Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway
Correspondence
Per Einar Granum
per.e.granum{at}veths.no
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ABSTRACT |
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Present address: Faculty of Medicine, Rikshospitalet, University of Oslo, N-0027 Oslo, Norway.
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INTRODUCTION |
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Not all the factors that render B. cereus pathogenic to humans are precisely known, although several potential virulence factors have been characterized. All but one, the dodecadepsipeptide cereulide (Agata et al., 1994), are proteins encoded by genes dispersed on the bacterial chromosome (Økstad et al., 1999
). The transcription of the genes encoding these potential virulence factors is controlled by the pleiotropic regulator PlcR (Agaisse et al., 1999
; Økstad et al., 1999
).
Two different three-component enterotoxins have been isolated from B. cereus. These are haemolysin BL (Hbl) (Beecher & MacMillan, 1991) and non-haemolytic enterotoxin (Nhe) (Lund & Granum, 1996
), both of which are positively regulated by PlcR (Gohar et al., 2002
). In addition to its enterotoxic activity, Hbl possesses haemolytic and dermonecrotic activities (Beecher & Wong, 1994
). The genes encoding the B, L1 and L2 components of Hbl have been cloned and sequenced from B. cereus strain F837/76 (Heinrichs et al., 1993
; Ryan et al., 1997
). Beecher & Macmillan (1991)
showed that all three components were required for complete lysis of sheep erythrocytes, and HblB was suggested to be the component that bound to the cell surface before the two L components could act to cause cell lysis. However, more recently, another model for the action of Hbl has been proposed, suggesting that the components of Hbl bind to target cells independently, and then constitute a membrane-attacking complex resulting in a colloid osmotic lysis mechanism (Beecher & Wong, 1997
).
The three-component enterotoxin Nhe was first isolated from an Hbl-negative strain of B. cereus, which was involved in a large food-poisoning outbreak in Norway in 1995 (Lund & Granum, 1996). The proteins were different from the components of Hbl and had no detectable haemolytic activity. It was initially suggested that a collagenase of 105 kDa (Lund & Granum, 1999
) was part of the Nhe complex (Lund & Granum, 1996
, 1997
), but this was later shown to be incorrect. Sequencing of the operon encoding NheA and NheB (Granum et al., 1999
) revealed a novel gene encoding a protein designated NheC, suggesting that this protein was part of the Nhe complex. NheC has previously not been detected in, or purified from, B. cereus culture supernatants.
The present study shows specifically that all three components are necessary for cytotoxic activity, and that the molar ratio between the components is of importance.
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METHODS |
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Purification of NheA and NheB from B. cereus culture supernatants.
The purification schemes for NheA and NheB were similar to those described in detail for purification of Hbl (Beecher & Wong, 1994) and Nhe (Lund & Granum, 1996
, 1997
). Proteins from 2 l culture supernatant were concentrated by precipitation with 70 % saturated (NH4)2SO4, resuspended in 25 ml water, and dialysed against 25 mM Bistris/HCl pH 5·9. Chromatography on a DEAE-Sephacel column (Amersham Biosciences), with Bistris/HCl and a gradient of 00·5 M NaCl, was followed by chromatography on a HA column (Bio-Rad), using a sodium phosphate gradient of 00·25 M at pH 6·8 for elution. Fractions were analysed on SDS-PAGE, and combinations of fractions were tested for cytotoxic activity. Protein concentration was estimated by the difference in absorbance at 235 and 280 nm (Whitaker & Granum, 1980
).
Expression of recombinant NheA (rNheA) and NheC (rNheC) in E. coli.
The shuttle vector pMS10 was constructed by cloning the kanamycin-resistance gene from pUC4K (Amersham Biosciences) into the SacINcoI site of pGK12 (Kok et al., 1984). The strong, constitutive promoter p32 from pMG36e (van de Guchte et al., 1989
) was cloned into the KpnIMluI site of pMS10, and the construct was designated pMS20. Primers nhexfmlu and nhexrmlu (Table 1
), with restriction enzyme recognition site sequences (MluI) at their ends, were used to PCR-amplify nheA and nheC. The PCR products were cloned into the MluI site of pMS20, and the constructs were named pMS21 (rNheA) and pMS23 (rNheC). E. coli XL10-Gold/pMS21/23 was grown for 6 h, then the cells were pelleted by centrifugation, resuspended in water and lysed by sonication. Cell debris was removed by centrifugation, and the supernatants were used in toxicity tests on Vero cells.
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Expression and purification of His6-NheC.
