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LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria, promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species

Balamurugan Jagadeesan, Ok Kyung Koo, Kwang-Pyo Kim, Kristin M. Burkholder, Krishna K. Mishra, Amornrat Aroonnual, Arun K. Bhunia

  • Molecular Food Microbiology Laboratory, Department of Food Science, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN 47907, USA
  • Correspondence
    Arun K. Bhunia
    Bhunia{at}purdue.edu

    • Microbiology 2010; 156(9):2782–2795 · https://doi.org/10.1099/mic.0.036509-0

    View at publisher PubMed

    Abstract

    Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (lmo1634), interacts with host-cell receptor Hsp60 to promote bacterial adhesion during the intestinal phase of Listeria monocytogenes infection. The LAP homologue is present in pathogens (L. monocytogenes, L. ivanovii) and non-pathogens (L. innocua, L. welshimeri, L. seeligeri); however, its role in non-pathogens is unknown. Sequence analysis revealed 98 % amino acid similarity in LAP from all Listeria species. The N-terminus contains acetaldehyde dehydrogenase (ALDH) and the C-terminus an alcohol dehydrogenase (ADH). Recombinant LAP from L. monocytogenes, L. ivanovii, L. innocua and L. welshimeri exhibited ALDH and ADH activities, and displayed strong binding affinity (KD 2–31 nM) towards Hsp60. Flow cytometry, ELISA and immunoelectron microscopy revealed more surface-associated LAP in pathogens than non-pathogens. Pathogens exhibited significantly higher adhesion (P<0.05) to Caco-2 cells than non-pathogens; however, pretreatment of bacteria with Hsp60 caused 47–92 % reduction in adhesion only in pathogens. These data suggest that biochemical properties of LAP from pathogenic Listeria are similar to those of the protein from non-pathogens in many respects, such as substrate specificity, immunogenicity, and binding affinity to Hsp60. However, protein fractionation analysis of extracts from pathogenic and non-pathogenic Listeria species revealed that LAP was greatly reduced in intracellular and cell-surface protein fractions, and undetectable in the extracellular milieu of non-pathogens even though the lap transcript levels were similar for both. Furthermore, a LAP preparation from L. monocytogenes restored adhesion in a lap mutant (KB208) of L. monocytogenes but not in L. innocua, indicating possible lack of surface reassociation of LAP molecules in this bacterium. Taken together, these data suggest that LAP expression level, cell-surface localization, secretion and reassociation are responsible for LAP-mediated pathogenicity and possibly evolved to adapt to a parasitic life cycle in the host.

    • ↵†Present address: Neogen Corporation, Lansing, MI 48912, USA.

    • ↵‡Present address: Department of Food Science, School of Agriculture and Life Sciences, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.

    • Supplementary material is available with the online version of this paper.

    Edited by: H. Ingmer

    INTRODUCTION

    The genus Listeria comprises six species, of which L. monocytogenes and L. ivanovii are pathogenic and L. seeligeri, L. welshimeri, L. innocua and L. grayi are considered non-pathogenic (Vazquez-Boland et al., 2001). Although rare, L. monocytogenes infection can cause fatal disease in immunocompromised hosts. Infection occurs predominantly through consumption of contaminated food or through feto-placental transmission in pregnant women. L. monocytogenes carries a unique set of virulence genes clustered in a 9.0 kb pathogenicity island consisting of prfA, plc, hlyA, mpl and actA (Freitag et al., 2009; Vazquez-Boland et al., 2001). Several additional pathogenicity genes are located outside the pathogenicity island, encoding proteins including members of the internalin multigene family (InlA, InlB, InlJ, etc.), Vip, bile-salt hydrolase, autolysin amidase (Ami), fibronectin-binding protein (Fbp), lipoteichoic acid and lipoprotein promoting entry protein (LpeA) (Bierne & Cossart, 2007; Sabet et al., 2008). Although cellular mechanisms of infection are becoming more clear (Hamon et al., 2006), we still have much to learn about the intestinal (Gahan & Hill, 2005; Pentecost et al., 2006), utero-placental (Bakardjiev et al., 2006; Lecuit et al., 2004) and neurological phases of infection (Drevets & Bronze, 2008).

