Negative regulation of PrfA, the key activator of Listeria monocytogenes virulence gene expression, is dispensable for bacterial pathogenesis

Sonya L. Greene1,2 and Nancy E. Freitag2,3

1 Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, USA
2 Seattle Biomedical Research Institute, 4 Nickerson St, Seattle, WA 98109, USA
3 Departments of Pathobiology and Microbiology, University of Washington, Seattle, WA 98195, USA

Correspondence
Nancy E. Freitag
nfreitag{at}sbri.org


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Listeria monocytogenes is a facultative intracellular bacterial pathogen that regulates the expression of virulence-associated gene products in response to specific host cell compartment environments. The PrfA protein of L. monocytogenes functions as a key regulatory factor required for the differential expression of bacterial virulence gene products within infected host cells. PrfA both positively and negatively regulates its own expression, and while PrfA positive regulation is required for cell-to-cell spread of L. monocytogenes and for full virulence in infected mice, a role for negative regulation has been of presumed importance but has yet to be established. To address the role of negative regulation of prfA expression in L. monocytogenes pathogenesis, prfA promoter mutations designed to reduce or eliminate negative regulation were introduced into L. monocytogenes and analysed for their effects on patterns of PrfA-dependent gene expression and virulence in murine models of infection. High level PrfA production resulting from the prfA promoter mutations produced significantly increased levels of PrfA-regulated gene expression in broth-grown cultures; however the apparent loss of negative prfA regulation had no deleterious effects on growth and spread of the bacteria within infected tissue culture cells or on virulence in mice. The results indicate that while negative regulation of prfA expression exists and provides a feedback system for the control of PrfA synthesis, this feedback system is dispensable for virulence.

Abbreviations: GUS, ß-glucuronidase; LLO, listeriolysin O


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The ability to coordinate the expression of virulence factors in response to specific environmental stimuli is an important facet of bacterial pathogenesis. Alterations in patterns of virulence gene expression have been observed in vitro in response to a variety of environmental parameters such as changes in temperature, pH, O2 concentrations, nutrient conditions and essential metal ion deficiency (Datta & Kothary, 1993; Bohne et al., 1996; Garcia Vescovi et al., 1996; Soncini et al., 1996; Cotter & DiRita, 2000; Swanson & Hammer, 2000; Wosten et al., 2000; Kreft & Vazquez-Boland, 2001). Regulation of pathogen virulence gene expression appears to be finely coordinated with environmental cues in that multiple stimuli may be required to trigger full activation of expression (Cotter & Miller, 1997). The patterns of regulation of bacterial gene expression observed for pathogens in vitro presumably reflects the ability of the bacteria to adapt to specific host cell and tissue environments in vivo. Experimental models of host infection indicate that bacterial virulence genes are regulated in a manner that allows the differential expression of virulence factors at specific phases of infection or within defined host cell compartments (Slauch et al., 1994; Mahan et al., 1995; Cotter & Miller, 1997; Bubert et al., 1999; Freitag & Jacobs, 1999; Lee & Camilli, 2000; Slauch & Camilli, 2000; Stanley et al., 2000).

We have been using the facultative intracellular bacterial pathogen Listeria monocytogenes as a model system to define the mechanisms used by a prokaryotic parasite to regulate virulence gene expression within host cells. L. monocytogenes is responsible for serious infections in immunocompromised individuals and pregnant women (Gray & Killinger, 1966; Gellin & Broome, 1989; Vazquez-Boland et al., 2001). The bacterium expresses a variety of virulence factors that enable it to invade many host cell types and to gain entry into the cell cytosol where bacterial replication occurs. L. monocytogenes regulates the expression of virulence genes in response to specific host cell compartment environments, including those of the phagosome and cytosol (Bubert et al., 1999; Freitag & Jacobs, 1999; Moors et al., 1999; Kreft & Vazquez-Boland, 2001). The majority of the L. monocytogenes virulence determinants that have been thus far identified are regulated by a transcriptional activator known as PrfA, a 27 kDa site-specific DNA binding protein that is essential for L. monocytogenes pathogenesis (Mengaud et al., 1991; Chakraborty et al., 1992; Freitag et al., 1993).

While it is clear that PrfA is a key regulatory element required for the control of virulence gene expression in L. monocytogenes, it is not clear what controls its activity or how prfA expression is regulated. Three distinct promoters contribute to prfA expression. Two promoters, prfAP1 and prfAP2, are located immediately upstream of the prfA coding region (Freitag & Portnoy, 1994) (Fig. 1). The third promoter is contributed by the upstream plcA gene through the generation of a bicistronic plcA–prfA transcript (Camilli et al., 1993; Freitag et al., 1993). PrfA positively regulates its own expression through the activation of plcA transcription, and the increase in PrfA synthesis resulting from the generation of the prfAplcA transcript is essential for spread of the bacteria from the initial infected cell to adjacent cells and for full virulence (Camilli et al., 1993; Freitag et al., 1993; Freitag & Portnoy, 1994). PrfA also appears to negatively regulate its own expression from the prfAP1 and prfAP2 promoters, as transcripts directed by these two promoters are significantly increased in the absence of functional PrfA (Freitag et al., 1993). It has been proposed that L. monocytogenes regulates the amounts of available PrfA protein through the use of a negative feedback loop that involves PrfA binding to the -35 region of prfAP2 and inhibition of transcriptional initiation at that promoter (and possibly at prfAP1) by an unknown mechanism (Freitag & Portnoy, 1994). The existence of this negative feedback loop has been postulated to be an important facet of L. monocytogenes virulence gene expression.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1. Construction of L. monocytogenes strains containing prfA promoter mutations. (a) Organization of the plcAprfA operon. The stem–loop structure denotes the rho-independent transcriptional terminator located downstream of plcA. The asterisks indicate the region shown in detail in (b). (b) Detailed depiction of the mutations introduced into the prfA upstream region. The stop codon for plcA and the start codon for prfA are underlined. The stem of the plcA terminator is indicated by the dashed line, and boxed sequences represent the sequences deleted for the plcA-{Delta}T and prfA{Delta}P2-35 mutants. Sequence within the heavy black box represents the PrfA DNA binding site from the hly promoter introduced into the prfAP2 promoter region. Small arrows indicate the start of transcription for transcripts originating from the plcA, prfAP1 and prfAP2 promoters. All mutations were introduced in single copy into the L. monocytogenes chromosome by allelic exchange and verified by DNA sequencing.

