Maturation of Lipoproteins by Type II Signal Peptidase Is Required for Phagosomal Escape of Listeria monocytogenes
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m307953200
ISSN1083-351X
AutoresHélène Réglier‐Poupet, Claude Fréhel, Iharilalao Dubail, Jean‐Luc Béretti, Patrick Berche, Alain Charbit, Catherine Raynaud,
Tópico(s)Escherichia coli research studies
ResumoLipoproteins of Gram-positive bacteria are involed in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding. Lipoproteins are also known to initiate both innate and adaptative immune responses. However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood. In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells. Among the putative surface proteins of L. monocytogenes, lipoproteins constitute the largest family. Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis. We identified a gene, lsp, encoding a SPase II in the genome of L. monocytogenes and constructed a Δlsp chromosomal deletion mutant. The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II. This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence. We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages. The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L. monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells. Lipoproteins of Gram-positive bacteria are involed in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding. Lipoproteins are also known to initiate both innate and adaptative immune responses. However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood. In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells. Among the putative surface proteins of L. monocytogenes, lipoproteins constitute the largest family. Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis. We identified a gene, lsp, encoding a SPase II in the genome of L. monocytogenes and constructed a Δlsp chromosomal deletion mutant. The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II. This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence. We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages. The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L. monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells. Listeria monocytogenes is a ubiquitous food-borne Gram-positive bacterium, responsible for life threatening infections in humans and animals (1.Farber J.M. Peterkin P.I. Microbiol. Rev. 1991; 55: 476-511Crossref PubMed Google Scholar). It is a facultative intracellular pathogen able to enter and multiply in both professional (2.Mackaness G.B. J. Exp. Med. 1962; 116: 381-406Crossref PubMed Scopus (821) Google Scholar) and non-professional phagocytes such as epithelial cells (3.Gaillard J.L. Berche P. Mounier J. Richard S. Sansonetti P. Infect. Immun. 1987; 55: 2822-2829Crossref PubMed Google Scholar) or hepatocytes (4.Wood S. Maroushek N. Czuprynski C.J. Infect. Immun. 1993; 61: 3068-3072Crossref PubMed Google Scholar). After entry, bacteria rapidly lyse the phagosomal membranes and gain access to the cytosol where they spread to adjacent cells by an actin-based motility process (5.Vazquez-Boland J.A. Kuhn M. Berche P. Chakraborty T. Dominguez-Bernal G. Goebel W. Gonzalez-Zorn B. Clin. Microbiol. Rev. 2001; 14: 584-640Crossref PubMed Scopus (1692) Google Scholar). Most of the virulence factors of L. monocytogenes, identified to date, are either secreted or surface-associated proteins (6.Cossart P. Bierne H. Curr. Opin. Immunol. 2001; 13: 96-103Crossref PubMed Scopus (87) Google Scholar). Several distinct mechanisms of cell wall attachment and display of surface proteins have been described in Gram-positive bacteria (7.Cabanes D. Dehoux P. Dussurget O. Frangeul L. Cossart P. Trends Microbiol. 2002; 10: 238-245Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Among them, bacterial lipoproteins form a subclass of exported proteins that are involved in a broad range of functions such as: (i) substrate-binding proteins, in ABC transporter systems; (ii) antibiotic resistance; (iii) cell signaling; (iv) protein export and folding; and (v) sporulation, germination, and conjugation (8.Sutcliffe I.C. Russell R.R. J. Bacteriol. 