    Our interest is in understanding the intestinal phase of listeriosis, particularly the role of Listeria adhesion protein (LAP) during infection. Previous research has demonstrated that LAP interacts with the cellular receptor Hsp60 (Wampler et al., 2004), and exhibits preferential affinity towards cells of intestinal origin (Jaradat et al., 2003; Kim et al., 2006; Pandiripally et al., 1999). We have recently shown that LAP secretion is essential for L. monocytogenes pathogenesis and that its secretion is SecA2-dependent (Burkholder et al., 2009). The L. monocytogenes lap (lmo1634) gene was identified as encoding an alcohol acetaldehyde dehydrogenase, a housekeeping enzyme (Kim, 2004). Participation of metabolic or housekeeping enzymes in pathogenic functions is not unprecedented (Pancholi & Chhatwal, 2003). In group III streptococci (Seifert et al., 2003) and Candida albicans (Gil-Navarro et al., 1997), glyceraldehyde-3-phosphate dehydrogenase is expressed on the cell surface and serves as a virulence factor. In Entamoeba histolytica, alcohol acetaldehyde dehydrogenase is an adhesion factor that is present primarily in pathogenic strains, but is absent or present in trace amounts in six non-pathogenic strains (Torian et al., 1990). Metabolic enzymes such as PavA (Holmes et al., 2001) and α-enolase (Bergmann et al., 2001) in Streptococcus pneumoniae are often referred to as ‘anchorless adhesins’ (Chhatwal, 2002) and promote adhesion to host cells. These adhesion molecules do not contain any cell-surface anchoring motif and probably reassociate on the bacterial surface after being secreted (Pancholi & Chhatwal, 2003). In Mycobacterium tuberculosis, the reassociation of the housekeeping enzyme malate synthase on the surface allows bacteria to adhere to host laminin and fibronectin during infection (Kinhikar et al., 2006). Similarly, secreted LAP has been shown to reassociate on the surface of L. monocytogenes cells prior to binding to mammalian cells (Burkholder et al., 2009). Although housekeeping enzymes of many pathogens act as virulence factors, often by secretion and reassociation on their surfaces, the role of these virulence factors in non-pathogenic species of the same genera is unknown.

    Our earlier work (Pandiripally et al., 1999) revealed that LAP is present in all Listeria species (except L. grayi), as demonstrated by Western blotting when probed with an anti-LAP monoclonal antibody; however, variation in LAP from different species could not be assessed by this antibody since it reacts with a specific epitope that may be common to LAPs from different Listeria species. Since a LAP homologue is present in both pathogenic and non-pathogenic Listeria, we hypothesized that LAP serves as a general adhesion/colonization factor irrespective of the pathogenic status of the organism and may have evolved in pathogens to perform pathogenesis-related functions. Therefore, we investigated the molecular and phenotypic properties of LAP from different Listeria species and determined their role in adhesion to purified Hsp60 and to enterocyte-like Caco-2 cells. In this paper we show that low LAP-mediated adhesion of non-pathogens is due to inadequate secretion and surface association of the protein.

    METHODS

    Bacterial strains, plasmids, primers, mammalian cells and antibodies.

    Bacterial cultures, plasmids and primers used in this study are listed in Table 1⇓. Bacterial cultures were cultured in Brain Heart Infusion broth (BHI, Difco) at 37 °C for 18–20 h with appropriate antibiotics.

    Table 1.

    Bacterial strains, plasmids and primers

    The enterocyte-like colon carcinoma cell line Caco-2 (HTB37: ATCC, Manassas, VA, USA) was maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma), containing 10 % fetal bovine serum (FBS, Gibco). Cells were used at passages of 30–35 and grown (at 37 °C in the presence of 7 % CO2) in 24-well plates for 10–14 days, at which point the monolayers were formed and differentiated.

    Anti-LAP antibody mAb-H7 (Pandiripally et al., 1999), anti-66 kDa antibody mAb-EM-7G1 (Bhunia & Johnson, 1992) and anti-InlA pAb (5 μg ml−1), developed in our laboratory against recombinant InlA (Schubert et al., 2002), were used in these experiments.

    Analysis of sequence variation in lap by Southern hybridization.

