INSERM-U570, Faculté de Médecine Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex-15, France
Correspondence
Catherine Raynaud
cathraynaud{at}yahoo.fr
Alain Charbit
charbit{at}necker.fr
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ABSTRACT |
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INTRODUCTION |
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Bacterial proteins that are exported from the cytoplasm through the general secretory pathway (Sec machinery) are synthesized as precursors with an amino-terminal signal peptide (Pugsley & Possot, 1993). The signal peptide is required for the targeting of precursor proteins to the cytoplasmic membrane, and for the initiation of their translocation across this membrane. During, or shortly after, the translocation process, most signal peptides are removed by signal peptidases (SPases), which is a prerequisite for the release of secretory proteins from the extracytoplasmic side of the membrane (Paetzel et al., 2000
). Thus, SPases play a key role in the transport of protein across membranes in all living organisms (van Wely et al., 2001
). In Gram-positive bacteria, the exported proteins are either secreted into the medium, or they remain associated by various means with the bacterial envelope. In silico analysis of the genome of L. monocytogenes EGD-e revealed that more than 5 % of the coding sequences carry a typical signal peptide cleavable by type I SPases (SPases I) (Cabanes et al., 2002
; Glaser et al., 2001
).
We have recently reported the identification of a locus containing three contiguous genes encoding three SPases I in the genome of L. monocytogenes (Bonnemain et al., 2004). The construction of single and multiple knockout mutants in the sip genes showed that SipX and SipZ perform distinct functions in pathogenicity, and that SipZ is the major SPase I of L. monocytogenes. Most biological membranes contain one or two SPases I for the removal of signal peptides from the secretory precursor proteins. In this respect, the Gram-positive bacteria L. monocytogenes and Bacillus subtilis seem to be exceptional, since they possess three and five chromosomally encoded SPases I, respectively (van Roosmalen et al., 2001
). To date, the largest number of SPases I has been found in Bacillus anthracis and Bacillus cereus, which contain six and seven paralogous enzymes, respectively (see van Roosmalen et al., 2004
for a recent review). The unique SPase I of Escherichia coli, also known as leader peptidase (Lep), is essential for cell viability, and SPase I limitation results in accumulation of precursors of exported proteins. Similarly, the SPases I SpsB, from Staphylococcus aureus, and Sec11p, of the yeast endoplasmic reticulum membrane, are enzymes that are essential for cell viability. In contrast, the SPase I SipS of B. subtilis is not essential for cell viability, and mutant B. subtilis strains with a disrupted sipS gene are still able to process secreted preproteins (Bolhuis et al., 1999
). This observation seems to indicate that multiple SPases I serve to guarantee a sufficient capacity for protein secretion under various conditions (Tjalsma et al., 1997
).
In the present work, we studied the regulation of L. monocytogenes sip gene expression in broth and in infected eukaryotic cells. We show that the three sip genes are under the control of two distinct promoter regions. Our data reveal that the regulation of sip gene expression is temporally controlled, and influenced by complex global regulatory circuits.
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METHODS |
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Real-time quantitative PCR.
Total RNA isolated from cultures of L. monocytogenes grown in two different conditions was converted into DNA with reverse transcriptase, and quantitative PCR using sets of primers from different genes was performed to evaluate the relative levels of expression. Quantification of expression level was performed on EGD-e grown in BHI at 37 °C. We also compared EGD-e growing inside cells [bone-marrow-derived macrophages (BMM) from BALB/c mice and human enterocyte Caco-2 cells] with EGD-e growing in RPMI medium. For RNA preparations, cells were broken in a solution of 1 ml Trizol (Invitrogen) containing mini glass beads using a Bead Beater (Savant). Total RNA was extracted with 300 µl chloroform/isoamyl alcohol (24 : 1, v/v), then precipitated with 2-propanol, and resuspended in diethyl-pyrocarbonate-treated water. Contaminating DNA was removed by digestion with DNase I.
The absence of contaminating DNA after DNase treatment was confirmed by including a control reaction without reverse transcriptase in the RT-PCR assay (data not shown).
