Design of a novel affinity probe using the cell wall-binding domain of a Listeria monocytogenes autolysin for pathogen detection

ABSTRACT Rapid identification of foods and food-processing environments contaminated with Listeria monocytogenes is crucial for controlling human listeriosis, a leading cause of fatality related to foodborne illnesses. The L. monocytogenes autolysin IspC anchors to the bacterial surface non-covalently via its C-terminal cell wall-binding domain (CWBD), which contains conserved Gly-Trp (GW) dipeptides. This study explored the binding property of CWBDIspC to design an affinity probe for detecting L. monocytogenes. Immunogold electron microscopy analysis of a ∆ispC mutant and its wild type (WT) revealed that exogenously added CWBDIspC bound to surface ligands that were evenly distributed and remained mostly unoccupied in the wild type. CWBDIspC detected, via recognizing the cell wall component lipoteichoic acid, L. monocytogenes strains primarily belonging to the serotype 4 group, as well as Listeria innocua, and Listeria welshimeri but did not detect three other Listeria spp. (Listeria seeligeri, Listeria ivanovii, and Listeria grayi) nor any non-Listeria spp. tested. CWBDIspC, when tagged with the enhanced green fluorescent protein, was capable of detecting L. monocytogenes by fluorescence microscopy, a colony lift filter assay, and a microtube binding assay. CWBDIspC, when covalently attached to magnetic beads, showed the ability to separate L. monocytogenes cells with a maximum capture rate of 90 to nearly 100% from 1 × 103 to 1 × 105 target cells. This study demonstrated for the first time that the CWBD derived from a GW surface protein IspC is a novel antibody alternative for the rapid detection and isolation of L. monocytogenes despite binding to two other Listeria species. IMPORTANCE Human listeriosis is caused by consuming foods contaminated with the bacterial pathogen Listeria monocytogenes, leading to the development of a severe and life-threatening foodborne illness. Detection of L. monocytogenes present in food and food processing environments is crucial for preventing Listeria infection. The L. monocytogenes peptidoglycan hydrolase IspC anchors non-covalently to the bacterial surface through its C-terminal cell wall-binding domain (CWBD), CWBDIspC. This study explored the surface binding property of CWBDIspC to design, construct, characterize, and assess an affinity molecular probe for detecting L. monocytogenes. CWBDIspC recognized a cell wall ligand lipoteichoic acid that remains evenly displayed and mostly unoccupied on the bacterial surface for interaction with the exogenously added CWBDIspC. CWBDIspC, when fused to the enhanced green fluorescent protein reporter or covalently conjugated onto magnetic beads, exhibited the functionality as an antibody alternative for rapid detection and efficient separation of the pathogen.

