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B-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes
Department of Food Science, Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA
Correspondence
Martin Wiedmann
mw16{at}cornell.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative B-dependent genes based on the presence of a predicted B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes 
sigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes sigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.
-glucuronidase; MU-, methylumbelliferone |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Symptoms of systemic listeriosis include septicaemia, meningitis and spontaneous abortion. Individuals with compromised immune systems, neonates and the elderly are at the highest risk of contracting listeriosis (Farber & Peterkin, 1991). L. monocytogenes is considered ubiquitous in nature. This organism survives and multiplies under a wide spectrum of environmental conditions, including in food-processing plants and in a variety of ready-to-eat food products. Growth of L. monocytogenes has been documented at temperatures ranging from -0·4 to +45 °C (Farber & Peterkin, 1991) and survival has been observed under acidic conditions (e.g. pH 2·5) and in solutions of high (10–20 % NaCl) osmolarity (Cole et al., 1990; Sleator et al., 2000). L. monocytogenes may encounter refrigeration temperatures, low pH and high osmolarity at various stages of food processing (Farber & Peterkin, 1991; Lou & Yousef, 1999). Following consumption of a contaminated food product by a potential host, L. monocytogenes encounters additional stressful environmental conditions during gastrointestinal passage, including exposure to proteolytic enzymes, the stomach's acidic environment (as low as pH 2·0), bile salts (Davenport, 1982) and an intestinal osmolarity equivalent to 0·3 M NaCl (Chowdhury et al., 1996).
Bacteria can alter gene expression patterns at appropriate times in response to changing environments and stressful conditions. Both Gram-positive and Gram-negative bacteria have the ability to regulate patterns of gene expression at the transcriptional level (Becker et al., 2000; Fang et al., 1992; Helmann et al., 2001). The holoenzyme RNA polymerase (RNAP) catalyses the transcription of DNA into mRNA (Burgess et al., 1969). A sigma factor is a protein subunit of RNAP that is required for recognition of specific promoter sequences and for initiation of transcription (Helmann & Chamberlin, 1988). The association of different alternative sigma factors with RNAP is one mechanism that enables a bacterial cell to rapidly induce expression of specific genes within a regulon in response to specific stimuli. The general stress-responsive alternative sigma factor S has been identified in many Gram-negative bacteria, including Escherichia coli, Salmonella spp. and Yersinia spp. (Badger & Miller, 1995; Fang et al., 1992; McCann et al., 1991). In both E. coli and Salmonella Typhimurium, S plays a crucial role in protection against conditions of starvation, hyperosmolarity, oxidative and acid stresses (Cheville et al., 1996; Small et al., 1994). For Gram-positive bacteria, the general stress-responsive alternative sigma factor B was first identified and characterized in Bacillus subtilis (Boylan et al., 1993). The B-dependent general stress regulon of B. subtilis consists of well over 100 genes that are induced by exposure to stressful conditions such as heat, acid, ethanol or high osmolarity, or by deprivation of glucose, oxygen or phosphate (Helmann et al., 2001; Petersohn et al., 2001; Price et al., 2001). Previous studies have demonstrated roles for stress-responsive sigma factors in regulating expression of virulence genes in some bacterial pathogens, including Staphylococcus aureus, Yersinia enterocolitica and Salmonella (Deora et al., 1997; Humphreys et al., 1999; Kullik et al., 1998), suggesting a link between stress response and virulence in these organisms.