Since free-existing NheC has not been detected in culture supernatants from B. cereus, NheC was expressed as a histidine-tagged fusion protein, His6-NheC, to facilitate the purification of the predicted protein. The primers nhecf and nhecr (Table 1) were used to PCR-amplify nheC, which was subsequently cloned into the pCR T7/NT-TOPO vector (Invitrogen). His6-NheC was purified using NiNTA agarose (Qiagen) under denaturing conditions (8 M urea), according to the procedure provided by the supplier. The urea was removed by stepwise dialysis against 3 M urea/20 mM Tris/HCl pH 7·6, 1 M urea/20 mM Tris/HCl pH 7·6, 0·1 M urea/20 mM Tris/HCl pH 7·6, and then 20 mM Tris/HCl pH 7·6.
SDS-PAGE, native PAGE and immunoblotting.
PAGE and Western immunoblotting were carried out using a Bio-Rad Mini-Protean II Dual Slab Cell, according to standard protocols (Harlow & Lane, 1988). The gels were silver stained according to Blum et al. (1987)
. The molecular mass of the proteins was estimated using Bio-Rad SDS-PAGE standards, and prestained kaleidoscopic standard (Bio-Rad) for Western blots. Monoclonal antibodies against NheA and NheB were obtained as a gift from Dr Erwin Märtlbauer (Ludwig-Maximilians-Universität, Munich, Germany). His6-NheC was detected on Western blots using monoclonal antibodies against the histidine tag (Invitrogen) in a dilution of 1 : 5000. Biotin-conjugated anti-mouse antibodies from goat (Amersham Biosciences) were used as secondary antibodies (1 : 10 000). A complex of streptavidin (Bio-Rad) and biotinylated alkaline phosphatase (Bio-Rad) was used at a dilution of 1 : 3000, prior to development with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad). Native PAGE was performed according to Yoshida et al. (2002)
, using a 10 % polyacrylamide separation gel without SDS in 187·5 mM Tris/HCl (pH 8·9). The gel was run for 4 h at 60 V in 82·6 mM Tris/HCl (pH 9·4) and 33 mM glycine.
Cytotoxicity test on Vero cells.
Cytotoxicity was tested using a Vero cell assay according to Sandvig & Olsnes (1982). The assay monitors inhibition of protein synthesis in Vero cells after addition of toxic proteins. Vero cells were grown in minimal essential medium, MEM (Gibco), with 10 % fetal calf serum added.
RNA isolation and analysis.
RNA was isolated from 10 ml samples as described by Igo & Losick (1986). Mapping of the 5' terminus of the nhe mRNA was performed on total RNA isolated from B. cereus 0075-95 and 1230-88, using a 5' end-labelled oligonucleotide, P5452 (Table 1
), located 28 bp downstream of the translational start site of nheA. Annealing and extension were performed according to Luna et al. (1994)
, using 10 U cloned AMV Reverse Transcriptase (Invitrogen) in the extension reaction. Products were resolved in 6 % polyacrylamide gels together with sequence reactions prepared according to the dideoxy chain-termination method using the Thermo Sequenase Radiolabelled Terminator Cycle Sequencing Kit (USB) according to protocol.
Reverse transcriptase PCR was performed using 1 µg total RNA treated with 2 U RNase-Free DNase (Promega) at 37 °C for 30 min. The extension reactions were carried out using 10 U cloned AMV Reverse Transcriptase (Invitrogen) in the supplied reaction buffer with 0·5 mM dNTPs (Finnzymes, Finland), all in a total volume of 20 µl at 45 °C for 1 h. The reverse primers used in the extension reactions (Table 1) were used in a final concentration of 1 pmol µl1. A 1 µl volume of the extension reaction was used as a template in standard PCR reactions.
PCR amplification.
All PCRs were carried out in an MJ Research Minicycler PTC-150 with heated lid, or an Eppendorf Mastercycler gradient. DyNAzyme II DNA polymerase (supplied with 10x buffer) and dNTP Mix from Finnzymes were used as instructed by the manufacturer. The primers used in the reactions are listed in Table 1. Depending on the primers, the standard programme was as follows: 95 °C for 1 min, 30 cycles of 95 °C for 1 min, 4852 °C for 1 min, and 72 °C for 1 min, then a final extension step of 72 °C for 7 min.
Binding of Nhe components to Vero cells.
Purified Nhe proteins (10 nM) were added to wells containing Vero cell monolayers, and incubated for 10 and 20 min at 37 °C. The monolayers were washed five times with MEM, and then suspended in 50 µl SDS-PAGE sample buffer. A 20 µl volume of each sample was applied to 12 % SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad) by Western immunoblotting.