L isteria monocytogenes, a widespread Gram-positive bacterium in natural environ ments, can cause a rare but serious foodborne illness referred to as listeriosis in humans, particularly susceptible individuals including pregnant women, newborns, older adults, and immunocompromised people, through consuming contaminated foods (1).Strains of this pathogenic microorganism are genetically diverse and are serologically differentiated into at least 13 serotypes and phylogenetically grouped into four distinct lineages (I, II, III, and IV) (2).Serotypes 1/2a, 1/2b, and 4b represent at least 95% of L. monocytogenes strains responsible for human cases of listeriosis (3).Rapid identification of food sources and food processing environments contaminated with L. monocytogenes is crucial for controlling the spread of the pathogen but remains challenging due to a number of interfering factors including the low level of the pathogen present, the complex food matrices, background microflora, the high genetic diversity of L. monocy togenes strains, and lack of suitable affinity probes for highly efficient detection of a wide spectrum of L. monocytogenes serotypes.In the past, studies have focused on the development and use of monoclonal antibodies (MAbs) to L. monocytogenes antigens for pathogen detection and isolation with some promising results (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).Some significant drawbacks of MAbs for the detection of L. monocytogenes are the use of animals in antibody production and the tedious, time-consuming process of MAb screening and identification despite the mature hybridoma technology used (11,13,(15)(16)(17).Specific molecular probes with high affinity for the cell surface ligands common to a wide range of L. monocytogenes strains are always desirable and sought as new alternatives to antibodies for the use in the development of rapid detection methods for L. mono cytogenes present in food, environmental, or clinical samples.One of the mechanisms employed by Gram-positive bacteria to anchor proteins to the cell surface is through the non-covalent interaction of cell wall-binding domains (CWBDs) with the cell wall components (18)(19)(20)(21).This study aims to design and evaluate a CWBD-based affinity probe for such intended applications.
In previous studies, we identified and characterized an immunogenic surface protein IspC as one of the targets of humoral immune response to L. monocytogenes infection (22).IspC is a surface peptidoglycan hydrolase (autolysin) composed of an N-terminal N-acetylglucosaminidase activity domain (aa ) and a C-terminal CWBD (aa 198-774) here referred to as CWBD IspC (23,24).It also plays a role in L. monocytogenes virulence (25).Discovery of the CWBD IspC involved in anchoring the IspC protein to the cell surface through non-covalent interaction with the cell wall (23) has prompted us herein to explore this binding property to design novel strategies for the detection and isolation of L. monocytogenes.In L. monocytogenes, the internalin B (InlB) protein was first demonstrated to contain a C-terminal CWBD InlB (aa 399-630) composed of 3 Gly-Trp (GW) modules (motifs) each having a ~80 aa sequence and containing a GW dipeptide (19,26,27).This type of GW-containing CWBD, interacting with the cell wall component lipoteichoic acid (LTA), functions as one of the non-covalent cell surface anchoring mechanisms in Gram-positive bacteria (18,19,27).However, contrasting results from a recent study show that galactosylated WTA is required and sufficient for the display of InlB on the cell surface of L. monocytogenes serotype 4b (28).The polyribitol phos phate-based wall teichoic acid (WTA) and the polyglycerol phosphate-based LTA are two different structural classes of surface exposed glycopolymers in the Listeria cell wall (28).WTA is covalently linked to the peptidoglycan, whereas LTA is anchored to the cytoplasmic membrane via a lipid moiety.IspC, together with other proteins such as InlB, Ami, and Auto, belongs to a class of cell wall-anchored, GW module-containing surface proteins in L. monocytogenes (18,19,21,23).A CWBD composed of a higher number of GW modules renders a stronger attachment of the protein to the cell wall, as illustrated by the eight GW module-containing Ami in comparison to the three GW module-con taining InlB (19,26).Staphylococcus species are another Gram-positive bacteria that use a similar cell wall-anchoring mechanism for the surface display of GW module-containing proteins (18)(19)(20) including several surface autolysins like AtlC from Staphylococcus caprae (29), AtlE from Staphylococcus epidermidis (30), and Aas from Staphylococcus saprophyti cus (31).
In some Gram-positive bacteria, including Streptococcus pneumoniae and Clostridium acctobutylicum, non-covalent anchoring of surface proteins is accomplished by the binding of the C-terminal domains to choline residues in the WTA or LTA (19,20).The CWBD of a S. pneumoniae autolytic amidase LytA is a C-terminal domain, composed of serval imperfect repeats of ~20 aa, which specifically recognizes choline residues (32).PspA, an important virulence factor and vaccine candidate anchored to the cell surface in S. pneumoniae, possessing a C-terminal proline-rich CWBD made up of 10 highly conserved 20-aa repeats (20), is another well-characterized choline-binding protein.C-terminal CWBDs are also present in several L. monocytogenes bacteriophage peptidoglycan hydrolases (endolysins) (33,34), thus bringing their N-terminal enzymatic domains close to the substrates by binding to the cell wall ligands.In contrast to the CWBDs of L. monocytogenes GW surface proteins such as IspC, InlB, Ami, and Auto, the C-terminal CWBDs of Listeria phage endolysins contain no GW modules or other sequence repeats, bind the cell wall devoid of LTA or proteins, and exhibit the specificity with a high binding affinity for the cell wall carbohydrates (33).Uncovering a variety of CWBDs in the surface proteins of Gram-positive pathogenic bacteria has left the door wide open to the possibility of developing CWBD-based innovative tools for rapid pathogen detection.The CWBDs derived from L. monocytogenes phage endolysins have found applications in the detection and isolation of the pathogen from food culture enrichment samples using magnetic separation (35)(36)(37).To date, no study has been conducted to explore the use of CWBDs derived from a class of L. monocytogenes GW surface proteins in bacterial detection and isolation.The C-terminal CWBD present in the IspC protein of L. monocytogenes strain LI0521 (serotype 4b) contains seven GW modules (23) and is hypothesized to confer strong non-covalent attachment to the cell surface, thereby serving as a new alternative to MAbs for the pathogen detection.The present investigation focused on the design, characterization, and evaluation of a CWBD IspC -based affinity probe, either fused with the enhanced green fluorescent protein (EGFP) reporter or covalently conjugated onto magnetic beads (MB), for the rapid detection of L. monocytogenes by using fluorescence microscopy (FM), microtube binding assay, or colony lift filter assay and efficient isolation of pathogenic bacteria from culture enrichment samples by using magnetic separation.Here, we demonstrate that CWBD IspC , the first example of a CWBD from a class of GW surface proteins, is suitable for the detection and isolation of important L. monocytogenes serotypes such as 4b with high efficiency despite research efforts still being needed to identify GW module-containing CWBDs that specifically recognize all serotypes of L. monocytogenes.