Mounting evidence also supports an association between the ability of L. monocytogenes to survive exposure to environmental stresses and to infect host cells. For example, B contributes to L. monocytogenes survival and growth under certain environmental stress conditions [e.g. acid stress, low-temperature stress, salt stress (Becker et al., 1998, 2000)], as well as to persistence within a host and to host cell infection (Nadon et al., 2002; Wiedmann et al., 1998). The stress-responsive compatible solute transporter opuCA has also been demonstrated to contribute to host infection in an animal model (Sleator et al., 2001). While transcription of selected L. monocytogenes genes (e.g. opuCA, lmo1421) has been shown to be reduced in a sigB null mutant background (Ferreira et al., 2003; Fraser et al., 2003), the temporal nature of B-dependent contributions to transcription induction of these genes has not yet been quantified. To test our hypothesis that B coordinates a rapid response that aids L. monocytogenes in survival of environmental and host-imposed stress conditions, we identified and confirmed the B dependence of L. monocytogenes genes that had previously been demonstrated to contribute to survival under these conditions and characterized their induction and expression patterns. Specifically, we confirmed the B dependence of L. monocytogenes opuCA, lmo1421 and bsh. These genes represent general stress-response genes (opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively) and a virulence gene [bsh encodes a conjugated bile salt hydrolase (BSH)]. We have shown that all three genes are expressed under conditions of environmental stress and that expression of opuCA and lmo1421 is induced following exposure to salt stress.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Listeria innocua wild-type strain DD680 was used in the BSH assay only.
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B-dependent expression of the target genes in stationary-phase cells, a L. monocytogenes wild-type strain (10403S) and a sigB null mutant (sigB) were grown for 12 h (30 °C, shaking at 250 r.p.m.) in Brain–Heart Infusion Broth (BHI). Stationary-phase cells grown in this manner were harvested and used for total RNA isolation and subsequent RT-PCR experiments as described below. To monitor target gene expression throughout growth, overnight cultures of the L. monocytogenes wild-type and sigB strains grown to stationary phase as described above were diluted 1 : 1000 into 10 ml of BHI broth and incubated at 30 °C (shaking at 250 r.p.m.). When these cultures reached an OD600 value of 0·4, they were diluted again (1 : 200) into a side-arm flask containing 150 ml of BHI and incubated at 30 °C with shaking at 250 r.p.m. When these cultures reached an OD600 value of 0·4, 0·8 or 0·8+1 h, 20 ml of broth were centrifuged (8800 g) and the harvested cells were used for total RNA isolation and RT-PCR experiments.
For detection of BSH activity, the L. monocytogenes wild-type, L. innocua wild-type (negative control) and sigB null mutant strains were spotted onto deMan, Rogosa and Sharpe (MRS) agar medium (BD Biosciences) containing 0·5 % (w/v) glycodeoxycholic acid (Sigma) as originally described by Dashkevicz & Feigner (Dashkevicz & Feighner, 1989; Dussurget et al., 2002).
Salt-stress conditions.
To monitor induction of target gene expression following exposure to salt stress, bacterial strains were grown to mid-exponential phase in BHI as described above. Specifically, 20 ml aliquots of cells grown to an OD600 value of 0·4 were centrifuged as described above, then each pellet was resuspended in 20 ml of 0·154 M NaCl (representing a physiological salt concentration of 0·9 %, w/v; pH 5·9), 0·5 M NaCl (pH 5·8) or 0·5 M KCl (pH 5·8). Following exposure times of 15, 30, 60 or 120 min, 1 ml samples of the wild-type and sigB opuCA–gus fusion strains (FSL S1-063 and FSL S1-059, respectively) were collected for -glucuronidase (GUS) activity measurement, as described below.
L. monocytogenes wild-type and sigB strains (10403S and FSL A1-254, respectively) were also exposed to 0·121 M KCl (0·9 %) and 0·5 M KCl as described above. Samples were collected at 5, 10 and 15 min post-exposure for total RNA isolation and RT-PCR.
Total RNA isolation.
For the RT-PCR experiments, total RNA was purified from cells collected during exposure to salt stress and throughout growth, as described above. Bacterial cells collected at the specified time points were centrifuged and immediately resuspended in 10 ml Trizol reagent (Invitrogen) per 30 ml of culture harvested. The resuspension was immediately placed on ice and sonicated for three 20 s intervals (output: 20 W) using a Sonicator 3000 (Misonix). A 20 ml aliquot of chloroform (Shelton Scientific) was added for each 10 ml of original cell culture. After vigorous vortexing and 10 min incubation at room temperature, tubes were centrifuged (2190 g) for 60 min. Nucleic acids from the aqueous layer were precipitated with an equal volume of 2-propanol and centrifuged (17 900 g). The resulting pellets were washed twice with 100 % ethanol, resuspended in RQ1 10x DNase Buffer and treated with RQ1 DNase (Promega). Nucleic acids were subsequently purified by phenol/chloroform extraction and ethanol precipitation with 0·3 M sodium acetate (Sambrook et al., 1989). DNase treatment, phenol/chloroform extraction and ethanol precipitation steps were repeated two additional times to remove any contaminating DNA. The final RNA pellet was resuspended in 60 µl diethyl pyrocarbonate (DEPC)-treated water (Invitrogen). Total nucleic acid concentrations were estimated using absorbance readings (260 nm/280 nm) on a DU Series 600 Spectrophotometer (Beckman Coulter).
