequence analysis and comparison of the cytK genes
The cytK PCR products of B. cereus strains 1230-88, FM-1 and 23 were DNA sequenced directly, thus obtaining 76 % of the gene sequences. Alignments of the deduced amino acid sequences revealed conserved parts of the protein. Furthermore, the identity between the obtained amino acid sequences from strains 1230-88, FM-1 and 23 was 9799 %, while the identity between these sequences and the original CytK-1 from strain 391-98 was only 89 %. We have named this new CytK-variant CytK-2. The CytK-2 sequences obtained were used to search the completed genomes of B. cereus ATCC 14579 and B. cereus ATCC 10987 using the BLAST algorithm (Altschul et al., 1997). A cytK-2 gene was identified in both strains (Fig. 1
).
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The mature CytK-2 proteins showed about 89 % identity to the CytK-1 from strain 391-98. The differences between the CytK-2 sequences and CytK-1 were clustered in specific regions (Fig. 1). The identities between the mature CytK-2 sequences of strains ATCC 14579, ATCC 10987, 1230-88, FM-1 and 23 were between 96 % and 100 %. The FM-1 CytK-2 had an additional amino acid at the C-teminal end as compared with the other CytK proteins, while the mature sequences of CytK-2 from strains ATCC 10987 and 23 were 100 % identical.
The PCR products obtained using the primers Fhly-II and Rhly-II (Table 2) with DNA from strains 1230-88 and FM-1 were sequenced. The sequences were 99 % identical to that deduced from the original hly-II (results not shown). This sequence was 37·5 % identical to CytK-1, and 39 % identical to CytK-2 (all containing 6 % gaps). The identity between the CytK proteins and
-haemolysin of S. aureus was 2930 %, with the differences between the proteins distributed throughout the amino acid sequences.
PlcR is a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. (Agaisse et al., 1999). It has previously been shown that the expression of CytK-2 was abolished in a plcR-deficient mutant of B. cereus ATCC 14579 (Gohar et al., 2002
). The recognition site for the positive transcriptional regulator PlcR in the promoter region of cytK-1 from strain 391-98 was 5'-TATGCAATTTCGCATA-3', thus con<1?show=[dh]>taining a one base mismatch (C11) compared to the PlcR binding site consensus sequence 5'-TATGNAN4TNCATA-3' (Agaisse et al., 1999
). In contrast, the PlcR boxes upstream from the cytK-2 genes in strains ATCC 14579, ATCC 10987, 1230-88 and 23 were identical to the highly conserved regulatory sequence. The location of the centre of the palindromic sequence of the PlcR box was at position 86 for cytK-1 from strain 391-98 and at position 89 for the cytK-2 genes, relative to the start codon. In comparing the promoter regions extending ten nucleotides upstream from the PlcR box through to the start codon, the identity between the cytK-1 promoter region in strain 391-98 and the corresponding cytK-2 promoter sequences in strains ATCC 14579, ATCC 10987, 1230-88 and 23 was 6673 %, containing 4·5 % gaps. The identity between the promoter regions of the four strains containing cytK-2 was 9299 %. The differences in the promoter regions explain the negative results obtained when PCRs were run with DNA from strains 1230-88, 68 and 23 using a primer made from the promoter region of cytK-1 from strain 391-98 (Lund et al., 2000
).
Protein isolation and comparison of the toxic activity of CytK-1 and CytK-2
To confirm that B. cereus strains 1230-88, FM-1 and 23 actually produce the CytK-2 toxins, supernatant proteins were precipitated with ammonium sulphate and purified on columns of DEAE, HA and ReQ. The CytK-2 proteins were eluted mainly in the void volume from the DEAE column at pH 5·9 and at approximately 100 mM sodium phosphate from the HA column (as for the original CytK-1). However, the original CytK-1 was eluted from ReQ at pH 8·1 at 20 mM NaCl, while the CytK-2 proteins were eluted at lower NaCl concentrations and not in a sharp peak. The CytK-2 protein of FM-1 seemed to be pure after fractionation on the HA column (Fig. 2), CytK-2 from strain 23 was pure after fractionation on the ReQ column, while the CytK-2 protein of strain 1230-88 (Fig. 2
) was still slightly contaminated with another protein after the last step on the ReQ column. The mobility of CytK-1 and the CytK-2 proteins on SDS-PAGE seemed to be identical, indicating that the size of the proteins is similar. The 20 first amino acid residues from the N-terminal ends of the CytK-2 proteins from strains 1230-88 and 23 were determined by amino acid sequencing, confirming the identity of the proteins.
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The volume of periplasmic extracts containing CytK-1 and CytK-2 necessary to obtain 50 % haemolytic activity towards bovine erythrocytes was determined. To normalize the two samples, their volumes were adjusted to contain the same amount of haemolytic activity. Then, the volume of the normalized periplasmic extracts needed to obtain 50 % inhibition of protein synthesis in Vero cells was determined. It was necessary to add five times the volume of periplasmic extract containing CytK-2 as compared to periplasmic extract containing CytK-1 to obtain the same degree of protein inhibition in Vero cells (mean values of duplicate experiments). Thus, relative to the haemolytic activity, CytK-1 from strain 391-98 was about five times more toxic against Vero cells than CytK-2 from strain 1230-88.