1995; 177: 1123-1128Crossref PubMed Scopus (331) Google Scholar). In addition, lipoproteins are known to initiate both innate and adaptative immune responses in activating nuclear factor-κB, cytokine production, and B-cell expansion through the interaction with human Toll-like receptor-2 (9.Aliprantis A.O. Yang R.B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1272) Google Scholar). However, the role of lipoproteins in the pathogenicity of intracellular bacteria is yet poorly understood. Two main genetic approaches can be undertaken to address the role of lipoproteins in pathogenesis. The first one consists of inactivating individually each of the genes encoding putative lipoproteins and study the properties of the mutant strains. This approach requires extensive work because the number of genes to inactivate is important (20 to >100) and may prove inefficient in the case of proteins with overlapping functions. The second approach consists of inactivating the gene encoding the signal peptidase responsible for their maturation. Indeed, the NH2-terminal signal sequences of lipoproteins are specifically processed by a unique signal peptidase, named type II signal peptidase (or SPase II). 1The abbreviations used are: SPase IIsignal peptidase type IIBHIbrain heart infusionBMbone marrow.1The abbreviations used are: SPase IIsignal peptidase type IIBHIbrain heart infusionBMbone marrow. Maturation of lipoproteins by SPase II, initially described in Gram-negative bacteria (10.Tokunaga M. Loranger J.M. Chang S.Y. Regue M. Chang S. Wu H.C. J. Biol. Chem. 1985; 260: 5610-5615Abstract Full Text PDF PubMed Google Scholar), has been recently studied in Gram-positive bacteria, and especially in Bacillus subtilis (11.Leskela S. Wahlstrom E. Kontinen V.P. Sarvas M. Mol. Microbiol. 1999; 31: 1075-1085Crossref PubMed Scopus (74) Google Scholar, 12.Tjalsma H. Kontinen V.P. Pragai Z. Wu H. Meima R. Venema G. Bron S. Sarvas M. van Dijl J.M. J. Biol. Chem. 1999; 274: 1698-1707Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). SPases II remove signal peptides from the lipoproteins first modified by a diacylglyceryltransferase (Lgt). This step could be inhibited by globomycin, a reversible and non-competitive peptide inhibitor (13.Giam C.Z. Hayashi S. Wu H.C. J. Biol. Chem. 1984; 259: 5601-5605Abstract Full Text PDF PubMed Google Scholar, 14.von Heijne G. Protein Eng. 1989; 2: 531-534Crossref PubMed Scopus (201) Google Scholar). After cleavage, the step of N-acylation of the cysteine observed in Escherichia coli was not described in B. subtilis, probably because of the absence of the lnt gene responsible of the lipoprotein aminoacyltransferase. signal peptidase type II brain heart infusion bone marrow. signal peptidase type II brain heart infusion bone marrow. Among the 133 putative surface proteins of the L. monocytogenes genome (15.Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. Charbit A. Chetouani F. Couve E. de Daruvar A. Dehoux P. Domann E. Dominguez-Bernal G. Duchaud E. Durant L. Dussurget O. Entian K.D. Fsihi H. Portillo F.G. Garrido P. Gautier L. Goebel W. Gomez-Lopez N. Hain T. Hauf J. Jackson D. Jones L.M. Kaerst U. Kreft J. Kuhn M. Kunst F. Kurapkat G. Madueno E. Maitournam A. Vicente J.M. Ng E. Nedjari H. Nordsiek G. Novella S. de Pablos B. Perez-Diaz J.C. Purcell R. Remmel B. Rose M. Schlueter T. Simoes N. Tierrez A. Vazquez-Boland J.A. Voss H. Wehland J. Cossart P. Science. 2001; 294: 849-852PubMed Google Scholar), lipoproteins constitute the largest family (i.e. 2.5% of all genes), with 68 members. The lipoprotein signal peptide is characterized by a well conserved lipobox preceding a cysteine residue. The signal sequences of the putative lipoproteins encoded by the genome of L. monocytogenes are listed in Table I.Table IPutative lipoproteins of L. monocytogenes Open table in a new tab Preliminary experimental data on the role of two of them have been recently obtained in our laboratory: (i) OppA that belongs to an oligopeptide ABC-like transporter, required for bacterial growth at low temperature (16.Borezee E. Pellegrini E. Berche P. Infect. Immun. 