 
To begin to address the role of negative regulation of prfA expression in L. monocytogenes pathogenesis, we describe here the introduction of several defined prfA promoter mutations that result in loss of prfA negative regulation and significantly increased synthesis of PrfA. Levels of PrfA synthesis in broth-grown cultures of the promoter mutants were found to be greater than the amounts of PrfA reported to be induced following entry of the bacteria into the host cell cytosol (Renzoni et al., 1999). High level PrfA production resulted in increased levels of PrfA-regulated gene expression in broth-grown cultures; however the apparent loss of negative prfA regulation had no deleterious effects on growth and spread of the bacteria within infected tissue culture cells or on virulence in mouse models of infection. Our results suggest that although negative regulation of prfA expression provides a feedback system to control PrfA levels, this feedback system is dispensable for virulence.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
The bacterial strains used in this study are listed in Table 1. L. monocytogenes (serotype 1/2a) is resistant to streptomycin and has a LD50 for mice of 2x104 (Freitag et al., 1993). L. monocytogenes was stored at -70 °C in brain–heart infusion broth (BHI, Difco Laboratories) containing 20 % (v/v) glycerol. Escherichia coli HB101 or DH5{alpha} was used as the host strain for recombinant plasmids. Antibiotics were used at the following concentrations unless otherwise noted: carbenicillin, 50 µg ml-1; chloramphenicol, 10 µg ml-1; streptomycin, 200 µg ml-1.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and relevant characteristics

 
The thermo-sensitive plasmid vectors pKSV7 (Smith & Youngman, 1992) and pCON1-{Delta}prfA7973 (Behari & Youngman, 1998) have been described previously.

Construction of L. monocytogenes prfA promoter mutants.
The introduction of targeted deletion and substitution mutations within the prfA promoter region was accomplished using the Kunkel method of site-directed DNA mutagenesis with M13 bacteriophage-derived vectors as previously described (Ausubel et al., 1991; Freitag & Portnoy, 1994). prfA mutant constructs were sequenced to verify desired mutations and then fragments containing the mutations were subcloned into the thermo-sensitive shuttle plasmid vector pKSV7 (Smith & Youngman, 1992). Mutations were introduced into the L. monocytogenes chromosome of strain NF-L476 [containing a transcriptional fusion of actA with the gus reporter gene (Shetron-Rama et al., 2002)] by allelic exchange as previously described (Freitag & Portnoy, 1994) to generate strains NF-L623 (containing plcA-{Delta}T +prfA{Delta}P2-35); NF-L625 (containing plcA-{Delta}T); NF-L627 [containing prfA{Delta}P2(hly)]; and NF-L629 (containing prfA{Delta}P2-35). Chromosomal mutations were confirmed by sequencing of PCR amplified products derived from genomic DNA isolated from each mutant strain.

Introduction of prfA-7973 (prfA*) into L. monocytogenes mutant strains.
L. monocytogenes strain NCTC 7973, a natural isolate, has been described (Park & Kroll, 1993; Millenbachs et al., 1997; Behari & Youngman, 1998). The NCTC 7973 prfA allele, which contains a serine in place of a glycine at position 145, produces increased expression of virulence genes in NCTC 7973 in comparison to wild-type L. monocytogenes and is thought to represent a transcriptionally active, cofactor-independent form of PrfA protein (PrfA*) (Ripio et al., 1997). The NCTC 7973 PrfA also has a second amino acid change (in comparison to 10403S PrfA), a Cys to Tyr change at position 229; however this substitution has not been demonstrated to influence PrfA-dependent gene expression (Behari & Youngman, 1998). pCON1-{Delta}prfA7973 was used to insertionally inactivate the wild-type prfA allele and to introduce the prfA* allele under the control of the wild-type prfA promoter (Behari & Youngman, 1998). pCON1-{Delta}prfA7973 conjugated into 10403S derivatives NF-L476, NF-L623, NF-L625, NF-L627 and NF-L629 to generate NF-L657 (prfA* actA–gus–plcB); NF-L653 [prfA*{Delta}P2(hly) actA–gus–plcB]; NF-L654 [plcA-{Delta}T prfA* actA–gus–plcB]; NF-L655 (plcA-{Delta}T+prfA*{Delta}P2-35 actA–gus–plcB); and NF-L656 (prfA*{Delta}P2-35 actA–gus–plcB). Transconjugants were isolated as described by Behari & Youngman (1998) with the following modifications: transconjugants were selected on BHI agar containing chloramphenicol (5 µg ml-1) after a 24 h incubation at 30 °C. Selected colonies were used to inoculate BHI media containing chloramphenicol and streptomycin, and grown overnight at 40 °C with shaking to force chromosomal integration of the vector. Overnight 40 °C cultures were then diluted 1 : 1000 into fresh BHI containing chloramphenicol and streptomycin, and again incubated at 40 °C overnight with shaking. Appropriate dilutions of overnight cultures were plated on BHI agar containing 5 µg chloramphenicol ml-1 and incubated at 40 °C. Integration of the vector into the chromosome and confirmation of the prfA7973 (prfA*) sequences were verified by PCR amplification of prfA from genomic DNA and sequencing of the PCR products.