    Genomic DNA was prepared (Sambrook et al., 1989) and purified using the Qiagen Gel Extraction kit. Genomic DNAs from Listeria species were digested with EcoRI, separated in agarose gel (1 %) and transferred to a nitrocellulose membrane using the Turboblotter system (S&S BioScience). An amplified PCR product of 999 bp, internal to the lap sequence (657–1657 bp from the start codon) of L. monocytogenes F4244 (accession no. AY561824) was used as a probe. The DIG labelling and chemiluminescent detection system from Roche was used as directed by the manufacturer.

    Sequence analysis of the lap gene from Listeria species.

    The genome sequence of L. monocytogenes EGD (Glaser et al., 2001) was used to generate primers for amplification of the entire 2.7 kb region containing complete lap (lmo1634; 2601 bp) or partial lap (999 bp internal sequence of lap) from different Listeria species. Initially the primer set P-LAP-F and P-LAP-R was employed to determine the presence of lap homologues in all Listeria species (Table 1⇑). Purified template DNA (100 ng), 25 pmol of each primer, 1 μl Taq polymerase (0.5 U μl−1; Applied Biosystems) and 10 mM dNTPs were mixed to a 50 μl final volume. The PCR amplification conditions were: hot start at 95 °C for 5 min followed by 30 cycles each consisting of denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1.5 min, and final extension at 72 °C for 10 min. Amplified DNA was resolved in 1 % agarose gel and visualized by ethidium bromide staining.

    For sequencing purposes, two primer sets were designed: E-LAP-F and E-LAP-R were used for amplification of the entire lap gene from L. monocytogenes F4244, L. innocua F4248, L. welshimeri ATCC 35897 and L. seeligeri SE31; and E-LAP-F and E-LAP-R2 were used to amplify lap from L. ivanovii SE98 (Table 1⇑). The PCR conditions were the same as above except with a 3 min extension time. PCR products were resolved in 1 % agarose gel and eluted with the Qiagen Gel Extraction kit. The TA-cloning vector pGEMT-Easy (Promega) was used for cloning the PCR products and the resulting plasmids were designated pLAP-mon (L. monocytogenes), pLAP-inn (L. innocua), pLAP-wel (L. welshimeri), pLAP-see (L. seeligeri) and pLAP-iva (L. ivanovii). The plasmids were purified using the Qiagen plasmid purification kit and the presence of lap was confirmed by restriction enzyme digestion (data not shown). Sequencing of each lap gene was carried out at Purdue University Genomics Center and sequences were submitted to GenBank. LAP sequences were aligned using megalign (DNASTAR) to determine alignment score and Prosite (ExPASY, Swiss Institute of Bioinformatics) was used for domain prediction.

    RNA isolation, cDNA synthesis and RT-PCR.

    Bacterial cultures (5 ml each) were grown in BHI broth to OD600 0.4. Cells were harvested, and total RNA was isolated using the RNeasy mini kit (Qiagen), treated with RNase-free DNase I and further purified using the RNeasy mini column (Qiagen). cDNA was synthesized using the iScript Reverse Transcriptase kit (Bio-Rad) and 2 μl of cDNA was used for PCR. Briefly, 20 μl reactions contained 1.0 μg total RNA, 4 μl 5× iScript reaction mix and 1 μl reverse transcriptase. Reactions were incubated sequentially for 5 min at 25 °C, 30 min at 42 °C and 85 °C for 5 min. A three-stage thermal profile was used to amplify products: (i) denaturation (94 °C for 5 min), (ii) PCR amplification (29 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 40 s, extension at 72 °C for 1 min kb−1), (iii) final extension (72 °C for 7 min).

    Preparation of recombinant LAP from L. ivanovii, L. welshimeri and L. innocua.

    Full-length lap of L. ivanovii, L. welshimeri and L. innocua was amplified by PCR using the primers designed from the respective lap sequence. Forward primer, E-LAPlis-F, was same for all species. Separate reverse primer sets were used for L. ivanovii (E-LAPiva-R), L. welshimeri (E-LAPwel-R) and L. innocua (E-LAPinn-R) (Table 1⇑). Amplified products were cloned into pGEMT- easy vector (Promega) and designated pGEMT-LAP-iva, pGEMT-LAP-wel and pGEMT-LAP-inn, respectively. Products were subcloned into pET28a using BamHI and SacI restriction sites introduced into the forward and reverse primers, resulting in pELAP-iva, pELAP-wel and pELAP-inn (Kim et al., 2006). Finally, all three constructs were transformed into Escherichia coli BL21(DE3) (Novagen) and overexpression of LAP was achieved by adding 1 mM IPTG during exponential growth. Recombinant (r) LAPiva (L. ivanovii), LAPwel (L. welshimeri) and LAPinn (L. innocua) proteins were purified on nickel affinity columns (EMD Chemicals) and examined by Western blotting (Kim et al., 2006).