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RESULTS |
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To elucidate the composition of the Nhe complex, different concentrations of the Nhe proteins were tested in the Vero cell assay. Representative results from inhibition of protein synthesis in Vero cells when altering the concentration of the individual Nhe components are presented in Table 2. The concentration of NheA was held constant at 1 nM, as this was the lowest concentration resulting in significant inhibition. The components were added one by one in the order NheA, NheB and His6-NheC. One nanomolar NheA, 1 nM NheB and 0·1 nM His6-NheC were the lowest concentrations of each Nhe component necessary for maximal inhibition in the linear part of the toxicity curve (linear between 30 and 70 % inhibition).
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Binding of NheB to Vero cells
To examine if any of the Nhe components were able to bind to the Vero cells, the individual Nhe components were incubated with Vero cells. Following incubation, the cells were washed five times, suspended in sample buffer, and applied to SDS-PAGE. Western immunoblots of the Vero cells incubated with NheA, NheB or His6-NheC, individually, showed that NheB was the binding component of the Nhe complex (Fig. 4). After both 10 and 25 min, NheB was the only single component attached to the Vero cell monolayer. When NheB was incubated with an equal amount of His6-NheC prior to addition to the Vero cells, no NheB was detected attached to the Vero cells, indicating that His6-NheC prevents NheB from binding to the Vero cell surface.
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Formation of complex between NheA/His6-NheC and NheB/His6-NheC
To investigate the association between the individual Nhe components, different combinations of the Nhe proteins were mixed in equal concentrations (1 : 1 : 1), incubated for 15 min at room temperature, subjected to native PAGE, blotted onto nitrocellulose membranes, and probed with Nhe or His antibodies. The Western immunoblot of the native gel showed that the individual Nhe bands disappeared when combining NheA/His6-NheC, NheB/His6-NheC and NheA/NheB/His6-NheC (Fig. 6), indicating the formation of complexes that were too large to enter the gel. Combining NheA and NheB did not result in the disappearance of the NheA band, indicating no (or weak) complex formation between these two proteins. The result shows that both NheA and NheB form complexes with His6-NheC, without the presence of the third component. His6-NheC appeared at the upper part of the native gel. Combining His6-NheC with NheA or NheB weakened the His6-NheC band, while combining all three components resulted in complete disappearance of the His6-NheC band (Fig. 6
).
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DISCUSSION |
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The transcriptional start site of the nhe operon in two B. cereus strains was identified at positions 62 and 66 bp upstream of the translational start site (Fig. 1). Variations in the location of transcriptional start sites between virulence genes of different B. cereus strains have been reported previously (Agaisse et al., 1999
; Lindbäck et al., 1999
). The putative 10 promoter sequence for both of the B. cereus strains examined in this study showed 50 % homology to the classical Pribnow box (TATAAT). In addition, the 35 region was conserved to 50/33 % homology with the classical 35 sequence (TTGACA). The 10 sequence of B. cereus 0075-95 was located 3 bp closer to the transcriptional start site than that of strain 1230-88, but this difference is most probably of no significance, as both strains are high producers of Nhe.
Since attempts to purify NheC from B. cereus culture supernatant have not succeeded, NheC was expressed in E. coli as a histidine-tagged fusion protein. The addition of His6-NheC to NheA and NheB was essential to obtain cytotoxic activity of the Nhe complex in the Vero cell assay. The maximum cytotoxic activity of the Nhe complex was obtained when the NheA : NheB : NheC ratio was 10 : 10 : 1, as determined by the Vero cell assay (Table 2). The accuracy of the Vero cell assay and protein concentration measurements is not precise enough to give a definite ratio between the three components, so a ratio down to 8 : 8 : 1 might be possible. The important observation is that a concentration of NheC higher than about 10 % of that of NheA and NheB inhibited the toxic activity. As the correlation between percentage inhibition of protein synthesis in Vero cells and concentration of toxin is linear in the range from about 30 to 70 %, the minimum cytotoxic concentrations presented in Table 2
were kept within this range.