RT-PCR.
Reverse transcription was performed using the Superscript First-Strand Synthesis RT-PCR System (Invitrogen) with 50 ng of total RNA for each reaction. Primers to amplify opuCA, lmo1421, bsh and rpoB, which were designed using PRIMEREXPRESS software (Applied Biosystems), are shown in Table 2. Reverse transcription reactions were cycled once at 42 °C for 50 min and then at 70 °C for 15 min. PCR amplification of cDNA was performed using 10 µl of each reverse transcriptase reaction and the AmpliTaq Gold DNA Polymerase system (Applied Biosystems). PCR cycling conditions included an initial 9 min hold at 95 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, 30 s at 72 °C, and a final hold of 5 min at 72 °C. Reverse transcription and PCR amplification reactions were performed in a GeneAmp 9600 (Perkin Elmer). To monitor for possible contamination by genomic DNA, an aliquot of each RT-PCR was run in the absence of Superscript II enzyme. RT-PCR products (10 µl) were subjected to gel electrophoresis using 3 % Metaphor (BioWhittaker Molecular Applications) agarose gels. The pGEM DNA ladder was used as a molecular mass marker and as a standard for PCR product quantification.
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B-dependent in L. monocytogenes, served as an internal control gene for these RT-PCR experiments (see Results). To account for variation between RT-PCRs and RNA collections, the DNA quantities from lmo1421, opuCA and bsh RT-PCR were each normalized to the quantities of rpoB RT-PCR products that were generated in each experiment. For RT-PCR analysis of opuCA throughout growth, opuCA transcripts were quantified as relative opuCA product quantities normalized to rpoB quantities at corresponding time-collection points. For the salt-stress induction experiments, DNA quantities for lmo1421, opuCA and bsh RT-PCR products were also normalized to rpoB RT-PCR product quantities to give relative RT-PCR product quantities. To measure induction, the normalized RT-PCR product quantities from cells exposed to salt stress (0·5 M KCl) were divided by the corresponding normalized RT-PCR product quantities for non-exposed cells (0·121 M KCl).
Quantitative GUS assay.
Assays for GUS activity were performed essentially as described by Youngman (Harwood & Cutting, 1990). Specifically, cells from 1 ml aliquots were pelleted and washed in 1 ml Buffer AB Light (60 mM K2HPO4, 40 mM KH2PO4, 0·1 M NaCl, pH 7·0). The washed cell pellet was resuspended in 400 µl of Buffer AB Light for GUS assay and standard plate counts on BHI agar. To quantify GUS activities, 100 µl of the cell suspension were thoroughly mixed with 100 µl of Buffer AB Plus (Buffer AB Light containing 0·2 % Triton X-100) and incubated at room temperature for 60 min to lyse the cells. In a black, flat-bottomed Packard OptiPlate 96-well plate (Perkin Elmer), 50 µl of the lysed cells were mixed with 10 µl of 4-methylumbelliferyl--D-glucuronide (4-MUG; Sigma) in 0·4 mg ml-1 DMSO (Fisher Scientific) and held at room temperature for at least 60 min. Exact incubation times were recorded to calculate activity units as described below. Fluorescence was measured in a Packard Fusion Instrument (Perkin Elmer) using an excitation filter of 360 nm and an emission filter of 460 nm. Fluorescence units were converted to picomoles of methylumbelliferone (MU-) using a standard curve of known MU- (Sigma) concentrations. GUS activities were expressed in activity units defined as picomoles of 4-MUG hydrolysed per millilitre of cells at OD600=1·0, per minute. For salt-stress experiments, percentage changes between GUS activities of the salt-exposed cells (0·5 M NaCl and 0·5 M KCl) and non-exposed cells (0·154 M NaCl) were calculated at each collection time point. The one-sample t-test was used to identify significant differences in activity units between samples exposed to the different test conditions at each time point. Normality of observations was satisfied using the Anderson–Darling test (P<0·05). All statistical analyses were performed using MINITAB version 13 (Minitab).