Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies.

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Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies Teela Boivin,a,b Cathie Elmgren,a Brian W. Brooks,a Hongsheng Huang,a Franco Pagotto,b,c Min Lina,b Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, Ontario, Canadaa; Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canadab; Listeriosis Reference Service, Bureau of Microbial Hazards, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canadac

ABSTRACT

Protein antigens expressed on the surface of all strains of Listeria monocytogenes and absent from nonpathogenic Listeria spp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains of L. monocytogenes for diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved in L. monocytogenes virulence and identified Listeria adhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present in L. monocytogenes strains and absent from strains of other Listeria spp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) of L. monocytogenes serotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface of L. monocytogenes cells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion antiLapB MAbs, can be targeted as a biomarker for the detection and isolation of various L. monocytogenes strains from contaminated foods. IMPORTANCE

Strains of L. monocytogenes are traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation of L. monocytogenes from foods. Bioinformatics analysis revealed that the gene coding for Listeria adhesion protein B (LapB), a surface protein involved in L. monocytogenes virulence, was present in L. monocytogenes strains and absent from other Listeria spp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface of L. monocytogenes. Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range of L. monocytogenes isolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation.

T

he Gram-positive bacterium Listeria monocytogenes is an intracellular pathogen that can cause a severe and life-threatening illness referred to as listeriosis in humans (1). This organism is ubiquitous in nature, is commonly found in water, soil, and vegetation, and can survive in harsh environments (2), making foods vulnerable to pathogen contamination during harvesting and/or processing. Consumption of contaminated foods is the most common route of transmission of L. monocytogenes to humans, posing an extremely high risk to newborns, the elderly, pregnant women, and immunocompromised individuals. Although the incidence of listeriosis is low, its high mortality rate of up to 50% (3, 4, 5, 6) continues to make L. monocytogenes a serious foodborne pathogen. Recent outbreaks of listeriosis in Canada, linked to contaminated ready-to-eat meat products resulting in 22 deaths (http: //news.gc.ca/web/article-en.do?nid⫽468909), and in the United States, attributed to cantaloupes causing 33 deaths (http://www .cdc.gov/listeria/outbreaks/cantaloupes-jensen-farms/082712 /index.html), highlight the importance of enhancing the capability to detect and identify this pathogen in food chains.

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L. monocytogenes belongs to the genus of Listeria, in which 17 species are recognized (7): L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri, and the recently described L. marthii, L. rocourtii, L. fleischmannii, L. weihenstephanensis, L. floridensis, L. aquatica, L. cornellensis, L. riparia, L. grandensis, L. booriae, and L. newyorkensis. Only strains of L. monocytogenes and

Received 29 June 2016 Accepted 6 September 2016 Accepted manuscript posted online 9 September 2016 Citation Boivin T, Elmgren C, Brooks BW, Huang H, Pagotto F, Lin M. 2016. Expression of surface protein LapB by a wide spectrum of Listeria monocytogenes serotypes as demonstrated with anti-LapB monoclonal antibodies. Appl Environ Microbiol 82:6768 –6778. doi:10.1128/AEM.01908-16. Editor: D. W. Schaffner, Rutgers, The State University of New Jersey Address correspondence to Min Lin, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.01908-16. © Crown copyright 2016.

Applied and Environmental Microbiology

November 2016 Volume 82 Number 22

Expression of LapB in L. monocytogenes

L. ivanovii are considered to be pathogenic, with L. ivanovii mainly infecting animals. Although atypical strains of L. innocua contain some virulence genes specific to L. monocytogenes (8, 9), there is no evidence that they are pathogenic. Conventional procedures for the isolation, detection, and identification of L. monocytogenes from food and environmental samples are laborious and timeconsuming, involving primary and secondary culture enrichment followed by plating on selective agar and biochemical and serological tests for confirmation, which can take 5 to 10 days to complete (10, 11). Although molecular methods such as PCR are available to expedite detection and identification, these methods are often hampered by inhibitory substances and a low number of target bacteria present in food samples and are thus frequently dependent on culture enrichment of the target or the availability of pure cultures (12). Because of these drawbacks, L. monocytogenes cells must, in many cases, be separated and concentrated from sample matrices prior to employing molecular detection methods. The lack of rapid and effective methods for L. monocytogenes isolation from food samples forms a major obstacle to the use of molecular detection and identification technologies designed to reduce the turnaround time from sampling to obtaining test results. Antibody (Ab)-based methods offer means for isolation and detection of L. monocytogenes that are faster than traditional methods (10, 13, 14) but face a considerable challenge, as at least 12 L. monocytogenes serotypes with antigenic variations are recognized (15–17). Monoclonal antibodies (MAbs) which bind specifically to strains of all L. monocytogenes serotypes and which do not cross-react with nonpathogenic Listeria spp. and other bacteria are still being sought for use in the development of rapid and reliable immunological tools for the isolation, detection, and identification of this pathogen. One strategy for the development of L. monocytogenes-specific MAbs is that of identifying surface proteins that are expressed by all serotype strains of L. monocytogenes grown in enrichment culture and targeting these proteins for the development of such antibodies. Listeria species adhesion protein B (LapB) is a L. monocytogenes LPXTG surface protein (Lmo1666) that is absent from nonpathogenic species and is one of the virulence adhesion molecules involved in entry of L. monocytogenes into eukaryotic cells (18). The aim of the present study was to investigate the expression of LapB in L. monocytogenes strains in a wide range of serotypes by developing and using MAbs against this protein. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. Escherichia coli DH5␣ was used as a host strain for molecular cloning. E. coli Rosetta (DE3)/pLysS was used as a protein expression host. E. coli strains were grown on Luria-Bertani (LB) agar or in LB broth supplemented as required with 50 ␮g/ml kanamycin at 37°C overnight. Listeria strains (Table 1) were grown on brain heart infusion (BHI) agar or in BHI broth at 37°C overnight. Listeria cell concentrations were determined using an optical density at 620 nm (OD620) of 0.61 (equivalent to 1 ⫻ 109 cells/ml) and confirmed by plating (19). E. coli O157:H7, E. coli American Type Culture Collection (ATCC) 25922, Salmonella, and Campylobacter strains were cultured and quantified for cell concentrations as described previously (19). Bioinformatic analysis. LapB (Lmo1666), encoded by the genome of L. monocytogenes EGDe serotype 1/2a, was first characterized as a LPXTG surface protein required for adhesion to and entry into mammalian cells and for virulence in a mouse model (18). To determine if LapB is well