ß-Glucuronidase (GUS) assays of bacteria grown in liquid culture.
For experiments using LB with and without added glucose or cellobiose, overnight cultures of bacteria grown in LB medium were diluted 1 to 10 into fresh LB buffered with 100 mM MOPS (pH 7·4) with or without glucose or cellobiose at the indicated concentrations and grown for 5 h with shaking at 37 °C. Bacterial pellets from 1 ml culture aliquots were collected following centrifugation and were quickly frozen on dry ice. Optical density (at 595 nm) was measured for each culture using a Spectronic 20 spectrophotometer (Milton Roy). For GUS enzymic assays, bacterial cell pellets were thawed, washed once with ABT buffer [0·1 M potassium phosphate (pH 7·0), 0·1 M NaCl, 0·1 % (v/v) Triton X-100] and resuspended in 200 µl ABT buffer. GUS activity was measured as described by Youngman (1987) with the substitution of 4-methylumbelliferyl ß-D-glucuronide in place of 4-methylumbelliferyl ß-D-galactoside.

Western analysis of PrfA protein levels.
Polyclonal rabbit antiserum was generated against amino-terminal histidine-tagged PrfA protein produced using the Qiagen pQE30 vector in E. coli and purified by nickel column chromatography as recommended by the manufacturer (Qiagen). For Western analysis, overnight cultures of L. monocytogenes grown in LB broth at 37 °C without shaking were diluted 1 : 100 into fresh LB broth buffered to pH 7·4 with 100 mM MOPS buffer and grown for 6 h at 37 °C with shaking. For experiments designed to measure PrfA protein levels in LB broth buffered to pH5·8 or in the presence of glucose or cellobiose, overnight cultures grown in LB were diluted 1 : 100 into fresh LB broth buffered with 100 mM MES to a pH of 5·8, or into LB broth buffered with 100 mM MOPS to pH7·4 plus 25 mM glucose or 25 mM cellobiose. A 20 ml aliquot of each culture was briefly centrifuged to recover the cell pellet; the pellet was resuspended in 500 µl Cell Lysis Buffer (50 mM Tris/HCl, pH7·5, 1 mM DTT, 0·1 % (v/v) Triton X-100). Bacterial suspensions were disrupted using a Mini-Beadbeater (Biospec Products) with 1 min cycles followed by 30 s cooling on ice, for a total of 3 cycles. Samples were then centrifuged at 13 000 g for 5 min to recover supernatants. Protein concentration was determined using the DC Protein Assay Kit as recommended by the manufacturer (Bio-Rad Laboratories). Equivalent amounts of total protein from each culture lysate were mixed with equal volumes of 2xSDS-PAGE buffer [60 mM Tris/HCl (pH6·8), 2 % (w/v) SDS, 10 % (v/v) glycerol, 10 % 2-mercaptoethanol, 0·01 % (w/v) bromophenol blue]. Samples were heated to 90 °C for 5 min prior to being subjected to SDS-PAGE on 10 % polyacrylamide gels. Proteins were visualized by staining the gel with Coomassie brilliant blue prior to transfer. Proteins were transferred to nitrocellulose membranes and incubated with a 1 : 200 dilution of polyclonal antibody generated against purified PrfA protein, followed by incubation with an alkaline phosphatase-coupled goat anti-rabbit antibody (Sigma). PrfA protein antigen was detected using the Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets, a colorimetric alkaline phosphate substrate.

For quantitative comparison of PrfA protein levels as visualized by Western analysis, 2 µg, 8 µg and 15 µg purified PrfA protein were compared with serial dilutions of protein extracts derived from the wild-type and prfA promoter mutant strains to determine the relative amount of PrfA produced by each mutant.

Assay for haemolytic activity.
Stationary phase bacteria were diluted 1 : 10 into BHI and grown at 37 °C for 5 h with shaking. The supernatant fluid was assayed for haemolytic activity as previously described (Camilli et al., 1989).

Plaque formation in L2 cells.
Plaque assays were performed as previously described by Sun et al. (1990). Plaque size was measured using a micrometer and the mean diameter of at least 10 plaques from three independent experiments was determined.

LD50 determinations.
The LD50 were determined in BALB/c mice by intravenous injection as previously described (Portnoy et al., 1988).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of L. monocytogenes strains containing mutations within prfA promoter regulatory regions
Mutations designed to alleviate negative regulation of prfA expression were introduced into the chromosome of L. monocytogenes by homologous recombination (Fig. 1 and Table 1). Mutations introduced were as follows. (1) A deletion of the motif resembling a degenerate PrfA binding site in the -35 region of the prfAP2 promoter, previously shown to contribute to down-regulation of prfAP1 expression (prfA{Delta}P2-35 mutation) (Freitag & Portnoy, 1994). (2) Replacement of the prfAP2 promoter -35 motif with a high affinity PrfA binding site derived from the hly promoter in an attempt to introduce a positive PrfA regulatory element in place of a negative one [prfA{Delta}P2(hly)]. (3) A 14 bp deletion designed to facilitate transcription through the plcA rho-independent terminator and to increase prfA expression through the augmented generation of plcA–prfA transcripts (plcA-{Delta}T mutation). The plcA-{Delta}T deletion also removed a small 7 bp motif that resembles one half of a PrfA DNA binding site. The plcA-{Delta}T deletion was introduced alone and in combination with the prfA{Delta}P2-35 deletion to determine if loss of both motifs resulted in levels of prfA expression that were higher than those observed for strains lacking a single motif. All mutations were introduced in single copy into the L. monocytogenes chromosome.