Culture of bacteria
Listeria monocytogenes strains and non-L.monocytogenes bacterial species (Table 1) were cultured in brain heart infusion (BHI) broth with gentle shaking or agar medium (Becton Dickinson, Sparks, MD, USA) at 37°C for 16-18 h for use in the study.L. monocytogenes strain LI0521 (serotype 4b), from which the CWBD IspC coding sequence was derived, was used as a control strain for the binding of CWBD IspC to bacterial cells.E. coli strains, DH5α and BL21(DE3)/pLysS used for molecular cloning and protein expression experiments, respectively, were grown in Luria-Bertani (LB) medium.

Generation of expression constructs
All DNA manipulations were essentially performed according to the established procedures (38).The genomic DNA of L. monocytogenes serotype 4b strain LI0521 was prepared from a 10 mL overnight BHI culture by using DNAZol (Invitrogen, Burlington, ON, Canada) as per the manufacturer's instructions (39).All of the oligonucleotide a Underlined sequences are additional nucleotides included at the 5′ end of each primer to meet the requirements for cloning into the pLIC-NHis vector; bold sequences added at the 5′ end are for generating the sequences coding for the fusion proteins EGFP-CWBD IspC or EGFP-CWBD InlB by using overlap extension PCR.
in a construct pCWBD IspC for the production of the recombinant CWBD IspC with an Nterminal 6× His tag.
To create an expression construct for CWBD IspC tagged with EGFP at the N-terminus, the coding sequence for the EGFP-CWBD IspC fusion was synthesized using a two-step PCR strategy (23).Briefly, the EGFP coding sequence (omitting the stop codon) was PCR amplified from the plasmid pEGFP-N1 (Clontech Laboratories, Santa Clara, CA, USA) using Platinum Pfx DNA polymerase and the primers P1086 and P1089, and the CWBD IspC coding sequence derived from the L. monocytogenes LI0521 genomic DNA by PCR using the primers P1088 and P1087.Then, the two PCR products were spliced by overlap extension PCR with the primers P1086 and P1087.As described above, the fused DNA fragment was cloned into pLIC-NHis to produce the recombinant plasmid pEGFP-CWBD IspC .For the production of the EGFP-CWBD InlB fusion protein, a similar PCR cloning strategy was employed to create the recombinant plasmid pEGFP-CWBD InlB using the primers P1086 and P1203 for the EGFP coding sequence, P1202 and P1201 for the coding sequence of CWBD InlB , the CWBD of InlB (aa399-630), and P1086 and P1201 for the EGFP-CWBD InlB fusion protein coding sequence.
All of the recombinant plasmids were propagated in E. coli DH5α and verified to contain the correct inserts by colony PCR as described previously (22) with a genespecific primer (Table 2) and the T7 terminator primer (5′-GCTAGTTATTGCTCAGCGG -3′) followed by Sanger DNA sequencing at the sequencing facility, McGill University (Montreal, QC, Canada).

Expression and purification of recombinant proteins
E. coli BL21(DE3)/pLysS harboring a recombinant plasmid (pCWBD IspC or pEGFP-CWBD IspC ) was used to express the encoded recombinant protein in LB broth culture as described (41).After growth to an OD at 600 nm of about 0.6 in 2-4 L of LB broth containing 50 µg/mL kanamycin, E. coli cells were induced by adding 1 mM isopropyl-βd-thiogalactopyranoside (final concentration) to express the target protein at 37°C for 3 h and then at room temperature (RT) for 1 h followed by overnight incubation at 4°C.The cells were harvested by centrifugation at 16,900 × g at 4°C for 20 min and frozen at −20°C until use.For protein purification, the cell pellets were resuspended in phosphate-buffered saline (PBS, pH 7.2) containing 1 mM phenylmethylsulfonyl fluoride and lysed by passing through a French press twice at 1,500 lb/in 2 .The cell homogenates were spun at 27,000 × g for 25 min at 4°C to collect the supernatant containing the soluble recombinant protein, which was purified by affinity liquid chromatography on a column (1 by 1.5 cm) of Ni-nitrilotriacetic acid superflow (Qiagen, Santa Clarita, CA.USA) as described (41).The concentration of purified proteins was determined by using the Bradford method (42) with bovine serum albumin (BSA) as a standard.

Immunogold labeling for electron microscopy
L. monocytogenes serotype 4b strain LI0521 and its ∆ispC mutant created in a previous study (25) were used to investigate the attachment of the recombinant CWBD ispC using the immunogold electron microscopy method described previously (11), together with a MAb M2773 specific for the CWBD of IspC (13,43).