2000; 68: 7069-7077Crossref PubMed Scopus (160) Google Scholar); and (ii) LpeA an adhesin-like protein favoring entry of L. monocytogenes into non-professional phagocytes (17.Reglier-Poupet H. Pellegrini E. Charbit A. Berche P. Infect. Immun. 2003; 71: 474-482Crossref PubMed Scopus (39) Google Scholar). Individual inactivation of these genes had no effect on bacterial virulence. Here, we focused on a gene, designated lsp, encoding a potential SPase II in the genome of L. monocytogenes. We constructed a Δlsp chromosomal deletion mutant and studied its characteristics. Our analyses demonstrate that the lsp gene product is a genuine SPase II involved in the processing of lipoproteins, and that this process is critical for efficient phagosomal escape and intracellular survival of L. monocytogenes. We also monitored the expression of lsp during the infectious process. The data suggest that maturation of lipoproteins might be temporally controlled by the regulation of Lsp production inside infected cells. Bacterial Strains, Plasmids, and Growth Conditions—We used the reference strain of L. monocytogenes EGD-e belonging to serovar 1/2a recently sequenced (15.Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. Charbit A. Chetouani F. Couve E. de Daruvar A. Dehoux P. Domann E. Dominguez-Bernal G. Duchaud E. Durant L. Dussurget O. Entian K.D. Fsihi H. Portillo F.G. Garrido P. Gautier L. Goebel W. Gomez-Lopez N. Hain T. Hauf J. Jackson D. Jones L.M. Kaerst U. Kreft J. Kuhn M. Kunst F. Kurapkat G. Madueno E. Maitournam A. Vicente J.M. Ng E. Nedjari H. Nordsiek G. Novella S. de Pablos B. Perez-Diaz J.C. Purcell R. Remmel B. Rose M. Schlueter T. Simoes N. Tierrez A. Vazquez-Boland J.A. Voss H. Wehland J. Cossart P. Science. 2001; 294: 849-852PubMed Google Scholar). Brain heart infusion (BHI; Difco Laboratories, Detroit, MI) and Luria-Bertani (LB, Difco) broth and agar were used to grow L. monocytogenes and E. coli strains, respectively. Strains harboring plasmids were grown in the presence of the following antibiotics: pCR™ derivatives, kanamycin (Km) 50 μg/ml; pAUL-A derivatives, erythromycin (Em) 150 (E. coli) and 5 μg/ml (L. monocytogenes). To analyze mutant bacteria, we studied 50 metabolic characters on API-50 strips (Biomerieux, Marcy l'Etoile, France). Genetic Manipulations—Chromosomal DNA, plasmid extraction, electrophoresis, restriction enzyme analysis, and amplification by PCR were performed according to standard protocols (18.Sambrook J. Fritsch E.F. Maniatis T. Nolan, C. Molecular Cloning: A Laboratory Manual. Vol. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 17.37-17.41Google Scholar). Restriction enzymes and ligase were purchased from New England Biolabs and used as recommended by the manufacturer. DNA was amplified with the AmpliTaq DNA polymerase of Thermus aquaticus from PerkinElmer, in a Gene Amp System 9600 thermal cycler (PerkinElmer Life Sciences). Nucleotide sequencing was carried out with Taq dideoxy terminators and the DyePrimer Cycling Sequence protocol developed by Applied Biosystems with fluorescently labeled dideoxynucleotides and primers, respectively (Invitrogen). Labeled extension products were analyzed on an ABI Prism 310 apparatus (Applied Biosystems). Construction of a Δlsp Mutant of L. monocytogenes—A lsp mutant was constructed by deletion of a 200-bp internal fragment of lsp. We inserted a 1,022-bp EcoRI-BamHI EGD-e DNA fragment (-800 to +200) and a 922-bp BamHI-HindIII EGD-e DNA fragment (+400 to +1,300), between the EcoRI and HindIII sites of the thermosensitive shuttle vector pAUL-A, as previously described (19.Lingnau A. Domann E. Hudel M. Bock M. Nichterlein T. Wehland J. Chakraborty T. Infect. Immun. 