Factors involved in the down-regulation of cytochrome P450 during Listeria monocytogenes infection

Int. J. lmmunopharmac., Vol. 16, No. 9, pp. 747-754, 1994 Elsevier Science Ltd Copyright © 1994 International Society for lmmunopharmacology Printed in Great Britain. All rights reserved 0192-0561/94 $7.00 + .00

Pergamon

0192 - 0561(94)E0036-M

FACTORS INVOLVED IN THE DOWN-REGULATION OF CYTOCHROME P450 DURING LISTERIA MONOCYTOGENES INFECTION STEVEN G. ARMSTRONG* and KENNETH W. RENTON Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 (Received 14 September 1993 and in final f o r m 10 March 1994)

Abstract -- The activation of host defense mechanisms has been shown to cause a depression in hepatic cytochrome P450-mediated metabolism in rodents and humans. In a previous study, it was demonstrated that the Gram-positive bacteria Listeria monocytogenes causes a down-regulation of hepatic cytochrome P450 and related substrate metabolism as a result of a pretranslational depression of apoprotein synthesis. The objectives of this study were to determine whether the effect of listeria on hepatocyte cytochrome P450 involves hepatic nonparenchymal cells and whether the hemolysin, secreted only by hemolytic forms of the bacteria, plays any part in mediating this effect. Total cytochrome P450 levels as well as ethoxyresorufin-Odealkylase (EROD*) and benzyloxyresorufin-O-dealkylase (BROD) activities were significantly reduced in hepatic microsomes isolated from mice infected in vivo for 48 h with 15U listeria, whereas the same dose of the avirulent non-hemolytic M3D strain had no effect. Listeria (15U) significantly depressed hepatocyte EROD and BROD activities after 24 h incubations with liver cell cultures containing hepatoeytes and nonparenchymal cells, as the result of both a direct effect on the hepatocyte and an interaction of listeria with hepatic nonparenchymal cells. The M3D strain of listeria had no effect on cytochrome P-450-mediated metabolism in isolated cells, confirming that hemolysin is an essential component of the mechanism responsible for the down-regulation of cytochrome P450 during listeria infections.

Many different immunoactive agents cause a downregulation of hepatic microsomal cytochrome P450 following their administration to rodents (Renton & Mannering, 1976a, b; Kato et al., 1963; Singh & Renton, 1981; Williams & Szentivanyi, 1985). There have also been several documented examples of compromised drug metabolism in infected humans (Fleetham et al., 1978; Clark & Boyd, 1979; Forsyth et al., 1982; Koren & Greenwald, 1985; Greenwald & Koren, 1990). The down-regulation of cytochrome P450 during viral infections is mediated through the production of interferon (Renton, 1981), whereas the depressant effect of endotoxin may involve one or more cytokines such as IL-1, TNF or IL-6 (Ghezzi et al., 1986a,b; Shedlofski et al., 1987; Bertini et al., 1988, 1989; Williams et al., 1991). The down-

regulation of P450 caused by dextran sulfate and latex beads indicates that a factor released from nonparenchymal cells likely affects P450 regulation in adjacent hepatocytes (Soyka et al., 1979; Williams et al., 1981; Peterson & Renton, 1984, 1986). We have previously shown that infection with the Gram-positive bacteria L i s t e r i a m o n o c y t o g e n e s depresses cytochrome P450-mediated metabolism in mice (Azri & Renton, 1987), and we have recently demonstrated that this is the result of a pretranslational depression of cytochrome P450 apoprotein synthesis (Armstrong & Renton, 1993). Neutrophils, Kupffer cells, monocytes and T-cells are involved in host defense responses that are initiated during listeria infection and cytokines such as interferon, IL-1, and TNF appear to play a role in the immune

*Author to whom correspondence should be addressed, at: Hamilton Regional Cancer Centre, 699 Concession Street, Hamilton, Ontario, Canada L8V 5C2. *Abbreviations - - BHI, brain-heart infusion broth; BROD, benzyloxyresorufin-O-dealkylase; CFU, colony forming units; EROD, ethoxyresorufin-O-dealkylase; PBS, phosphate-buffered saline; poly(rI.rC), polyriboinosinic.polyribocytidylic acid; TSA, tryptic soy agar. 747

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S. G. ARMSTRONGand K. W. RENTON

response that is mounted against the bacterium (Kauffmann, 1987; Havell, 1987; Petit et al., 1988). The objectives of this study were to determine whether the effect of listeria on cytochrome P450 results from a direct effect on the hepatocyte or involves hepatic non-parenchymal cells, and to determine the role played by the hemolysin secreted only by hemolytic forms of the bacteria.

