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Previous Article | Next Article 
Infection and Immunity, January 2003, p. 474-482, Vol. 71, No. 1
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.1.474-482.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
INSERM U570, Faculté de Médecine Necker-Enfants Malades, 75730 Paris cedex 15, France
Received 5 July 2002/ Returned for modification 12 September 2002/ Accepted 22 October 2002
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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/ß, interleukin 6, and tumor necrosis factor alpha in murine and human monocytes (4, 32). During the intimate contact of bacteria with endothelial cells, listeriolysine O and phospholipases can also elicit host cell responses, including the generation of lipid second messengers (48, 49), NF-
B activation (29, 46), stimulation of cytokines and chemokines, and induction of cell adhesion molecule expression (17, 24, 29). During invasion, Listeria bacteria are engulfed within phagocytic vacuoles and disrupt the phagosome membranes to be free in the cytoplasm. Listeriolysine O and phospholipases are involved in the escape from the phagosomal compartment. Through ActA, a bacterial membrane-anchored protein, intracytoplasmic bacteria become surrounded with actin filaments that form an actin tail allowing bacteria to move inside the cytoplasm and to spread from cell to cell (reviewed in reference 51). In this work, we searched for new virulence factors implicated in the entry of L. monocytogenes into eukaryotic cells. We identified in silico from the recently completed sequence of the genome of L. monocytogenes (23) a gene encoding a PsaA-like protein belonging to the LraI family. PsaA is a lipoprotein previously implicated in the cell adherence of Streptococcus pneumoniae (44) and related species (10). By constructing a mutant in which this gene is deleted, we show that this PsaA-like protein is required for entry of L. monocytogenes into eukaryotic cells, not into macrophages. This protein, designated LpeA (for lipoprotein promoting entry), promotes entry and facilitates intracellular survival in infected macrophages.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genetic manipulations. Chromosomal DNA, plasmid extraction, electrophoresis, restriction enzyme analysis, and amplification by PCR were performed according to standard protocols (43). 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 Perkin-Elmer (Branchburg, N.J.) in a Gene Amp System 9600 thermal cycler (Perkin-Elmer). Nucleotide sequencing was carried out with Taq DiDeoxy terminators and by the DyePrimer cycling sequence protocol developed by Applied Biosystems (Perkin-Elmer) with fluorescently labeled dideoxynucleotides and primers, respectively (Life Technologies). Labeled extension products were analyzed on an ABI Prism 310 apparatus (Applied Biosystems).
Construction of a mutant of L. monocytogenes lacking the lpeA gene
An lpeA mutant was constructed by deletion of a 270-bp internal fragment of lpeA and insertion of a promoterless aphA-3 gene conferring resistance to Km by double recombination, as previously described (6). The deletion replacement mutant of lpeA was constructed by inserting a 1,067-bp EcoRI-BamHI EGD DNA fragment (-38 to +1,017), an 855-bp BamHI Enterococcus faecalis DNA fragment carrying aphA-3, and a 1,062-bp BamHI-HindIII EGD DNA fragment (+1,293 to +2,336) between the EcoRI and HindIII sites of the thermosensitive shuttle vector pAUL-A, as previously described (6), to yield pAUL-lpeA 
aphA3. These three DNA fragments were amplified by PCR from L. monocytogenes (EGD-e) genomic DNA by using the following primers pairs: papEco (5'-GGAATTCCGCAGCGGGGGTGTAAGAGTTGTTGTTTTTA-3') and papBamas (5'-CGCGGATCCACGCCAACCAGGGGTACAATAC-3'); km1 (5'-CGGGATCCCGACTAACTAGGAGGAATA-3') and km2 (5'-CGGGATCCCGGGTCATTATTCCCTCC-3'); and papBams (5'-CTTGTAAAAGCGGATCCAGACAATGCGGA-3') and papHind (5'CCCAAGCTTGGGGGGGGCCTTTGGGACGGAGACAATTGCGGC-3'). Oligonucleotides were synthesized by Genset (Paris, France). The two amplified double-stranded DNA fragments were first cloned into the pCRTM cloning vector by using the TA cloningTM kit (Invitrogen Corporation, San Diego, Calif.). Plasmid pAUL-lpeA aphA3 was electroporated into EGD, and transformants were selected for Em resistance at 30°C. Allelic exchange was obtained by homologous recombination by 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 Northern blot analysis. Hybridizations of total RNA extracted during exponential phase at 37°C from wild-type EGD were done as described previously (41). A specific probe (length, 1,062 bp) used for hybridization was generated with the following primers pair: psa1 (5'-CGCACCGAAACAAGGCTTGCTATTTTC-3') and pap2 (5'-CCGGGTTCGTAAAACGGAGCAAAAAC-3').