Native PAGE showed that both NheA and NheB formed complexes with His6-NheC, which were probably too large to enter the gel (Fig. 6). Combining rNheA and rNheB in the Vero cell assay did not result in any inhibition of protein synthesis, strongly indicating that NheC is needed for toxic activity of the Nhe complex. The presence of the histidine tag did not seem to affect the properties of the NheC protein, as rNheC could replace the His6-NheC in the Vero cell assay, with identical results. In some experiments, combination of NheA and NheB from different batches of purified proteins resulted in up to 30 % inhibition of protein synthesis in Vero cells, without the addition of His6-NheC/rNheC. This was probably due to minor contamination of NheC, as NheC can be associated with both NheA and NheB.
Results from cell-binding experiments followed by Western immunoblotting showed that NheB was the only individual component that bound to the Vero cells. Neither NheA nor His6-NheC was observed attached to the Vero cells. In the Vero cell assay, the addition of His6-NheC in concentrations equal to NheA and NheB resulted in complete loss of cytotoxic activity of the Nhe complex. When Vero cells were incubated with a high enough concentration of NheB prior to addition of NheA:NheB:NheC in the ratio 1 : 1 : 1, the cytotoxic activity was restored (Fig. 5). This was, however, not observed when the Vero cells were incubated with NheA prior to addition of the Nhe complex. The inhibitory effect of increasing the NheC concentration was probably due to complex formation between NheB and NheC, which prevented the binding of NheB to the membrane. Free His6-NheC bound to NheB, and NheB in complex with His6-NheC was not able to bind to the surface of the Vero cells. Pre-incubation of the components of the Nhe complex prior to addition to the Vero cell assay was not necessary in order to obtain maximum cytotoxic activity. Furthermore, differences in the temperature (22 and 37 °C) used in the pre-incubation of the Nhe components prior to addition to Vero cells did not influence the level of inhibition obtained.
RT-PCR products obtained using internal primers in nheC, in addition to a forward primer in nheA and a reverse primer in nheB (Fig. 2a), showed that intact nhe mRNA was present in the reaction mixture (Fig. 2b
). RT-PCR products covering the intergenic region between nheA and nheB indicate no, or easily dissolved, secondary structure in the mRNA between these two genes. All attempts to amplify RT-PCR products of the correct size containing the section between nheB and nheC failed. This indicated that the predicted structural conformation between the two genes (Granum et al., 1999
) prevented amplification of complete cDNA during the reverse transcriptase reaction. The efficiency of translational initiation and regulation is strongly correlated with the structure of the ribosome-binding site (RBS) (Schlax & Worhunsky, 2003
). The primary interaction between mRNA and the 30S subunit rRNA requires local single-stranded mRNA (Draper, 1987
, 1996
; Gualerzi & Pon, 1990
; Draper et al., 1998
). Secondary structure in the RBS can lower translational efficiency through a competitive mechanism, where cis-mRNA sequences compete with the 30S subunit for binding to the RBS (de Smit & van Duin, 1994
; de Smit, 1998
). We suggest that the low level of expression of NheC is caused by translational repression due to the structure of the RBS or the sequence surrounding the RBS. Alternatively, the 5' end of the mRNA could be more susceptible to degradation, as the predicted secondary structure between nheB and nheC might protect against degradation. A combination of both possibilities cannot be excluded. The formation of an active enterotoxin complex requires the concentration of NheC to be lower than that of NheA and NheB. This observation is supported by the results obtained by Gohar et al. (2002)
, where NheA and NheB, but not NheC, were identified on a two-dimensional SDS-PAGE, and we suggest that NheC was not identified because of its low concentration.
According to Granum et al. (1999), two transmembrane regions in NheB were predicted from the nucleotide sequence, while the predictions for NheA and NheC were no and one transmembrane region, respectively. By comparison with Hbl, where L1 has two transmembrane regions, while B, the binding component, and L2 have one and none, respectively, we expected NheC to be the binding component. Final binding studies of the Hbl components to Vero cells (or other epithelial cells) have not yet been carried out.
The composition of the enterotoxin described in this report is unique. The concentration of each Nhe component is of importance for the function of the enterotoxin, and NheA and NheB together have negligible activity. An increase in the concentration of NheC results in a decrease in toxic activity, possibly due to the binding of NheC to NheB inhibiting the binding of NheB to the receptors. The function of NheC is not yet elucidated, but it seems likely that it functions as some kind of catalyst, either by bringing NheA and NheB together (after binding of NheB to the target cells), or by enhancing conformational changes. The structure of the Hbl complex has not been resolved, although a 1 : 1 : 1 ratio has been postulated, so it would be of great interest to further study the difference between the two three-component enterotoxins produced by B. cereus.
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ACKNOWLEDGEMENTS |
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Received 25 May 2004;
revised 10 August 2004;
accepted 30 August 2004.
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