Fluorescence microscopy
The binding of the EGFP-CWBD ispC fusion protein to the cell surface of L. monocyto genes or other bacterial cells was examined by fluorescence microscopy on an Olympus BX60 fluorescence microscope essentially as described (15).Briefly, approximately 2 × 10 8 bacterial cells from an overnight BHI culture were harvested by centrifugation, washed twice with PBS, and then stained with 800 nM EGFP-CWBD ispC in PBS containing 0.1% (vol/vol) Tween-20 (polyethylene glycol sorbitan monolaurate) (PBS-T) at room temperature for 5 min.Cells were collected by centrifugation at 13,200 × g for 60 s, washed twice with 500 µL of PBS-T, and resuspended in 50 µL of PBS-T.Ten microliter of cell suspension was placed on a glass slide and viewed at 100× magnification on the microscope with an excitation 490 nm/emission 510 nm filter set.Fluorescence images of bacterial cells complexed with EGFP-CWBD ispC were captured with a charge-coupled device camera.

Microtube binding assay with EGFP-CWBD IspC
The microtube binding assay was performed by collecting approximately 2 × 10 8 cells of L. monocytogenes or other bacterial cells from an overnight BHI culture into a microtube followed by centrifugation at 13,200 × g for 1 min, washing twice with PBS, and then staining with 800 nM EGFP-CWBD ispC in PBS-T at room temperature for 5 min.Stained cells were washed twice with 500 μL of PBS-T, pelleted by centrifugation, and then macroscopically imaged under a GFP-MDS-96/BN excitation stand equipped with eight light sources of 96 ultra-bright blue LEDs (BLS Ltd., Budapest, Hungary).The fluorescent images were captured with a Canon digital camera (5D Mark ii) through an FHS/EF-3GY1 filter (BLS Ltd.) attached to the camera lens.

Binding of CWBD ispC to proteinase K-treated L. monocytogenes cells
Cells of L. monocytogenes strain LI0521 were harvested from a 10-mL aliquot of overnight BHI broth culture, resuspended in 100 µL of PBS, and digested with 100 µL of proteinase K (20 mg/mL) at 50°C for 30 min.After the treatments, cells were washed extensively in PBS and stained with 800 nM EGFP-CWBD IspC followed by detection with the microtube binding assay described above.

Colonies of L. monocytogenes cells grown on BHI agar medium or BBL CHROMagar
Listeria medium (BD Biosciences, Mississauga, ON, Canada) were lifted onto an Optitran nitrocellulose membrane (Schleicher & Schuell, NH, USA).The membrane was air dried, incubated with 5% (wt/vol) BSA in PBS for 60 min at RT, rinsed twice with PBS-T, and then probed with 800 nM EGFP-CWBD IspC in PBS-T for 5 min.After washing twice with PBS-T, the membrane was imaged under a GFP-MDS-96/BN excitation stand (BLS Ltd.) using a Canon digital camera (5D Mark ii) through an FHS/EF-3GY1GFP filter (BLS Ltd.).

Competitive binding analysis
The CWBD IspC protein was analyzed for the ability to compete with CWBD InlB for binding to the cell wall LTA in L. monocytogenes strain LI0521 by forming a complex of bacterial cells with CWBD IspC followed by fluorescent staining with the EGFP-CWBD InlB fusion protein.Briefly, about 2 × 10 8 formalin-killed cells were incubated with CWBD IspC (0-5.2 µM) in PBS at RT for 1 h with gentle rotation, washed twice with PBS-T, stained with 3.2 µM EGFP-CWBD InlB in PBS for 1 h, and again washed twice with PBS-T.Fluores cence signals emitted from the surface of stained cells were detected by fluorescence microscopy as above.Alternatively, macroscopic imaging for the stained cell pellets in a microtube was performed under a GFP-MDS-96/BN excitation stand (BLS) as described above.

Conjugation of the CWBD IspC protein to magnetic beads
Covalent immobilization of the recombinant CWBD IspC to M-270 Epoxy Dynabeads (Dynal, Oslo, Norway) was performed as per the manufacturer's instructions.Lyophilized beads were suspended in diethyleneglycol-dimethyl ether to a final concentration of 30 mg/mL.A 270 µL aliquot of bead suspension was washed twice each with 800 µL of PBS and then resuspended in 100 µL of PBS.The CWBD IspC protein of various quantities (0, 5, 20, 25, 50, 150, 200, 250, 300, 400, and 500 µg) each in 100 µL of PBS was mixed with 100 µL of bead suspension in PBS, and 100 µL of 3 M (NH 4 ) 2 SO 4 (pH 7.4).The mixtures were incubated in a rotating mixer at 10 rpm for 16 h at 4°C, and then for 6 h at ambient temperature (22°C).Residual epoxy groups on beads were blocked by washing the beads four times with PBS containing 0.1% (wt/vol) BSA.CWBD-conjugated magnetic beads, at a concentration of ~2.0 × 10 9 beads/mL in PBS containing 0.1% (wt/vol) BSA and 0.02% NaN 3 , were stored at 4°C until use.

