SvpA, a novel surface virulence-associated protein required for intracellular survival of Listeria monocytogenes

Elise Borezée1, Elisabeth Pellegrini1, Jean-Luc Beretti1 and Patrick Berche1

Laboratoire de Microbiologie, Institut National de la Santé et de la Recherche Médicale U411, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France1

Author for correspondence: Patrick Berche. Tel: +33 1 40 61 53 71. Fax: +33 1 40 61 55 92. e-mail: berche{at}necker.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A previously unknown protein, designated SvpA (surface virulence-associated protein) and implicated in the virulence of the intracellular pathogen Listeria monocytogenes, was identified. This 64 kDa protein, encoded by svpA, is both secreted in culture supernatants and surface-exposed, as shown by immunogold labelling of whole bacteria with an anti-SvpA antibody. Analysis of the peptide sequence revealed that SvpA contains a leader peptide, a predicted C-terminal transmembrane region and a positively charged tail resembling that of the surface protein ActA, suggesting that SvpA might partially reassociate with the bacterial surface by its C-terminal membrane anchor. An allelic mutant was constructed by disrupting svpA in the wild-type strain LO28. The virulence of this mutant was strongly attenuated in the mouse, with a 2 log decrease in the LD50 and restricted bacterial growth in organs as compared to the wild-type strain. This reduced virulence was not related either to a loss of adherence or to a lower expression of known virulence factors, which remained unaffected in the svpA mutant. It was caused by a restriction of intracellular growth of mutant bacteria. By following the intracellular behaviour of bacteria within bone-marrow-derived macrophages by confocal and electron microscopy studies, it was found that most svpA mutant bacteria remained confined within phagosomes, in contrast to wild-type bacteria which rapidly escaped to the cytoplasm. The regulation of svpA was independent of PrfA, the transcriptional activator of virulence genes in L. monocytogenes. In fact, SvpA was down-regulated by MecA, ClpC and ClpP, which are highly homologous to proteins of Bacillus subtilis forming a regulatory complex controlling the competence state of this saprophyte. The results indicate that: (i) SvpA is a novel factor involved in the virulence of L. monocytogenes, promoting bacterial escape from phagosomes of macrophages; (ii) SvpA is, at least partially, associated with the surface of bacteria; and (iii) SvpA is PrfA-independent and controlled by a MecA-dependent regulatory network.

Keywords: microbial pathogenicity, surface protein, bacterial competence

The GenBank accession number for the sequence reported in this paper is AF282221.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Listeria monocytogenes is a ubiquitous Gram-positive, facultative intracellular bacterium responsible for severe food-borne infections in humans and many animals (Gray & Killinger, 1966 ). The virulence of this pathogen is due to its capacity to invade and multiply within host cells, including macrophages, hepatocytes, epithelial and endothelial cells. Early after internalization, bacteria disrupt the phagosomal membrane and access the cytoplasm where they polymerize actin and spread from cell to cell (Gaillard et al., 1987 ; Tilney & Portnoy, 1989 ). Each step of the intracellular parasitism is dependent upon the production of virulence factors, including internalin (InlA), listeriolysin O (LLO), phospholipases and ActA, which are controlled by the pleiotropic transcriptional activator PrfA (Cossart & Lecuit, 1998 ).

Like other intracellular pathogens, L. monocytogenes is exposed to hostile conditions during intracellular survival, including starvation, low pH, chemical and enzymic stresses and elevated temperature. L. monocytogenes has evolved a complex adaptive network to maintain cell viability under stress and ensure persistence and growth both in the environment and occasionally in host tissues. In this network, stress proteins such as Clp (caseinolytic proteins) play a crucial role. Clp possess an ATPase activity and belong to the 100 kDa heat-shock protein (HSP100) Clp family of highly conserved and universal molecular chaperones (Schirmer et al., 1996 ). They are involved in the folding and assembly of proteins, and in the regulation of ATP-dependent proteolysis, ultimately promoting stress-induced resolubilization of aggregates (Gottesman & Maurizi, 1992 ; Schirmer et al., 1996 ; Squires & Squires, 1992 ). In Escherichia coli, ClpP alone degrades peptides less than 7 aa long (Woo et al., 1989 ). When associated with ClpA or ClpX, ClpP expresses protease activity against specific substrates determined by the ATPase subunit (Gottesman et al., 1998 ; Larsen & Finley, 1997 ; Wang et al., 1997 ). In L. monocytogenes, we previously found that two Clp, ClpC and ClpE, play an important role in stress tolerance and in vivo intracellular survival (Nair et al., 1999 ; Rouquette et al., 1996 , 1998 ). We also identified a stress-induced serine protease ClpP, required for growth under stress conditions and playing a crucial role in intracellular survival of L. monocytogenes (Gaillot et al., 2000 ). ClpP modulates the production of LLO under stress conditions (Gaillot et al., 2000 ).

