1 Institut Pasteur, Génétique et Physiologie des Bacillus Pathogènes, Département de Microbiologie Fondamentale et Médicale, 25 rue du Dr Roux, 75724 Paris cedex 15, France
2 INRA, Unité Génétique Microbienne et Environnement, La Minière, 78285 Guyancourt cedex, France
Correspondence
Didier Lereclus
lereclus{at}jouy.inra.fr
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ABSTRACT |
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Present address: INRA Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
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INTRODUCTION |
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Food-borne diseases caused by B. cereus are classified as emetic and diarrhoeal syndromes. The emetic syndrome is due to one toxin, the emetic toxin (cereulide), which causes vomiting (Agata et al., 1995a). The diarrhoeal syndrome might be caused by several enterotoxins. The HBL enterotoxin is a three-component haemolysin that consists of one binding component (B) and two lytic proteins (L1 and L2) (Beecher & Wong, 1997
). This toxin has haemolytic and dermonecrotic activities, and it increases vascular permeability and causes fluid accumulation in rabbit ileal loops (Beecher et al., 1995
). Another important enterotoxin, NHE (non-haemolytic enterotoxin), is a three-component complex that was originally identified in a B. cereus strain responsible for a food-poisoning outbreak (Lund & Granum, 1996
). Other enterotoxin genes, bceT, entFM, entS and entI, have also been described (Agata et al., 1995b
; Asano et al., 1997
). However, it was recently suggested that the bceT gene product does not contribute to food-borne diseases (Choma & Granum, 2002
) and may in fact be a cloning artefact (Hansen et al., 2003
). B. cereus produces several other secreted proteins, including phospholipases and proteases, that may contribute to B. cereus pathogenicity associated with food-borne diseases. The expression of most of these putative virulence factors is controlled by the pleiotropic transcriptional activator PlcR (Agaisse et al., 1999
; Gohar et al., 2002
). Its recognition site is believed to be a highly conserved palindromic sequence: TATGNAN4TNCATA. This global regulator has been shown to contribute to B. cereus virulence in mice, in insects (Salamitou et al., 2000
) and in rabbit endophthalmitis (Callegan et al., 2003
).
Cytotoxin K (CytK) may also be involved in B. cereus food poisoning. It was first characterized in B. cereus strain 391-98, a strain isolated from cases of food-borne disease and responsible for the death of three people (Lund et al., 2000). Interestingly, none of the other, commonly described, enterotoxin genes (for example hbl and nhe) was detected in this strain, further implicating CytK as a major virulence factor. However, several B. cereus isolates possess the cytK gene, as is the case for the hbl or nhe genes (Guinebretière et al., 2002
). Thus, the presence of a particular gene presumably involved in virulence is not in itself sufficient to confer pathogenicity. Presumably, the transcription level of the gene is important in virulence.
The cytotoxin CytK is a pore-forming toxin that belongs to a family of -barrel channel-forming toxins (including Staphylococcus aureus leucocidins and Clostridium perfringens
-toxin). It is necrotic and haemolytic (Lund et al., 2000
), and also cytotoxic for intestinal epithelia and therefore probably causes diarrhoeal syndrome (Hardy et al., 2001
). The cytK promoter region of B. cereus 391-98 contains a putative PlcR box which, however, does not conform to the consensus as currently defined (Lund et al., 2000
).
To understand the strong pathogenicity of B. cereus strain 391-98, in which cytK is the only known enterotoxin gene (Lund et al., 2000), we studied the transcriptional activity of the cytK promoter region. We also found that PlcR regulated the cytK promoters of both this strain and the reference strain ATCC 14579 whether the PlcR box was the native sequence or carried mutations.
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METHODS |
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E. coli and B. cereus cells were routinely grown in Luria broth (LB) medium with vigorous agitation at 37 °C. The antibiotic concentrations used for bacterial selection were: 100 µg ampicillin ml1 (for E. coli), 10 µg erythromycin ml1 (for B. cereus) and 200 µg kanamycin ml1 (for B. cereus). Bacteria with the Lac+ phenotype were identified on LB agar containing 40 µg X-Gal ml1.
DNA manipulation.