    Expression of lap of L. monocytogenes in L. innocua F4248.

    Plasmid pMGS-101 carrying the lap gene of L. monocytogenes (pCLAP-67) was transferred into electrocompetent L. innocua F4248. The positive transformants were selected by plating on BHI supplemented with chloramphenicol (7 μg ml−1). A stable transformant, L. innocua AKB-204 (LinnLAPLm), was further characterized for LAP expression by Western blotting and adhesion to Caco-2 cells.

    Analysis of the surface expression of LAP on Listeria cells

    Flow cytometry.

    Aliquots of washed bacterial suspensions (∼1×108 cells ml−1) were incubated with anti-LAP mAb-H7 (4 μg ml−1) for 1 h at 37 °C. Cells were washed three times in 20 mM phosphate-buffered saline, pH 7.0 (PBS), and incubated with FITC-conjugated secondary antibody (4 μg ml−1) (Jackson ImmunoResearch) for 1 h at 37 °C, followed by three final PBS washes. Samples were analysed on a Cytomics FC 500 (Beckman-Coulter). For each cell type, a preset fixed forward scatter and side scatter gating and fixed amplification were used. Cells were analysed for LAP expression after the addition of propidium iodide (PI) for detection of dead or lysed cells, with a gating for PI exclusion in FL-4 (675 nm band path). FITC fluorescence was detected in FL-1 (525 nm band path).

    Indirect ELISA.

    Washed Listeria cells were suspended in 0.1 M sodium carbonate buffer (pH 9.6), immobilized in wells of 96-well microtitre plates (Dynex) at ∼108 cells per well, and incubated overnight at 4 °C. Plates were washed four times with PBS-Tween 20 (0.5 %) and blocked with 2 % BSA (Sigma). Purified Hsp60 (1 μg ml−1: Assay Design) was then added (100 μl per well) and incubated at 37 °C for 1 h. Binding of Hsp60 to LAP was detected by adding anti-Hsp60 PAb (1.4 μg ml−1) (Assay Design), goat anti-rabbit peroxidase-conjugated secondary antibody (0.2 μg ml−1) (Jackson ImmunoResearch Laboratories) and ο-phenylenediamine with H2O2 substrate (Bhunia et al., 1991).

    Western blot analysis to determine LAP expression in Listeria cell fractions.

    Listeria cells were harvested from 200 ml cultures, and secreted, cell-wall-associated and intracellular proteins were extracted (Jonquieres et al., 1999). Absence of intracellular or membrane protein contamination with cell-surface protein fraction was verified by PepC assay (Schaumburg et al., 2004). Protein concentration was estimated by the BCA method (Pierce). Proteins were separated by SDS-PAGE (4–15 % acrylamide gradient gel; Bio-Rad), transferred to Immobilon-P membrane (Millipore), and probed with anti-LAP antibody (10 μg ml−1) or anti-InlA pAb (5 μg ml−1). A chemiluminescence substrate (Pierce) was used to develop the membranes. The level of secreted, cell-wall-associated and intracellular LAP in L. monocytogenes and L. innocua was compared by measuring band intensity using Quantity One software (Bio-Rad). To ensure uniform loading of protein preparations, the exact quantity of proteins used in Western blots was resolved similarly by SDS-PAGE (4–15 % acrylamide) and stained with Coommassie blue.

    Transmission electron microscopy.

    Transmission electron microscopy was performed to compare the distribution patterns and localization of LAP in L. monocytogenes and L. innocua cells and immunoprobed with anti-LAP mAb-H7 (0.1 mg ml−1) and goat anti-mouse IgG conjugated to 12 nm gold particles (1 : 100) (Jackson ImmunoResearch) as described previously (Jaradat et al., 2003).

    Adhesion of Listeria to Caco-2 cells.