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Proteins from extracts were prepared from cultures of bacteria grown in BHI broth at 37°C (optical density at 600 nm, 0.6). The bacterial pellets were suspended in 10 mM Tris-1 mM EDTA, and bacteria were disrupted with a Fastprep FP120 apparatus (BIO101; Ozyme) by three pulses of 30 s at a speed of 6.5 m/s. Bacterial debris were removed by centrifugation, and the resulting supernatant consisted of the cytoplasmic proteins. Electrophoresis and Western blotting were carried out as described previously in 10% sodium dodecyl sulfate-polyacrylamide minigels (Mini Protean II; Bio-Rad) (38). Nitrocellulose sheets were probed with anti-ScaA monoclonal antibody kindly provided by P. E. Kolenbrander (Bethesda) and antirabbit horseradish peroxidase-conjugated secondary antibody. Mouse monoclonal antibodies directed against InlA, InlB, or ActA obtained from P. Cossart (Institut Pasteur, Paris) were also used as previously described (38). 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).
Infection of macrophages and cell lines. Bone marrow-derived macrophages from C57/BL6 mice (IFFA-CREDO, Grenoble, France) were cultured and infected for growth curves at a cell-to-bacterium ratio of 1 to 1, as previously described (6). After 15 min of bacterial adherence on ice, macrophages were exposed for 15 min at 37°C (time zero). The numbers of intracellular bacteria were estimated in cell lysates at selected intervals (from time zero to 8 h postinfection). We also used the human colon carcinoma cell line Caco-2 (ATCC HTB37) and the murine embryonic hepatocyte cell line TIB73 (ATCC TIB73) from the American Type Culture Collection (Manassas, Va.). Cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing N-acetyl-L-alanyl-L-glutamine (Gibco Laboratories, Grand Island, N.Y.) and supplemented with 10% fetal bovine serum (Gibco Laboratories). Cells were maintained in 10% CO2 at 37°C without antibiotics. Cells were seeded at a density of 105/cm2 in 24-well tissue culture plates (Falcon Labware; Becton Dickinson & Co., Lincoln Park, N.J.). Monolayers were used 24 to 48 h after seeding. The invasion assays were carried out as described previously (37). Briefly, cells were inoculated with bacteria at a multiplicity of infection of approximately 100 bacteria/cell. They were incubated for 1 h and then washed three times with DMEM and overlaid with fresh DMEM containing gentamicin (10 mg/liter) to kill extracellular bacteria. At 2 and 4 h, cells were washed and lysed by addition of cold water. Viable bacteria released from the cells were plated onto BHI plates. Each experiment was carried out in triplicate and repeated three times, and results were expressed as means ± standard deviations. We compared the differences between the curves by Student's t test.
Confocal microscopy. Infected cells were examined at progressive times by confocal microscopy. Double fluorescence labeling of F-actin and bacteria was performed as described previously (30) by using phalloidin coupled to Oregon Green 488 (Molecular Probes, Eugene, Oreg.) and a rabbit anti-Listeria serum (J. Rocourt, Institut Pasteur, Paris, France) revealed with an anti-immunoglobulin G antibody coupled to Alexa 546 (Molecular Probes). Images were scanned on a Zeiss LSM 510 confocal microscope.