1995; 63: 3896-3903Crossref PubMed Google Scholar), to give pAUL-Δlsp. These two DNA fragments were amplified by PCR from the EGD-e genomic DNA using the following primers pairs: Ecolsp5′ (5′-GGAATTCCTTATTCGGAAAATTCCAGGCGCTTTCTTATGG-3′) and Bamlsp3′ (5′-CGCGGATCCGCGACAACAACAGTAATAAGATAGAAAAACC-3′); Bamlsp5′ (5′-CGCGGATCCGCGTTGGTGTCGTACTAATGCTCGTGTATG-3′) and HIIIlsp3′ (5′-CCCAAGCTTGGGTCATTCGTTGTCGGATGATCAAATCCTA-3′). Oligonucleotides were synthesized by Genset (Paris, France). The 2 amplified double-stranded DNA fragments were first cloned into the pCR™ cloning vector using the Invitrogen TA Cloning™ kit (Invitrogen). Plasmid pAUL-Δlsp was electroporated into EGD-e and transformants were selected for Em resistance at 30 °C. Allelic exchange was obtained by homologous recombination using a two-step procedure: at 40 °C, a single crossing-over event integrated the entire plasmid into the chromosome; the plasmid was then excised by subculture at 30 °C. The deletion was confirmed by PCR sequence analysis of chromosomal DNA from the mutant. RNA Isolation and Real-time Quantitative PCR Assays—To extract RNA of L. monocytogenes grown in Caco-2 cell or bone marrow (BM) macrophages from BALB/c mice, cells were infected at a multiplicity of infection of 10, for 30 min, 30 min, and 1 h, respectively. Cells were lysed with 4 ml of 0.1% Triton X-100, the supernatant containing the bacteria was centrifuged for 10 min at 4,000 × g and the pellet was washed twice with saline buffer. Bacteria were broken in a solution of Trizol (1 ml) (Invitrogen) with miniglass beads using a Bead Beater apparatus (Polylabo) set at maximum speed. RNA was extracted with 300 μl of chloroform:isoamyl alcohol. After 10 min of centrifugation at 13,000 × g, the aqueous phase was transferred to a tube containing 270 μl of isopropyl alcohol. Total RNA was then precipitated overnight at 4 °C and washed with 1 μl of a 75% ethanol solution before suspension in diethyl pyrocarbonate-treated water. Contaminating DNA was removed by digestion with DNase I, according to the manufacturer's instructions (Roche Diagnostics). As a negative control, the same experiment was performed on non-infected cells. Real-time quantitative PCR was carried out on the ABI Prism 7700 sequence detection system using Taqman Universal PCR master mixture (PE Applied Biosystems). The primers were designed using the Primer Express software and obtained from PE Applied Biosystems. Sequences were as follows: lsp primers, forward, TATGCCAAAGGAAAGCGACTATT; and reverse, ACCCGGTCGATAAAATTACCAA; gyrA primers, forward, AAATGCGGACATCATTCCTAGACT, and reverse, TTTAACCCGTCACGAACATCAG. Reverse transcription-PCR experiments were carried out with 1 μg of RNA and 2.5 pmol of specific primers for lsp, gyrA, in a volume of 8 μl. After denaturation at 65 °C for 10 min, 12 μl of the mixture containing 2 μl of dNTP (25 mm), 4 μlof4× buffer, 2 μl of dithiothreitol, 1 μl of RNasin (Promega), and 1.5 μl of 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. Samples were diluted with 40 μl of H2O and stored at -20 °C. PCR conditions were identical for all reactions. The 25-μl reactions consisted of 12.5 μl of PCR master mixture (PE Applied Biosystems) containing Sybr Green, 4 μl of template, 5 pmol of each primer. The reactions were carried out in sealed tubes. Results were normalized to the amount of gyrA mRNA (31.Autret N. Raynaud C. Dubail I. Trieu-Cuot P. Berche P. Charbit A. Infect. Immun. 2003; 71: 4463-4471Crossref PubMed Scopus (124) Google Scholar). The gene gyrA was chosen because its expression remained constant under the different experimental conditions used here. Each assay was performed in triplicate. SDS-PAGE and Western Blot Analyses—Protein extracts were prepared from cultures of bacteria grown in BHI broth at 37 °C (at A600 of 0.6 for exponential phase; and after overnight incubation, at A600 of 1.2 for stationary phase). The bacterial pellets were suspended in Tris (10 mm)/EDTA (1 mm) and bacteria were disrupted using a Fastprep FP120 apparatus (BIO 101 Inc., Ozyme) by 2 pulses of 30 s at a speed of 6.5. Lysates were centrifuged and the pellet suspended in 1× SDS-PAGE sample buffer. Electrophoresis and Western blotting were carried out as described previously in SDS-10% polyacrylamide minigels (Mini Protean II; Bio-Rad). Nitrocellulose sheets were probed with anti-ScaA antibody, kindly