    This assay was performed as described before (Kim et al., 2006; Wampler et al., 2004). Briefly, fresh Listeria cells were harvested, washed in PBS and used at an m.o.i. of 10 : 1 (bacteria : Caco-2 cells) for 1 h. Caco-2 monolayers were treated with Triton X-100 (0.1 %), serially diluted, and bacterial adhesion was enumerated by plating (Kim et al., 2006; Pandiripally et al., 1999). To determine the effect of Hsp60 on Listeria adhesion to Caco-2 cells, bacterial cells were reacted with Hsp60 (1 μg per ml PBS: Assay Design) for 30 min at room temperature with agitation, and excess Hsp60 was removed by washing. As a control, Listeria cells treated with Hsp70 (1 μg ml−1: Assay Design) were used.

    To determine if secreted LAP is able to reassociate on the surface of L. innocua cells, we exposed this bacterium to a LAP preparation from L. monocytogenes and examined adhesion to Caco-2 cells as described before (Burkholder et al., 2009). Briefly, LAP was prepared from stationary-phase culture supernatant of L. monocytogenes F4244 and filtered (0.22 μm). Freshly harvested and washed L. innocua cells were incubated with 1 ml L. monocytogenes LAP preparation at room temperature for 2 h. L. innocua cells were harvested and washed, and adhesion to Caco-2 cells was examined. L. monocytogenes lap mutant KB208 was used as a control.

    Binding kinetics of purified rLAPiva, rLAPwel and rLAPinn to Hsp60.

    Binding of recombinant LAPs from L. ivanovii, L. welshimeri and L. innocua to purified human receptor Hsp60 was analysed in a surface plasmon resonance (SPR) biosensor (IAsys sensor; Affinity Sensors) as described previously (Kim et al., 2006). Briefly, 14.5 μg purified human Hsp60 (Assay Design) was covalently coupled to the N-hydroxysuccinimide (NHS)/N-ethyl-N-(diethylaminopropyl)carobidiimide (EDC)-activated carboxylate surface of the cuvette (Lathrop et al., 2003). The association and dissociation phases of rLAP binding to Hsp60 were determined (Kim et al., 2006). Monoclonal anti-Hsp60 antibody (Assay Design), rLAPmono and recombinant InlB were used as controls in binding experiments (Kim et al., 2006). Observed rate constant values (kon) were plotted against their respective ligand (rLAP) concentrations. The slope of the line represents the association rate constant ka (M−1 s−1) and the intercept of the line represents the dissociation rate constant kd (s−1). The dissociation equilibrium constant KD (M), a measure of the binding strength, was calculated using the formula KD=kd/ka.

    Binding of rLAP from different Listeria species to Hsp60 was also investigated by ELISA. Purified rLAP (100 μl of 25 μg ml−1) was diluted in carbonate coating buffer (pH 9.6) and immobilized in the wells of 96-well microtitre plates. Binding of Hsp60 was monitored as described above, using phosphatase-conjugated secondary antibody and 4-methylumbelliferyl phosphate (4-MUP: Sigma) as substrate. Fluorescence intensity was measured using a spectrofluorometer (Spectramax) at excitation wavelength 360 nm and emission wavelength 440 nm.

    Analysis of enzyme activity of rLAP from different Listeria species.

    Recombinant E. coli BL21(DE3) strains harbouring pELAP-mon, pELAP-iva, pELAP-wel and pELAP-inn were grown in LB broth at 37 °C in a shaker incubator to mid-exponential phase (OD600 0.5), and IPTG (1 mM) was added to induce overexpression of LAP. Cell pellets were harvested (9820 g for 10 min at 4 °C), and were washed once with 0.1 vol. 50 mM potassium phosphate buffer, pH 7.4 (PPB) and centrifuged (3900 g for 5 min at 4 °C). The pellets were resuspended in PPB, lysed using a sonicator (Branson Sonifier 150D), centrifuged (63 222 g for 1 h at 4 °C), and supernatants were freeze-dried for future use. Activity of alcohol dehydrogenase (ADH) was determined with reaction mixtures containing 80 mM Tris/HCl buffer (pH 8.8), 2 mM NAD+, 0.8 mM ethanol and 0.95 mg crude enzyme (Blandino et al., 1997). For acetaldehyde dehydrogenase (ALDH) activity, reaction mixtures contained 80 mM Tris/HCl buffer (pH 8.0), 2 mM NAD+, 0.089 mM acetaldehyde, 0.1 mM CoASH, 0.27 mM dithiothreitol and 0.95 mg crude enzyme preparation (Gupta et al., 2000). NADH production was determined spectrophotometrically at 340 nm for 15 min at 37 °C. One unit was defined as the amount of enzyme required to catalyse the transformation of 1 μmol NADH min−1 (ϵ=6.22×103 M−1 cm−1) (Jelski et al., 2006; Waseem et al., 2006).