Immunogold labeling. Bacteria were grown overnight in BHI broth and processed as previously described (20). The grids were incubated for 1 h with rabbit anti-Listeria or rabbit anti-ScaA antibodies and further incubated with goat anti-rabbit immunoglobulin G conjugated to 10-nm gold particles.
Mouse virulence assay. Six- to 8-week-old female Swiss mice (Janvier, Le Geneset St-Isle, France) were inoculated intravenously (i.v.) with various doses of bacteria. Mortality was monitored over a 14-day period for groups of five mice. The 50% lethal doses (LD50) were determined by the probit method. Bacterial growth in organs (spleen and liver) of mice infected i.v. with 105 bacteria was monitored as previously described (38). For growth in organs, we compared the data obtained with mutants to the data obtained with wild-type strains by a multifactorial variance analysis.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By BLAST search, we also found a PsaA-like homologue in L. innocua, sharing 94% identity with LpeA (orf 3387.1) (23). Using primers specific for lpeA of L. monocytogenes, we showed by Southern blotting that these genes were present in all 17 tested strains of L. monocytogenes and other Listeria species (L. ivanovii, L. innocua, and L. seeligeri) (data not shown), suggesting that lpeA is highly conserved in the genus Listeria.
lpeA of L. monocytogenes belongs to an operon resembling those of a putative ATP-binding cassette (ABC) transporter family. Analysis of the region of the L. monocytogenes genome comprising lpeA revealed the presence of three overlapping ORFs in the same orientation (orf 3390.3, orf 3389.1, and orf 3387.1), surrounded by two terminators (Fig. 1 A). The first orf encodes a putative ATP-binding protein of 240 amino acids, with a consensus nucleotide-binding site for ATP (GPNGAGKST) starting at position 33, corresponding to the consensus sequence (GXXGXGKS/T) in the glycine-rich loop of ATP-binding enzymes (52). In addition, a glutamine-glycine-rich motif (LSGGQLQR) could function as a peptide linker joining different domains of the protein. The second orf encodes a putative transmembrane protein of 279 residues, with a 16-amino-acid sequence (ALQTVGIILVVAMLITP) in position 182, also present in several hydrophobic membrane proteins involved in the transport of peptides and other small molecule transports (39).
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Construction and phenotypic analysis of an lpeA mutant of L. monocytogenes.
We constructed an lpeA mutant of L. monocytogenes strain EGD-e by deletion of an internal fragment of lpeA gene (270 bp) and chromosomal integration by allelic replacement (EGD
lpeA). Inactivation of lpeA was confirmed by Western blot analysis of bacterial extracts from the wild-type EGD-e or from the lpeA mutant by using antibodies directed against ScaA of S. gordonii, a protein sharing 57% of peptide identity with LpeA of L. monocytogenes. We found that this antiserum recognizes LpeA as a single 35-kDa band in bacterial extracts from the wild-type strain, in contrast to the mutant (Fig. 1B).
We did not find any difference between the mutant and the wild-type strain EGD-e with respect to microscopic morphology, motility, colony aspect, hemolysis on blood agar plates, metabolic profiles on API strips, and growth in BHI broth at 4, 37, or 42°C (data not shown). In contrast to previous data on the phenotype of a psaA mutant of S. pneumoniae (13), growth of the lpeA mutant of L. monocytogenes was not impaired on previously defined synthetic medium F70 (41). Since LpeA might be a putative factor involved in bacterial invasion, we confirmed by Western blot analysis that the proteins InlA, InlB, and ActA were produced at the same levels in the mutant and the wild-type bacteria (data not shown). Using the anti-ScaA serum, we also detected LpeA on the bacterial surface of the wild-type strain by immunogold labeling, in contrast to the absence of such labeling with the lpeA mutant, showing that LpeA is a surface-exposed protein (Fig. 2).