Capture of L. monocytogenes with CWBD-conjugated magnetic beads
The ability of CWBD IspC to capture L. monocytogenes cells from BHI broth culture samples was assessed with CWBD IspC -conjugated magnetic beads in three independent experiments.CWBD IspC -coated beads (4.0 × 10 7 beads in 20 µL) were mixed with 1 mL of bacterial cells (10 3 , 10 4 , or 10 5 cells/ml) prepared by diluting an overnight BHI culture in PBS.The mixtures were rotated at 10 rpm for 45 min at 4°C.After magnetic separation, beads were washed with 800 µL of PBST for 15 min at room temperature, resuspended in 450 µL of PBST, and vortexed vigorously for 1 min.Three 100 µL aliquots from each bead captured cell suspension were plated, respectively, on BHI agar media, incubated at 37°C for 20-24 h, and examined for the number of colony-forming units (CFUs).The rate of bacterial recovery was estimated by calculating the ratio of the input cell number to the number of CFUs based on the assumption that each CFU on the BHI agar plate is derived from a free single bacterial cell in the captured cell suspension.For comparison, a Listeria Capture Kit for Plate (Hyglos GmbH, Bernried, Germany) was used in parallel to capture L. monocytogenes cells according to the manufacturer's instructions.

Even distribution of the surface ligand recognized by CWBD IspC
Immunogold labeling with a CWBD IspC -specific MAb M2773 revealed that IspC expressed in L. monocytogenes was targeted to the cell surface through CWBD IspC interacting with the cell wall ligand (Fig. 1a).IspC localized only to certain regions of the cell surface, primarily the polar regions.Probing L. monocytogenes cells with CWBD IspC followed by immunogold labeling with M2773 showed that the exogenously added CWBD IspC decorated the cell surface evenly (Fig. 1b), indicating that only a small number of the ligands formed a complex with IspC expressed in the bacterial cells and the majority of ligands were freely available for interaction with CWBD IspC .As expected, immunogold labeling of the ispC deletion (∆ispC) mutant with M2773 showed no gold particles on the cell surface (Fig. 1c) but displayed an even distribution of gold particles on the surface of the mutant cells pre-probed with CWBD IspC (Fig. 1d).The results of the immunogold labeling experiments demonstrate that ligands serving as the binding sites for CWBD IspC are evenly distributed on the cell surface.

Non-protein nature of the cell surface ligand involved in binding CWBD IspC
To understand the molecular nature of the surface ligand non-covalently bound with CWBD IspC , live L. monocytogenes that had been treated with proteinase K to digest surface proteins were probed with EGFP-CWBD IspC .Fluorescence microscopy showed that protease-treated cells did not appear to alter the binding of the CWBD IspC protein to the cell surface, compared to the untreated control (data not shown).This result indicated that the cell surface component functioning as the binding ligand of CWBD IspC was of a non-protein nature.

Inhibition of CWBD InlB binding to the cell wall by CWBD IspC
The InlB protein is known to be anchored to the cell surface via the non-covalent interaction of CWBD InlB , the C-terminal region (aa 399-630) composed of three GW modules, with the cell wall component LTA (26,27).To demonstrate that CWBD IspC recognizes the same cell wall component, the CWBD IspC protein at various concentra tions was tested for the ability to block the binding of the EGFP-tagged CWBD InlB to L. monocytogenes.Fluorescence microscopy revealed that live L. monocytogenes that had been pre-decorated with CWBD IspC showed a reduction in fluorescence signals to almost none as the concentration of exogenously added CWBD IspC was increased, compared to the untreated cells (Fig. 2A).Consistent results were also obtained with the microtube binding assay (Fig. 2B), showing that decoration of live L. monocytogenes by EGFP-CWBD InlB was blocked near completely by pre-treatment of bacterial cells with increasing concentrations of CWBD IspC.The EGFP alone showed no effect on the cell

Rapid detection of L. monocytogenes using CWBD IspC -based binding assays
The application of CWBD IspC in the detection of L. monocytogenes was demonstrated in two binding assay formats.The EGFP-CWBD IspC fusion protein was able to detect the target cells from a broth culture in a microfuge tube binding assay (Fig. 4).Alternatively, colonies of L. monocytogenes cells grown on an agar medium were detected by EGFP-CWBD IspC after blotting onto a nitrocellulose membrane in a colony lift filter assay (Fig. 3C).