EXPERIMENTAL PROCEDURES

Materials Ethoxyresorufin was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.) and benzyloxyresorufin was purchased from Molecular Probes Inc. (Eugene, OR, U.S.A.).

Animals and treatments Male Swiss BALB/c mice ( 2 5 - 3 5 g) were obtained from Charles River Laboratories (Montreal, Quebec, Canada). The mice were maintained (five/cage) on clay chip bedding and allowed to acclimatize in our facility for at least 1 week before being used for experiments. Animals were fed standard Purina Rat Chow and water ad libitum. During the period of infection, mice were maintained in cages covered with protective filter bonnets in a laminar flow hood. Listeria monocytogenes strain 15U (serotype 4b), isolated from an infected patient, was supplied by Dr Robert Bortolussi (Bortolussi et al., 1984), Izaak Walton Killam Hospital (Halifax, Nova Scotia, Canada). The non-hemolytic strain of the bacteria (M3D) was supplied by Dr S. Kathariou (Kathariou et al., 1987), Centres for Disease Control (Atlanta, GA, U.S.A.). Aliquots of listeria were stored at - 7 0 ° C in b r a i n - h e a r t infusion broth (BHI) containing 20°7o glycerol. For each experiment, an aliquot of listeria was thawed and 50 tal was added to 10 ml of BHI and grown for 18 h at 37°C. The bacterial suspension was then centrifuged at 1000 x g for 15 min and the bacteria were resuspended in sterile PBS, pH 7.4. The bacteria were then washed three times using 5 rain centrifugation periods. Bacterial concentrations were determined by measuring the absorbance of a suspension using a wavelength of 620 nm; 0.6 absorbance units is equivalent to 5 x 108 colony forming units (CFU)/ml. Mice were infected by the intraperitoneal (i.p.) administration of listeria suspended in PBS.

Microsomal drug biotransformation Hepatic microsomes were prepared as previously described (el Defraway el Masry & Mannering, 1974), suspended (50o7o suspension by liver weight) and homogenized in glycerol buffer (25% 200 mM KH2PO4, pH 7.4, 20% glycerol, 0.23% KCI) and then stored until use at - 7 0 ° C . Cytochrome P450 content was determined by the method of Omura & Sato (1964), and protein by the method of Lowry et al. (1951). Ethoxyresorufin-O-dealkylase, and benzyloxyresorufin-O-dealkylase activities were determined by the method of Burke et al. (1985). Histological examination o f liver sections Centrilobular sections of liver were removed from both control and infected mice. The sections were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for approximately 4 h. The sections were then post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate for 1 h. Sections were dehydrated using graded alcohols and embedded in TAAB embedding resin (Marivac Limited, Halifax, Nova Scotia, Canada). Sections (60 nm) were stained with uranyl acetate and lead citrate and were examined using a Phillips 300 electron microscope. In vitro incubation o f microsomes with 15U listeria Pooled hepatic microsomes from control mice were incubated at 37°C with 1 x 10 ' CFU of 15U listeria or PBS for 12 h, after which penicillin (100U/ml) and streptomycin (100~g/ml) were added. After a total of 24 h of incubation, the microsomes were analyzed for protein content, P450, EROD, and BROD as described above. Hepatocyte isolation and culture Hepatocytes were prepared by the method of Renton (1987). Aliquots of freshly isolated liver cells were placed in 75 cm 2 culture flasks (5 x 106 viable hepatocytes/flask along with non-parenchymal cells) in DMEM media supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.5 g/1 albumin, 8 mg/1 insulin, and 30 mg/1 moxalactam. The remaining portion of the cell preparation was centrifuged four times at 50 x g x 1 min to obtain a hepatocyte-enriched fraction at a hepatocyte purity greater than 90%, which was then split into two flasks (5 x 106 viable hepatocytes/flask). After 3 h of incubation in DMEM media, non-adherent cells were discarded, and treated flasks were infected with 1 X 1 0 4 CFU of 15U listeria or 1 x 105 CFU of