conserved among strains of L. monocytogenes, the LapB homolog (Lmof2365_1690) from L. monocytogenes strain F2365 serotype 4b (20) was used as a reference to perform a similarity search within the public databases using the BLASTp search tool. Similar proteins identified by the BLSATp search were retrieved from the databases and compared by multiple-sequence alignments using the MegAlign module of the Lasergene software package (DNAStar, Madison, WI). LapB expression construct. All DNA manipulations were essentially performed according to established procedures (21). Genomic DNA was extracted from L. monocytogenes strain L10521 serotype 4b using DNAzol (Invitrogen) according to the manufacturer’s instructions and was used as a template to synthesize the DNA fragment coding for the mature LapB (amino acids [aa] 26 to 1682) by PCR. The primers, p912(F) (TTTA AGAAGGAGATATAAGTCATGAAATCGGTGGGAGATATTTC) and p913(R) (AGTGGTGGTGGTGGTGGTGAGTCCCGCTTTCATTTGTA GCGGTA), were designed to allow the PCR-derived lapB gene fragment to be cloned into the pLIC-CHis vector using a ligation-independent cloning procedure (22). This resulted in an expression construct, designated pLapB, which was propagated in E. coli DH5␣. The presence of the correct insertion in pLapB was confirmed by PCR and DNA sequencing with T7 promoter and T7 terminator primers. The recombinant LapB (rLapB) expressed from pLapB in E. coli Rosetta (DE3)/pLysS contained a 6⫻His tag at the C terminus. Expression and purification of recombinant LapB. A colony of E. coli Rosetta (DE3)/pLysS cells containing pLapB was inoculated into 5 ml of LB broth containing 50 ␮g/ml kanamycin and incubated at 37°C with continuous shaking for 16 h. The 16-h culture (5 ml) was added to 500 ml of LB broth containing 50 ␮g/ml kanamycin and incubated at 37°C with continuous shaking until an OD at 595 nm of 0.5 to 0.8 was obtained. The expression of rLapB was induced by the addition of 1 mM isopropyl-␤-Dthiogalactopyranoside (IPTG) followed by incubation for an additional 3 h. The cells were harvested by centrifugation at 8,000 ⫻ g for 20 min at 4°C and stored frozen at ⫺80°C until use. Frozen cell pellets were thawed and suspended in 10 ml of phosphatebuffered saline (PBS; pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspensions were passed through a French press (SLM Instruments Inc.) twice at 1,500 lb/in2. Lysed cells were centrifuged at 27,000 ⫻ g for 20 min at 4°C, and the supernatant (designated crude protein extract) was collected. The extract was mixed with 1 volume of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and then applied at 1.0 ml/min to a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (1 cm by 2.5 cm) which had been preequilibrated with 30 ml of wash buffer. The column was washed with 30 ml of wash buffer at 1 ml/min followed by 30 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Fractions of eluted protein were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) with an anti-His tag MAb (Qiagen). Fractions containing rLapB were pooled and concentrated by centrifugation at 3,270 ⫻ g for 30 to 60 min using Amicon filter centrifugal devices (Millipore). Protein concentrations were quantified using the Bradford method (23) with bovine serum albumin (BSA) as the standard. Preparation of killed Listeria cells for ELISA. Killed cells of the Listeria strains were prepared for use as coating antigens in an enzyme-linked immunosorbent assay (ELISA). One colony of each Listeria strain, maintained on brain heart infusion (BHI) agar at 4°C, was inoculated into BHI broth and incubated at 37°C overnight. Cell concentrations were determined based on the OD620 value as described above. Cells were centrifuged at 8,000 ⫻ g for 10 min and washed 3 times with PBS. Pelleted cells were killed by treatment with 0.3% (vol/vol) formalin for 24 h at room temperature. Killed bacteria were centrifuged and washed as described above. Cells were resuspended in PBS, mixed with an equal volume of glycerol to give a final concentration of 109 cells/ml, and stored at ⫺20°C until use.


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TABLE 1 Number of strains of L. monocytogenes and other organisms grown in brain heart infusion broth that were positive on ELISA with selected MAbs No. (%) of strains positivea on ELISA with indicated MAb or mean OD414 ⫾ SD Organism L. monocytogenes Lineage I

Lineage II

Lineage III/IV

Serotype

Total no. of strains

M3484

M3495

M3500

M3509

M3517

M3519

M3524

1/2b 3b 3c 4ab 4b 4d 4e Total Mean OD414 ⫾ SDb 1/2a 1/2c 3a Total Mean OD414 ⫾ SD 4a 4c Total Mean OD414 ⫾ SD

