INTRODUCTION

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Listeria monocytogenes is a gram-positive, facultative intracellular bacterium that causes food-borne infections in animals and humans with severe implications, especially for newborns and immunocompromised individuals. The initial site of entry into the host normally occurs in the gut following ingestion of Listeria-contaminated food. Subsequently invading bacteria can breach the intestinal, placental, or blood-brain barrier, leading to systemic infections. During this process, the bacterium must also be capable of invading nonphagocytic parenchymal, epithelial, and endothelial cells (14, 19, 21). Early electron microscopic studies demonstrated the ability of this pathogen to invade epithelial cells of the cornea and the intestine in vivo (33, 34). A variety of nonphagocytic cell lines from different tissues, including epithelial cells, fibroblasts, and hepatocytes, can be infected in vitro with L. monocytogenes (5, 17, 26). Like Salmonella, Shigella, and Yersinia species, L. monocytogenes actively triggers its entry into these nonphagocytic cells. This process, also termed induced phagocytosis, involves host cell signalling pathways leading to rearrangements of the cortical actin cytoskeleton (2, 13).

Transposon-induced mutagenesis enabled the isolation of noninvasive mutants of L. monocytogenes and subsequently led to the identification of a genetic locus coding for the internalin A (InlA) and internalin B (InlB) polypeptides, which were identified as proteins with molecular weights of 88,000 and 65,000, respectively (17, 26). Monoclonal antibodies (MAbs) generated against either internalin detected both the InlA and InlB polypeptides in sodium dodecyl sulfate (SDS) cell wall extracts and culture supernatants of L. monocytogenes. The expression of both polypeptides was shown to be strongly dependent on growth temperature and the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that these genes are transcribed both in an operon as well as individually by PrfA-dependent and -independent mechanisms (9, 10, 26).

Evidence that InlA is involved in invasion of nonphagocytic cells stems from genetic complementation studies, in which InlA when expressed in noninvasive Listeria innocua rendered this strain invasive for the human enterocyte cell line Caco-2 (17). InlA mediates entry into Caco-2 and other cell lines expressing its receptor, the cell adhesion molecule E-cadherin (29). Entry of bacteria requires the surface-bound form of InlA, which is tethered to the bacterial cell wall by a 20-amino-acid C-terminal region harboring an LPXTG motif followed by a membrane-spanning region of about 20 amino acids and a few positively charged amino acid residues (9, 35).

Unlike InlA, InlB is highly enriched in cell wall extracts and only weakly detectable in culture supernatants of L. monocytogenes (26). Despite its presence in cell wall extracts of these bacteria, InlB is unusual because its primary sequence harbors neither a C-terminal membrane anchor nor a cell wall anchoring motif, both of which are present in the InlA polypeptide (8, 9, 17). Recently, it has been shown that the 230-amino-acid C-terminal region comprising about three 80-amino-acid repeats that start with the motif Gly-Trp (GW) is responsible for the association of InlB with the bacterial cell wall (3).

By constructing isogenic chromosomal deletion mutants, it was recently demonstrated that InlB is also a crucial virulence factor for L. monocytogenes. In mice infected intraperitoneally with inlB deletion mutants, such strains were attenuated for virulence in comparison to the wild-type strain (26). Dramsi and colleagues (8) reported that the InlB polypeptide was essential for entry into hepatocytes but not for invasion of epithelial Caco-2 cells. Nevertheless, heterologous expression of inlB in L. innocua failed to promote entry of this recombinant strain into hepatocytic cell lines, suggesting that additional products of L. monocytogenes are involved in the uptake (8). Also, significant impairment of inlB2 deletion mutants was observed with respect to entry into different epithelial-like cells, such as the human HEp-G2, HeLa, or A549 cells (7, 26), and human umbilical vein endothelial cells (32).

In this study, we sought independent experimental evidence that the InlB polypeptide does indeed mediate bacterial adherence and internalization. Here we report on a simple procedure to purify the native inlB gene product of L. monocytogenes in large quantities for biochemical and functional analysis. Purified InlB was found to be highly active and promoted entry into two cell lines when added externally to noninvasive Listeria strains.