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B-dependent expression of opuCA, lmo1421 and bshB-dependent promoters upstream of opuCA, which encodes a component of an osmoprotectant ABC (ATP binding cassette) transporter (Fraser et al., 2000), and upstream of lmo1421, which encodes a putative ABC transporter (Fraser et al., 2003), had been identified previously by sequence analyses. Our group applied a Hidden Markov model to identify putative B-dependent promoters (Kazmierczak et al., 2003) in the published L. monocytogenes EGD genome (Glaser et al., 2001). The results of the search confirmed the presence of predicted B-dependent promoters upstream of opuCA and lmo1421 and identified a predicted B-dependent promoter upstream of bsh. RACE (Rapid Amplification of cDNA Ends) PCR analysis performed in our laboratory confirmed the existence and location of these B-dependent promoters upstream of opuCA, lmo1421 and bsh in L. monocytogenes 10403S (Kazmierczak et al., 2003). All three promoters exhibit high similarity to previously identified B-dependent promoters in L. monocytogenes, B. subtilis and S. aureus (Table 3). RT-PCR primers targeting opuCA, lmo1421 and bsh were designed to allow semi-quantitative evaluation of mRNA expression levels for these three genes. In addition, RT-PCR primers were designed for amplification of rpoB, which does not exhibit an upstream, predicted B-dependent promoter. RT-PCR on total RNA isolated from stationary-phase L. monocytogenes wild-type and sigB cells clearly demonstrated that opuCA, lmo1421 and bsh transcript levels are much higher in the L. monocytogenes wild-type strain (10403S) than in the sigB strain (Fig. 1). RT-PCR amplification of rpoB showed visually indistinguishable rpoB transcript levels for both stationary-phase wild-type and sigB L. monocytogenes cells.
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sigB) were used to independently measure expression of opuCA in the presence and absence of an intact sigB, respectively. At stationary phase, strain FSL S1-063 exhibited a GUS activity of 754 pmol MU- ml-1 min-1 (log c.f.u.)-1, while the sigB null mutant strain showed virtually no GUS activity [4·5 pmol MU- ml-1 min-1 (log c.f.u.)-1].
BSH assay
To confirm the role of B in expression of bsh, which encodes a conjugated BSH (Dussurget et al., 2002), L. monocytogenes wild-type 10403S, L. innocua DD608 (as a negative control) and the sigB strain FSL A1-254 were spotted onto MRS medium agar containing 0·5 % glycodeoxycholic acid. A heavy white precipitate of free bile salt was observed only around the L. monocytogenes wild-type strain, but not around the sigB or the L. innocua strains, indicating the presence of BSH activity only in the wild-type strain, but not in the others (Fig. 2).
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). Briefly, opuCA expression increases in a growth-phase-dependent fashion as wild-type L. monocytogenes 10403S approaches stationary phase. opuCA-directed GUS activity is essentially abolished in a sigB strain (Ferreira et al., 2003). In this report, RT-PCR was used as an independent strategy for examining the role of B in L. monocytogenes opuCA expression throughout growth and in stationary phase. opuCA transcript levels for L. monocytogenes 10403S increased in a growth-phase-dependent manner, as assessed by the increase in RT-PCR product band intensity when RNA was harvested from wild-type cells at OD600 values of 0·4, 0·8 and 0·8+1 h during growth in BHI (Fig. 3). No RT-PCR product was visible for the opuCA RT-PCR from RNA isolated from the L. monocytogenes sigB strain grown to an OD600 value of 0·8+1 h, further confirming the B-dependent expression of opuCA. The products from the rpoB RT-PCRs from L. monocytogenes wild-type and sigB cells exhibited consistently strong bands under all conditions (Fig. 3). A semi-quantitative analysis, in which the band intensities for each opuCA RT-PCR were normalized to rpoB band intensities at corresponding collection time points, further confirmed the increase in relative opuCA expression throughout growth. Relative opuCA RT-PCR band intensities increased from 0·67 at OD600=0·4, to 0·83 at OD600=0·8, to 1·15 at OD600=0·8+1 h.