9 2 1 2 10 1 2 27

9 2 1 2 10 1 1 26 (96.3) 1.6 ⫾ 0.5 10 5 6 21 (100) 1.3 ⫾ 0.5 0 0 0 (0) 0.1 ⫾ 0.0

9 2 1 2 10 1 1 26 (96.3) 1.5 ⫾ 0.5 9 5 6 20 (95.2) 1.2 ⫾ 0.5 0 0 0 (0) 0.1 ⫾ 0.0

9 2 1 2 10 1 1 26 (96.3) 1.4 ⫾ 0.5 9 5 6 20 (95.2) 1.1 ⫾ 0.5 0 0 0 (0) 0.1 ⫾ 0.1

9 2 1 2 10 1 1 26 (96.3) 1.2 ⫾ 0.4 9 5 6 20 (95.2) 1.0 ⫾ 0.4 0 0 0 (0) 0.1 ⫾ 0.0

9 2 1 2 10 1 1 26 (96.3) 1.6 ⫾ 0.5 9 5 6 20 (95.2) 1.3 ⫾ 0.5 0 0 0 (0) 0.1 ⫾ 0.0

9 2 1 2 10 1 1 26 (96.3) 1.4 ⫾ 0.4 9 5 6 20 (95.2) 1.1 ⫾ 0.4 0 0 0 (0) 0.1 ⫾ 0.0

7 2 0 2 10 1 1 23 (85.2) 0.4 ⫾ 0.2 1 0 0 1 (4.8) 0.1 ⫾ 0.1 0 0 0 (0) 0.1 ⫾ 0.0

1 1 1 1 1 5

0 0 0 1 0 1 (20) 0.1 ⫾ 0.1

0 0 0 1 0 1 (20) 0.1 ⫾ 0.1

0 0 0 0 0 0 (0) 0.1 ⫾ 0.1

0 0 0 1 0 1 (20) 0.2 ⫾ 0.0

0 0 0 1 0 1 (20) 0.1 ⫾ 0.1

0 0 0 0 0 0 (0) 0.1 ⫾ 0.1

0 0 0 0 0 0 (0) 0.1 ⫾ 0.0

5 5

0 0 (0) 0.1 ⫾ 0.0

0 0 (0) 0.0 ⫾ 0.0

0 0 (0) 0.0 ⫾ 0.0

0 0 (0) 0.1 ⫾ 0.0

0 0 (0) 0.0 ⫾ 0.0

0 0 (0) 0.0 ⫾ 0.0

0 0 (0) 0.0 ⫾ 0.0

L. innocua L. ivanovii L. grayi L. seeligeri L. welshimeri Total Mean OD414 ⫾ SD Other bacteriac Total Mean OD414 ⫾ SD

10 5 6 21 2 3 5

OD414 ⫽ ⬎0.2. ELISA mean OD414 ⫾ standard deviation. c E. coli O157:H7, E. coli, S. Typhimurium, S. California, C. jejuni. a b

Extraction of L. monocytogenes total cellular proteins. A colony of L. monocytogenes strain LI0521 serotype 4b from a BHI agar plate was inoculated into 50 ml of BHI broth and incubated with shaking at 225 rpm for 16 h at 37°C. Cells were centrifuged at 8,000 ⫻ g for 10 min and resuspended in 500 ␮l of 0.5 M Tris-HCl (pH 6.8) containing 10% (wt/vol) SDS and 1 mM PMSF. The cell suspension was transferred to a Lysing Matrix B tube (MP Biomedicals) and homogenized using a FastPrep FP120 cell disrupter (Thermo Electron) for 40 s at speed level 6. A 500-␮l volume of a 2⫻ SDS-PAGE sample buffer was added, and the mixture was boiled for 10 min. The supernatant was kept at ⫺20°C for analysis by SDS-PAGE and WB. SDS-PAGE and Western blotting. SDS-PAGE was carried out as described by Laemmli (24), using a 4% (wt/vol) stacking gel and a 10% (wt/vol) separating gel. Following electrophoresis, the separated proteins were either stained with Coomassie blue or electrotransferred onto a nitrocellulose membrane for WB (19). Prior to WB, successful transfer of proteins was confirmed by staining the membrane with 0.1% (wt/vol) Ponceau S–5% (vol/vol) acetic acid for 2 min. The membrane was then blocked with PBS-TT (PBS, 0.05% [vol/vol] Tween 20, 0.2% [vol/vol] Triton X-100) containing 3% (wt/vol) BSA for 1 h at room temperature or overnight at 4°C. WB detection of proteins was performed using specific MAbs followed by peroxidase-conjugated goat anti-mouse IgG (H⫹L) (Jackson ImmunoResearch Laboratories) as described previously (19).

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Production of LapB PAbs. Polyclonal antibodies (PAbs) were raised by immunizing rabbits with rLapB. A blood sample was collected prior to immunization. Purified rLapB (200 ␮g in 0.5 ml PBS) was emulsified with an equal volume of incomplete Freund’s adjuvant (IFA) and used to inject subcutaneously each of two New Zealand White female rabbits (Charles River Laboratories, Senneville, Quebec, Canada) on day 0 (total volume ⫽ 1 ml for each rabbit). Booster inoculations were given with a mixture of 100 ␮g of the purified protein and 0.5 ml PBS emulsified with an equal volume of IFA on days 14 and 28. After a test bleed performed on day 42, rabbits were exsanguinated on day 46 to collect the maximal volume of blood samples. PAbs against LapB were also obtained from mice immunized with rLapB for production of MAbs (see below). All experiments involving animals were approved by the local Animal Care Committee under the guidelines of the Canadian Council on Animal Care. Production of LapB monoclonal antibodies. Six-week-old Swiss Webster (CFW) mice and BALB/c mice (3 of each strain; Charles River Laboratories) were immunized with purified rLapB at scheduled intervals over a 2-month period. The mice were bled prior to immunization. On day 0, each mouse was injected subcutaneously with rLapB protein (30 ␮g) emulsified with an equal volume of complete Freund’s adjuvant (total volume ⫽ 100 ␮l). Booster inoculations were given to each mouse intraperitoneally with 20 ␮g and 10 ␮g of the protein