Clp also play regulatory functions in Bacillus subtilis, a genetically related soil Gram-positive bacterium. The B. subtilis ClpC protein, which is highly homologous to ClpC of L. monocytogenes, negatively controls bacterial competence, a physiological state allowing bacteria to efficiently internalize exogenous DNA. Indeed, ClpC of B. subtilis forms a ternary complex with MecA and ComK, leading to the sequestration and degradation of ComK, the key transcriptional activator of competence (Turgay et al., 1998 ). Recently, we identified a MecA homologue in L. monocytogenes. This protein mimics the regulatory function of B. subtilis MecA, repressing transcription of comK, and subsequently comG, when introduced in B. subtilis (Borezée et al., 2000a ). Although we failed to demonstrate any competence in L. monocytogenes, many homologues of late competence gene products and competence regulatory proteins of B. subtilis are present in this pathogen (Borezée et al., 2000a ). Interestingly, we found that a 64 kDa secreted protein (p64) of L. monocytogenes accumulates in the supernatant of mecA, clpC and clpP mutants. These results suggested that MecA of L. monocytogenes might belong to a signal transduction network involved in the regulatory processes of this pathogen (Borezée et al., 2000a ).

In this work, we studied the role of this secreted protein by constructing an allelic mutant of the gene encoding this protein. We found that the 64 kDa protein is required for intracellular survival and virulence of L. monocytogenes. This protein is a novel PrfA-independent factor implicated in the virulence of L. monocytogenes. It was designated SvpA for surface virulence-associated protein, encoded by the gene svpA.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, growth conditions and transformation.
We used L. monocytogenes reference strain LO28 and E. coli DH5. All strains were routinely grown in Brain Heart Infusion (BHI) medium. Antibiotics were used at the following concentrations: ampicillin, 100 µg ml-1 for E. coli; erythromycin (Em), 8 µg ml-1 for L. monocytogenes and 200 µg ml-1 for E. coli; kanamycin (Km), 50 µg ml-1; colistin, 10 µg ml-1; nalidixic acid, 50 µg ml-1. Constructs were introduced into Listeria strains by conjugation or electroporation as described previously (Poyart et al., 1993 ). Bacterial growth and phenotype analysis of strains were performed as described by Rouquette et al. (1996) . The metabolic profiles were determined on an API strip (50 substrates; BioMérieux).

DNA manipulations, RNA extraction and reverse transcription (RT) PCR.
Chromosomal DNA, plasmid extraction, electrophoresis, restriction enzyme analysis, hybridizations and amplification by PCR were performed according to standard protocols (Sambrook et al., 1989 ). DNA sequencing was performed with the ABI-Prism 310 Sequencer (Perkin Elmer). Total RNA was extracted as described by Celli & Trieu-Cuot (1998) from L. monocytogenes cultures grown until mid-exponential phase in BHI broth at 37 °C and subjected to RT-PCR, as described previously (Borezée et al., 2000b ). The following primers were used to amplify svpA and LiM00807.1 from chromosomal DNA or total RNA of LO28 and the svpA mutant strains: svpa (5'-CGGGATCCAAGGGGGATTATAATGAAGAAATTATGG-3') and svpb 5'-TCTAGATAACCCCGACCTAACTCGC-3'); 807a (5'-GGGGCCAAATTTGATCGTA-3') and 807b (5'-GTGCTTCTGGCGCATTC-3'), respectively.

Construction of an svpA-deleted mutant.
An svpA mutant (LO28 svpA{Omega}aphA3) was constructed by deletion of a 79 bp internal fragment of svpA (nt 559–638) and insertion of a promoterless aphA-3 gene conferring resistance to Km (Menard et al., 1993 ) by double recombination. The deletion/replacement mutant of svpA was constructed by inserting a 964 bp EcoRI–BamHI LO28 DNA fragment (-397 to +557), an 855 bp BamHI E. faecalis DNA fragment carrying aphA-3 and a 1119 bp BamHI–XbaI LO28 DNA fragment (+634 to +1746), between the EcoRI and XbaI sites of the thermosensitive shuttle vector pAUL-A (Chakraborty et al., 1992 ) to give plasmid pAUL-svpA{Omega}aphA3. Positions are given relative to the translation initiation codon of svpA. These three DNA fragments were generated by PCR using the following primers: mut1 (5'-GAATTCGGGCCTATGGGTTGAAGGGAACGC-3') and mut2 (5'-GGATCCGAAAGAGTCACAGGTGTTG-3'); km1 (5'-CGGGATCCCGACTAACTAGGAGGAATA-3') and km2 (5'-CGGGATCCCGGGTCATTATTCCCTCC-3'); mut3 (5'-GGATCCTGCTTGGTTGAAAGTG-3') and svpb. pAUL-svpA{Omega}aphA3 was introduced into LO28 by electroporation and transformants were selected for Em resistance at 30 °C. We used a gene replacement procedure described by Chakraborty et al. (1992) to obtain an isogenic mutant carrying the disrupted svpA gene on the chromosome. The genotype of the mutant was confirmed by PCR sequencing and Southern blotting.