    Statistical analysis.

    Data were analysed using the generalized linear model (GLM) procedure of SAS (SAS Institute) and significant differences among different treatments were determined using least significant difference (lsd) or Tukey's test at P≤0.05.

    RESULTS

    lap is present in all Listeria species

    PCR analysis targeting a 999 bp internal sequence of the 2.6 kb lap gene indicated that lap is present in all Listeria species except L. grayi (Fig. 1a⇓), which is in agreement with our earlier report (Pandiripally et al., 1999). To detect potential genetic variability of the lap locus in different species of Listeria, Southern hybridization was performed using EcoRI-digested genomic DNA and the 999 bp probe obtained from the internal sequence of lap from L. monocytogenes. Presence of a 1.3 kb band in L. monocytogenes, L. seeligeri and L. ivanovii confirmed two EcoRI sites in the ORF of these species (Fig. 1b⇓). The 2 kb band in L. monocytogenes indicates the presence of an additional EcoRI site upstream of the start codon in the lap locus, which is unique for the L monocytogenes sequence. The data confirmed the presence of lap in all Listeria species (except L. grayi) but indicated some sequence heterogeneity. Similar heterogeneity has been reported for another well-known Listeria cell wall hydrolase protein, p60, encoded by iap, which is present in all Listeria species except for L. grayi, where it was expressed only under restricted growth conditions (Bubert et al., 1992; Pilgrim et al., 2003).

    Figure image not available in archive
    Fig. 1.

    (a) PCR amplification of lap in different Listeria species using a primer pair designed to amplify partial lap (999 bp). (b) Southern hybridization of genomic DNA from different Listeria species with the 999 bp lap probe. EcoRI-digested genomic DNA was hybridized with the DIG-labelled partial lap gene (999 bp) probe from L. monocytogenes F4244. (c) Amino acid sequence comparison of LAP from different Listeria species. Acetaldehyde dehydrogenase (ALDH) is located at the N-terminus (amino acids 1–452) and alcohol dehydrogenase (ADH) at the C-terminus (amino acids 458–866). The putative NAD+-binding domain is located at amino acids 427–432 and the Fe2+-binding domain is located at amino acids 724–742. Dark shaded boxes indicate the conserved domains of ADH, ALDH, and the NAD+- and Fe2+-binding sites. Vertical lines represent non-identical amino acid sequences.

    Sequence analysis of the lap gene from Listeria species

    Nucleotide and amino acid sequences for lap were determined. Their GenBank accession numbers are AY561824 (L. monocytogenes F4244), AY561825 (L. innocua F4248), AY561826 (L. ivanovii SE98), AY561827 (L. seeligeri SE31) and AY561828 (L. welshimeri ATCC 35897). LAP from all these Listeria spp. is 866 amino acids in length. The LAP sequence alignment score between L. monocytogenes F4244 and L. innocua F4248 was 99. An identical sequence score was obtained when published sequences from L. monocytogenes EGD (Glaser et al., 2001) and L. innocua CLIP 11262 (Glaser et al., 2001) were used for the calculation. The alignment score for the remaining Listeria species was 97–98.

    Domain prediction analysis of LAP indicated the presence of an ALDH at the N-terminus (amino acids 1–452) and an ADH at the C-terminus (amino acids 458–866), similar to alcohol acetaldehyde dehydrogenases of other bacteria (Koo et al., 2005) (Fig. 1c⇑). A putative NAD+-binding domain is located at position Gly427–Gly432, and an Fe2+-binding domain is located at position Gly724–Gly742 (Membrillo-Hernandez et al., 2000). The ALDH (Glu111–Asn141 and Lys247–Cys255), ADH (Ile639–Glu666), and NAD+- and Fe2+-binding domains are conserved in all the LAPs (Fig. 1c⇑).

