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
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ABSTRACT |
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Keywords: microbial pathogenicity, surface protein, bacterial competence
The GenBank accession number for the sequence reported in this paper is AF282221.
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INTRODUCTION |
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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.
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METHODS |
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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 svpAaphA3) was constructed by deletion of a 79 bp internal fragment of svpA (nt 559638) 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 EcoRIBamHI LO28 DNA fragment (-397 to +557), an 855 bp BamHI E. faecalis DNA fragment carrying aphA-3 and a 1119 bp BamHIXbaI 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
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
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 (10500 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 immunoglobulinperoxidase 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 50100 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)
.
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RESULTS |
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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 545563) 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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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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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 34 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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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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DISCUSSION |
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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 proteinprotein 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.
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ACKNOWLEDGEMENTS |
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Received 20 April 2001;
revised 5 July 2001;
accepted 10 July 2001.