Efficient L. monocytogenes capturing using CWBD IspC -based magnetic separation
Preparations of CWBD IspC conjugated to the surface of M-270 Epoxy Dynabeads at various amounts of the protein to a fixed amount of beads were assessed for their capability to capture L. monocytogenes strain LI0521 (serotype 4b) from three input quantities of target cells (1 × 10 3 , 1 × 10 4 , and 1 × 10 5 cells).In all cases, the beads without CWBD IspC coupling (control) showed approximately 0% cell recovery, while an increase in the amount of the protein used for conjugation with a fixed quantity of beads led to an increase in the capture efficiency with a maximum of 90 to near 100% at a ratio of 200-300 µg CWBD IspC to 8 mg beads (Fig. 5).Continuing to increase the amount of the CWBD IspC protein used in the conjugation after reaching a maximum of capture efficiency showed no improvement in the bacterial capture.At the optimum ratios (wt/wt) of the CWBD IspC protein to magnetic beads, CWBD IspC resulted in a higher capture efficiency than a commercial Listeria capture kit (Fig. 5).It is worth noting that CWBD IspC beads can capture at least two out of six L. monocytogenes LI0521 cells in 1 mL PBS (M.Lin and H. Dan, unpublished data).

DISCUSSION
This investigation derived the GW module-containing CWBD IspC from the L. monocyto genes surface autolysin IspC and demonstrated the suitability of this domain as a new alternative to antibodies for rapid detection of pathogenic bacteria.CWBD IspC , via the non-covalent association with the cell wall non-protein component LTA, is responsible for anchoring IspC to the cell surface.As demonstrated here, the construction of an EGFP-tagged CWBD IspC fusion protein allowed direct detection of the target pathogen in various assay configurations such as a fluorescence microscopy-based assay, a microtube binding assay, or a colony lift filter assay.It is reasonably foreseeable that CWBD IspC could be applied in substitution for antibody in the development of other assays for pathogen detection, including flow cytometry assay and immunochromatography assay.These CWBD IspC -based detection methods are efficient and fast compared to conven tional antibody-based immunological assays for pathogen detection because they do not require secondary antibodies and thus require fewer steps than the immunological assays.CWBD IspC , when coated onto magnetic beads for bacterial separation, showed a striking ability to capture L. monocytogenes cells from culture samples (strain LI0521 serotype 4b) with a recovery efficiency of up to near 100% (Fig. 5).In addition, recombi nant production of the EGFP-CWBD IspC chimera or the CWBD IspC in vitro using E. coli cells for the diagnostic applications is important and significant because this approach does not involve the use of live animals for antibody development and permits scaling up of production of antibody alternatives.Anchoring of the endogenously expressed IspC to the cell surface via the non-cova lent association of CWBD IspC with its binding ligand (23) necessarily occupies the CWBD IspC binding sites and may decrease the effectiveness of detecting or capturing L. monocytogenes cells using exogenously added recombinant CWBD IspC .It is crucial to assess the availability of the CWBD IspC -binding sites on the cell surface of live L. monocytogenes to explore the practical application of CWBD IspC in pathogen detection.Examination of the wild-type and the ∆ispC mutant cells of L. monocytogenes by electron microscopy with the CWBD IspC protein and an MAb specific for CWBD IspC showed an even distribution of the CWBD IspC binding sites on the entire cell surface, while decoration of the binding sites by the IspC expressed in L. monocytogenes primarily clustered within certain surface regions such as the poles and septal regions.This observation implies that the majority of binding ligands are freely available on the surface to serve as targets for detecting L. monocytogenes with exogenously expressed CWBD IspC .The use of the CWBDs derived from bacteriophage-encoded peptidogly can hydrolases (endolysins) to capture and separate L. monocytogenes from selective enrichment cultures of artificially contaminated food samples was successfully demon strated in previous studies (35)(36)(37).Instead of binding WTA or LTA, the CWBDs of bacteriophage endolysins specifically recognize the cell wall carbohydrate component peptidoglycan in L. monocytogenes, and WTA spatially limits access of phage endolysins to the peptidoglycan component (33,44).The CWBD IspC , similar to the CWBD InlB in GW module organization, was shown to recognize the membrane-anchored cell wall ligand LTA as it was capable of inhibiting the LTA-binding CWBD derived from InlB from binding to the cell surface of L. monocytogenes.Thus, CWBD IspC , representing a structural class of CWBD different from the CWBDs of phage endolysins involved in the mechanism of anchoring surface protein, is being explored for the detection of L. monocytogenes in the present investigation.Given that teichoic acids are the most abundant cell wall component of Listeria existing as two different structural classes of glycopolymers WTA and LTA (28), specific targeting of LTA by a CWBD such as the CWBD IspC of L. monocy togenes is expected to have a diagnostic value when CWBD IspC is incorporated into laboratory tests for the pathogen detection and separation.
Tagging the reporter EGFP to CWBD IspC allowed for the visualization of surface decoration of L. monocytogenes with CWBD IspC by fluorescence microscopy, as similarly reported for the CWBDs of some bacteriophage endolysins (33).The CWBD of a Staphylococcus aureus autolysin when fused with the green fluorescent protein (GFP) was capable of decorating S. aureus cells visualized by fluorescence microscopy (45).These observations of the cell surface decoration with an EGFP-or GFP-CWBD fusion protein have created motivation for the development of GFP fusion protein-based assays for the rapid detection of L. monocytogenes, as successfully illustrated here with a microtube binding assay and a colony lift filter assay.These assay strategies may be extended to design assays for the rapid detection of any Gram-positive bacterial pathogens using the CWBDs derived from non-covalent cell wall-associated surface proteins encoded by bacterial genomes.