emulsified with incomplete Freund’s adjuvant (1:1 [vol/vol]; total volume ⫽ 200 ␮l) on days 21 and 42, respectively. Mice were bled on day 64 for the assessment of the anti-LapB antibody response by ELISA. One CFW mouse and one BALB/c mouse that exhibited high titers of antibodies to rLapB were selected for MAb production, and each was inoculated intraperitoneally with 0.5 ␮g epinephrine and 100 ␮g dehydroepiandrosterone sulfate and intravenously with 5 ␮g of rLapB– 100 ␮l PBS 5 days before splenectomy was performed. Blood was also collected on the day of splenectomy. Mouse Sp2/0-Ag14 myeloma cells were fused with spleen cells from two mice as previously described (19). Undiluted tissue culture fluid (TCF) samples of parent hybridomas from the two fusions were screened by an indirect ELISA procedure (19) for antibody reactivity to purified rLapB protein coated on Nunc 96-well microtiter plates (Thermo Scientific) at a concentration of 1 ␮g/ml (100 ␮l/well) mixed with 60 mM carbonate buffer (pH 9.6). TCF samples from positive hybridomas were further screened by ELISA against killed L. monocytogenes cells (serotypes 4b, 1/2a, and 1/2c) coated on microtiter plates at 37°C overnight at 5 ⫻ 108 cells/ml (100 ␮l/well) mixed with PBS. The subclass of immunoglobulin secreted by the hybridoma cell lines was determined by ELISA using commercial reagents according to the instructions of the manufacturer (SBA Clonotyping system; Southern Biotech). In addition, immunofluorescence microscopy (IFM) analysis (see below) was performed to evaluate binding of MAbs to the cell surface of L. monocytogenes serotype 4b strain L10521 cells. Based on the results of ELISA, IFM analysis, and isotyping, hybridomas were selected for single-cell cloning and expansion. Immunofluorescence microscopy analysis. IFM was performed as previously described (19). Bacterial cells were probed with rabbit antiserum (1:500 dilution) or mouse MAbs (TCF at 1:25 dilution) followed by immunofluorescence staining with Dylight 488 goat antirabbit IgG (1:50 dilution) or anti-mouse IgG (1:100 dilution) (Jackson ImmunoResearch) mixed with PBS–5% (wt/vol) BSA. Phase contrast and fluorescence images of bacteria were viewed and captured with a fluorescence microscope (Olympus BX60) equipped with a chargecoupled camera. ELISA analysis of LapB expression. Indirect ELISA was performed with anti-LapB MAbs as described above to evaluate the expression of LapB in 53 L. monocytogenes strains (Table 1) and other bacterial species: L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri, E. coli O157:H7 ATCC 43889, E. coli ATCC 25922, Salmonella enterica serovar Typhimurium DT104 SA03-1907, S. enterica serovar California, and Campylobacter jejuni NCTC11168. Microtiter plates were coated with bacteria at 2 ⫻ 108 cells/ml (100 ␮l/well) mixed with PBS. An ELISA OD414 value of ⱖ0.2 was considered to represent a positive result. LapB expression in enrichment culture. To assess the expression of LapB during enrichment in media used in a diagnostic procedure for the isolation of L. monocytogenes and other Listeria spp. from food and environmental samples (MFHPB-07 method; the Compendium of Analytical Methods, Health Canada [http://www.hc-sc.gc.ca/fn-an/res-rech/analy -meth/microbio/volume2-eng.php]), a single colony of each of 4 selected Listeria strains (3 L. monocytogenes strains and 1 L. innocua strain) was inoculated into 7 ml of Palcam broth (Oxoid) and incubated at 35°C with shaking at 225 rpm overnight. A 1-ml volume of the overnight culture was transferred into 9 ml of fresh UVM broth (BD Diagnostics) and incubated at 30°C with shaking at 225 rpm overnight. Bacterial cells were then centrifuged and washed 3 times with PBS and used to coat microplate wells (Nunc Maxisorp plates; Thermo Scientific) at 2 ⫻ 108 cells/ml (100 ␮l/ well) overnight at 37°C and evaluated for the expression of LapB by ELISA with MAbs as described above.

RESULTS

LapB protein expression in L. monocytogenes. To characterize the cell surface expression of LapB protein in L. monocytogenes and investigate if LapB could be targeted for the development of MAbs