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B-dependent genes after exposure to salt-stress conditions. RT-PCR was used to monitor expression patterns of opuCA, lmo1421 and bsh in the L. monocytogenes wild-type 10403S strain following exposure to salt stress (0·5 M KCl) as compared to a physiological salt concentration (0·121 M KCl) environment. Total RNA for RT-PCR analysis was obtained from exponential-phase bacterial cells exposed to either 0·5 or 0·121 M KCl for 5, 10 or 15 min. RT-PCR product band intensities for these genes were first normalized to RT-PCR band intensities for rpoB, which was used as a control gene. Normalized RT-PCR band intensities for the three B-dependent target genes were then used to calculate relative induction coefficients, which represented the normalized RT-PCR band intensity for RNA from exposed cells (0·5 M KCl) relative to the normalized RT-PCR band intensity for the non-exposed cells (0·121 M KCl) collected at the same time point. Fig. 4 shows the results from two independent salt-stress experiments for the opuCA and lmo1421 RT-PCRs and three independent salt-stress experiments for the bsh RT-PCRs. While these RT-PCR data show a consistent pattern of increased opuCA and lmo1421 mRNA levels after exposure to 0·5 M KCl, we did not observe a reproducible increase in bsh mRNA levels following exposure to 0·5 M KCl.
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shows the mean relative activities calculated from all three experiments. Significant differences (P<0·05) were observed following 15 and 30 min exposure to 0·5 M NaCl, when compared to non-induced cells (Fig. 5). Similarly, cells exposed to 0·5 M KCl for 15 min exhibited a significantly (P<0·05) higher GUS activity relative to non-exposed cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B plays an important role in the general stress response of the food-borne pathogen L. monocytogenes (Becker et al., 2000; Wiedmann et al., 1998). Although sigB null mutants have been characterized phenotypically (Becker et al., 1998, 2000; Ferreira et al., 2001; Nadon et al., 2002; Wiedmann et al., 1998), only three putative B-dependent promoter regions (e.g. promoters for rsbV, opuC and lmo1421) had been described previously in L. monocytogenes (Becker et al., 1998; Fraser et al., 2003). While a putative B-dependent promoter has been found upstream of betL, which encodes a sodium-dependent secondary betaine transporter, both reporter fusion and preliminary RT-PCR experiments have shown that betL expression is not B-dependent (Fraser et al., 2003). Stress-responsive induction has been demonstrated only for rsbV (Becker et al., 1998) and upon entry into stationary phase for opuCA (Ferreira et al., 2003). The objectives of this research were to confirm the B-dependent expression of three putative B-dependent genes (opuCA, lmo1421 and bsh) using RT-PCR and reporter fusions and to monitor the induction and expression patterns of these genes under specific stress conditions, including entry into stationary phase and exposure to salt stress. We confirmed the B dependence of these genes, each of which contributes to the L. monocytogenes general stress response, to virulence, or to both. Our data provide evidence for a broad role of B in L. monocytogenes virulence and stress response and also provide new insight into expression profiles of selected members of the L. monocytogenes B regulon.