	MATERIALS AND METHODS

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Bacterial strains, cultivation, and reagents. The wild-type L. innocua strain (NCTC 11288), L. monocytogenes EGD (serotype 1/2) and the isogenic EGD inlB2 deletion mutant, and the L. monocytogenes actA2/pERL3 50-1 strain have been described previously (20, 26). All Listeria strains were grown in brain heart infusion broth (Difco, Detroit, Mich.) overnight at 37°C and with erythromycin (5 µg/ml) in the case of L. monocytogenes actA2/pERL3 50-1. All chemical reagents were purchased from Sigma, Deisenhofen, Germany, unless indicated otherwise.

Extraction of bacterial cell wall proteins with SDS, Triton X-100, CHAPS, and Tris-Cl. Exponentially growing bacterial cultures were harvested by centrifugation (6,000 × g for 10 min) and washed with phosphate-buffered saline (PBS) twice at room temperature. Pelleted bacteria were immediately resuspended in approximately 0.5% of the original culture volume, using PBS containing either 2% (wt/vol) SDS, 1% (vol/vol) Triton X-100, or 16.2 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (27) or in Tris-Cl buffer at different concentrations and pH values. Resuspended bacteria were incubated for 15 min at 37°C with gentle shaking, except in the case of Tris-Cl extractions, for which bacteria were incubated for 60 min on ice. Bacterial suspensions were centrifuged at 12,000 × g for 10 min; the supernatants were aliquoted and stored at 70°C.

Extraction with mutanolysin. The bacterial pellet was resuspended in an excess of ice-cold acetone following 10 min of incubation on ice (15). After centrifugation (12,000 × g, 10 min), the pellets were resuspended in 50 mM Tris-Cl (pH 6.8); 10 U of mutanolysin per ml was added, followed by overnight incubation at 37°C with gentle shaking. After centrifugation (12,000 × g, 10 min), the supernatants were aliquoted and stored at 70°C.

SDS-PAGE and immunoblotting. SDS-polyacrylamide gel electrophoresis (PAGE) was done with 10% polyacrylamide gels. For staining of the gels, a silver staining kit (Bio-Rad, Munich, Germany) was used. Immunoblotting was performed by a semidry method using Immobilon P membranes (Millipore, Eschborn, Germany). After incubation with horseradish peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany), the blots were reacted by using a sensitive enhanced chemiluminescence-based immunoblot assay (Amersham Buchler, Braunschweig, Germany) as instructed by the vendor.

MAbs. Tris-Cl extracts of L. monocytogenes actA2/pERL3 50-1 (see above) were dialyzed against PBS, and approximately 100 µg of protein was used for repeated immunizations of three BALB/c mice. The immunization and fusion protocols have been described previously (31). Hybridoma supernatants were tested by enzyme-linked immunosorbent assay (ELISA) on separate microtiter plates coated with Tris-Cl extracts of EGD or the isogenic mutant EGD inlB2. Clones that were positive on EGD extracts and negative on EGD inlB2 extracts were further analyzed by immunoblotting prepared with Tris-Cl extracts of the EGD strain. Positive clones were subcloned twice by limiting dilution, and immunoglobulin subclasses were determined by using an isotyping kit (Medac, Hamburg, Germany). Five InlB-specific MAbs of the immunoglobulin G (IgG) subclass were further processed (see below). The InlB-specific MAb IC100 has been described previously (26).

Purification of InlB. (i) Gel filtration and phenyl-Sepharose chromatography. Tris-Cl extracts were concentrated by ultrafiltration (cutoff, 30 kDa) and dialyzed extensively against 20 mM Tris-Cl (pH 7.6) containing 1 M NaCl. After high-speed centrifugation (100,000 × g, 30 min, 4°C), the sample (3 ml) was loaded onto a gel filtration column at 5 ml/min, using Sephacryl S-100 (XK; 1.9 by 90 cm; Pharmacia, Uppsala, Sweden) equilibrated with the same buffer. For phenyl-Sepharose chromatography, concentrated Tris-Cl extracts were dialyzed against 3 M NaCl-20 mM Tris-Cl (pH 7.6). The sample (5 ml) was loaded on a 1 ml phenyl-Sepharose HP column (Pharmacia) equilibrated with the same buffer. InlB was eluted by a continuous salt gradient starting with 3 M NaCl.