Listeria-mediated Depression of Hepatocyte Cytochrome P450 M3D listeria; control flasks received an equal volume of sterile PBS. The reason for using a higher initial dose of the M3D strain was that it grows at a slower rate than the 15U strain in DMEM media at 37°C. After a 12 h incubation period, a combination of penicillin (200 U/ml) and streptomycin (200/ag/ml) was added; gentamicin (200/ag/ml) was also added to M3D-containing flasks (and the corresponding control flasks) since this non-hemolytic mutant is streptomycin resistant (Kathariou et al., 1987). The incubations were terminated 24 h after the addition of the listeria and cells were removed from the surface of the flasks with a solution of 0.125°70 trypsin and 0.5 mM EDTA. The trypsinized cells were then added to DMEM media containing 10°70 fetal calf serum and centrifuged at 50 x g x 2 rain. The supernatant was discarded and the hepatocyte pellet was resuspended in PBS. The cells were centrifuged twice more at 50 × g x 2 min and the final hepatocyte pellet was resuspended in PBS and used for the determination of EROD and BROD activities. Cellular DNA content was determined by the method of Labarca & Paigen (1980), using calf thymus DNA as a standard.

Hepatocyte E R O D and BROD activities Aliquots of 0.2 x 106 viable hepatocytes, infected with 15U (1 x 104 C F U ) o r M3D (1 x 105 CFU) listeria for 24 h, were diluted to 3.2 ml with PBS, pH 7.4. Ethoxyresorufin (40/al of 31 /~M in 10070 DMSO) was added to give a final substrate concentration of 0.4taM and a final DMSO concentration of 0.1%. This cell solution was incubated at 37°C for 2 rain after which the reaction rate was monitored at an excitation wavelength of 510 nm and an emission wavelength of 586 nm, during constant gentle mixing of the cells with a stirring device that fitted on to the top of the glass cuvettes; reaction rates were determined in the presence of 0.4 mM NADPH (in PBS, pH 7.4). EROD activities were expressed as nmoles resorufin formed/per min//ag D N A . BROD activity in the cell culture experiments was determined as follows: 0.1 x 10 6 viable hepatocytes infected with 15U (1 × 104 C F U ) o r M3D (1 x 105 CFU) listeria for 24 h were diluted to 3.2 ml with PBS, pH 7.4. Benzyloxyresorufin (50/al of 10/aM in 10070 DMSO) was added to give a final substrate concentration of 0.15/aM and a final DMSO concentration of 0.1°70. An identical procedure to that used for assessing EROD activity (as noted above) was then followed, except that the excitation wavelength was 530nm. BROD

activities were expressed formed/per min//ag DNA.

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Determination o f the liver burden o f bacter& After the specified infectious period, livers were homogenized, serial dilutions of the homogenates were prepared in PBS, and 100/al of each dilution was added to a 5°/o TSA (tryptic soy agar) blood agar plate. The plates were incubated at 37°C for 24 h and the number of CFU of listeria was counted. To determine liver burden in the cell culture experiments, 100/al of cell culture media was obtained from each flask, immediately before the addition of penicillin and streptomycin, and diluted 1 : 10,000 with sterile PBS. A 100/al aliquot of this solution was added to a 5°70 TSA blood agar plate and colonies were enumerated 24 h later. Stat&tical methods The unpaired Student's t-test was used for statistical comparison between the treated group and the corresponding control group for all in vivo experiments. A paired t-test was used for statistical comparison in all in vitro cell culture experiments. The statistical comparison of differences in hepatocyte viability was carried out using a paired-sample t-test of the arcsine transformed data. A P-value less than 0.05 was considered significant. All error bars on the graphs represent the S.E.M.