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lpeA, or an inlAB mutant as a control for invasiveness. Cells were then washed and incubated in the presence of gentamicin (10 mg/liter) to eliminate extracellular bacteria. Bacteria were counted at time zero and after 2 and 4 h of incubation. No significant difference between EGD-e and EGDlpeA was observed by time zero in either cell line, indicating that LpeA does not influence adherence to cells (Fig. 3). As previously described (21), the rate of infection of Caco-2 cells by wild-type bacteria reached about 5% after 4 h, compared to a low infection rate of TIB73 cells (<0.3%) exposed to the same conditions. We found that the invasion by the lpeA mutant was significantly reduced in both cell lines, compared to that by wild-type EGD-e (P < 0.01; Student's t test). This defect of invasion was less pronounced in Caco-2 cells than that of the inlAB mutant (Fig. 3).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main finding of this work is that LpeA is a surface-exposed protein promoting cell invasion by L. monocytogenes. Using a mutant lacking lpeA, we observed that LpeA-defective bacteria efficiently adhere to eukaryotic cells but fail to penetrate in vitro into epithelial and hepatocyte cells, as confirmed by confocal microscopy showing that very few lpeA mutant bacteria penetrate inside cells and then rapidly grow in the cytoplasm (Fig. 4). In contrast, an S. pneumoniae psaA mutant expresses a reduced adherence to A549 lung cells in vitro (3). These results demonstrate that (i) L. monocytogenes LpeA is not an adhesin and behaves as an invasin and that (ii) LpeA is not required to escape from the phagosomal compartment. This is the first demonstration that a putative lipoprotein promotes invasion by the pathogen L. monocytogenes.
The LpeA-dependent reduced invasion of epithelial and hepatocyte cell lines contrasts with the rapid invasion of bone marrow macrophages by lpeA mutant bacteria, resulting in a moderately higher bacterial intracellular load than with wild-type bacteria (Fig. 5A). This was confirmed by the finding of a moderate exacerbation of virulence in the absence of LpeA. Indeed, the mutant was fully virulent in the mouse with an LD50 estimated at 104.3, moderately lower than the LD50 value of the wild-type strain (104.6). Under the same infecting-challenge conditions, we observed higher bacterial loads in organs, associated with an early mortality (Fig. 5B and C). This result is surprising, since internalin-defective Listeria mutants express a weakly reduced level of virulence after i.v. inoculation (21). Moreover, an S. pneumoniae psaA mutant displays reduced virulence in mice infected by intranasal and intraperitoneal routes (3). Our results suggest that virulent bacteria at the initial phase of i.v. infection do not require invasins to directly spread in vivo from cell to cell. Circulating bacteria might mainly target resident or mobile macrophages, including dendritic cells, and then spread to adjacent cells, ultimately resulting in rapid growth in organs. This also suggests that direct invasion of hepatocytes or epithelial cells is not required for the expression of virulence in this model. To our knowledge, the exacerbation of virulence has never yet been observed with a deletion mutant of L. monocytogenes. The reasons for this virulence phenotype remain unknown. LpeA might act directly through a cell receptor or indirectly through its hypothetical function of the ABC transporter, which might influence the cell sensing or an intracellular cascade associated to cell entry through the uptake of polypeptides, oligopeptides, and multiple sugars. Our results clearly show that the putative lipoprotein LpeA of L. monocytogenes is a novel invasin involved in the entry process, but not in intracellular survival.
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ACKNOWLEDGMENTS |
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This work was supported by INSERM, The University of Paris V, two grants from the European Commission (contracts ERBCHRXCT 94-0451 and CT980036), and a grant from the DGA (no. 0034069).
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FOOTNOTES |
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, wild-type EGD-e; 
, lpeA mutant. (A) Growth of L. monocytogenes in bone marrow-derived macrophages from C57/BL6 mice. The cells were exposed for 15 min (time zero) to bacteria (1 bacterium per cell), and bacterial survival was monitored for 6 h after the infection. Entry was very efficient in both strains, and mutant bacteria repeatedly grew faster in macrophages (*, P < 0.001). (B and C) Growth of L. monocytogenes in the spleen (B) and the liver (C) of Swiss mice inoculated i.v. with 105 bacteria. Mutant bacteria grew faster in organs (*, P < 0.007) than did wild-type bacteria, and they induced an early mortality in mice by day 3.