(ii) Affinity purification of InlB. The InlB-specific MAbs were purified from hybridoma culture supernatants on protein A-Sepharose, immobilized on CNBr-activated Separose 4B (Pharmacia), and incubated overnight at 4°C on a rotating device (batch method) with Tris-Cl extracts of L. monocytogenes actA2/pERL3 50-1 previously dialyzed against PBS. After several PBS washes, portions of the Sepharose affinity matrix were boiled with SDS sample buffer and analyzed by SDS-PAGE as previously described (31). For scaling up the affinity purification, the affinity matrix was filled into an appropriate column, washed with PBS, and then washed with 1.5 M NaCl in PBS to remove nonspecifically bound material. Before the elution was started, the Sepharose was washed with 5 volumes of PBS. Finally, the bound antigen was eluted with 0.1 M sodium citrate (pH 4.0). The eluted fractions were immediately neutralized with 1 M Tris-Cl (pH 8.9) and analyzed by SDS-PAGE. Relevant fractions were pooled, dialyzed against PBS, and concentrated by repeated centrifugation in Centriprep-10 concentrators (Amicon, Witten, Germany) at 4°C, using PBS.

Amino acid sequence analysis. For N-terminal amino acid sequence analysis, samples of trichloroacetic acid-precipitated fractions were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Problot; Applied Biosystems, Weiterstadt, Germany) with a mini trans-blot electrophoretic transfer cell (Bio-Rad) for 2 h at 150 mA. The blot was then stained with amido black, and the regions corresponding to the proteins of interest were excised and subjected to analysis on an Applied Biosystems gas-phase sequenator (model A470) equipped with an on-line phenylthiohydantoin amino acid analyzer.

Analytical ultracentrifugation analysis. To determine the native form of purified InlB, sedimentation-diffusion equilibrium runs were performed in a Beckman model F analytical ultracentrifuge. Samples with InlB at 230, 490, and 700 µg/ml in PBS were run to equilibrium in a 12-mm charcoal-filled Epon double-sector cell with sapphire windows in a two-place AN-H rotor. Two independent runs were performed. After 20 and 24 h, the samples were scanned at 280 nm and evaluated as previously described (16).

Reassociation of externally added InlB to bacteria. One milliliter of an overnight culture of bacteria grown in brain heart infusion broth was pelleted by centrifugation in an Eppendorf tube. The resulting pellet was washed three times in PBS (pH 7.4) before addition of various concentrations of purified InlB (4 and 10 µg/ml). The mixture was incubated for 30 min at 30°C with continuous shaking (Thermomixer; Eppendorf, Hamburg, Germany), pelleted and washed five times in PBS, resuspended in Dulbecco modified Eagle medium, and used in the invasion assay as described previously (7).

	RESULTS AND DISCUSSION

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Selective extraction of the native InlB from bacterial pellets. The inlB gene product was easily identified as a distinct protein band of 65 kDa in SDS extracts of L. monocytogenes EGD and was also present in small amounts in concentrated culture supernatants, where it showed extensive degradation (26). We tested various buffer combinations containing nondenaturing detergents or enzymes to extract the inlB gene product in its native form from the surface of L. monocytogenes.

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Analysis of extracts generated by treatment of L. monocytogenes with different detergents and other reagents. (A) Silver-stained SDS-gel (10%). Extracts were obtained by treating bacterial pellets (strain actA2/pERL3 50-1) with 2% SDS (lane 1), 16.2 mM CHAPS (lane 2), 1% Triton X-100 (lane 3), mutanolysin (lane 4), and 1 M Tris-Cl (pH 7.5) (lane 5). (B) Corresponding immunoblot reacted with InlB MAb IC100. Note that InlB was selectively extracted by Tris-Cl in lane 5. The arrow indicates the InlB protein band. Molecular mass markers (bars on the left), from top to bottom: 97, 66, 45, and 31 kDa.