    Pathogenic Listeria species exhibit greater adhesion to Caco-2 cells than non-pathogenic species

    We next examined whether LAPs from different species are involved in adhesion to Caco-2 cells. Three strains representing each species were tested. Strains of L. monocytogenes and L. ivanovii exhibited significantly greater adhesion (P<0.05) to Caco-2 cells compared to the non-pathogenic Listeria species (Fig. 2a⇓). In our previous study, mammalian cell-associated Hsp60 was shown to be a receptor for LAP (Wampler et al., 2004). In the present study, we employed a competitive assay, where adhesion was examined in bacteria pretreated with purified Hsp60. Blocking the bacterial surface with Hsp60 resulted in significantly (P<0.05) reduced adhesion for L. monocytogenes F4244 (wild-type) (68 % reduction), lap-complemented CKB208 (92 % reduction), and L. ivanovii (47 % reduction) but had little or no effect on the L. monocytogenes lap mutant KB208 and other Listeria species (Fig. 2b⇓). Hsp60 also caused about 50 % reduction in adhesion of inlA or inlAB mutant strains to Caco-2 cells (see Supplementary Fig. S1, available with the online version of this paper). Pretreatment of Caco-2 cells with Hsp70 did not affect adhesion of L. monocytogenes to Caco-2 cells (data not shown). These data indicate that pathogenic Listeria (L. monocytogenes and L. ivanovii) show greater LAP-mediated binding than non-pathogenic Listeria to Caco-2 cells. Adhesion was reduced when bacteria were pretreated with Hsp60, indicating that prior occupation of LAP on the surface of these pathogens by exogenous Hsp60 hindered bacterial interaction with target cells; however, exogenous Hsp60 had no effect on adhesion of non-pathogens.

    Figure image not available in archive
    Fig. 2.

    (a) Adhesion of Listeria to Caco-2 cells. Three strains from each Listeria species were examined. Means±sd are plotted. Different letters (a, b, c) indicate significant differences among strains (P<0.05). (b) Effect of exogenous Hsp60 on Listeria adhesion to Caco-2 cells. Bacterial cells were treated with purified Hsp60 at 1 μg ml−1 before addition to Caco-2 cells at a ratio of 10 : 1 (bacteria : Caco-2 cells). Means±sd are plotted. * Significant (P<0.05) difference in adhesion between Hsp60-treated and untreated Listeria. Lm, L. monocytogenes; KB208, L. monocytogenes lap-deficient strain; CKB208, lap-complemented KB208 strain; Liv, L. ivanovii; Linn, L. innocua; Lwel, L. welshimeri, Lg, L. grayi; Lsee, L. seeligeri.

    Two possibilities exist to explain the lack of LAP-mediated adhesion for non-pathogenic Listeria: (i) LAPs of non-pathogenic Listeria species may be structurally different from the LAPs of pathogenic species, rendering them unable to interact with the Hsp60 or (ii) inadequate expression, secretion and surface reassociation of LAP molecules in bacterial cells may limit interaction with Caco-2 cells. To investigate the first possibility, we purified recombinant LAP of L. monocytogenes (rLAPmon), L. innocua (rLAPinn), L. welshimeri (rLAPwel) and L. ivanovii (rLAPiva) from recombinant E. coli cultures (Fig. 3⇓) and determined their binding affinity to purified human Hsp60 (see below).

    Figure image not available in archive
    Fig. 3.

    Western blot showing reaction of anti-LAP antibody with purified recombinant LAP from L. monocytogenes (rLAPmon), L. innocua (rLAPinn), L. welshimeri (rLAPwel) and L. ivanovii (rLAPiva). Protein preparation from parental E. coli/pET-28a without the lap insert did not show any reaction with antibody.

    SPR and ELISA indicate strong affinity of purified LAP from both pathogenic and non-pathogenic Listeria species to the Hsp60 receptor

    In the SPR sensor, binding of purified LAP from two representative pathogenic (L. monocytogenes and L. ivanovii) and two non-pathogenic (L. innocua and L. welshimeri) species to Hsp60 showed a concentration-dependent increase for all LAPs tested. The ka values of rLAP of L. ivanovii, L. welshimeri and L. innocua were calculated to be 3.12×107 M−1 s−1, 9.5×109 M−1 s−1 and 8.79×107 M−1 s−1, respectively (Table 2⇓); these values were equivalent to those for rLAP of L. monocytogenes (Kim et al., 2006). The KD values for rLAP of L. ivanovii and L. innocua were 1.97×10−9 M and 3.12×10−8 M, respectively. The KD value for rLAP of L. welshimeri could not be calculated, as the intercept was negative when kon was plotted against its respective concentration (Kim et al., 2006). The binding kinetics results for a positive control (anti-Hsp60 antibody) and a negative control (rInlB) were similar to the results obtained in the previous study (Kim et al., 2006).