Overproduction, purification of His6-SvpA and His6-MecA, and antibody production.
Plasmids pET-14b and pET-20b(+) were used for protein overexpression and purification (Novagen). The L. monocytogenes svpA and mecA genes were amplified by PCR using primers: svp1 (5'-CATATGAAGAAATTATGGAAAAAAGGCTTAGTAGC-3') and svp2 (5'-GGATCCTTAACTCAATCTTTTACGTTTTAATCG-3'), mec1 (5'-CCATGGAAATTGAACGAATTAATGAGG-3') and mec2 (5'-GGATCCGAGAAGTGTTTTCTAATTTGCTTTAATG-3'), respectively. The svpA and mecA PCR products were then cloned into the NdeI/BamHI sites of plasmid pET-14b and the NcoI/BamHI sites of pET20b(+), to generate pETsvpA and pETmecA, respectively. These plasmids were used to transform E. coli strain BL21DE3 in which the T7 RNA polymerase gene is under the control of the inducible lacUV5 promoter. The recombinant strains were grown in LB medium at 37 °C to mid-exponential exponential phase (OD600=0·7). IPTG (1 mM) was then added and incubation continued for 2 h. The cells were centrifuged, resuspended in 1/50 of the culture volume of PBS, disrupted by sonication and cell debris removed by centrifugation. E. coli crude extracts were loaded on a 1 ml Poly-His Protein Purification Resin column (Roche) previously equilibrated with PBS and the His6-tagged proteins were eluted with an imidazole gradient (10–500 mM). The eluted fractions were subjected to SDS-PAGE as described by Laemmli (1970) . Protein concentrations were determined using the Bio-Rad protein assay (Bradford, 1976 ). Molecular size references markers were obtained from Life Technologies. The His6-tagged proteins were used for custom antibody production in rabbits (Centre de Production Animale, Olivet, France).

Western blot analysis.
Cultures of LO28 and mutant strains in the exponential growth phase were pelleted and supernatants collected and filtered. Supernatant protein extracts were prepared by TCA precipitation as described by Sambrook et al. (1989) and further concentrated with ultrafree columns (Millipore). The bacterial pellet was resuspended in 1/20 of the culture volume of Tris (10 mM)/EDTA (1 mM) and sonicated as described by Rouquette et al. (1998). Bacterial debris was removed by centrifugation and the resulting supernatant consisted of the cytoplasmic proteins. Supernatant and cytoplasmic protein extracts were analysed by Western blotting as described by Geoffroy et al. (1991) . The membranes were incubated with rabbit polyclonal antibodies for His6-SvpA and His6-MecA, or with mouse mAbs directed against purified LLO, ActA, InlA, InlB, PC-PLC (obtained from P. Cossart, Institut Pasteur, Paris, France). Anti-rabbit or anti-mouse immunoglobulin–peroxidase conjugates were used for immunodetection (Sigma). Enzymic activity was revealed by the addition of diamino-benzidine tetrahydrochloride (Sigma) supplemented with hydrogen peroxide (0·1%).

Infection of macrophages.
Macrophages were infected at a bacterium/macrophage ratio of 1:1 and 15:1 for growth curves and microscopic studies, respectively. Bone-marrow-derived macrophages from C57/BL6 mice were cultured and infected as previously described (Gaillot et al., 2000 ). After 15 min of bacterial adherence on ice, macrophages were exposed for 15 min at 37 °C (time 0). The number of intracellular bacteria was estimated in cell lysates at selected intervals (from 0 to 8 h post-infection). Double fluorescence labelling of F-actin and bacteria was performed as described by Kocks et al. (1992) using phalloidin coupled to Oregon Green 488 (Molecular Probes) and a rabbit anti-Listeria serum (J. Rocourt, Institut Pasteur, Paris, France) revealed with an anti-IgG antibody coupled to Alexa 546 (Molecular Probes). Images were scanned on a Zeiss LSM 510 confocal microscope.