RESULTS

The effect o f in vivo lister& infection on hepatic cytochrome P450 The time course for the loss of hepatic microsomal cytochrome P450 during an infection with 1 x 10 6 colony forming units (CFU) of a hemolytic and virulent strain of L. monocytogenes (15U) is shown in Fig. 1. A significant reduction in cytochrome P450 was observed after 48 h of infection and this suppression was maintained for at least 3 days. An avirulent non-hemolytic strain of listeria (M3D) had no effect on microsomai cytochrome P450 content after 48 h of infection, as shown in Fig. 2. EROD and BROD were also significantly reduced to approximately 40 - 50% of control values by the 15U strain but the M3D strain had no significant effect on these parameters (Fig. 2). Although the same dose of the two strains of listeria was administered in each of the in vivo experiments, the respective bacterial liver loads at the time of sacrifice were significantly

750

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Fig. 1. Time course of the effect of infection with 15U listeria on hepatic cytochrome P450 levels. Mice were infected with 1 x 106 CFU of 15U listeria for 2 4 - 9 6 h; control mice received an equal volume of sterile PBS. Cytochrome P450 levels were determined in hepatic microsomes and are expressed as nmoles P450/mg protein. Statistical analysis was carried out on the raw data using an unpaired Student's t-test. *Significantly different from corresponding control, P<0.05 (four or five mice/group). different. A f t e r 48 h of infection with the 15U strain, the average n u m b e r o f b a c t e r i a per liver was 2.0 × 104 C F U , whereas no detectable colonies o f the M 3 D strain were observed after the same infectious period. W h e n the i n o c u l a t i o n dose of the M 3 D strain was increased to 1.0 × 108 C F U , the levels o f P450 r e m a i n e d u n a f f e c t e d a n d the average liver load of the b a c t e r i a was 9.0 × 1 0 3 C F U / | i v e r . After 48 h of in vivo infection with 1 x 1 0 6 C F U o f 15U listeria the mice did n o t d e m o n s t r a t e o b v i o u s b e h a v i o r a l changes a n d previous studies h a v e s h o w n t h a t the mice were not febrile (Azri & R e n t o n , 1987). N o c h a n g e in b o d y weight or liver weight occurred a n d histological e x a m i n a t i o n of liver sections t a k e n f r o m mice after 65 h o f infection did not indicate a n y pathological changes. T h e direct i n c u b a t i o n o f 15U listeria (1 × 104 C F U ) with hepatic microsomes for 24 h at 37°C h a d n o effect o n total c y t o c h r o m e P450, or E R O D or B R O D activities (data not shown).

The effect o f in vitro listeria infection on hepatocyte cytoehrome P450-mediated metabolism A h e p a t o c y t e cell culture system was established to c o m p a r e the effects o f the hemolytic (15U) a n d n o n hemolytic (M3D) f o r m s of listeria at c o m p a r a b l e levels o f bacterial b u r d e n , in the presence a n d absence of hepatic n o n - p a r e n c h y m a l cells. T h e 15U

15U

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Fig. 2. Effect of 48 h of infection with listeria on hepatic microsomal cytochrome P450 and related substrate metabolism. Mice were infected with 1 x 106 CFU of either 15U or M3D listeria for 48 h. Hepatic microsomal cytochrome P450 content, EROD, and BROD activities were determined and expressed as a °70 of the mean control levels determined in mice treated for the same period with an equal volume of sterile PBS. The mean control levels were: cytochrome P450 = 0.73 _+ 0.04 nmoles/mg protein; EROD = 45.8 _+ 8.2 pmoles resorufin/mg/min; and BROD = 4.5 _+ 0.4 pmoles resorufin/mg/min. Statistical analysis was carried out on the raw data using an unpaired Student's t-test. *Significantly different from corresponding control, P<0.05 (four or five mice/group). strain of listeria h a d no effect o n E R O D activity in hepatocyte-enriched cultures b u t evoked a significant loss o f E R O D activity w h e n i n c u b a t e d with cultures c o n t a i n i n g hepatocytes a n d n o n p a r e n c h y m a l cells (Fig. 3). The M 3 D strain h a d n o effect on E R O D activity in either type o f prep a r a t i o n (Fig. 3). T h e 15U strain of listeria evoked a reduction in B R O D activity in hepatocyte-enriched cultures a n d in cultures containing hepatocytes along with n o n p a r e n c h y m a l cells, whereas the M 3 D strain h a d no effect o n B R O D activity in either type o f culture (Fig. 4). T h e average n u m b e r of Listeria bacteria present in liver cell cultures (at t = 12 h) was c o m p a r a b l e between the two strains; 8.8 x 104 C F U / m l for the 15U strain a n d 5.1 x 104 C F U / m l for the M 3 D strain, with neither f o r m p r o d u c i n g a change in viability or total cell protein. DISCUSSION A variety o f infections a n d m o d e s of i m m u n e s t i m u l a t i o n have b e e n s h o w n to affect c y t o c h r o m e P450-mediated m e t a b o l i s m . These effects appear to