recognizing various L. monocytogenes strains, polyclonal antibodies were first raised against the recombinant form of LapB for use as a probe. Figure 1A shows the expression in E. coli of the rLapB (aa 26 to 1682) carrying a C-terminal 6⫻His tag from the construct pLapB, as detected by WB with an anti-His MAb. The rLapB protein migrated to a position with an apparent mass close to the calculated value of 180 kDa. The presence of rLapB in the E. coli whole-cell lysates was not evident by SDS-PAGE analysis (Fig. 1B), indicating a low level of rLapB expression upon induction with IPTG. For PAb generation, an adequate amount of soluble rLapB was purified from pLapB-transformed E. coli cells using Ni-NTA agarose affinity chromatography. Eluted fractions that were confirmed to contain the rLapB protein by WB using anti-His MAb (Fig. 1C) were pooled and concentrated to approximately 1.0 mg/ ml. SDS-PAGE and WB revealed that the concentrated rLapB preparation had only minimal contamination with E. coli proteins (Fig. 1D and E). Sera from rabbits immunized with rLapB were demonstrated to contain specific anti-LapB PAbs by ELISA and WB (data not shown). The surface expression of LapB protein on live L. monocytogenes cells was probed with PAbs to rLapB in IFM analysis. The PAbs bound to the surface of L. monocytogenes serotype 4b L10521 cells, and no fluorescence signal was detected with the preimmune serum (Fig. 2). This result demonstrated that LapB is surface expressed in L. monocytogenes, suggesting that LapB could be explored as a candidate antigen against which MAbs could be raised for pathogen detection and capture by immunological methods. Consistent with this observation was the finding that antiserum from mice immunized with rLapB recognized the native protein in the total protein extract of L. monocytogenes on WB (see Fig. S1 in the supplemental material). Development of MAbs to LapB. A total of 48 stable hybridoma clones secreting MAbs to rLapB were obtained. Of these MAbs, 41 were Ig subclass G1 (M3478 to M3491, M3493 to M3495, M3497 to M3511, M3514 to M3519, M3521, M3524, and M3525) and 7 were Ig subclass G2a (M3492, M3496, M3512 and M3513, M3520, M3522, and M3523). All 48 MAbs, with the exception of M3511, reacted to rLapB in ELISA with an OD414 of ⬎2.0, and all but one MAb (M3511) did not react with soluble proteins of E. coli BL21(DE3)/pLysS (with no pLapB expression vector) (see Fig. S2 in the supplemental material). Binding of anti-LapB MAbs to the surface of L. monocytogenes serotype 4b. The ELISA results (Fig. 3) showed that 11 of the 48 MAbs reacted strongly (OD414 ⫽ ⬎1.0) with L. monocytogenes serotype 4b L10521 cells. Seven (M3484, M3495, M3500, M3509, M3517, M3519, and M3524) of the 11 MAbs bound strongly to the surface of the serotype 4b L10521 strain as revealed with IFM, while four MAbs (M3482, M3493, M3498, and M3499) had a negative to weak immunofluorescence signal (Fig. 4). Reactivity of anti-LapB MAbs with various strains of L. monocytogenes grown in BHI broth. The seven MAbs that bound to the surface of L. monocytogenes serotype 4b L10521 cells were further tested by ELISA with 53 L. monocytogenes strains representing 12 serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4a, 4b, 4c, 4d, and 4e). The reactivity of each MAb with all L. monocytogenes strains tested was given a score (⫺ to ⫹⫹⫹) based on the mean OD414 values from three separate experiments (see Table S1 in the supplemental material). As shown in Table 1, six of the seven anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and


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FIG 1 SDS-PAGE and Western blot (WB) analysis of rLapB protein expression in E. coli Rosetta (DE3) cells. (A) WB analysis. Lane 1, prestained protein standards with their molecular masses indicated (in kilodaltons) on the left; lane 2, lysate from uninduced E. coli cells; lane 3, lysate from IPTG-induced E. coli cells. (B) SDS-PAGE. Lane 1, prestained protein standards with their molecular masses indicated (in kilodaltons) on the left; lane 2, lysate from uninduced E. coli cells; lane 3, lysate from IPTG induced E. coli cells. (C) WB. Lane 1, prestained protein standards with their molecular masses indicated (in kilodaltons) on the left; lanes 2 to 6, fractions 1 to 5 of LapB protein eluted from a Ni-NTA agarose affinity column. (D) WB. Lane 1, prestained protein standards with their molecular mass in kilodalton on the left; lane 2, concentrated pool of eluted fractions of purified rLapB protein. (E) SDS-PAGE. Lane 1, prestained protein standards with their molecular masses indicated (in kilodaltons) on the left; lane 2, concentrated pool of eluted fractions of rLapB protein. The blot or gel shown in panels A and B was spliced along the narrow white gap between lane 1 and lane 2 to remove the lanes irrelevant to this work. Arrows indicate the rLapB protein at ⬃195 kDa, close to the theoretical molecular mass of 180 kDa for LapB protein.

M3519) detected 46 (86.8%) of 53 strains, which represented 10 of the 12 L. monocytogenes serotypes tested. M3524 showed weaker reactivity to the majority of strains and recognized only 24 of 53 strains (Table 1). None of the anti-LapB MAbs reacted with the three 4c strains or the two 4a strains tested. Overall, M3484 reacted with the highest number of L. monocytogenes strains (47 of 53) and had the highest reactivity scores among the seven MAbs (see Table S1). As shown in Fig. 5, M3484 reacted with the majority of the L. monocytogenes strains tested but not with non-L. monocytogenes strains except for L. seeligeri. Cross-reactivity of anti-LapB MAbs with nonpathogenic Listeria spp. and other microorganisms. ELISA results showed that none of the seven MAbs (M3484, M3495, M3500, M3509, M3517, M3519, and M3524) reacted with the L. innocua, L. ivanovii, L. grayi, or L. welshimeri strains tested (Table 1). Three of the MAbs (M3500, M3519, and M3524) did not react with a L. seeligeri

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strain, whereas two MAbs (M3495 and M3509) reacted very weakly (OD414 values of 0.2 to 0.3) and two (M3484 and M3517) reacted moderately (OD414 values of 0.3 to 1.0) with this strain. None of these seven MAbs reacted with the E. coli O157, Salmonella enterica, or C. jejuni strains tested (Table 1). LapB expression in L. monocytogenes under culture enrichment conditions. L. monocytogenes serotype 4b strains L10521 and HPB5906 and serotype 1/2a strain HPB6036 and L. innocua strain HPB583 were cultured in enrichment broths (i.e., first in Palcam and then in UVM), and the cells were tested by ELISA for LapB expression using MAbs M3484, M3495, M3500, M3509, M3517, and M3519. All three L. monocytogenes strains, but not the L. innocua strain, were detected by each of the six anti-LapB MAbs (Fig. 6). As observed with the Listeria cells grown in BHI broth (Table 1), these anti-LapB MAbs reacted more strongly with L.