CWBD IspC did not display specificity for any of the non-Listeria bacterial species tested, regardless of whether they were Gram-positive or Gram-negative.This observa tion is consistent with the findings that the CWBDs from L. monocytogenes bacterio phage endolysins generally did not bind to Gram-positive or -negative bacteria other than Listeria cells (33).One exception to this specificity was that the CWBD of a L. monocytogenes bacteriophage endolysin recognized Bacillus megaterium (33).In contrast to the CWBD IspC , the CWBD derived from a S. aureus autolysin, which is composed of three repeated sequences (150 aa each), binds to a wide range of Gram-positive bacteria but not to most Gram-negative bacteria (45).These findings underscore the importance of understanding the bacterial cell-binding specificity of a given CWBD in order to explore its diagnostic value in pathogen detection.The CWBD IspC recognized three out of the six Listeria spp.tested (L.monocytogenes, L. innocua, and L. welshimeri), suggesting these three species display a structurally similar binding site within the membrane-bound cell wall component LTA on the surface.The CWBD IspC was specific for strains of L. monocytogenes, largely restricted to the serotype 4 group, suggesting that a structural variation in LTA exists among L. monocytogenes strains.Strains of L. monocy togenes, L. innocua, and L. welshimeri reactive with CWBD IspC were found to encode protein homologs having near or greater than 90% sequence identity with IspC (43).This suggests that the IspC homolog encoded by the genome of a Listeria strain allows to make a prediction about the reactivity of this strain with CWBD IspC .Listeria strains are classified into various serotypes based on the serological reaction of O-antigenic factors with rabbit antisera (46,47).Interestingly, all CWBD IspC -reactive non-4b strains of L. monocytogenes or other Listeria spp.such as L. innocua and L. welshimeri contain the O-antigenic factor VII. Lack of the VII factor in L. seeligeri, L. ivanovii, and L. grayi serotypes appears to correlate with the observation that CWBD IspC did not react with these Listeria spp.In spite of the specificity and limitation of CWBD IspC , this domain is still a useful affinity probe for the detection of L. monocytogenes because the detection of other Listeria spp., especially a certain level of L. innocua, is a good indicator of possible L. monocytogenes contamination or poor sanitation in food processing environments (48)(49)(50).Nonetheless, CWBD IspC serves as a promising model of GW module-containing CWBDs being explored as a new antibody alternative for the rapid detection of L. monocytogenes.It is worth making continuous efforts to screen other non-covalent cell wall-anchored proteins encoded by the genome of L. monocytogenes to possibly discover CWBDs specifically recognizing L. monocytogenes, given that a number of surface proteins containing GW modules or LysM domains are identified in L. monocyto genes (18,51).
MAbs coated onto magnetic beads via covalent linkages were demonstrated to be successful in capturing and separating L. monocytogenes from pure cultures or selective enrichment cultures of artificially contaminated foods; however, low rates of bacterial capture were generally obtained (5,52).This study showed CWBD IspC as a new antibody alternative for capturing L. monocytogenes from pure culture when covalently coupled to magnetic beads.A much higher capture efficiency of 90 to near 100% with CWBD IspC than those (2.0-3.7 %) with MAbs (52) showed a considerable strength of CWBDs for L. monocytogenes capture and separation.Similarly, the CWBDs of L. monocytogenes bacteriophage endolysins and of a S. aureus autolysin were demonstrated to be very efficient in the magnetic bead-based separation of target pathogenic bacteria (35)(36)(37)45).Several factors including high binding affinity and high binding site density may have strengthened the ability of CWBD IspC to overcome the limitation of conventional MAbs to achieve an excellent efficiency in the separation and detection of L. monocyto genes.The CWBD IspC presumably has a high affinity for the cell surface ligand LTA due to its seven GW modules (23), since the L. monocytogenes autolysin Ami containing eight GW modules exhibited a much stronger cell surface attachment in comparison to the three GW module-containing InlB (19,26,27,53).The CWBDs of L. monocytogenes bacteriophage endolysins were shown to have high affinity with equilibrium dissociation constant K D values in the nanomolar range (1/3 × 10 −8 -1/6 × 10 −8 M) (33), while the CWBD of a S. aureus autolysin bound to the cells with a K D of 1.5 × 10 −8 M (45).Electron microscopy semi-quantitatively revealed a high density of binding sites for CWBD IspC , which is consistent with the observation that a L. monocytogenes cell displays 4 × 10 4 -8 × 10 4 CWBD-binding sites on the cell surface for bacteriophage endolysins (33).Another element that may contribute to the effectiveness of using CWBD IspC as an antibody alternative is that its binding ligand LTA is a major non-protein constituent of the cell wall, in contrast to the surface protein antigens recognized by anti-L.monocytogenes MAbs, which may be expressed at a low level or down-regulated under certain environ mental conditions.To realize the full potential of a CWBD IspC -based detection method, it is necessary to assess its application to the detection of target L. monocytogenes serotypes in naturally or artificially contaminated food samples.
In conclusion, this study demonstrated the development of CWBD IspC , a GW module-containing CWBD derived from a L. monocytogenes peptidoglycan hydrolase IspC, as a novel alternative to traditional MAbs for the pathogen detection and isolation.The CWBD IspC exhibited a much higher efficiency in bacterial detection and separation in comparison to MAbs, presumably due to the high affinity of CWBD IspC for bacterial cell recognition, the high density of binding sites on the cell surface, and the specificity for a major non-protein cell wall component LTA.Some limitations are associated with the use of CWBD IspC for L. monocytogenes detection as this CWBD recognizes two other Listeria species, L. innocua and L. welshimeri, besides L. monocytogenes and binds L. monocytogenes strains primarily belonging to the serotype 4 group.Because of these limitations, future research efforts need to be focused on the discovery of novel L. monocytogenes-specific CWBDs for diagnostic applications through bioinformat ics analysis of the genome sequences of various L. monocytogenes strains available in the public databases.In this way, more GW surface proteins and other non-covalent surface-anchored proteins can be identified for intended use.