RT-PCR experiments were done with 1 µg RNA, with 2·5 pmol specific primers for the genes gyrA (gyrase, constitutively expressed), and sipX, sipY and sipZ, in a volume of 8 µl. After denaturation at 65 °C for 10 min, 12 µl of the mixture containing 2 µl dNTP (25 mM), 4 µl 4x buffer (provided with the Superscript II), 2 µl DTT, 1 µl RNasin (Promega), and 1·5 µl Superscript II (Invitrogen), was added. Samples were incubated for 60 min at 42 °C, heated at 75 °C for 15 min, and then chilled on ice. PCR conditions were identical for all reactions. The 25 µl reactions, performed in sealed tubes, consisted of 12·5 µl PCR master mix (PE Applied Biosystems) containing Sybr Green, 4 µl template, and 5 pmol each primer. For real-time quantitative PCR, we used the ABI Prism 7700 sequence detection system with TaqMan Universal PCR master mix (PE Applied Biosystems).
For the infection experiments, three different infections were performed, and each point corresponds to three measurements. The human colon carcinoma cell line Caco-2 (ATCC HTB37) was propagated in Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum at 37 °C with 5 % CO2. Cells were seeded at approximately 2x105 cells ml1 in tissue culture plates. Monolayers were used 24 h after seeding. Cells were infected with bacteria grown at 37 °C overnight at a multiplicity of 50 bacteria per cell. In experiments involving Caco-2 cells, the induction was measured in infected cells cultivated in RPMI, as compared with L. monocytogenes grown in RPMI.
Data analyses.
Results were normalized to the amount of gyrase mRNA, which was constant under our growth conditions (Réglier-Poupet et al., 2003). For example, to compare sipZ gene expression at pH 7 and 5, for each experiment (carried out three times) we calculated the ratio: (sipZ pH 5/gyrA pH 5)/(sipZ pH 7/gyrA pH 7), and then we calculated the means and standard deviations of the three values obtained.
-Galactosidase assays.
We used pTCV-lac, a mobilizable shuttle vector that enables transcriptional fusion to -galactosidase in a wide range of Gram-positive bacteria, in which it replicates at a low copy number per bacterium (Poyart & Trieu-Cuot, 1997
). All oligonucleotides were designed to add EcoRI and BamHI sites upstream and downstream of the amplified potential promoters (Table 1
). The amplified fragments were digested with EcoRI and BamHI, and cloned into pTCV-lac. Bacteria were grown overnight in BHI broth containing kanamycin. The cultures were then diluted 50-fold in BHI broth supplemented with kanamycin, and cells were collected at different time points during the growth phase.
-Galactosidase was assayed as described by Miller (1972)
, except that the cells were permeabilized by treatment with 0·5 % toluene and 4·5 % ethanol. The
-galactosidase specific activities, determined in three separate experiments, were expressed as [103x(OD420 of the reaction mixture1·75 OD550 of the reaction mixture)] divided by [reaction time (min) xOD600 of the quantity of cells used in the assay].
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Gel-mobility-shift DNA-binding assays.
EcoRI/BamHI DNA fragments, corresponding to the promoter regions of different genes, were generated by PCR, and labelled radioactively by treatment with the Klenow fragment of DNA polymerase I in the presence of a mixture of dGTP, dATP, dTTP (0·08 mM) and [-32P]dCTP (40 µCi; 1·48 MBq). Binding of protein to DNA was carried out in a 20 µl reaction mixture containing 32P-labelled DNA, 1 µg poly(dI-dC) (Pharmacia), 25 mM sodium phosphate (pH 7), 150 mM NaCl, 0·1 mM EDTA, 2 mM MgSO4, 1 mM DTT, 10 % (v/v) glycerol. The DNA-binding reaction was initiated by the addition of total crude protein, and incubated at room temperature for 30 min. Samples were then loaded directly onto an SDS-6 % polyacrylamide gel (50 mM Tris, 400 mM glycine, 1·73 mM EDTA) for electrophoresis (10 V cm1). After overnight electrophoresis, the gels were dried, and scanned with a Molecular Dynamics PhosphorImager. The autoradiographs shown correspond to a 24 h exposure.