Culture of Caco-2 cells and adhesion assays.
The human colon carcinoma Caco-2 cell line was propagated as described by Gaillard & Finlay (1996) . All incubations were carried out in a 10% CO2 atmosphere at 37 °C. Cells were seeded at 105 cells cm-2 onto 12 mm diameter glass coverslips in 24-well plates. Monolayers were used 24 h after seeding. Bacteria were grown for 16 h in BHI broth, pelleted, washed once and diluted appropriately in DMEM. Cells were inoculated with bacteria at a ratio of 100 bacteria per cell and incubated for 1 h. They were subsequently washed, fixed and processed for immunolabelling as described by Milohanic et al. (2000) . Coverslips were mounted on slides and examined by fluorescence microscopy. Each assay was carried out in triplicate and repeated twice. Adherent bacteria were counted by examining 50 cells in randomly selected microscope fields.

Processing for electron microscopy.
Macrophages were infected at a bacterium/macrophage ratio of 15:1, fixed for 1 h at room temperature and processed as described previously (de Chastellier & Berche, 1994 ). The percentage of intraphagosomal or intracytoplasmic bacteria was determined at selected intervals post-infection (0, 4, 6 h) for 50–100 different cell profiles (about 100 bacteria were examined per time point). For immunogold labelling, bacteria were grown overnight in BHI broth and processed as described previously (Gaillard et al., 1991 ). The grids were incubated for 1 h with rabbit anti-Listeria or rabbit anti-SvpA antibodies and further incubated with goat anti-rabbit IgG conjugated to 10 nm gold particles.

Mouse virulence assay.
Six- to eight-week-old female Swiss mice (Janvier) were inoculated intravenously (i.v.) with various doses of bacteria (Gaillard et al., 1996 ). Mortality was followed over a 14-d period on groups of five mice. The LD50 was determined by the probit method. Bacterial growth was followed in organs (spleen and liver) of mice infected i.v. with 8x105 bacteria as described by Nair et al. (1999) .


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The svpA gene encodes a secreted protein
We previously identified a secreted p64 protein of 569 aa (63·4 kDa) of unknown function (Borezée et al., 2000a ), encoded by a gene of 1707 nt designated svpA (GenBank accession no. AF282221). The genetic organization of the svpA region of LO28 is similar to that of strain EGD-E recently sequenced by the Listeria Genome Consortium. svpA corresponds to ORF LiM00806.1 of EGD-E and is located upstream of ORF LiM00807.1, separated by a 114 bp non-coding region. There is neither an obvious promoter nor any prfA boxes detectable in the upstream region of svpA. A transcriptional analysis by Northern blotting failed to detect svpA transcripts in the parental strain LO28 and in the mecA mutant, presumably due to unstable transcripts. However, we detected in LO28 specific svpA transcripts by RT-PCR using the primers svpa and svpb (data not shown).

As previously reported (Borezée et al., 2000a), the SvpA protein is identical to the product of ORF LiM00806.1 of strain EGD-E from the Listeria genome project. SvpA shares several repeated homologies with an ORF of 82 aa encoded by the virulence plasmid pXO1 from Bacillus anthracis (~33% identity). Analysis of the peptide sequence of SvpA revealed three distinct regions: a peptide leader sequence with a predicted signal peptidase cleavage site between residues 28 and 29, a large region containing a proline-rich domain (residues 316 to 348), forming a predicted strong secondary structure and a stretch of 19 hydrophobic amino acids, presumed to form a transmembrane helix at the C-terminal extremity (residues 545–563) of the protein (Fig. 1). The hydrophobic region is followed by a tail of six residues, most of which are positively charged. The presence of a predicted peptide signal associated with a cleavage site is in accordance with the fact that SvpA is secreted in the culture supernatant. Nevertheless, it is possible that SvpA might reassociate to the bacterial surface by its predicted C-terminal transmembrane region.



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Fig. 1. Schematic representation of the SvpA protein of L. monocytogenes. The SvpA peptide sequence contains a signal peptide at the N terminus (N), a large region with a central proline-rich domain and a C-terminal region (C) composed of a transmembrane helix and a positively charged tail. The amino-acid sequence of the C-terminal region is indicated below and its hydrophobic domain is underlined. The amino acid sequence of the proline-rich domain is also indicated; proline residues are in bold.

 
Construction and phenotypic analysis of a deleted svpA mutant
An allelic mutant was constructed by deletion of an internal fragment of svpA and insertion of an aphA-3 cassette by double-crossover into the chromosomal DNA of strain LO28, using a two-step procedure described by Chakraborty et al. (1992) . The mutant was verified by Southern blotting and PCR sequencing of the svpA region. A transcriptional analysis was performed in wild-type and svpA mutant strains by RT-PCR using specific primers of svpA and of the downstream ORF LiM00807.1 mentioned above. In this mutant, the svpA transcript was larger in size due to the aphA-3 cassette insertion. The ORF LiM00807.1 transcript was present in both strains (data not shown), indicating that there is no polar effect due to the aphA-3 gene insertion in the mutant.