Identification of B-dependent genes and promoters
Semi-quantitative RT-PCR and opuCA–gus reporter fusions confirmed opuCA, lmo1421 and bsh as members of the L. monocytogenes B regulon. Previous investigations by Fraser and co-workers identified a putative B-dependent promoter sequence upstream of the opuC operon (Fraser et al., 2000) and lmo1421 and provided initial evidence for B-dependent transcription of these genes (Fraser et al., 2003). Confirmation of opuCA and lmo1421 as B-dependent genes further illustrates the importance of B in regulating transcription of osmotic stress genes in L. monocytogenes. In L. monocytogenes, OpuC is one of several compatible solute transporters (e.g. transporters encoded by betL, gbu) which confer increased osmotolerance to L. monocytogenes under osmotic stress environments (Fraser et al., 2000; Ko & Smith, 1999; Sleator et al., 1999). For example, under chill- and salt-stress conditions (0·5 M KCl), an opuCB mutant strain of L. monocytogenes 10403S exhibited a reduced ability to accumulate carnitine as compared to the wild-type strain (Angelidis et al., 2002). lmo1421 is predicted to encode the ATPase subunit of another ABC compatible solute (choline) transporter, based on sequence similarities to known transporter genes in L. monocytogenes and B. subtilis (Fraser & O'Byrne, 2002). Based on the high sequence homology with the B. subtilis opuBA, which encodes a choline transporter, other groups also have referred to lmo1421 as opuBA (Sleator et al., 2003; Wemekamp-Kamphuis et al., 2002). OpuB has been demonstrated in B. subtilis to contribute to choline uptake (Kappes et al., 1999) and thus is likely to contribute to osmotolerance in L. monocytogenes, although Sleator et al. (2003) proposed a possible role for OpuB in carnitine uptake in L. monocytogenes strain LO28. The demonstrated B dependence of opuCA and lmo1421 is consistent with the previous observation that a L. monocytogenes sigB null mutant shows a reduced ability to accumulate betaine and carnitine under osmotic- and cold-stress conditions (Becker et al., 1998, 2000). Interestingly, opuE, which also encodes an osmoprotectant, is regulated by B in B. subtilis (von Blohn et al., 1997). In combination, these observations support a broad role for B in regulation of osmolyte uptake systems in low-G+C-content Gram-positive bacteria.
In addition to opuCA and lmo1421, we also experimentally identified and confirmed bsh as a B-dependent gene using a semi-quantitative RT-PCR approach. bsh, which encodes a conjugated BSH, was first identified in L. monocytogenes by Dussurget et al. (2002). Using the assay described by Dussurget et al. (2002), we demonstrated that only the L. monocytogenes wild-type strain hydrolysed bile salt, while no visually apparent bile salt hydrolysis was observed in the sigB strain (Fig. 2
). Thus, we phenotypically confirmed the importance of an intact sigB on expression of BSH activity in L. monocytogenes. Since a functional bsh was shown to be required for full virulence in guinea pig infections (Dussurget et al., 2002), our results provide evidence for a role of B in virulence gene expression and virulence in L. monocytogenes. The role of bsh as a virulence gene is further supported by the observation that its expression is regulated by PrfA, a general activator of virulence gene expression in L. monocytogenes (Dussurget et al., 2002).
In combination with other studies, our results provide further evidence for a broad role of L. monocytogenes B during host–pathogen interactions (Milohanic et al., 2003). Specifically, previous work has shown that B directly contributes to the regulation of prfA transcription (Nadon et al., 2002), and that both opuCA and bsh contribute to intra-host survival by L. monocytogenes (Dussurget et al., 2002; Sleator et al., 2001; Wemekamp-Kamphuis et al., 2002). An intact opuC was required for wild-type colonization of the mouse upper small intestine following peroral inoculation by L. monocytogenes LO28 (Sleator et al., 2001; Wemekamp-Kamphuis et al., 2002). Dussurget et al. (2002) described a reduction in the recovery of a L. monocytogenes EGD bsh null mutant as compared to its wild-type parent strain in guinea pig stools 48 h after intragastric inoculation (Dussurget et al., 2002). While bsh appears to be specific to L. monocytogenes and is absent from the non-pathogenic L. innocua and B. subtilis, similarity searches for opuCA and lmo1421 show that homologues for both these genes exist in both B. subtilis strain 168 and L. innocua CLIP11262 (Glaser et al., 2001). Thus, B appears to be involved in the transcriptional regulation of both classical virulence genes (e.g. bsh, prfA) and of general stress-response genes, which are important for bacterial survival during host–pathogen interaction as well as for survival in non-host environments. During gastrointestinal passage, pathogens such as L. monocytogenes experience dramatic changes in environmental conditions, such as exposure to low pH in the stomach and to high osmolarity in the small intestine (Davenport, 1982). As B fulfils functional roles in both virulence and general stress response and as the gastrointestinal environment encountered by L. monocytogenes during passage through a host imposes a set of physiological stresses on bacteria, we hypothesize that B contributes to ensuring appropriate gene expression enabling bacterial survival under these conditions.