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RESULTS |
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We tested the role of ClpC and ClpP in the expression of the sip locus by introducing plasmid pTCV-lac-PsipX or pTCV-lac-PsipZ into the two knockout mutants clpC and
clpP of L. monocytogenes. Since the mutations had been constructed in strain LO28, we used wild-type EGD-e and LO28 strains as controls. The activities of PsipX and PsipZ were essentially identical in EGD-e and LO28 (approx. 40
-galactosidase units). The promoter activities of PsipX and PsipZ were three- to fourfold higher in the clpC and clpP mutants (174±2 and 162±2·6
-galactosidase units for PsipX in the clpC and clpP mutants, respectively; 158±0·3 and 167±3
-galactosidase units for PsipZ in the clpC and clpP mutants, respectively) compared with the wild-type strains. These results suggest that, as in B. subtilis, expression of ClpC and ClpP downregulates transcription of the sip genes.
PrfA, the central virulence regulator of L. monocytogenes, has been shown to exert a negative control on ClpC expression at the transcriptional level, reflecting the existence of crosstalk between these two systems (Ripio et al., 1998). Moreover, in B. subtilis, ClpC controls the expression of competence by forming a complex with MecA to negatively regulate ComK (Bockmann et al., 1996
). We therefore tested whether MecA and PrfA could also modulate the expression of the sip genes, by monitoring PsipX- and PsipZ-dependent lacZ transcription in
prfA and
mecA mutant backgrounds. In these two backgrounds, the activity of both PsipX and PsipZ was two- to threefold lower than in the wild-type strains (approx. 20 and 10
-galactosidase units for PsipX in the mecA and prfA mutants, respectively; approx. 15 and 10
-galactosidase units for PsipZ in the mecA and prfA mutants, respectively). Thus, while ClpC and ClpP are involved in the negative regulation of sip gene expression, PrfA and MecA are involved in the positive regulation of their expression.
Proteins bind specifically to the promoter regions PsipX and PsipZ
We performed different gel-mobility-shift DNA-binding assays to the visualize the binding of proteins to the promoter regions PsipX and PsipZ. First, we used total protein extract from L. monocytogenes cells in DNA-binding assays, with 200 bp double-stranded DNA fragments corresponding to PsipX and PsipZ. All the assays were performed in the presence of an excess of non-specific competitor DNA [1 µg poly(dI-dC)]. Radiolabelled probes corresponding to PsipX and PsipZ were incubated with increasing amounts of total protein extract. The displacement of radiolabelled fragment was proportional to the amount of protein extract added, and was complete at a concentration of 15 µg total protein (Fig. 3). The same experiment was performed with a radiolabelled probe corresponding to PsipY (a region showing no promoter activity in the previous experiment), and no displacement of radiolabelled fragment among the amount of protein extract was observed (data not shown). Cold competitor chase experiments (as described by Chastanet et al., 2003
) were used to demonstrate the specificity of protein binding. The addition of increasing amounts of unlabelled DNA fragments corresponding to PsipX or PsipZ efficiently prevented binding of the radiolabelled probes (Fig. 3a, b
). These results indicate that one or several proteins bind directly to the promoter regions of sipX and sipZ.
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These preliminary observations will need to be followed up with additional experiments to define the actual regulatory mechanisms involved.
Expression of the sip genes is upregulated in infected cells
We monitored quantitatively the transcription of each sip gene by real-time PCR, upon infection by L. monocytogenes of either mouse bone marrow-derived macrophages (BMM) or the human enterocyte Caco-2 cell line (Fig. 4). In both cell types, an increase in the transcription of the three sip genes was observed shortly after infection. In mouse BMM (Fig. 4a
), a sevenfold induction of sipZ was recorded after 30 min of infection; induction levels of sipX and sipY were similar to each other, and slightly lower (approximately fivefold induction). In Caco-2 cells (Fig. 4b
), significant induction of sip gene transcription was also observed after 30 min of infection (approximately fivefold induction of sipZ, and three- to fourfold induction of sipX and sipY). Our electron microscopy observations suggest that, at this early stage of the infection, the majority of bacteria are still localized inside phagosomes (data not shown; Réglier-Poupet et al., 2003
). In contrast, after 1 h of infection, about 65 % of the bacteria were surrounded by a meshwork of polymerized actin. After 1 h of infection, the induction of each of the three sip genes decreased in the two cell types, but was still about twofold higher than that recorded in broth. These assays suggest that sip gene expression might be preferentially upregulated during the intraphagosomal stage of the infectious cycle of L. monocytogenes.