As compared with the parental strain LO28, the phenotypic analysis of the svpA mutant did not reveal any difference with respect to microscopic morphology (Gram staining), aspect of colonies, motility at 22 °C, the profiles of 50 metabolic characters using API-CH50, or the haemolytic activity on horse-blood agar plates (data not shown). Ultra-thin sections of bacteria were also examined by electron microscopy, which detected neither morphological difference nor any alteration in the bacterial cell wall (data not shown). Growth of the svpA mutant in BHI broth was moderately delayed at 37 °C, reaching a lower bacterial density at the stationary phase as compared to the wild-type strain (Fig. 2).



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Fig. 2. Growth of L. monocytogenes LO28 wild-type ({bullet}) or svpA mutant ({blacksquare}) in BHI broth at 37 °C. Wild-type or svpA bacteria were grown at 37 °C and OD600 was measured at various intervals.

 
SvpA is a surface-exposed protein
Although SvpA is detected in culture supernatants, its predicted structure indicates a C-terminal sequence consisting of a 19 aa hydrophobic domain and a positively charged tail. This situation is reminiscent of that of other L. monocytogenes proteins, i.e. InlA (Gaillard et al., 1991 ) or ActA (Domann et al., 1992 ; Kocks et al., 1992 ; Lebrun et al., 1996 ), which are secreted and reassociated with the bacterial surface. We therefore tested the hypothesis that SvpA could be partially exposed on the bacterial surface. Wild-type and svpA mutant bacteria were immunolabelled using anti-SvpA serum. Whole bacteria were stained by the immunogold method and examined by electron microscopy. As a control, bacteria were also labelled with an anti-Listeria serum, revealing a strong labelling surrounding wild-type and svpA mutant bacteria (Fig. 3a, b). With the anti-SvpA serum, wild-type bacteria were efficiently stained (about 102 gold particles per bacterium) (Fig. 3c), whereas a background labelling was seen with the svpA mutant (about 25 gold particles per bacterium) as illustrated in Fig. 3(d). We conclude that the secreted SvpA might at least partially associate to the bacterial surface.



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Fig. 3. Immunogold labelling of wild-type strain (a and c) and svpA mutant strain (b and d), using an anti-Listeria serum (a and b) or an anti-SvpA serum (c and d). In these representative pictures SvpA was detected on the surface of wild-type bacteria, but svpA mutant bacteria were not significantly labelled.

 
As the SvpA protein is present at the surface of L. monocytogenes, it might be implicated in the binding to non-professional phagocytic host cells. We tested the role of SvpA in the binding of L. monocytogenes to Caco-2 cells, using a method described by Milohanic et al. (2000). We did not find any difference in the capacity to adhere to Caco-2 cells between the wild-type (32±11 bacteria per cell islet) and svpA mutant strains (36±12 bacteria per cell islet), suggesting that SvpA itself does not play a role in adhesion.

SvpA is required for the virulence of L. monocytogenes
We studied the role of the secreted SvpA protein in the virulence of L. monocytogenes. The LD50 of LO28 and an svpA mutant were determined by inoculating (i.v.) Swiss mice with increasing doses of bacteria. The LD50 value of the svpA mutant was 107·2 bacteria per mouse, indicating a 2 log decrease compared to the wild-type strain (105). Bacterial survival in organs was monitored over a period of 3 d by following the number of viable bacteria in the spleen and the liver of mice infected i.v. with 105·6 bacteria (Fig. 4). In contrast to the wild-type strain, which rapidly grew in organs until the death of mice, mutant bacteria were progressively eliminated in the liver and the spleen over the 3-d period. The growth of the svpA mutant was severely restricted in the spleen and the liver, with a 3–4 log difference compared to the wild-type strain by day 3 post-infection (Fig. 4). We examined the expression of the main virulence factors in supernatants of wild-type and mutant bacteria (InlA, InlB, LLO, ActA, PC-PLC). They were not altered in the svpA mutant, as shown by Western blotting analysis with specific anti-sera (Fig. 5a). These results show the role of SvpA as a novel secreted factor required for virulence of L. monocytogenes.



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Fig. 4. Growth of L. monocytogenes LO28 wild-type ({bullet}) or svpA mutant ({blacksquare}) in the spleen (a) or the liver (b) of mice inoculated i.v. with 105·6 bacteria.