provided by Dr. Kolenbrander (Bethesda) and anti-rabbit horseradish peroxidase-conjugated secondary antibody. Antibodies were used at a final dilution of 1:1,000. Antibody binding was revealed by adding 0.05% diaminobenzidine tetrahydrochloride (Sigma) and 0.03% hydrogen peroxide (Sigma). Protein Sequencing—Wild-type EGD-e and EGDΔlsp were grown in BHI overnight with agitation at 37 °C. The bacterial pellets were suspended in Tris (10 mm)/EDTA (1 mm) and bacteria were disrupted using a Fastprep FP120 apparatus (BIO 101 Inc., Ozyme) by 2 pulses of 30 s at a speed of 6.5. Denatured proteins were separated by SDS-PAGE and then transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore). Protein bands were visualized by staining the membrane with Coomassie Blue. For NH2-terminal sequencing, the two major unknown protein bands observed in the EGDΔlsp were cut from the polyvinylidene difluoride membranes and sequenced on an Applied Biosystems Procise sequencer (J. d'Alayer, Laboratoire de Séquencage des Protéines, Institut Pasteur, Paris). Intracellular Growth in Macrophages—Bone marrow-derived macrophages from BALB/c mice (IFFA-CREDO, Grenoble, France) were cultured and infected for growth curves at a cell/bacterium ratio of 10:1. Macrophages were exposed for 15 min at 37 °C and non-ingested bacteria were removed by several washings. Infected cells were re-fed with fresh medium (time 0). Confocal Microscopy—Infected cells were examined at 0, 1, 2, and 4 h post-infection. Double fluorescence labeling of F-actin and bacteria was performed as described (20.Lety M.A. Frehel C. Dubail I. Beretti J.L. Kayal S. Berche P. Charbit A. Mol. Microbiol. 2001; 39: 1124-1140Crossref PubMed Google Scholar), using phalloidin coupled to Oregon Green 488 (Molecular Probes, Eugene, OR) and a rabbit anti-Listeria serum (J. Rocourt, Institut Pasteur, Paris), revealed with an anti-IgG antibody coupled to Alexa 546 (Molecular Probes). Images were scanned on a Zeiss LSM 510 confocal microscope. The percentage of phagosomal escape was calculated as described previously (20.Lety M.A. Frehel C. Dubail I. Beretti J.L. Kayal S. Berche P. Charbit A. Mol. Microbiol. 2001; 39: 1124-1140Crossref PubMed Google Scholar). The number of bacteria per infected cell and the number of infected cells containing bacteria surrounded by polymerized actin were quantified on a mean of 50-100 infected cells, at 1, 2, and 4 h of the infection. Electron Microscopy—At selected intervals following infection (0, 1, 2, and 4 h), BM macrophages were fixed and processed as described previously (20.Lety M.A. Frehel C. Dubail I. Beretti J.L. Kayal S. Berche P. Charbit A. Mol. Microbiol. 2001; 39: 1124-1140Crossref PubMed Google Scholar). Thin sections were stained with 2% uranyl acetate and lead citrate. We quantified the percentage of bacteria surrounded by: (i) an intact phagosomal membrane; (ii) a partially damaged membrane; or (iii) polymerized actin. For this purpose, BM macrophages were infected at a ratio of 100:1, to have a sufficient number of bacteria per thin section. Fifty to 100 phagosomes were studied, corresponding to about 50 infected cells. Infection of Caco-2 Cells—We also used the human colon carcinoma cell line Caco-2 (ATCC HTB37) from the American Type Culture Collection (Manassas, VA). The invasion assays were carried out as described previously (17.Reglier-Poupet H. Pellegrini E. Charbit A. Berche P. Infect. Immun. 2003; 71: 474-482Crossref PubMed Scopus (39) Google Scholar). Viable bacteria released from the cells were plated onto BHI plates. Each experiment was carried out in triplicate, repeated three times and expressed as mean ± S.D. Mouse Virulence Assays—Six- to eight-week-old female Swiss mice (Janvier, Le Geneset St-Isle, France) were inoculated intravenously (iv) with various doses of bacteria. Mortality was followed over a 14-day period on groups of 5 mice. The lethal doses 50 (LD50) were determined by the Probit method. Bacterial growth was followed in organs (spleen and liver) of mice infected intravenously with 105 bacteria, as previously described (21.Autret N. Dubail I. Trieu-Cuot P. Berche P. Charbit A. Infect. Immun. 