Analysis of the effects of prfA promoter mutations on PrfA protein levels
The prfA{Delta}P2-35 mutation has been previously demonstrated to increase levels of transcripts initiating at the prfAP1 promoter (Freitag & Portnoy, 1994). However, PrfA protein levels were not examined in this strain, and it was important to determine whether increased mRNA levels directly correlated with an increase in protein levels. To directly compare the levels of PrfA present in each of the prfA promoter mutant strains, extracts derived from wild-type L. monocytogenes and the promoter mutants were examined by Western analysis using a rabbit polyclonal antibody directed against purified PrfA protein. As shown in Fig. 2, all of the L. monocytogenes strains containing prfA promoter mutations produced dramatically higher levels of PrfA protein than the wild-type strain. Each of the prfA promoter mutants produced at least five times more PrfA protein as determined by comparison of extract dilutions with known amounts of purified PrfA (S. Greene & N. Freitag, data not shown). It has been previously reported that a threefold increase in PrfA protein levels occurs in bacteria that reside within the host cytosol, and that this increase appears sufficient for full induction of intracellular gene expression (Renzoni et al., 1999). The prfA promoter mutations therefore appeared to result in increased synthesis of PrfA protein in broth-grown cultures to levels equivalent to those observed for intracellular bacteria via the alleviation of negative regulation of prfA expression.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Western analysis of PrfA protein levels produced by the L. monocytogenes prfA promoter mutant strains. Soluble bacterial cell extracts were prepared from L. monocytogenes cultures grown in buffered LB broth for 6 h. Equal amounts (91 µg) of total protein in SDS-PAGE sample buffer were run for each sample, and PrfA was detected using a rabbit polyclonal antibody directed against purified His-tagged PrfA. The arrow indicates the position of PrfA.

 
In vitro expression of PrfA-regulated genes by prfA promoter mutant strains
To examine the effects of increased synthesis of PrfA protein on PrfA-dependent gene expression, two differentially regulated virulence genes were selected. hly, encoding the haemolysin listeriolysin O (LLO), is normally expressed during bacterial growth in standard broth culture and LLO activity can be easily detected in culture supernatants. actA encodes a gene product necessary for cell-to-cell spread of L. monocytogenes and in contrast to hly, is expressed at low to undetectable levels in broth-grown bacterial cultures but is highly induced following entry of L. monocytogenes into the host cell cytosol (Brundage et al., 1993; Bubert et al., 1999; Freitag & Jacobs, 1999; Moors et al., 1999). The prfA promoter mutations were introduced into L. monocytogenes strains containing actA transcriptional fusions to the gus reporter gene to facilitate measurement of actA expression. The actA–gus fusions do not affect intracellular growth or cell-to-cell spread of L. monocytogenes in tissue culture models of infection, or bacterial virulence in murine models of infection (Shetron-Rama et al., 2002).

Despite the high levels of PrfA protein observed in each of the mutant strains, none of the mutants produced significantly increased levels of LLO (Table 1). In contrast, significant increases in the levels of actA expression were observed for all prfA promoter mutant strains following growth in LB broth, with the sole exception of mutant plcA-{Delta}T which had levels of expression equivalent to those observed for wild-type L. monocytogenes (Fig. 3). The prfA{Delta}P2(hly) mutation resulted in the highest levels of actA expression, approximately eightfold greater than the levels observed for wild-type bacteria grown in broth culture. These results suggest that increased synthesis of PrfA protein influences a subset of PrfA-regulated genes (such as actA) and does not result in increased levels of all PrfA-dependent gene products (such as LLO).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Measurement of actA expression levels in prfA promoter mutants containing transcriptional actA–gus reporter gene fusions. GUS activity was measured following 5 h growth in LB broth buffered to pH 7·4 in the presence and absence of either 25 mM glucose or 25 mM cellobiose, or in LB broth buffered to pH 5·8. Units of GUS activity were normalized for optical density at 595 nm as described by Youngman (1987) for the measurement of ß-galactosidase activity but with the appropriate substrate substitution of 4-methylumbelliferyl ß-D-glucuronide. Each assay was done in duplicate, and the data represents the mean and SE for three separate experiments. Part (b) provides an enhanced view of the data obtained in the presence of cellobiose or glucose shown in (a). Black bars, WT; dark grey bars, plcA-{Delta}T; bars shaded with straight lines, prfA{Delta}P2-35; light grey bars, plcA-{Delta}T + prfA{Delta}P2-35; bars shaded with curved lines, prfA{Delta}P(hly).