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DISCUSSION |
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It is tempting to suggest that L. monocytogenes has evolved this regulation of SPase I expression as an additional means to control its production of virulence factors. However, it is possible that L. monocytogenes simply adapts the synthesis of SPases according to its needs.
PrfA and other factors are involved in the regulation of sip gene expression
In agreement with our earlier observations (Bonnemain et al., 2004), we found that the three sip genes were regulated by two different promoters, PsipX and PsipZ. Expression of the sip genes appeared to be coordinated by complex regulatory signals, including the pleiotropic transcriptional activator PrfA, and stress proteins involved in L. monocytogenes pathogenicity. Clp ATPases are believed to form part of post-translation regulatory networks that ensure survival in stress conditions, presumably by acting as molecular chaperones that mediate the repair or scavenging of damaged proteins (Wawrzynow et al., 1996
). In B. subtilis, Clp ATPases mediate adaptive responses to stress conditions, as well as to many other different processes, including the development of competence, sporulation, exoenzyme synthesis and cell-cycle regulation. Of particular interest, the inactivation of clp genes has been shown to upregulate the transcription of three SPase I genes, sipS, sipT and sipV, and of the lipoprotein signal peptidase gene lsp, suggesting that Clp proteins control a step in the secretion pathway that is common to both non-lipoproteins and lipoproteins (Pummi et al., 2002
).
In L. monocytogenes, we found that inactivation of clpC or clpP increased sip gene expression, suggesting that Clp proteins act to downregulate sip expression, and we found that inactivation of MecA downregulated sip expression, suggesting that MecA could act as both a negative and a positive regulator. We also observed that PrfA influenced sip expression, while a large-scale transcriptomic analysis of PrfA-dependent promoters (Milohanic et al., 2003) failed to identify the sip genes. Thus, either sip gene regulation was below the detection threshold determined by the transcriptomic assay, or, under the studied conditions, the sip genes were not regulated.
To date, the interconnection between the PrfA regulon and the regulatory pathways that control stress responses in L. monocytogenes is not clearly understood. ClpC expression is negatively controlled at the transcriptional level by the central virulence regulator PrfA. Gel-mobility DNA-binding assays, performed here with total protein extracts devoid of PrfA, ClpC, ClpP or MecA, confirmed the absence of direct action of these proteins on the sip promoters, suggesting complex control mechanisms of sip gene expression in L. monocytogenes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bolhuis, A., Tjalsma, H., Stephenson, K., Harwood, C. R., Venema, G., Bron, S. & van Dijl, J. M. (1999). Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants. J Biol Chem 274, 1586515868.
Bonnemain, C., Raynaud, C., Réglier-Poupet, H., Dubail, I., Frehel, C., Lety, M. A., Berche, P. & Charbit, A. (2004). Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes. Mol Microbiol 51, 12511266.[CrossRef][Medline]
Borezee, E., Msadek, T., Durant, L. & Berche, P. (2000). Identification in Listeria monocytogenes of MecA, a homologue of the Bacillus subtilis competence regulatory protein. J Bacteriol 182, 59315934.