View larger version (73K): [in this window] [in a new window]   FIG. 2.   Effects of varying pH values on the extraction of InlB from bacterial pellets. The pH of the Tris-Cl buffer was adjusted to 7.0 (lane 1), 7.25 (lane 2), 7.5 (lane 3), 8.0 (lane 4), and 9.0 (lane 5). (A) Silver-stained extracts; (B) corresponding immunoblot reacted with InlB-specific MAb IC100. Molecular mass markers (bars on the left) indicate 66 kDa.

Purification of InlB and generation of new InlB-specific MAbs. When we tried to isolate InlB from the bacterial Tris-Cl extracts with biochemical methods, the rather unusual nature of the protein complicated the application of standard chromatography material. To apply ion-exchange chromatography (Mono-Q and Mono-S), the Tris-Cl extract was dialyzed against buffers with low ionic strength (20 mM Tris-Cl or 20 mM sodium phosphate [pH 7.5]). Under these conditions, InlB was readily sedimentable by low-speed centrifugation.

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Purification of InlB from Tris-Cl extracts of L. monocytogenes by immunoaffinity chromatography. (A) Silver-stained SDS-gel (10%) of the Tris-Cl extract of L. monocytogenes actA2/pERL3 50-1 (lane 1) which was incubated with immobilized MAb IF32. After extensive washing of the matrix, InlB was eluted with 0.1 M sodium citrate, pH 4.0 (lane 2). (B) Corresponding immunoblot reacted with MAb IC100. Molecular mass markers (bars on the left), from top to bottom: 97, 66, and 45 kDa.

Purified InlB promotes efficient internalization of noninvasive listeriae. It was recently shown that prior incubation of spent culture supernatants of InlB-expressing bacteria confers invasiveness to an inlB mutant (3). This study was recently extended by assessing the ability of purified recombinant InlB proteins expressed in Escherichia coli to promote invasion of an inlB mutant and noninvasive L. innocua (4). We used this assay to assess the activity of the purified InlB protein by examining the ability of externally added purified InlB to promote the invasivity of the wild-type L. monocytogenes EGD strain, an isogenic inlB2 mutant, and a noninvasive L. innocua strain. Preincubation of bacteria with purified InlB resulted in strong association of the protein with the bacterial cell wall, which was readily visible in Coomassie blue-stained SDS extracts of the inlB2 mutant strain (Fig. 4A). Specific association of InlB to the bacterial surface was confirmed by using MAb IF32, specific for InlB (Fig. 4B). In invasion assays, the addition of increasing amounts of purified InlB to noninvasive strains rendered them highly competent for entry into the Vero tissue culture cell line. Thus, in the experiment presented, prior addition of InlB at 4 µg/ml increased invasion of the inlB2 mutant 17-fold and that of a noninvasive L. innocua strain greater than 300-fold (Fig. 5). No increase in invasion was observed using the wild-type strain for this cell line. The results were more dramatic when invasion was examined in the epithelial-like cell line HeLa. Thus, externally added InlB at 4 µg/ml increased invasion of the wild-type strain, the inlB2 mutant, and L. innocua 6-, 3,700-, and 9,000-fold, respectively (Fig. 5). These results demonstrate that the purified native InlB isolated in this study is highly active and promotes invasion of noninvasive bacteria into two different cell lines.

View larger version (67K): [in this window] [in a new window]   FIG. 4.   Purified InlB binds to the surface of L. monocytogenes. Bacteria (108/ml) of the L. monocytogenes EGD inlB2 mutant were incubated with 4 µg of purified InlB for 30 min at room temperature as described in Materials and Methods and analyzed following SDS-PAGE by Coomassie blue staining (A) or immunoblotting using InlB-specific MAb IC100 (B). SDS extracts from the L. monocytogenes EGD inlB2 mutant (lane1) and the same mutant preincubated with 4 µg of purified InlB (lane 2). The position of InlB in panel A is indicated by an arrow. Molecular mass markers (bars on the left), from top to bottom: 97, 66, 45, 31, 21, and 14 kDa.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Externally added purified InlB protein enhances entry of invasive and noninvasive Listeria strains into Vero (A) and HeLa (B) cell lines. Bacteria were either not treated or preincubated with InlB at 4 µg/ml before performance of the invasion assay as described previously (10). Values along the vertical axis are given relative to the invasion of wild-type strain EGD, which is arbitrarily fixed at 100. Bars indicate standard deviations.