FIG 2 Cell surface expression of native LapB protein in L. monocytogenes serotype 4b cells observed with immunofluorescence microscopy. L. monocytogenes cells were incubated with (A) preimmune serum and (B) antiserum from a rabbit immunized with rLapB protein and then with a goat anti-rabbit fluorescent IgG conjugate. Phase contrast images are shown in the panels on the left, and fluorescence images are shown in the panels on the right (magnification, ⫻100).

monocytogenes strains HPB5906 and HPB6036 than with the L10521 immunizing strain. DISCUSSION

LapB is a LPXTG surface protein encoded by lapB, a L. monocytogenes-specific gene that has not previously been found in nonpathogenic Listeria species (18). The expression of LapB in L. monocytogenes would provide a unique surface marker for the detection, isolation, and identification of this pathogen by immunological methods. The objective of the present study was to examine if the LapB protein was expressed in a wide range of L. monocytogenes strains and to evaluate if this protein can serve as a suitable target in the antibody-based diagnostic methods. Through producing a recombinant form of LapB (rLapB) and raising polyclonal antibodies against this protein, we confirmed

with IFM that LapB is surface localized in L. monocytogenes. This result is consistent with previous findings that LapB was detected in the cell wall proteome of L. monocytogenes (25) and was absent in the surface extract of a L. monocytogenes ⌬srtA mutant (18). The surface exposure characteristic of LapB meets the minimum requirement for the use of LapB as a biomarker for the antibodybased detection and isolation of L. monocytogenes. Production and characterization of a number of mouse MAbs against the LapB protein revealed that several of these MAbs were capable of recognizing strains of 10 L. monocytogenes serotypes and thus have the potential for use in the detection and isolation of L. monocytogenes in food, clinical, and environmental sources. In the present study of 58 Listeria strains, using MAbs raised against rLapB, the LapB protein was shown to be present and exposed on the surface of L. monocytogenes strains of lineage I (serotypes 1/2b, 3b, 3c, 4ab, 4b, 4d, and 4e) and lineage II (serotypes 1/2a, 1/2c, and 3a) (26). LapB was not detected in the rare lineage III strains (serotypes 4a and 4c) (26) or the L. innocua, L. ivanovii, L. grayi, or L. welshimeri strains tested but was detected in the L. seeligeri strain. These results are consistent with the findings of some previous investigations. In an analysis of surface proteinencoding genes, Cabanes et al. (27) provided evidence that L. monocytogenes protein Lmo1666, later renamed LapB (18), contains an LPXTG sequence motif, indicating that this protein is covalently linked to the cell wall through sortase A (SrtA) and an Arg-Gly-Asp (RGD) motif which is found in proteins mediating adhesion to eukaryotic cells. Doumith et al. (28) examined 113 Listeria strains using comparative genomics and DNA hybridization techniques and showed that the lmo1666 gene is present in L. monocytogenes lineages I and II and in one of the two L. seeligeri strains studied but absent from L. monocytogenes lineage III, L. innocua, L. ivanovii, L. welshimeri, and L. grayi. Calvo et al. (25) demonstrated that Lmo1666 is present in the cell wall proteome of L. monocytogenes but absent from that of L. innocua, and Reis et al. (18) found no similarity between the N-terminal part of the LapB protein (amino acids 25 to 839) and that of other proteins by a BLAST search of the public sequence databases. Some anti-LapB MAbs were unable to detect all the L. mono-

FIG 3 Indirect ELISA reactivity of 48 anti-LapB MAbs with L. monocytogenes serotype 4b strain L10521 cells grown in BHI broth. Microplate wells were coated

with L. monocytogenes cells at a concentration of 5 ⫻ 108 cells/ml. Positive and negative controls were anti-IspC MAb M2799 and 10% fetal calf serum, respectively. ELISA OD414 values represent averages of duplicate determinations for each MAb. The dashed line indicates the negative-positive threshold OD414 value of 0.20.



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FIG 4 Analysis of binding of anti-LapB MAbs to the surface of L. monocytogenes 4b strain L10521 cells using immunofluorescence microscopy. L. monocytogenes cells were probed with (A) mouse preimmune serum (1:50), (B) MAb M3484, (C) MAb M3495, (D) MAb M3500, (E) MAb M3509, (F) MAb M3517, (G) MAb M3519 (H) MAb M3524, (I) MAb M3482, (J) MAb M3493, (K) MAb M3498, and (L) MAb M3499 hybridoma tissue culture fluid (1:25 dilution). Both phase contrast and fluorescence images of bacterial cells were captured at a magnification of ⫻100.

cytogenes strains of the same serotype. A possible explanation for this observation is the absence of the lapB gene in certain strains of the same serotype. Consistent with this explanation is the high genetic diversity among L. monocytogenes isolates, which has formed the basis of grouping isolates of the same serotype into genetically distinct lineages (26). In fact, Doumith et al. showed the absence of lapB in several strains of serotypes 4c and 4a (28). Another possible explanation is differential expression of the lapB gene among strains of the same serotype. It has been shown that a number of genes found in both lineages I and II are expressed differentially among isolates of both lineages at 37°C (26). Comparative genomics and gene expression studies are necessary to provide a definitive explanation for the observed reactivity of antiLapB MAbs with isolates of the same serotype. As LapB is highly specific for L. monocytogenes and appears to be conserved in the serotypes most frequently associated with foodborne infections, the anti-LapB MAbs developed in this study are potentially valuable for the development of improved antibody-based methods for detection and isolation of this organism