 
Expression of virulence genes in L. monocytogenes has been shown to be dramatically influenced by available carbon sources (Park & Kroll, 1993; Millenbachs et al., 1997) and by low pH (Behari & Youngman, 1998). Readily metabolized carbon sources, such as glucose, fructose and cellobiose, repress the expression of hly and actA, as does media buffered to pH 5·8. We examined the effects of overexpression of prfA on catabolite and pH-mediated repression of actA gene expression (Fig. 3). Similar to wild-type expression patterns, expression of actA was subject to repression by low pH in all of the mutant strains; however the absolute level of actA expression remained above wild-type levels for mutants prfA{Delta}P2-35, plcA-{Delta}T+prfA{Delta}P2-35 and prfA{Delta}P2(hly). All of the mutants were extremely sensitive to catabolite repression of gene expression by both glucose and cellobiose, although again the absolute level of actA expression remained above wild-type levels for mutants prfA{Delta}P2-35, plcA-{Delta}T + prfA{Delta}P2-35 and prfA{Delta}P2(hly) (Fig. 3b). The prfA{Delta}P2(hly) mutant exhibited the highest levels of repression in the presence of glucose with a 34-fold reduction in actA expression (compared to a ninefold reduction for wild-type). Interestingly, Western analysis of L. monocytogenes prfA{Delta}P2(hly)-derived extracts still showed higher levels of PrfA protein in comparison to the amounts observed for the wild-type strain following growth in media containing glucose or cellobiose (Fig. 4). These data are consistent with previously reported observations that indicate that PrfA protein can be present but inactive (Millenbachs et al., 1997; Renzoni et al., 1997). It further demonstrates that PrfA-dependent gene expression remains sensitive to repression by low pH and readily metabolized sugars despite the presence of high levels of PrfA.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Western analysis of PrfA protein levels produced by L. monocytogenes wild-type and prfA{Delta}P2(hly) promoter mutant strains. Soluble bacterial cell extracts were prepared from L. monocytogenes cultures grown in LB broth buffered to pH 7·4 in the presence of either 25 mM glucose or 25 mM cellobiose, or in LB broth buffered to pH 5·8. Equal amounts (121 µg) of total protein in SDS-PAGE sample buffer were run for each sample, and PrfA was detected using a rabbit polyclonal antibody directed against purified His-tagged PrfA. The arrow indicates the position of PrfA.

 
Introduction of PrfA*, a constitutively activated form of PrfA, into L. monocytogenes prfA promoter mutant strains
A growing body of evidence strongly suggests that PrfA requires post-translational modification or the presence of a cofactor for full activity (Ripio et al., 1997; Vega et al., 1998; Kreft & Vazquez-Boland, 2001; Vazquez-Boland et al., 2001). Thus, the effects of increased prfA expression in L. monocytogenes are likely to be reduced in broth culture where the protein is not in its fully activated state. Ripio et al. (1997) have reported that L. monocytogenes strains that constitutively express high levels of several virulence gene products contain a prfA allele that encodes a serine in place of a glycine at position 145 within the protein. PrfA shares significant homology with the cAMP receptor protein (CRP) of E. coli (Lampidis et al., 1994; Sheehan et al., 1995; Vega et al., 1998) and the prfA G145S mutant allele, also known as prfA*, appears analogous to crp* mutants in which CRP functions as a transcriptional activator in the absence of its cAMP cofactor (Garges & Adhya, 1985; Harman et al., 1986; Kolb et al., 1993). To test if the effects of high level PrfA synthesis would be amplified by the presence of a constitutively activated form of the protein, the prfA* allele was introduced into the prfA promoter mutants via the integration of a temperature-sensitive plasmid (see Table 1). Integration of the prfA*-containing plasmid resulted in the inactivation of the wild-type prfA allele and the expression of prfA* under the control of the wild-type prfA promoter or the mutant prfA promoters. PrfA* levels were measured in each of the mutant backgrounds using Western analysis with polyclonal rabbit antiserum directed against PrfA (Fig. 5). PrfA* levels in the plcA-{Delta}T (prfA*), prfA*{Delta}P2-35 and prfA*{Delta}P2(hly) mutant strains were found to be approximately fivefold higher than the levels observed in the presence of the wild-type promoter or in the plcA-{Delta}T +prfA*{Delta}P2-35 mutant. The overall levels of PrfA* protein were similar to the levels observed in the wild-type and mutant strains containing the wild-type prfA allele (see Fig. 2), indicating that the mere presence of PrfA* did not dramatically increase levels of protein expression.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Western analysis of PrfA* protein levels produced by the L. monocytogenes prfA* promoter mutant strains. Soluble bacterial cell extracts were prepared from L. monocytogenes cultures grown in buffered LB broth for 6 h. Equal amounts (91 µg) of total protein in SDS-PAGE sample buffer were run for each sample, and PrfA* was detected using a rabbit polyclonal antibody directed against purified His-tagged PrfA. The arrow indicates the position of PrfA.

 
Analysis of the effects of the L. monocytogenes prfA* promoter mutations on virulence gene expression
Supernatants derived from prfA* promoter mutant strain cultures were assayed for secreted LLO activity. While the introduction of the prfA* allele did lead to a significant increase in LLO activity in comparison to wild-type strains, no additional increase was observed for the prfA* promoter mutants (Table 1). Similarly, the introduction of the prfA* allele yielded an overall increase in actA expression for all strains examined, but the presence of the prfA promoter mutations did not significantly increase actA expression above the levels observed for the wild-type promoter (Fig. 6). Levels were increased over wild-type for several of the mutants at pH 5·8 and in the presence of cellobiose. All of the prfA* strains were significantly more resistant to glucose-mediated repression. The prfA*{Delta}P2(hly) mutant, for example, had a twofold decrease in actA expression in the presence of glucose, in contrast to the 34-fold decrease observed for the prfA{Delta}P2(hly) strain. The relative resistance of strains containing PrfA* to glucose-mediated repression was observed at glucose concentrations at least 10 times higher than those found to repress wild-type gene expression (Fig. 7). Taken together, these results indicate that no significant increase in actA expression results from an increase in synthesis of PrfA*, suggesting that for actA transcriptional activation, saturating amounts of PrfA* are already present in strains containing the wild-type promoter. Strains containing the prfA* allele were much less sensitive to repression mediated by low pH, cellobiose or glucose (as previously reported by Ripio et al., 1997; Behari & Youngman, 1998; Vega et al., 1998) and this reduction in sensitivity was only slightly augmented by the presence of the prfA promoter mutations.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6. Measurement of actA expression levels in prfA* promoter mutants containing transcriptional actA–gus reporter gene fusions. GUS activity was measured following 5 h growth in LB broth buffered to pH7·4 in the presence and absence of either 25 mM glucose or 25 mM cellobiose, or in LB broth buffered to pH5·8. Units of GUS activity were normalized for optical density at 595 nm as described by Youngman (1987) for the measurement of ß-galactosidase activity but with the appropriate substrate substitution of 4-methylumbelliferyl ß-D-glucuronide. Each assay was done in duplicate, and the data represent the mean and SE for three separate experiments. Black bars, WT; white bars, prfA*; dark grey bars, plcA-{Delta}T prfA*; bars shaded with straight lines, prfA*{Delta}P2-35; light grey shading, plcA-{Delta}T + prfA*{Delta}P2-35; bars shaded with curved lines, prfA*{Delta}P(hly).