Growth-phase-dependent activation of B
Through both a reporter fusion strategy (Ferreira et al., 2003) and semi-quantitative RT-PCR, opuCA was demonstrated to be expressed in a growth-phase-dependent manner. Maximal opuCA-directed GUS activity was observed at entry into the stationary phase of growth (Ferreira et al., 2003), which generally corresponds with cellular exposure to nutrient-limiting and other stress conditions (Kolter et al., 1993). Our results are consistent with those of Becker et al. (1998), who used primer extension analysis of the B-dependent rsbV promoter to show induction of B activity following entry into stationary phase. Studies in B. subtilis have also shown induction of B activity upon entry into stationary phase (Boylan et al., 1993; Varon et al., 1996). Growth-phase-dependent expression of opuCA thus follows the typical B-dependent expression profiles previously established for the B-dependent rsbV in L. monocytogenes and for B-dependent genes in other Gram-positive bacteria.
Induction of B-dependent genes during salt stress
To further examine induction of B-dependent genes under osmotic-stress conditions, we used opuCA–gus transcriptional gene fusions as well as semi-quantitative RT-PCR to monitor transcription of opuCA, bsh and lmo1421. Salt-stress conditions were selected for this study, as L. monocytogenes is likely to experience stress of this nature under food-processing conditions (e.g. in brines) and in food products associated with L. monocytogenes contamination (e.g. smoked fish and brined meat products) as well as during intra-host survival (Davenport, 1982). RT-PCR assays showed a rapid induction of opuCA and lmo1421 under salt-stress conditions (i.e. within 5 min of salt-stress exposure). opuCA induction was also further confirmed by GUS reporter fusion assays. These data are consistent with preliminary primer extension experiments by Becker et al. (1998), who showed increased transcription of the B-dependent rsbV promoter following salt stress in L. monocytogenes. Osmotic stress has also been shown to induce B activity and transcription of the B regulon in B. subtilis (Petersohn et al., 2001). Interestingly, even though bsh clearly showed B-dependent transcription, we were not able to observe consistent induction of bsh transcription following exposure of exponential-phase cells to salt-stress conditions. These results may indicate that bsh requires additional co-factors (other than the B–RNA polymerase holoenzyme complex) for transcription induction, which would be consistent with the fact that BSH may only be required under specific environmental conditions (i.e. the presence of bile salts). The existence of B-dependent genes that require additional B-independent stress induction mechanisms has recently been demonstrated in B. subtilis. In fact, at least 24 of 125 genes in the B. subtilis regulon may require additional B-independent stress-induction mechanisms (Petersohn et al., 2001). Further studies on the regulation of opuCA, lmo1421 and bsh will provide important insight into the complex mechanism(s) that likely govern expression of these stress-response genes and the L. monocytogenes B regulon under different stress conditions, including those associated with intra-host environments.
Conclusions
L. monocytogenes has the unique ability to survive and grow under adverse environmental conditions including low pH, high osmolarity and low temperatures, and also to cause food-borne infections in mammalian hosts through survival and multiplication in the intracellular environment of host cells (Cole et al., 1990; Farber & Peterkin, 1991; Lou & Yousef, 1999). Our data support an emerging model that proposes a link between environmental survival and virulence in the food-borne pathogen L. monocytogenes. We have identified and confirmed three B-dependent genes, including two osmotolerance genes (opuCA and lmo1421) and a virulence gene (bsh). Together with the previous description of a B-dependent promoter contributing to the transcription of prfA (Nadon et al., 2002), which encodes a key positive regulator of virulence gene expression in L. monocytogenes, and the description of a set of PrfA-regulated genes preceded by putative B-dependent promoters (Milohanic et al., 2003), our data suggest that B may broadly respond to host-associated bacterial stress conditions by directing appropriate gene expression patterns. Further use and refinement of the reporter fusion and RT-PCR tools for monitoring B activity in L. monocytogenes described here will allow us to enhance our understanding of the contributions of B to the pathogenesis of food-borne listeriosis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 May 2003;
revised 18 July 2003;
accepted 21 July 2003.
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