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Fig. 3. Effect of 15U and M3D listeria on EROD activities in liver cell cultures. Hepatocyte-enriched cultures or cultures of hepatocytes along with hepatic nonparenchymal cells were prepared as described in Experimental Procedures. After a 24 h incubation with 15U (1 × 104 CFU) or M3D (1 × 105 CFU) listeria, EROD activity was determined in the listeria-treated hepatocytes and expressed relative to the corresponding control hepatocytes. The mean control hepatocyte EROD activities were 1.6 _+ 0.3 and 1.8 _+ 0.3 pmoles//ag DNA/min in hepatocyte-enriched and hepatocyte + nonparenchymal cell cultures, respectively. Statistical analysis was carried out on the raw data using a paired t-test. *Significantly different from corresponding control, /)<0.05 (four experiments for 15U and three experiments for M3D).

Fig. 4. Effect of I5U and M3D listeria on BROD activity in liver cell cultures. Hepatocyte-enriched cultures or cultures of hepatocytes along with hepatic nonparenchymal ceils were prepared as described in Experimental Procedures. After a 24 h incubation with 15U (1 x i 0 4 CFU) or M3D (1 × 105 CFU) listeria, the BROD activity was determined in the listeria-treated hepatocytes and expressed relative to the corresponding control hepatocytes. The mean control hepatocyte BROD activities were 0.34 +_ 0.06 and 0.32 _+ 0.10 pmoles/gg DNA/min in hepatocyte-enriched and hepatocyte + nonparenchymal cell cultures, respectively. Statistical analysis was carried out on the raw data using a paired t-test. *Significantly different from corresponding control, P<0.05 (four experiments for 15U and three experiments for M3D).

involve cytokines and a down-regulation of P450 synthesis at a pretranslational step (Renton, 1981; Ghezzi et al., 1986a, b; Shedlofski et al., 1987; Bertini et al., 1988, 1989; Renton & Knickle, 1990; M o r g a n & N o r m a n , 1990; Craig et al., 1990; Wright & Morgan, 1990; Morgan, 1991). We have recently shown that the P450 down-regulation that occurs during listeria infections also involves a p r o f o u n d loss in m R N A and a depression in the rate of apoprotein synthesis (Armstrong & Renton, 1993). The present study attempted to identify some of the factors that lead to this effect. Histological examination of liver sections during listeria infection did not indicate any pathological change, and the direct incubation of 15U listeria with hepatic microsomes did not result in a destruction of cytochrome P450 apoproteins, consistent with previous reports that have shown that interferon and endotoxin also have no effect when incubated directly with hepatic microsomes (Parkinson et al., 1982; Egawa & Kasai, 1979). These observations indicate that the mechanism by which 15U iisteria decreases P450-mediated metabolism cannot be due to a direct

destruction of P450 in the endoplasmic reticulum, nor is it merely an extension of overt liver damage. The hemolysin secreted by the 15U strain of the bacteria is thought to be a principal virulence factor as a result of its ability to disrupt the phagocytic membrane allowing escape of the bacterium into the cytoplasm of host cells and subsequent spread to neighboring cells. A dose of 1 × 1 0 6 C F U of 15U listeria resulted in a significant depression of total cytochrome P450 content, E R O D , and B R O D activities after 48 h of in vivo infection, but the nonhemolytic avirulent M3D strain had no effect. The average liver load of the 15U strain at the time of sacrifice was 2.0 × 104 C F U / l i v e r , but the bacterial liver burden was undetectable following the same dose of the M3D strain. It is therefore possible that the maintenance of a critical liver burden of bacteria is a prerequisite for an effect on cytochrome P450. We were unable to determine whether the lack of effect on cytochrome P450 during infection with the M3D strain was due to its rapid clearance or the lack o f hemolysin production. Attempts to achieve equal in vivo liver loads of the two strains of the bacteria