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 7. Repression of L. monocytogenes actA expression by glucose. GUS activity was measured following 5 h growth in LB broth buffered to pH 7·4 alone or in the presence of increasing amounts of glucose. Units of GUS activity were normalized for optical density at 595 nm as described by Youngman (1987) for the measurement of ß-galactosidase activity but with the appropriate substrate substitution of 4-methylumbelliferyl ß-D-glucuronide. Each assay was done in duplicate, and the data represent the mean and SE for three separate experiments. (a) Measurement of glucose-mediated repression of actA expression in the presence of the wild-type prfA allele. (b) Measurement of glucose-mediated repression of actA expression in the presence of prfA*.

 
Loss of negative prfA regulation does not affect L. monocytogenes virulence
To assess the effects of loss of prfA negative regulation within infected host cells, the prfA promoter mutant strains were examined for their ability to replicate within infected tissue culture cell monolayers. The capacity of L. monocytogenes to escape from a vacuole and spread from cell to cell can be measured by the ability of the bacteria to form plaques on monolayers of mouse L2 cells (Sun et al., 1990). Mutations that interfere with the function of virulence-related gene products can be identified by the inability of the mutants to form plaques equal in size to those formed by wild-type bacteria (Sun et al., 1990; Camilli et al., 1993; Freitag & Portnoy, 1994; Jones & Portnoy, 1994; Marquis et al., 1995; Smith et al., 1995). All of the prfA promoter mutant strains were found to form plaques of approximately the same size and with the same efficiency as wild-type L. monocytogenes (Table 1), indicating that the high level PrfA synthesis observed in broth culture did not adversely affect intracellular growth or cell-to-cell spread in vitro. Interestingly, strains containing the prfA* allele formed plaques that were smaller in size than those formed by wild-type L. monocytogenes, but the strains containing the prfA* promoter mutations did not differ in size from the prfA* parent strain. These results may indicate that the presence of the constitutively activated prfA* allele reduced either intracellular growth or cell-to-cell spread of L. monocytogenes in L2 cells; however it should be noted that the mere presence of pKSV7 integrated within the L. monocytogenes chromosome can reduce plaque size by 30–40 % (Camilli et al., 1993).

Intracellular growth of the prfA promoter mutants was also examined in a variety of host cell types, including mouse macrophage-like tissue culture cell lines. No defect in intracellular growth or cell-to-cell spread was detected (S. Greene & N. Freitag, unpublished data). Loss of prfA negative regulation also resulted in no significant defect in virulence following intravenous injection of mice for any of the mutant strains (LD50 2x104 c.f.u. for parent strain NF-L476, and <105 c.f.u. for each mutant).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The regulation of virulence gene expression by the PrfA transcriptional activator is a complex but essential process for L. monocytogenes virulence. It has been demonstrated that an increase in PrfA protein levels is necessary for the full induction of virulence gene expression within the host (Camilli et al., 1993; Freitag et al., 1993; Freitag & Portnoy, 1994), and previous reports have demonstrated that a threefold increase in PrfA protein synthesis occurs during infection of mammalian cells (Renzoni et al., 1999). Negative regulation of prfA expression has been implicated as an important feedback regulatory tool, but its role in L. monocytogenes virulence has never been examined. In this report, we show that it is possible to increase PrfA protein levels more than fivefold via the introduction of specific promoter mutations that interfere with negative regulation of prfA expression. Strains lacking prfA negative regulation had significantly increased levels of actA expression, but surprisingly no significant effects were observed for intracellular growth, cell-to-cell spread or virulence in mouse models of infection. These results confirm the existence of negative regulation of prfA expression, but show that this feedback system is dispensable for bacterial virulence.

We had anticipated that the loss of prfA negative regulation would be amplified in the presence of the constitutively activated PrfA* form of the protein. The G145S substitution within PrfA* has been postulated to produce a conformational change in the protein that results in activation of PrfA in the absence of cofactor binding (Ripio et al., 1997; Vega et al., 1998). As it has been demonstrated that an approximately threefold induction of PrfA synthesis occurs during infection of mammalian cells (Renzoni et al., 1999), the fivefold increase in PrfA* observed for the promoter mutants might have been sufficient to induce actA expression to the levels observed in cytosolic bacteria (Shetron-Rama et al., 2002). However, the prfA* promoter mutants did not show a significant increase in actA expression in comparison to strains containing prfA* in the presence of the native promoter, and the approximately tenfold induction of actA expression measured for the prfA* strains was still well below the greater than 200-fold induction observed for bacteria located within the host cytosol (Moors et al., 1999; Shetron-Rama et al., 2002). These results strongly support the premise that other factors besides PrfA are required for optimal induction of actA expression in L. monocytogenes and that the expression of these factors is likely to be PrfA-independent. Work in progress has indicated that it is possible to isolate L. monocytogenes mutants with increased actA expression in broth culture which contain mutations mapping outside of the prfA regulon (L. Shetron-Rama & N. Freitag, unpublished results). The participation of additional regulatory factors in the induction of L. monocytogenes gene expression within infected host cells further illustrates the resources used by the bacterium to ensure that specific gene products are synthesized within the proper host cell environment.