Plasmid DNA was extracted from E. coli and B. cereus by a standard alkaline lysis procedure on QIAprep spin columns (Qiagen), with the following modification in the first step of the lysis procedure for B. cereus: incubation at 37 °C for 1 h with 5 mg chicken egg white lyzosyme (14 300 U mg1). Chromosomal DNA was extracted from B. cereus cells harvested in mid-exponential phase as described previously (Msadek et al., 1990). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs). Oligonucleotide primers were synthesized by Proligo-Genset. PCR was performed in a GeneAmp PCR system 2400 thermal cycler (Perkin-Elmer), using Pwo DNA polymerase (Roche) or Pfx DNA polymerase (Invitrogen), both high-fidelity polymerases. Amplified DNA fragments were purified by using the QIAquick PCR purification Kit (Qiagen) and separated on 0·7 % agarose gels after digestion. Digested DNA fragments were extracted from agarose gels with a centrifugal filter device (montage DNA gel extraction kit; Millipore). All constructions were confirmed by DNA sequencing.
Electroporation was used to transform E. coli (Dower et al., 1988) and B. cereus (Lereclus et al., 1989
) as previously described.
Construction of the cytK'lacZ transcriptional fusion.
The cytK'lacZ transcriptional fusions were constructed by cloning a BamHI/HindIII DNA fragment harbouring the putative cytK promoter between the BamHI and HindIII sites of pHT304-18'Z (Agaisse & Lereclus, 1994). The 402 bp (B. cereus ATCC 14579) and 381 bp (B. cereus 391-98) DNA fragments were generated by PCR amplification of chromosomal DNA with the primers Pc79-F and Pc79-R, and Pc98-F and Pc98-R, respectively (Table 1
). The recombinant plasmids, designated Pc79'-Z (cytK promoter originating from B. cereus ATCC 14579) and Pc98'-Z (cytK promoter originating from B. cereus 391-98), were introduced into B. cereus ATCC 14579 wild-type and
plcR mutant strains by electroporation. The transformants were resistant to erythromycin (10 µg ml1).
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Mean values of -galactosidase activity measured 2 h after the entry in stationary phase of growth for three to five distinct cultures were analysed by the Student t-test in order to determine P values for differences in expected values versus actual values.
Mapping of the 5' end of cytK mRNA by primer extension.
Total RNA was extracted from wild-type B. cereus ATCC 14579 and 391-98 cells grown in LB at 37 °C with shaking, as described previously (Agaisse & Lereclus, 1996). The cytK transcription start site of both strains was determined by primer extension using ExtsnCytK79 and ExtsnCytK98 oligonucleotides (Table 1
), as described previously (Agaisse & Lereclus, 1996
). DNA sequencing was performed by the dideoxy chain-termination method with the primers ExtsnCytK79 and ExtsnCytK98, and using the corresponding PCR product as the template, with the T7 sequenase PCR product sequencing kit (USB).
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RESULTS |
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Expression of cytK is regulated by PlcR
The B. cereus 391-98 cytK promoter region contains a putative PlcR box (Fig. 1): TATGCAATTTCGCATA (the underlined nucleotides are the most highly conserved in all the PlcR boxes) (Lund et al., 2000
). However, this sequence is not palindromic and does not conform to the consensus as currently defined, TATGNAN4TNCATA (Agaisse et al., 1999
). In contrast, the cytK promoter in B. cereus strain ATCC 14579 contains a PlcR recognition site conforming to the consensus (Fig. 1
).
To determine whether this divergence of the PlcR box has an effect on regulation by PlcR, we introduced the cytK transcriptional fusions into the B. cereus ATCC 14579 plcR mutant. No transcriptional activity above 10 Miller units was detected for either Pc79'-Z or Pc98'-Z (data not shown). This indicates that cytK transcription is PlcR-dependent in both cases.
To elucidate the role played by the PlcR box in the difference of cytK expression between strains, we introduced substitutions as shown in Fig. 1.