 


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Fig. 5. (a) Western blot analysis of culture supernatant extracts of wild-type and svpA mutant L. monocytogenes revealed with anti-InlA, anti-InlB, anti-LLO, anti-ActA and anti-PC-PLC antibodies. The expression of the known virulence factors LLO, ActA, PC-PLC, InlA and InlB is not altered in the svpA mutant. (b) Western blot analysis of culture supernatant extracts of wild-type, svpA, mecA, clpC, clpP and prfA mutant L. monocytogenes using anti-SvpA antibodies. The expression of SvpA is controlled by MecA, ClpC and ClpP but does not depend on PrfA. (c) Western blot analysis of cytoplasmic extracts of wild-type, mecA, svpA, clpC and clpP mutant L. monocytogenes revealed with anti-MecA antibodies. The MecA protein of L. monocytogenes accumulates in the clpC and clpP mutants.

 
The expression of SvpA is controlled by MecA, ClpC and ClpP, and is PrfA-independent
Using SDS-PAGE analysis, we previously found that SvpA (formerly termed p64) was overexpressed in the supernatants of the mecA, clpP and clpC mutants (Borezée et al., 2000a). This finding was confirmed by Western blotting analysis using a rabbit anti-SvpA serum raised against purified His-tagged SvpA. SvpA was not detected in the supernatant of the svpA mutant, as opposed to the wild-type strain, thus confirming that the expression of SvpA is abrogated in the mutant (Fig. 5b). SvpA is overexpressed in the mecA, clpP and clpC mutants (Fig. 5b). In contrast, the expression of SvpA is similar in LO28 and in a prfA mutant of LO28, showing that PrfA is not necessary to induce expression of svpA (Fig. 5b). We also purified His6-MecA with the His-tag system and prepared a rabbit anti-MecA serum. The expression of MecA was similar in cytoplasmic extracts of LO28 and of the svpA mutant (Fig. 5c), showing that SvpA does not regulate MecA. On the other hand, MecA accumulated in the clpC and clpP mutants (Fig. 5c). This result is consistent with the apparent control of the B. subtilis MecA level by the ClpC/ClpP protease complex (Msadek et al., 1998 ; Turgay et al., 1998 ).

One might expect that the overexpression of SvpA could exacerbate the level of virulence of L. monocytogenes. So, we compared the level of virulence of LO28 with that of the mecA mutant. No significant difference was found, as determined by LD50 and the growth curves in the spleen and liver over a 3 d period as described above (data not shown). Conversely, we also tested the virulence of LO28 transformed with a multicopy plasmid harbouring mecA, which down-regulates the expression of SvpA. The virulence was attenuated in this strain as shown by the LD50 value estimated at 107 per mouse as compared to 105 for the wild-type strain. Altogether, these results indicate that: (i) SvpA is down-regulated by MecA, ClpC and ClpP; (ii) SvpA is not controlled by the transcriptional activator PrfA controlling virulence genes; and (iii) the level of MecA is also controlled by ClpC and ClpP.

SvpA is required for intracellular growth in macrophages
The behaviour of the svpA mutant and LO28 were studied in bone-marrow-derived macrophages from C57/BL6 mice. Macrophages were exposed to a bacterium/cell ratio of 1:1. As shown in Fig. 6, a similar amount of bacteria associated with macrophages was found at the onset of infection, suggesting that SvpA is not involved in the uptake by macrophages. After a latent phase of 2 h, wild-type bacteria rapidly multiplied inside macrophages, inducing cellular lysis 6 h post-infection. In contrast, svpA mutant bacteria were partially killed in the first 2 h post-infection (decrease of 0·5 log), suggesting that the svpA mutant might be more susceptible to the bactericidal activity of phagosomes than wild-type bacteria. Surviving bacteria grew slowly until 6 h post-infection. Then, the number of viable bacteria remained stationary, without significant lysis of macrophages. These results indicate that SvpA plays an important role in the intracellular survival of L. monocytogenes.



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Fig. 6. Growth of L. monocytogenes LO28 wild-type ({bullet}) or svpA mutant ({blacktriangleup}) in macrophages. Bone-marrow-derived macrophages from C57/BL6 mice were exposed for 15 min (time 0) to bacteria (1 bacterium per cell) and bacterial survival was followed for 8 h post-infection.

 
SvpA facilitates the bacterial escape from phagosomes of macrophages
The intracellular fate of wild-type and svpA mutant bacteria was then studied by confocal microscopy in bone-marrow-derived macrophages (bacterium/cell 15:1). Infected cells were examined at various intervals (0, 4 and 8 h) after double staining with an anti-Listeria antibody and with ß-phalloidin to visualize F-actin. As found above, the early bacterial uptake (time 0) was similar for the wild-type and mutant bacteria (Fig. 7a and d). At 4 and 8 h post-infection, most wild-type bacteria were associated with polymerized actin, either surrounded by sheaths of actin, or, more often, forming comets tails (Fig. 7b and c). In contrast, at 4 h post-infection, most mutant bacteria were apparently confined within phagosomes, with few bacteria associated with polymerized actin and comets (Fig. 7e). At 8 h post-infection, the total number of intracellular and intracytoplasmic mutant bacteria associated with actin or comets slowly increased, but remained much lower than those seen with wild-type bacteria (Fig. 7c and f).