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, 323336.[CrossRef][Medline]
Cabanes, D., Dehoux, P., Dussurget, O., Frangeul, L. & Cossart, P. (2002). Surface proteins and the pathogenic potential of Listeria monocytogenes. Trends Microbiol 10, 238245.[CrossRef][Medline]
Chastanet, A., Fert, J. & Msadek, T. (2003). Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria. Mol Microbiol 47, 10611073.[CrossRef][Medline]
Cossart, P. (2004). Bacterial invasion: a new strategy to dominate cytoskeleton plasticity. Dev Cell 6, 314315.[CrossRef][Medline]
Dussurget, O., Pizarro-Cerda, J. & Cossart, P. (2004). Molecular determinants of Listeria monocytogenes virulence. Annu Rev Microbiol 58, 587610.[CrossRef][Medline]
Gaillot, O., Pellegrini, E., Bregenholt, S., Nair, S. & Berche, P. (2000). The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol Microbiol 35, 12861294.[CrossRef][Medline]
Glaser, P., Frangeul, L., Buchrieser, C. & 52 other authors (2001). Comparative genomics of Listeria species. Science 294, 849852.
Miller, J. (1972). Assay of -Galactosidase. In Experiments in Molecular Genetics, pp. 352355. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Milohanic, E., Glaser, P., Coppee, J. Y., Frangeul, L., Vega, Y., Vazquez-Boland, J. A., Kunst, F., Cossart, P. & Buchrieser, C. (2003). Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol Microbiol 47, 16131625.[CrossRef][Medline]
Nair, S., Derre, I., Msadek, T., Gaillot, O. & Berche, P. (2000). CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes. Mol Microbiol 35, 800811.[CrossRef][Medline]
Paetzel, M., Dalbey, R. E. & Strynadka, N. C. (2000). The structure and mechanism of bacterial type I signal peptidases. A novel antibiotic target. Pharmacol Ther 87, 2749.[CrossRef][Medline]
Poyart, C. & Trieu-Cuot, P. (1997). A broad host-range mobilizable shuttle vector for the construction of transcriptional fusions to -galactosidase in Gram-positive bacteria. FEMS Microbiol Lett 156, 193198.[CrossRef][Medline]
Pugsley, A. P. & Possot, O. (1993). The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex. Mol Microbiol 10, 665674.[Medline]
Pummi, T., Leskela, S., Wahlstrom, E., Gerth, U., Tjalsma, H., Hecker, M., Sarvas, M. & Kontinen, V. P. (2002). ClpXP protease regulates the signal peptide cleavage of secretory preproteins in Bacillus subtilis with a mechanism distinct from that of the Ecs ABC transporter. J Bacteriol 184, 10101018.
Réglier-Poupet, H., Frehel, C., Dubail, I., Beretti, J. L., Berche, P., Charbit, A. & Raynaud, C. (2003). Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes. J Biol Chem 278, 4946949477.
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, 4550.[CrossRef][Medline]
Sheehan, B., Klarsfeld, A., Ebright, R. & Cossart, P. (1996). A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. Mol Microbiol 20, 785797.[Medline]
Tjalsma, H., Noback, M. A., Bron, S., Venema, G., Yamane, K. & van Dijl, J. M. (1997). Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities. Constitutive and temporally controlled expression of different sip genes. J Biol Chem 272, 2598325992.
van Roosmalen, M. L., Jongbloed, J. D., Dubois, J. Y., Venema, G., Bron, S. & van Dijl, J. M. (2001). Distinction between major and minor Bacillus signal peptidases based on phylogenetic and structural criteria. J Biol Chem 276, 2523025235.
van Roosmalen, M. L., Geukens, N., Jongbloed, J. D., Tjalsma, H., Dubois, J. Y., Bron, S., van Dijl, J. M. & Anne, J. (2004). Type I signal peptidases of Gram-positive bacteria. Biochim Biophys Acta 1694, 279297.[CrossRef][Medline]
van Wely, K. H., Swaving, J., Freudl, R. & Driessen, A. J. (2001). Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol Rev 25, 437454.[CrossRef][Medline]
Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W. & Gonzalez-Zorn, B. (2001). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584640.
Wawrzynow, A., Banecki, B. & Zylicz, M. (1996). The Clp ATPases define a novel class of molecular chaperones. Mol Microbiol 21, 895899.[CrossRef][Medline]
Received 24 March 2005;
revised 30 June 2005;
accepted 6 July 2005.
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