-Galactosidase expression was followed in three cultures for each of these constructs (mean values at T2 are presented in Fig. 3
). Both the single point mutation (Pc79M1'-Z) and the exchange of two adjacent nucleotides (Pc79M2'-Z, such that the sequence is the same as that of the PlcR box in Pc98'-Z) in the PlcR box of cytK promoter region from B. cereus strain ATCC 14579 substantially reduced, but did not abolish, cytK expression (Fig. 3a
).
In contrast, restoration of the consensus as currently defined for the PlcR box of Pc98'-Z (Pc98M1'-Z) and substitution with a PlcR box similar to that found in the reference strain (Pc98M2'-Z) did not significantly affect expression (Fig. 3b).
cytK promoter characterization
Primer extension experiments were performed in both strains to determine the cytK transcription start point (Fig. 4). We then determined the putative 10 and 35
A boxes of both cytK promoter regions (Fig. 5
). The putative 10
A boxes in the two strains are identical, and are in accordance with the 10
A consensus of Bacillus subtilis. However, the 35
A boxes of the two cytK promoter regions are different. The 35
A region of the cytK promoter from B. cereus strain 391-98 is very similar to the consensus for B. subtilis but that from B. cereus ATCC 14579 is not (Fig. 5
).
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DISCUSSION |
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This difference in cytK transcription is in accordance with the greater amount of CytK detected in the extracellular proteome of B. cereus 391-98 (6·9 % of total exported protein; M. Gohar, personal communication) than that of ATCC 14579 (0·4 %) as previously reported (Gohar et al., 2002). However, this high CytK production is not a general characteristic of clinical B. cereus isolates. Indeed, the amount of CytK protein was determined by proteomic approaches in a set of 25 B. cereus strains of various origins (clinical and non-clinical strains). In the 391-98 strain, CytK amount is at least three times higher than in the other strains (M. Gohar & R. Gravelline, unpublished results).
Lund et al. (2000) have shown that in B. cereus 391-98, the cytK promoter region contains a putative PlcR box. However, this sequence did not match the currently defined consensus: TATGNAN4TNCATA (Agaisse et al., 1999
; Økstad et al., 1999
). In contrast, the cytK promoter region in B. cereus ATCC 14579 contains a PlcR recognition site that matches the consensus. The kinetics of expression from the two promoter regions are characteristic of PlcR-regulated genes (Fig. 2
), and in both cases, expression was shut off in a PlcR-deficient mutant. This indicates that cytK transcription is PlcR-dependent in the two B. cereus strains. Thus, the PlcR box in the cytK promoter region in B. cereus 391-98 is functional, despite diverging from the currently defined consensus.
Our identification of a non-palindromic PlcR box is consistent with what has been found for the inhA2 gene, which has also been shown to be PlcR regulated, despite not conforming to the currently defined consensus (Fedhila et al., 2003); the divergence in the inhA2 gene is the first nucleotide of the PlcR box. Thus, the PlcR recognition-site consensus is not necessarily palindromic and PlcR may recognize divergent sequences. Consequently, the PlcR regulon of B. cereus is probably much larger than that proposed by Ivanova et al. (2003)
.
Introduction of substitutions into the PlcR box of cytK promoter region from B. cereus strain ATCC 14579 reduced significantly, but did not abolish, expression from the cytK promoter (Fig. 3a). The standard PlcR box found in the promoter region of cytK from ATCC 14579 is therefore more efficient than the one containing substitutions. However, restoration of the consensus in the PlcR box of Pc98'-Z did not significantly change the expression level (Fig. 3b
). This indicates that the higher transcription from the promoter region of cytK from 391-98 is not due to the sequence of the PlcR box. The difference in transcriptional activity between the two cytK promoter regions may be due to the differences between 35
A boxes.
In conclusion, our work indicates that B. cereus 391-98, responsible for an infectious outbreak, produces large amounts of cytotoxin CytK due to high levels of transcription. This high CytK expression may account for the high virulence of strain 391-98. However, there is only 88 % of identity between the CytK amino acid sequence of the two strains. We therefore can not rule out that such differences may also play a role in cytotoxic activity. Finally, we also found that the PlcR recognition site is more heterogeneous than previously believed, allowing the possibility to identify other PlcR-regulated virulence factors.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 January 2004;
revised 21 April 2004;
accepted 21 May 2004.
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