Why does negative regulation of prfA expression exist if it does not contribute to virulence in mammals? L. monocytogenes is a ubiquitous bacterium that occupies a variety of habitats. PrfA has been reported to negatively influence the expression of genes required for environmental stress, such as clpC (Ripio et al., 1998) and has been implicated in the down-regulation of stress resistance mechanisms during exponential growth (Herbert & Foster, 2001). Genes required for bacterial motility, such as motA, have also been reported to be downregulated by PrfA (Michel et al., 1998). It is possible that negative regulation of prfA expression plays an important role in facilitating bacterial survival outside host cells by ensuring that gene products required for survival in the extracellular environment are expressed. It is tempting to speculate, therefore, that while positive regulation of prfA expression is of paramount importance during mammalian infection, negative regulation has evolved to sustain bacterial survival in the outside environment. Confirmation of this hypothesis awaits detailed studies of factors contributing to L. monocytogenes survival within its ubiquitous habitats.


   ACKNOWLEDGEMENTS
 
We thank Archie Bouwer for LD50 determinations of the prfA promoter mutant strains, and Jai Behari and Phil Youngman for providing plasmid pCON1-{Delta}prfA7973. We also would like to thank members of the Freitag lab and Jerry Cangelosi for helpful discussions, and express our appreciation for comments made by the reviewers of this manuscript.

This work was supported by Public Health Service grant AI41816 to N. E. F.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (1991). Current Protocols in Molecular Biology. New York: Greene Publishing Associates.

Behari, J. & Youngman, P. (1998). Regulation of hly expression in Listeria monocytogenes by carbon sources and pH occurs through separate mechanisms mediated by PrfA. Infect Immun 66, 3635–3642.[Abstract/Free Full Text]

Bohne, J., Kestler, H., Uebele, C., Sokolovic, Z. & Goebel, W. (1996). Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA. Mol Microbiol 20, 1189–1198.[Medline]

Brundage, R. A., Smith, G. A., Camilli, A., Theriot, J. A. & Portnoy, D. A. (1993). Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc Natl Acad Sci U S A 90, 11890–11894.[Abstract]

Bubert, A., Sokolovic, Z., Chun, S.-K., Papatheodorou, L., Simm, A. & Goebel, W. (1999). Differential expression of Listeria monocytogenes virulence genes in mammalian host cells. Mol Gen Genet 261, 323–336.[CrossRef][Medline]

Camilli, A., Paynton, C. R. & Portnoy, D. A. (1989). Intracellular methicillin selection of Listeria monocytogenes mutants unable to replicate in a macrophage cell line. Proc Natl Acad Sci U S A 86, 5522–5526.[Abstract]

Camilli, A., Tilney, L. G. & Portnoy, D. A. (1993). Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol 8, 143–157.[Medline]

Chakraborty, T., Leimeister-Wachter, M., Domann, E., Hartl, M., Goebel, W., Nichterlein, T. & Notermans, S. (1992). Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol 174, 568–574.[Abstract]

Cotter, P. A. & DiRita, V. J. (2000). Bacterial virulence gene regulation: an evolutionary perspective. Annu Rev Microbiol 54, 519–565.[CrossRef][Medline]

Cotter, P. A. & Miller, J. F. (1997). A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens. Mol Microbiol 24, 671–685.[CrossRef][Medline]

Datta, A. R. & Kothary, M. H. (1993). Effects of glucose, growth temperature, and pH on Listeriolysin O production in Listeria monocytogenes. Appl Environ Microbiol 59, 3495–3497.[Abstract]

Freitag, N. E. & Jacobs, K. E. (1999). Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria. Infect Immun 67, 1844–1852.[Abstract/Free Full Text]

Freitag, N. E. & Portnoy, D. A. (1994). Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo. Mol Microbiol 12, 845–853.[Medline]

Freitag, N. E., Rong, L. & Portnoy, D. A. (1993). Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread. Infect Immun 61, 2537–2544.[Abstract]

Garcia Vescovi, E., Soncini, F. C. & Groisman, E. A. (1996). Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence. Cell 84, 165–174.[Medline]

Garges, S. & Adhya, S. (1985). Sites of allosteric shift in the structure of cyclic AMP receptor protein. Cell 41, 745–751.[Medline]

Gellin, B. G. & Broome, C. V. (1989). Listeriosis. J Amer Med Assoc 261, 1313–1320.[CrossRef][Medline]

Gray, M. L. & Killinger, A. H. (1966). Listeria monocytogenes and listeric infections. Bacteriol Rev 30, 309–382.[Medline]

Harman, J. G., McKenney, K. & Peterkofsky, A. (1986). Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein. J Biol Chem 261, 16332–16339.[Abstract/Free Full Text]

Herbert, K. C. & Foster, S. J. (2001). Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components. Microbiology 147, 2275–2284.[Abstract/Free Full Text]

Jones, S. & Portnoy, D. A. (1994). Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect Immun 62, 5608–5613.[Abstract]

Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749–795.[CrossRef][Medline]

Kreft, J. & Vazquez-Boland, J. A. (2001). Regulation of virulence genes in Listeria. Int J Med Microbiol 291, 145–157.[Medline]

Lampidis, R., Gross, R., Sokolovic, Z., Goebel, W. & Kreft, J. (1994). The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators. Mol Microbiol 13, 141–151.[Medline]