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from clinical specimens and from environmental and food samples. Six MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted with at least 46 (95.8%) of the 48 L. monocytogenes lineage I and II strains tested; isolates of these two lineages are known to be most frequently associated with human clinical cases (26). The reason that the six MAbs did not react or reacted weakly with one serotype 4e strain and one serotype 1/2a strain is not clear, but possible explanations include a lack of expression or minimal expression of LapB under the culture conditions used, an absence of the lmo1666 gene, or a mutation in the lmo1666 gene resulting in the expression of an altered or nonfunctional epitope. None of the six MAbs reacted with a strain of E. coli, S. enterica, or C. jejuni. Characterization with other Gram-positive bacteria such as Brochothrix (in the same family of Listeriaceae as Listeria), Enterococcus, and Bacillus species is needed to further assess the specificity of these anti-LapB MAbs and to address this limitation of the study. Given that these MAbs showed a lack of cross-reactivity with several non-L. monocytogenes species, it is unlikely that they would detect any other genetically distinct Gram-negative or




FIG 5 Indirect ELISA reactivity of anti-LapB MAb M3484 with 53 strains of L. monocytogenes representing 12 serotypes and 10 non-L. monocytogenes bacterial species. Bacterial cells were coated on 96-well microplates at a concentration of 1 ⫻ 108 cells/ml. Error bars represent 1 standard deviation from the mean OD414 of the results of three independent experiments. The dashed line indicates the cutoff for a negative reaction (OD414 ⬍ 0.20).

-positive bacteria, although this remains to be experimentally investigated. MAbs to Listeria have also been described in previous studies. In contrast to the anti-LapB MAbs that were shown here to react with the L. moncytogenes serotypes most frequently associated with foodborne outbreaks, some of which (M3500, M3519, and M3524) are highly specific for L. monocytogenes, the majority of those anti-Listeria MAbs previously reported in the literature are not specific for L. monocytogenes or do not recognize a wide spectrum of L. monocytogenes serotypes. Most of them recognize antigens of unknown identity. Butman et al. (29) reported that 15 MAbs recognizing a 30-to-38-kDa protein reacted with L. monocytogenes (1, 1a, 1b, 1/2a, 1/2b, 2, 3, 3a, 3b, 4b, 4c, 4d, and 4e), L. denitrificans (reclassified as Jonesia denitrificans), L. grayi, L. innocua, L. ivanovii, and L. seeligeri. Siragusa and Johnson (30) showed that MAb P5C9 recognized a 18.5-kDa antigen and was capable of reacting with 31 L. monocytogenes strains, L. innocua, and L. welshimeri but not with other Listeria spp. MAbs 20-10-2, 36-6-12, and 59-9-16 (31) were shown to react with L. monocytogenes, L. innocua, L. ivanovii, and L. seeligeri; the antigen recognized by these MAbs was not identified. MAb C11E9 (32) reacted with L. monocytogenes and L. innocua but not with L. ivanovii, L. grayi, L. welshimeri, L. seeligeri, or L. murrayi; these MAbs detected


several proteins of 76, 66, 56, and 52 kDa in L. monocytogenes and 66, 56, and 52 kDa in L. innocua. MAb EM-7G1 (33) recognized a 66-kDa cell surface protein and detected all 34 L. monocytogenes strains tested but not L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. murrayi, or L. ivanovii. MAb EM-6E11 (32) reacted with all 7 Listeria spp. and recognized 43-kDa and 94-to 97-kDa proteins. MAb 55-44 (34) reacted with all 11 L. monocytogenes 4b strains tested, with some L. monocytogenes 1/2b, 3c, 4a, 4ab, and 4d strains, and with L. innocua and L. welshimeri; this MAb detected 90-to-95-kDa and 35-kDa proteins. MAb B4 (35) reacts with viable L. monocytogenes (65 strains) and L. innocua (14 strains) but not with heat-killed cells of these bacteria or with L. ivanovii, L. seeligeri, L. welshimeri, or L. grayi. MAb p6007 (36), an antibody recognizing a 60-kDa p60 murein hydrolase protein (secreted in large quantity into the growth medium), detected L. monocytogenes but not L. innocua, L. grayi, L. welshimeri, L. ivanovii, or L. seeligeri. Another anti-p60 MAb, p6017 (36), reacted with L. monocytogenes, L. innocua, L. grayi, L. welshimeri, L. ivanovii, and L. seeligeri. MAb mAb2B3 (37) was shown to detect L. monocytogenes 1/2a, 1/2c, 4a, and 4b (the only serotypes tested) but not L. innocua, L. welshimeri, L. seeligeri, or L. ivanovii; the antigen was an 80-kDa protein assumed to be internalin A. MAb 22D10 (38) recognized a 66-kDa protein and was capable of detecting live and


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FIG 6 Indirect ELISA reactivity of six LapB MAbs with cells of three L. monocytogenes strains and one L. innocua strain cultured in an enrichment condition. Bacterial cells were grown first in Palcam broth and then in UVM broth prior to being coated on 96-well microplates at 2 ⫻ 108 cells/ml. The cells were probed with six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) and anti-IspC MAb M2799 specific to L. monocytogenes serotype 4b (positive control) and 10% fetal calf serum (FCS) (negative control). L. monocytogenes serotype 4b strains L10521 (L10521-4b) and HPB4906 (49064b), L. monocytogenes serotype 1/2a strain HPB6036 (6036-1/2a), and L. innocua strain HPB583 (583-Li) were tested. ELISA OD414 readings represent the averages of duplicate determinations. The ELISA OD414 values for L. innocua ranged from 0.062 to 0.106. The dashed line indicates the cutoff for a negative reaction (OD414 ⬍0.20). Similar results were obtained with a repeat of the experiment.