2001; 69: 2054-2065Crossref PubMed Scopus (90) Google Scholar). For the oral infections, 5-6-week-old-female BALB/c mice were starved for 24 h (water allowed) and were inoculated orally with 4 × 1010 bacteria (in 0.2 ml). This dose was chosen based on previous studies (22.Barbour A.H. Rampling A. Hormaeche C.E. Microbiol. Pathog. 1996; 20: 247-253Crossref PubMed Scopus (25) Google Scholar). After 3 days, the small intestines were recovered and rinsed in Dulbecco's modified Eagle's medium (Invitrogen) to remove the intestinal content. Intestines were then incubated at 20 °C for 2 h in Dulbecco's modified Eagle's medium containing 100 mg/liter gentamicin to kill extracellular bacteria from the intestinal lumen and rinsed three times in Dulbecco's modified Eagle's medium. Intestinal tissues were finally homogenized in 0.15 m NaCl and plated on BHI agar. The lsp Gene of L. monocytogenes Encodes a Lipoprotein-specific Signal Peptidase—We identified an orf, designated lsp, in the genome of L. monocytogenes EGD-e (15.Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. Charbit A. Chetouani F. Couve E. de Daruvar A. Dehoux P. Domann E. Dominguez-Bernal G. Duchaud E. Durant L. Dussurget O. Entian K.D. Fsihi H. Portillo F.G. Garrido P. Gautier L. Goebel W. Gomez-Lopez N. Hain T. Hauf J. Jackson D. Jones L.M. Kaerst U. Kreft J. Kuhn M. Kunst F. Kurapkat G. Madueno E. Maitournam A. Vicente J.M. Ng E. Nedjari H. Nordsiek G. Novella S. de Pablos B. Perez-Diaz J.C. Purcell R. Remmel B. Rose M. Schlueter T. Simoes N. Tierrez A. Vazquez-Boland J.A. Voss H. Wehland J. Cossart P. Science. 2001; 294: 849-852PubMed Google Scholar), encoding a putative SPase II. The protein has 57.5% amino acid identity with Lsp of B. subtilis and is of identical size (154 amino acids in length). Lsp is highly conserved in L. monocytogenes serovar 4b, as well as in the non-pathogenic species Listeria innocua, with 99 and 98.1% amino acid identity, respectively (preliminary sequence data on the 4b strain was obtained from The Institute for Genomic Research website). 2www.tigr.org. Lsp of EGD-e contains the five conserved domains present in all other known SPases II. In particular, the Asn, Asp, and Ala residues, in domains III and V, which are critical for the activity of B. subtilis SPase II (Ref. 23.Tjalsma H. Zanen G. Venema G. Bron S. van Dijl J.M. J. Biol. Chem. 1999; 274: 28191-28197Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and references therein) are conserved (not shown). An lsp transcript was detected by reverse transcriptase-PCR in bacteria grown in broth ("Experimental Procedures"), suggesting that lsp encodes a functional protein. Construction of a Chromosomal Δlsp Deletion—We constructed an lsp knockout mutant of L. monocytogenes (EGDΔlsp) by deletion of a 200-bp internal fragment of lsp, and chromosomal integration by allelic replacement (see "Experimental Procedures" for details). The deletion had no effect on bacterial viability in BHI, at all the temperatures tested (i.e. 4, 30, 37, and 42 °C). No difference between the mutant and wild-type strain was observed with respect to microscopic morphology, motility, colony aspect, hemolysis on blood agar plates, metabolic profiles on API strips, or antibiotic resistance profiles (not shown). These data indicate that the lsp gene product of L. monocytogenes is not essential for viability and not required for normal growth in broth. Processing of the Lipoprotein LpeA—We first tested the expression of LpeA, a recently identified lipoprotein of L. monocytogenes (17.Reglier-Poupet H. Pellegrini E. Charbit A. Berche P. Infect. Immun. 2003; 71: 474-482Crossref PubMed Scopus (39) Google Scholar), in wild-type EGD-e and in the Δlsp mutant. For Western blot analyses, we used an antibody directed against ScaA of Streptococcus gordonii (a protein sharing 57% amino acid identity with LpeA of L. monocytogenes) shown to cross-react with LpeA (17.Reglier-Poupet H. Pellegrini E. Charbit A. Berche P. Infect. Immun. 