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Fig. 7. Confocal microscopy of bone-marrow-derived mouse macrophages infected (15 bacteria per cell) with LO28 (a–c) or svpA mutant (d–f). Macrophages were observed at 0 (a, d), 4 (b, e) and 8 h (c, f) post-infection. F-actin was stained with phalloidin (green). Bacteria were labelled with an anti-Listeria serum (red). The majority of svpA mutant bacteria are not associated with actin and the intracellular growth is restricted.

 
We performed a quantitative electron microscopic study on macrophages infected by LO28 or the svpA mutant under the conditions described above (see Methods). Intracytoplasmic wild-type bacteria reached 69 and 92% of the total number of intracellular bacteria 4 and 6 h post-infection, respectively. In contrast, only 22 and 41% of svpA mutant bacteria were visible inside the cytoplasm 4 and 6 h post-infection. Typical aspects of intracellular wild-type and mutant bacteria 4 and 6 h post-infection are illustrated in Fig. 8(a–f). As expected, many wild-type bacteria escaped from the phagosomes at 4 h post-infection (Fig. 8a) and most bacteria were located inside the cytoplasm and surrounded with polymerized actin after 6 h (Fig. 8c, e). In contrast, a majority of svpA mutant bacteria remained confined in phagosomes where they were progressively destroyed (Fig. 8b, d, f). Rare bacteria surrounded by polymerized actin (Fig. 8b, d) and actin comets (Fig. 8d) were seen for the mutant 4 and 6 h post-infection, respectively. These results indicate that SvpA promotes the phagosomal escape of intracellular bacteria and the subsequent access to the cytoplasm of macrophages.



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Fig. 8. Distribution of L. monocytogenes within macrophages. Bone-marrow-derived mouse macrophages were infected with the wild-type (a, c, e) or svpA mutant (b, d, f) strains and processed for electron microscopy at selected times post-infection (a–f). Ultra-thin sections of macrophages 4 (a, b) and 6 h (c–f) post-infection. Arrows indicate intracytoplasmic bacteria. About 70 and 90% of wild-type bacteria are free in the cytoplasm 4 (a) and 6 h (c, e) post-infection, respectively. Cytoplasmic bacteria are surrounded by actin sheets and many of them by a double membrane, a result typical of cell-to-cell spreading in adjacent cells. In contrast, the majority of svpA mutant bacteria are enclosed within phagosomes (b, d, f). The small fraction of mutant bacteria accessing the cytoplasm could polymerize actin, as shown by an actin sheet (b, d) and a typical actin comet (d).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study we identified a novel secreted protein, SvpA, involved in the virulence of L. monocytogenes. The role of this protein was studied by constructing an svpA-deleted mutant. The in vitro phenotypic analysis of this mutant revealed that it closely resembles the wild-type strain, except that its growth was impaired at 37 °C (Fig. 2). The most important finding is that the virulence of a deleted svpA mutant is strongly attenuated with a 2 log drop of the LD50 value (107·2 versus 105). This loss of virulence was due to the restriction of bacterial growth in the spleen and liver of infected mice receiving a lethal challenge. We showed that SvpA plays an important role in the intracellular survival of L. monocytogenes. Mutant bacteria remained confined in phagosomes of bone-marrow macrophages, in contrast to wild-type bacteria, which rapidly escaped from phagosomes. This longer exposure to bactericidal activity of phagosomes in macrophage might explain that mutant bacteria were partially killed in the first 2 h post-infection, as compared to wild-type bacteria (Fig. 6). As a result, the intracellular growth was reduced in the mutant, with few bacteria escaping the phagosomes and multiplying in the cytoplasm of macrophages, without significant cellular lysis 6 h post-infection (Fig. 6). By confocal microscopy (Fig. 7) and quantitative electron microscopy, we confirmed that SvpA facilitates the bacterial escape from phagosomes since only 41% of mutants were seen in the cytoplasm 6 h post-infection, compared to 92% for wild-type bacteria. Many mutant bacteria remained confined in phagosomes (Fig. 8). These results indicate that SvpA promotes intracellular survival by facilitating the phagosomal escape of bacteria in macrophages.