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S.G. ARMSTRONGand K. W. RENTON

by increasing the dose of the M3D form of listeria were not successful. This was due to the rapid elimination of this non-hemolytic strain that has been documented previously (Kathariou et al., 1987; Gaillard et al., 1986; Berche et al., 1987). The average reversion rate (spontaneous loss of the transposon and return to the hemolytic phenotype) of this mutant was determined to be 3 × l0 4 (Kathariou et al., 1987), and therefore the administration of doses greater than 1 × l08 CFU carries the risk of the production of enough hemolytic revertants to initiate an infection and immune response characteristic of the hemolytic strain. For this reason, in vivo experiments with the M3D strain using higher doses could not be carried out and experiments that investigated the role of bacterial liver burden and hemolysin production were carried out in a liver cell culture system, in which the number of bacteria added to the cells could be controlled and maintained. The hemolytic 15U strain of listeria resulted in a significant loss of EROD and BROD activities in liver cell cultures containing hepatocytes and nonparenchymal cells, but a comparable bacterial burden of the M3D strain had no effect. This suggested that the presence of the hemolysin is an essential component of the mechanism. The 15U strain of listeria produced a 25% loss of EROD activity when incubated with cultures containing hepatocytes and non-parenchymal cells, but had no effect on EROD activity in hepatocyte-enriched cultures. The loss of EROD activity is therefore most likely due to an indirect effect of 15U listeria and/or the hemolysin it secretes on the non-parenchymal cells. The adherent non-parenchymal cells are predominantly Kupffer cells and thus these data are consistent with a mechanism that involves the release of a factor from Kupffer cells. In contrast, BROD activity was decreased by 25 - 30% by the 15U strain in cultures containing hepatocytes and non-parenchymal cells as well as in hepatocyte-enriched cultures. BROD activity can therefore be depressed as a result of a direct action of the bacterium and/or

the hemolysin it secretes on the hepatocyte itself. The M3D form of listeria had no effect on EROD or BROD activities in either type of preparation. These studies establish that listeria is capable of suppressing cytochrome P450-mediated metabolism in the hepatocyte by both direct and indirect mechanisms. The differential effect suggests varying susceptibilities of different isozymes of cytochrome P450. Neutrophils, Kupffer cells, monocytes and T-cells are involved in host defense responses that are initiated during listeria infection, and cytokines such as interferon, IL-1, and TNF appear to play a role in the immune response that is mounted against the bacterium (Kauffmann, 1987; Havell, 1987; Petit et al., 1988). Similarly, monocyte or macrophagederived cytokines such as interleukin-1, TNF or IL-6 are involved in the depression of cytochrome P450 that is observed after immune stimulation with agents such as endotoxin (Ghezzi et al., 1986a; Bertini et al., 1989; Dinarello, 1984; Sujita et al., 1990; Beulter et al., 1986; Heinrich et al., 1990). Previous studies from our laboratory have demonstrated that factors released from phagocytic cells after stimulation with dextran sulfate and latex beads are also capable of suppressing cytochrome P450 in hepatocytes (Peterson & Renton, 1984, 1986). This accumulation of evidence suggests that many factors released from phagocytic cells are capable of suppressing drug metabolism in hepatocytes, and supports the idea that infective agents can lower cytochrome P450 in the hepatocyte by an indirect action on other cell types. Our study supports this dogma but also shows that listeria can depress the activity of at least one isozyme as a result of a direct effect on the hepatocyte. Acknowledgements - - This research was supported by the Medical Research Council of Canada. Dr Armstrong is a recipient of a Studentship from the Faculty of Graduate Studies, Dalhousie University. We wish to thank Elaine Wright for excellent technical assistance and Drs Robert Bortolussi and Sophia Kathariou for supplying us with Listeria monocytogenes.

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