a
NA, not available.b Bacterial strains selected for the study are those of the 13 recognized L. monocytogenes serotypes, other Listeria spp., foodborne bacterial pathogens, Gram-positive and -negative bacteria frequently found in foods and the environment.These strains were collected by our laboratory over the years.c Positive results (+) indicate fluorescence emitted from bacterial cells that have been complexed with EGFP-CWBD IspC ; negative results (−) indicate no or negligible fluorescence signal emitted from bacterial cells following the reaction with EGFP-CWBD IspC .Negative results (−) were also observed with purified EGFP.Inconclusive results are indicated by (±).The results shown here are reproducible with three biological replicates.

FIG 1 9 FIG 2
FIG 1 Immunogold labeling of CWBD IspC -decorated L. monocytogenes cells for scanning electron microscopy.The WT or ΔispC mutant cells of L. monocytogenes strain LI0521 serotype 4b were decorated with the recombinant CWBD IspC at 800 nM and then immunogold labeled (black dots in the cell images) with a MAb M2773 against CWBD IspC .Bacterial cells without CWBD IspC decoration were used as the control for comparison.(a) WT cells probed with M2773; (b) WT cells probed with CWBD IspC followed by M2773; (c) mutant cells probed with M2773; and (d) mutant cells probed with CWBD IspC followed by M2773.The figure depicts one representative of at least two independent experiments that yield reproducible results.

FIG 3
FIG 3 Binding of EGFP-CWBD IspC to the surface of L. monocytogenes LI0521 serotype 4b.(A) A fluorescence microscopy image shows EGFP-CWBD IspC binds to live L. monocytogenes cells.(B) A phase-contrast image of bacterial cells in the same field as in panel (A).(C) Colony lift filter assay shows a fluorescent image of EGFP-CWBD IspC binding to L. monocytogenes colonies.(D) A microtube binding assay shows fluorescence emitted from EGFP-CWBD IspC binding to live L. monocytogenes cells (tube 1) and to live E. coil O157:H7 cells (tube 2).The figure exemplifies one representative of at least two independent experiments that yield reproducible results.

FIG 5
FIG 5 Efficiency of capturing L. monocytogenes strain LI0521 serotype 4b by CWBD IspC conjugated to MB. CWBD-coupled magnetic beads were prepared by using various quantities of CWBD (0-500 µg) and a fixed amount of M-270 Epoxy Dynabeads (8.0 mg).Each conjugate preparation (20 µL) was evaluated for the efficacy to capture bacterial cells from 1 mL of samples, prepared from the dilution of a BHI culture in PBS to contain 1 × 10 3 (A), 1 × 10 4 (B), and 1 × 10 5 (C) cells.The data represented by each bar are the mean ± SD calculated from three experimental replicates with three technical replicates.The data obtained in parallel with a commercial Listeria capture Kit are shown for comparison (Hyglos Kit).Beads without CWBD IspC conjugation serve as a control for bacterial capture.

TABLE 1
Examination of the EGFP-CWBD IspC fusion protein binding to the cell surface of various bacterial species by FM

TABLE 1
Examination of the EGFP-CWBD IspC fusion protein binding to the cell surface of various bacterial species by FM (Continued)

TABLE 2
Oligonucleotide primers used in the study