Although the 64 kDa SvpA is released in the culture supernatant (Fig. 5), this protein is also associated and exposed to the bacterial surface, as demonstrated by immunogold labelling (Fig. 3). However, SvpA is apparently not involved in bacterial adhesion to bone-marrow macrophages and Caco-2 cells, since an svpA mutant was efficiently internalized by macrophages and adhered normally to Caco-2 cells. The SvpA protein might be anchored to the bacterial membrane through its C-terminal hydrophobic region. Indeed, analysis of the peptide sequence revealed that SvpA possesses a peptide leader sequence, a large region containing a proline-rich domain and a predicted transmembrane segment followed by a short, positively charged tail at the C terminus (Fig. 1). This structure has similarity with some domains of ActA and InlA. Interestingly, these proteins are secreted and reassociated with the surface by their C-terminal domain. The hydrophobic region of InlA is preceded by the consensus motif LPXTG, required for the covalent anchoring of the protein to the cell wall (Lebrun et al., 1996 ). Nevertheless, surface proteins from Gram-positive bacteria exist which are not covalently anchored to the cell wall. This is the case for ActA, a virulence factor involved in actin assembly and intracellular movement, which possesses a hydrophobic domain of 22 residues followed by 4 charged residues, without the LPXTG motif (Kocks et al., 1992 ). This surface protein is assumed to be anchored to the cytoplasmic membrane via its C-terminal transmembrane helix (Chakraborty, 1999 ). Similarly, SvpA could be tethered to bacteria without an LPXTG motif. Another interesting feature is that SvpA also possesses a proline-rich region of 32 aa. ActA also contains a central domain with four proline-rich repeats involved in the direct binding to cytoskeletal components of infected cells (Chakraborty, 1999 ; Niebuhr et al., 1997 ). It is known that these proline-rich motifs are often involved in protein–protein interactions (Kay et al., 2000 ). Although the significance of the proline-rich domain of SvpA is unknown, it is predicted to form a secondary structure that might interact with host-cell components during the intracellular parasitism of L. monocytogenes. Since SvpA presumably acts inside phagosomes, one could speculate that SvpA might interact with the components of the phagosomal membrane to promote bacterial escape from this cellular compartment. However, the function of SvpA remains unknown.

By studying the regulation of SvpA, we found that it is not controlled by the transcriptional activator controlling virulence genes, PrfA: a prfA mutant produced the same amount of SvpA in culture supernatants as that of wild-type bacteria (Fig. 5b). On the other hand, SvpA was down-regulated by MecA, ClpC and ClpP. Using an antiserum raised against purified SvpA, we found by Western blotting analysis that SvpA accumulates in the culture supernatants of mecA, clpC and clpP mutants, as compared to the wild-type bacteria (Fig. 5b). SvpA might be part of a MecA-dependent regulatory network proposed previously (Borezée et al., 2000a ). We previously found by heterologous complementation in B. subtilis that MecA of L. monocytogenes mimics the regulatory function of the B. subtilis MecA protein, presumably forming a complex with ClpC and ClpP to sequester and degrade ComK, a transcriptional activator of the late competence genes in B. subtilis (Turgay et al., 1997 , 1998 ). The regulation of SvpA by MecA might be indirect and the expression of SvpA could be regulated by an unknown factor repressed by the MecA/ClpC/ClpP complex. We also found in the present study that L. monocytogenes MecA accumulates in the cytoplasm of the clpC and clpP mutants (Fig. 5c), suggesting that MecA might also be controlled by the ClpC/ClpP protease complex in this pathogen. In B. subtilis, MecA acts as a linker protein targeting ComK for proteolysis and is regulated by the same proteolytic complex (Msadek et al., 1998 ; Turgay et al., 1998 ). Although no competence could be detected in L. monocytogenes (Borezée et al., 2000a ; Rouquette et al., 1998 ), homologues for many competence factors of B. subtilis exist in L. monocytogenes. The MecA-dependent network of this pathogen might play an important role in bacterial physiology, including the down-regulation of SvpA, a factor implicated in virulence.

In conclusion, our results suggest that SvpA is a novel PrfA-independent factor required for intracellular survival and belongs to a regulatory network involving MecA, ClpC and ClpP. SvpA facilitates bacterial escape from phagosomes. SvpA might protect intraphagosomal bacteria from bacterial killing, and/or act synergistically with other virulence factors (listeriolysin O, phospholipases) or even directly to promote bacterial escape from phagosomes.


   ACKNOWLEDGEMENTS
 
We kindly thank S. Nair for critical reading of the manuscript and Y. Goureau for technical assistance with confocal microscopy. E.B. received a fellowship from the Ministère de l’Education Nationale de la Recherche et de la Technologie. This work was supported by INSERM, The University of Paris V, and two grants from the European Commission (contracts ERBCHRXCT 94-0451 and CT980036).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 20 April 2001; revised 5 July 2001; accepted 10 July 2001.