heat-killed L. monocytogenes cells of most serotypes except 4c and 4e and L. innocua, L. ivanovii, L. murrayi, L. seeligeri, and L. welshimeri. We recently reported the production and characterization of MAbs to a surface autolysin (IspC) of L. monocytogenes 4b with an apparent molecular mass of 77 kDa and possessing N-acetylglucosaminidase activity (39–41); some of these MAbs (M2774, M2775, M2780, M2790, and M2797) are specific for L. monocytogenes 4b strains. LapB is a surface adhesion molecule of L. monocytogenes required for entry into eukaryotic cells and virulence (18), is expected to be conserved in a wide range of pathogenic strains of L. monocytogenes, and therefore may serve as a useful diagnostic marker for the detection and isolation of this deadly pathogen by the anti-LapB MAbs developed in this study. Expression of a protein antigen on the surface of L. monocytogenes under different environmental conditions is an essential requirement for the protein to be a useful diagnostic marker for the antibody-based detection and isolation of this pathogen. The antiLapB MAbs were able to detect the expression of LapB on L. monocytogenes cells grown in BHI broth at 37°C, Palcam broth at 35°C, and UVM broth at 30°C; Palcam and UVM media are routinely used for primary and secondary culture enrichment at 35°C and 30°C, respectively (MFHPB-07 method). These steps are necessary to increase the cell number of L. monocytogenes in food samples to a level suitable for isolation and detection. Expression of LapB in L. monocytogenes grown under these culture enrichment conditions points to the feasibility of using anti-LapB MAbs to isolate and detect L. monocytogenes from food samples following

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culture enrichment. This is in contrast to the few previous studies performed with two MAbs (EM-6E11 and C11E9) that do not detect L. monocytogenes cells grown in UVM broth (42, 43). Expression of the lapB gene is presumably directed by a ␴B-dependent promoter predicted 67 bp upstream of its translational start codon (44) and was reported to be positively regulated by transcriptional activator PrfA (18, 45). The regulation of lapB by PrfA suggests a role of LapB in virulence, which has been demonstrated recently (18). ␴B regulation suggests that LapB is involved in stress response. The multicopy small RNA (sRNA) LhrC was recently shown to negatively regulate lapB expression at the posttranscriptional level (46). Given that expression of LapB is highly regulated, it may be possible to identify some enrichment conditions that promote the expression of LapB at a high level for the sensitive detection and isolation of the pathogen by anti-LapB MAbs in an immunomagnetic separation (IMS) procedure. This study demonstrated expression of LapB in L. monocytogenes cultured in a limited number of enrichment media described in one Canadian official method (MFHPB-07). It becomes necessary to determine whether LapB is expressed in other commonly used enrichment broths such as Listeria enrichment broth (LEB—UVM1 formulation) and in modified Fraser broth (MFB) in the other Canadian official method (MFHPB-30) (http://www.hc-sc.gc.ca/fn-an /res-rech/analy-meth/microbio/volume2-eng.php) or half-Fraser broth in the ISO method (47). As well, practical use of anti-LapB MAbs in the isolation and detection of L. monocytogenes remains to be demonstrated with food matrices. ACKNOWLEDGMENTS We thank K. Amoako and J. Guan for providing constructive comments and suggestions for the manuscript. We acknowledge the assistance from the Monoclonal Antibody Unit (K. Arnold, J. Widdison) at the Canadian Food Inspection Agency (CFIA) in the production of monoclonal antibodies. This work was supported by the funding of the CFIA Research Partnership Strategies program (M. Lin, H. Huang, B. W. Brooks). T. Boivin was supported in part by the Walkerton Clean Water Centre (WCWC) scholarship.

FUNDING INFORMATION This work, including the efforts of Min Lin, was funded by Canadian Food Inspection Agency (CFIA) (OLF (1011) 01).

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Evaluation of monoclonal antibodies to Listeria monocytogenes flagella by checkerboard ELISA and cluster analysis.

Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen.

The gene coding for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria monocytogenes.

Inhibition of different serotypes of Listeria monocytogenes by enterocins in solid and liquid media.

Purification of a surface-specific soluble antigen from Listeria monocytogenes.

Development and characterization of a monoclonal antibody specific for Listeria monocytogenes and Listeria innocua.

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools.

[Infections by Listeria monocytogenes].

Expression of chitinases in Listeria monocytogenes is influenced by lmo0327 that encodes an internalin-like protein.

Serotypes and Pulsotypes diversity of Listeria monocytogenes in a beef-processing environment.

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri.

Fournier's gangrene caused by Listeria monocytogenes as the primary organism.

Colovesical fistula presenting as Listeria monocytogenes bacteraemia.

Infection of the brainstem by Listeria monocytogenes.

Surface Listeria monocytogenes carbohydrate-binding components revealed by agglutination with neoglycoproteins.

Biofilm-Forming Abilities of Listeria monocytogenes Serotypes Isolated from Different Sources.

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Characterization of a Listeria monocytogenes-specific protein capable of inducing delayed hypersensitivity in Listeria-immune mice.

Measurement of antibody-dependent infection enhancement of four dengue virus serotypes by monoclonal and polyclonal antibodies.

A Case of Leukocytoclastic Vasculitis Caused by Listeria monocytogenes Bacteremia.

Evaluation of HER2 Protein Expression Using 2 New Monoclonal Antibodies.

Phenotypic expression of genetically controlled host resistance to Listeria monocytogenes.

Heterologous expression of thuricin CD immunity genes in Listeria monocytogenes.

Complete Genome Sequences of vB_LmoS_188 and vB_LmoS_293, Two Bacteriophages with Specificity for Listeria monocytogenes Strains of Serotypes 4b and 4e.