2003; 71: 474-482Crossref PubMed Scopus (39) Google Scholar). Two bands were detected in the Δlsp mutant: a minor band with the same apparent molecular weight as that of the single band detected in the wild-type strain; and a major band with a slightly higher apparent Mr (Fig. 1A). This upper band, which likely to corresponds to the precursor form of LpeA, was detected both in the exponential and stationary phases of growth. This assumption was confirmed by testing the expression of LpeA in the wild-type strain, grown in rich medium containing globomycin, an inhibitor of SPases II (see "Experimental Procedures"). Two forms of LpeA were detected after globomycin treatment: a lower band corresponding to mature LpeA; and an upper band corresponding to the unprocessed form of LpeA, with the same apparent Mr as the upper band of the Δlsp mutant. In agreement with these observations, previous studies have shown that B. subtilis cells lacking SPase II accumulate not only the precursor form, but also alternatively processed mature-like forms of lipoproteins PrsA (12.Tjalsma H. Kontinen V.P. Pragai Z. Wu H. Meima R. Venema G. Bron S. Sarvas M. van Dijl J.M. J. Biol. Chem. 1999; 274: 1698-1707Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). L. monocytogenes Expresses a PrsA-like Protein Processed by Lsp—In B. subtilis, the folding catalyst PrsA is a major lipoprotein that is essential for life (24.Kontinen V.P. Sarvas M. Mol. Microbiol. 1993; 8: 727-737Crossref PubMed Scopus (166) Google Scholar). The chromosome of L. monocytogenes encodes a putative PrsA-like protein (Lmo2219) sharing 45% identity with PrsA of B. subtilis. We tested whether an anti-B. subtilis PrsA antibody (kindly provided by Vesa Kontinen), could cross-react with PrsA of L. monocytogenes in Western blot. A single band was detected in wild-type EGD-e. One band with a slightly higher apparent Mr, likely to correspond to the unprocessed precursor form of PrsA, was detected in the Δlsp mutant (Fig. 1B). As with LpeA, two forms of PrsA were detected after globomycin treatment of the wild-type strain. As expected, the detection of PrsA from B. subtilis extracts (used as a control) was significantly stronger. These results show, thus that: (i) L. monocytogenes expresses a PrsA-like protein antigenically related to PrsA of B. subtilis; and (ii) Lsp is responsible for the maturation of this PrsA homolog. Together, the Western blot analyses demonstrate that the protein encoded by lsp is a genuine SPase II involved in the maturation of lipoproteins, including LpeA and PrsA. Variations in Envelope Protein Composition—The total protein composition was monitored in envelope fractions and culture supernatants, by Bradford protein assay. It was similar in EGD-e and in the Δlsp mutant, in both fractions. To visualize specific effects of lsp inactivation on protein expression, we compared the envelope fractions of the two strains by SDS-PAGE. The two protein patterns were globally similar (Fig. 2), except in the 30-kDa range where several differences were clearly visible. In particular, two major protein bands were detected in the Δlsp mutant extract, and missing in the wild-type extract. NH2-terminal protein sequence analyses ("Experimental Procedures") revealed that the upper band corresponded to the precursor form of a conserved lipoprotein of as yet unknown function (encoded by lmo2637). Notably, this 299-amino acid long lipoprotein shows significant similarity to a 15-kDa lipoprotein precursor of Treponema pallidum (25.Centurion-Lara A. Arroll T. Castillo R. Shaffer J.M. Castro C. Van Voorhis W.C. Lukehart S.A. Infect. Immun. 1997; 65: 1440-1444Crossref PubMed Google Scholar), a major membrane immunogen in syphilis infection. Peptidic alignment of the two sequences suggests that Lmo2637 is a duplicated form of the 15-kDa antigen (with 44% identity between residues 50 to 168 of Lmo2637 and 22 to 138 of T. pallidum lipoprotein; and 50% identity between residues 184-290 of Lmo2637 and 31-138 of T. pallidum lipoprotein). Lmo2637 also has
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