Analysis of the pore-forming ability of CytK-1 and CytK-2 using planar lipid bilayer recordings
The difference in activity between batches of purified CytK-1 and CytK-2 proteins was examined using planar lipid bilayers to test for pore formation. Five preparations were examined and the results are summarized in Fig. 3. For CytK-1 isolated from B. cereus 391-98 supernatant, two samples were tested: one active preparation, and one sample that had lost cytotoxicity upon purification and storage. Addition of up to 10 µg protein of the active CytK-1 protein to the cis bilayer chamber resulted in the appearance of rectangular current fluctuations. The time taken for single-channel current steps to appear was generally less than 15 min. The active CytK-1 protein purified from B. cereus 391-98 (i.e. haemolytic and cytotoxic) yielded single-channel events with a mean single-channel conductance of 143±3 pS (Fig. 3a
), similar to that described previously (Hardy et al., 2001
). The pore-forming ability of CytK-2 proteins was examined using CytK-2 purified from B. cereus 1230-88 supernatant. The purified protein sample was haemolytic but not cytotoxic towards Vero cells. As shown in Fig. 3(d)
, the CytK-2 protein was able to form pores in lipid bilayers but the distribution of pore sizes was distinct from that of the active CytK-1 protein, with 63 % of the channels having a conductance less than 100 pS. Recombinant CytK-1 and CytK-2 proteins from the same strains isolated from the periplast of E. coli were examined for channel conductance. Fig. 3(b)
shows the distribution of channel conductances obtained from recombinant CytK-1 (r391-98), showing that 75 % of the channels were between 101 and 220 pS, and thus were of similar size as the channels formed by the active CytK-1 protein isolated from B. cereus. Recombinant CytK-2 protein from strain 1230-88 (Fig. 3e
) yielded a spread of channel conductances, but the predominant conductance values were less than 100 pS.
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DISCUSSION |
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There are differences between CytK-1 and the CytK-2 proteins in six positions in the putative pore-forming region (Fig. 1). The substituted residues are clustered in the region that constitutes the base of the stem, and are predicted to line the exterior of the membrane channel. However, the amino acid substitutions were mainly conservative, indicating that the pore of the CytK-2 proteins will resemble that of the original CytK-1 protein. Nonetheless, the possibility that the differences in this region could have biological effect cannot be excluded.
The reduction in cytotoxic activity in the CytK-1 and CytK-2 proteins upon purification and storage may be attributed to spontaneous oligomerization of monomers. Chattopadhyay & Banerjee (2003) reported that Vibrio cholerae haemolysin A (HlyA), a
-barrel channel-forming toxin with 22 % identity to the CytK proteins, spontaneously and irreversibly transformed from the haemolytically active monomer to a haemolytically inactive heptamer when kept in water for days.
The pleiotropic transcriptional activator PlcR is involved in the control of extracellular virulence factor expression in B. cereus (Agaisse et al., 1999). The recognition site for PlcR, a conserved palindromic sequence (the PlcR box; Agaisse et al., 1999
), was found upstream of the cytK-2 genes, strongly indicating that PlcR regulates their expression. Gohar et al. (2002)
examined the extracellular proteome of the wild-type B. cereus strain ATCC 14579 and a mutant strain with disrupted plcR. CytK-2 was one of the proteins identified in the culture supernatant of the wild-type strain, which was not present in the mutant strain. It is interesting to note that the recognition site for PlcR in the promoter of cytK-1 from strain 391-98 was atypical, displaying a one-nucleotide mismatch (C11 versus T11) compared with the original sequence reported by Aigasse et al. (1999)
. A functional PlcR box that differs from the consensus sequence has previously only been described for the zinc-requiring metalloproteinase InhA-2, found in both B. thuringiensis and B. cereus (Fedhila et al., 2003
).
The CytK-2 proteins show relatively high sequence similarity (39 % identity) to the deduced product of hly-II (Baida et al., 1999). However, in aligning the deduced sequences of cytK and hly-II, 6 % gaps are present, whereas none are present when comparing CytK-1 and the CytK-2 proteins (with the exception that the FM-1 CytK-2 was one amino acid longer than the other CytK proteins). The presence of a lysine residue, K125, at the end of the possible transmembrane loop in the product of hly-II may cause cleavage of this protein by trypsin and thus result in the protein being inactivated in the small intestine. Its contribution to the toxicity of B. cereus strains is therefore probably not of any significant importance. The overlapping part of the sequence of haemolysin IV (only the 30 N-terminal amino acids were determined; Beecher et al., 2000
) and the CytK-2 protein of B. cereus ATCC 14579 are identical. It is therefore reasonable to include haemolysin IV, which is suggested to be an important virulence factor, in the CytK group of toxins. In summary, there seem to exist two subclasses of
-barrel pore-forming toxins in B. cereus and B. thuringiensis. The product of hly-II sequenced by Baida et al. (1999)
occurs widely in B. thuringiensis strains, while the other subclass, CytK toxins (including haemolysin IV), seems to be common in B. cereus strains.
The enterotoxin complexes Nhe (Lund & Granum, 1996) and Hbl (Beecher & Wong, 1994
), or the genes encoding them, have been shown to be present in some of the strains containing CytK-2 proteins (Lund & Granum, 1997
; Stenfors & Granum, 2001
). For instance, B. cereus 1230-88, which caused a severe food poisoning (although without bloody diarrhoea), expresses a CytK-2 toxin, Nhe and Hbl, and contains hly-II. Thus, several toxins may contribute to the enterotoxicity of certain B. cereus strains. In contrast, strain 391-98 only expresses CytK-1. It will be of interest to test the hypothesis that the CytK toxins form ulcerative and haemorrhagic lesions in the intestine whereas the other enterotoxin genes produce watery diarrhoea.
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ACKNOWLEDGEMENTS |
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Received 9 December 2003;
revised 28 April 2004;
accepted 7 May 2004.
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