Lee, S. H. & Camilli, A. (2000). Novel approaches to monitor bacterial gene expression in infected tissue and host. Curr Opin Microbiol 3, 97–101.[CrossRef][Medline]

Mahan, M. J., Tobias, J. W., Slauch, J. M., Hanna, P. C., Collier, R. J. & Mekalanos, J. J. (1995). Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host. Proc Natl Acad Sci U S A 92, 669–673.[Abstract]

Marquis, H., Doshi, V. & Portnoy, D. A. (1995). The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect Immun 63, 4531–4534.[Abstract]

Mengaud, J., Dramsi, S., Gouin, E., Vazquez-Boland, J. A., Milon, G. & Cossart, P. (1991). Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol Microbiol 5, 2273–2283.[Medline]

Michel, E., Mengaud, J., Galsworthy, S. & Cossart, P. (1998). Characterization of a large motility gene cluster containing the cheR, motAB genes of Listeria monocytogenes and evidence that PrfA downregulates motility genes. FEMS Microbiol Lett 15, 341–347.

Millenbachs, A. A., Brown, D. P., Moors, M. & Youngman, P. (1997). Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Mol Microbiol 23, 1075–1085.[Medline]

Moors, M. A., Levitt, B., Youngman, P. & Portnoy, D. A. (1999). Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect Immun 67, 131–139.[Abstract/Free Full Text]

Park, S. F. & Kroll, R. G. (1993). Expression of listeriolysin and phosphatidylinositol-specific phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes. Mol Microbiol 8, 653–661.[Medline]

Portnoy, D. A., Jacks, P. S. & Hinrichs, D. J. (1988). Role of hemolysin for the intracellular growth of Listeria monocytogenes. J Exp Med 167, 1459–1471.[Abstract]

Renzoni, A., Klarsfeld, A., Dramsi, S. & Cossart, P. (1997). Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes can be present but inactive. Infect Immun 65, 1515–1518.[Abstract]

Renzoni, A., Cossart, P. & Dramsi, S. (1999). PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts. Mol Microbiol 34, 552–561.[CrossRef][Medline]

Ripio, M.-T., Dominguez-Bernal, G., Lara, M., Suarez, M. & Vazquez-Boland, J.-A. (1997). A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes. J Bacteriol 179, 1533–1540.[Abstract]

Ripio, M. T., Vazquez-Boland, J. A., Vega, Y., Nair, S. & Berche, P. (1998). Evidence for expressional crosstalk between the central virulence regulator PrfA and the stress response mediator ClpC in Listeria monocytogenes. FEMS Microbiol Lett 158, 45–50.[CrossRef][Medline]

Sheehan, B., Klarsfeld, A., Msadek, T. & Cossart, P. (1995). Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator. J Bacteriol 177, 6469–6476.[Abstract]

Shetron-Rama, L. M., Marquis, H., Bouwer, H. G. A. & Freitag, N. E. (2002). Intracellular induction of Listeria monocytogenes actA expression. Infect Immun 70, 1087–1096.[Abstract/Free Full Text]

Slauch, J. M. & Camilli, A. (2000). IVET and RIVET: use of gene fusions to identify bacterial virulence factors specifically induced in host tissues. Methods Enzymol 326, 73–96.[CrossRef][Medline]

Slauch, J. M., Mahan, M. J. & Mekalanos, J. J. (1994). In vivo expression technology for selection of bacterial genes specifically induced in host tissues. Methods Enzymol 235, 481–492.[Medline]

Smith, K. & Youngman, P. (1992). Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74, 705–711.[CrossRef][Medline]

Smith, G. A., Marquis, H., Jones, S., Johnston, N. C., Portnoy, D. A. & Goldfine, H. (1995). The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect Immun 63, 4231–4237.[Abstract]

Soncini, F. C., Garcia Vescovi, E., Solomon, F. & Groisman, E. A. (1996). Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes. J Bacteriol 178, 5092–5099.[Abstract]

Stanley, T. L., Ellermeier, C. D. & Slauch, J. M. (2000). Tissue-specific gene expression identifies a gene in the lysogenic phage Gifsy-1 that affects Salmonella enterica serovar typhimurium survival in Peyer's patches. J Bacteriol 182, 4406–4413.[Abstract/Free Full Text]

Sun, A. N., Camilli, A. & Portnoy, D. A. (1990). Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect Immun 58, 3770–3778.[Medline]

Swanson, M. S. & Hammer, B. K. (2000). Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54, 567–613.[CrossRef][Medline]

Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J. & Kreft, J. (2001). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584–640.[Abstract/Free Full Text]

Vega, Y., Dickneite, C., Ripio, M.-T., Böckmann, R., Gonzalez-Zorn, B., Novella, S., Gominguez-Bernal, G., Goebel, W. & Vazquez-Boland, W. (1998). Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA. J Bacteriol 180, 6655–6660.[Abstract/Free Full Text]

Wosten, M. M., Kox, L. F., Chamnongpol, S., Soncini, F. C. & Groisman, E. A. (2000). A signal transduction system that responds to extracellular iron. Cell 103, 113–125.[Medline]

Youngman, P. (1987). Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other Gram-positive bacteria. In Plasmids: a Practical Approach, pp. 79–103. Edited by K. Hardy. Oxford: IRL Press.

Received 23 April 2002; revised 20 September 2002; accepted 26 September 2002.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Greene, S. L.
Articles by Freitag, N. E.
Articles citing this Article
PubMed
PubMed Citation
Articles by Greene, S. L.
Articles by Freitag, N. E.
Agricola
Articles by Greene, S. L.
Articles by Freitag, N. E.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2003 Society for General Microbiology.