    Table 2.

    Binding kinetics of purified rLAP from different Listeria species to purified Hsp60 as measured in an SPR biosensor

    Binding of purified Hsp60 to immobilized purified recombinant LAP from L. monocytogenes (relative fluorescence units, RFU 0.5±0.08) and L. innocua (RFU 0.56±0.07) was similar, as confirmed by immunoassay. Collectively, these data indicate that purified rLAP from both pathogenic and non-pathogenic Listeria species can interact with Hsp60, thus further substantiating our second speculation that non-pathogenic Listeria species may lack secreted or cell wall-associated LAP.

    Flow cytometry and ELISA analyses indicate reduced surface LAP on whole cells of non-pathogenic Listeria

    Flow cytometry (Fig. 4a⇓) and ELISA (Fig. 4b⇓) indicated that the surface LAP expression and binding of Hsp60 were significantly greater (P<0.05) in pathogens, L. monocytogenes wild-type and CKB208, and L. ivanovii, compared to non-pathogens. These data provide strong evidence for direct involvement of cell-surface-associated LAP only from pathogens in the interaction with Hsp60.

    Figure image not available in archive
    Fig. 4.

    Analysis of surface LAP expression in Listeria spp. Whole bacterial cells were reacted with (a) anti-LAP antibody or (b) Hsp60 to detect LAP expression on the surface of cells by flow cytometry and ELISA, respectively. Means±sd are plotted. Bars labelled with different letters (a, b, c, d) are significantly different at P<0.05.

    Enhanced surface localization of LAP on pathogenic Listeria

    We investigated the level of secreted and cell-wall-associated LAP in the pathogenic species L. monocytogenes and non-pathogenic L. innocua by immunoblotting using anti-LAP antibody (Fig. 5a⇓). Previous studies have shown that secreted proteins play an important role in pathogenesis (Braun et al., 1997; Lenz et al., 2003) and we recently reported that secreted LAP promotes L. monocytogenes adhesion to intestinal cells (Burkholder et al., 2009). Here, we found that secreted LAP is undetectable in L. innocua, while abundant in L. monocytogenes. Furthermore, the amount of cell-wall-associated LAP was about twofold greater in L. monocytogenes than in L. innocua. There was also more intracellular LAP in L. monocytogenes than in L. innocua (Fig. 5a⇓). PepC assay was negative for secreted and cell-wall-associated LAP preparations, indicating the absence of contamination with intracellular LAP (data not shown). InlA was used as a positive control to verify the assay; its presence was observed in the secreted fraction (abundant), cell-wall-associated fraction and intracellular fraction of L. monocytogenes but it was absent in L. innocua protein fractions (Fig. 5a⇓). We further examined if the overall reduced expression of LAP in L. innocua is due to the reduced transcription of lap. However, RT-PCR data revealed no significant differences in the amount of lap mRNA among the three strains of L. monocytogenes and of L. innocua (data not shown), suggesting that a post-transcriptional mechanism may be responsible for lower LAP accumulation in L. innocua.

    Figure image not available in archive
    Fig. 5.

    (a) Western blot of LAP expression in secreted, cell-wall-associated and intracellular fractions of L. monocytogenes F4244 (Lm) and L. innocua F4248 (Lin) cells. Proteins loaded per well in SDS-PAGE: 25 μg for secreted and 2.5 μg each for cell wall-associated and intracellular fractions. Expression of InlA was monitored in each fraction as a control. (b) Transmission immunoelectron microscopy showing localization and distribution patterns of LAP in L. monocytogenes F4244 and L. innocua F4248. Arrows point to surface-associated LAPs. Bars, 0.1 μm.

    Reduced or absent surface association and intracellular LAP in L. innocua compared to L. monocytogenes was further confirmed by transmission electron microscopy (Fig. 5b⇑). Enumeration of anti-LAP antibody-conjugated gold nanoparticles on L. monocytogenes cells (n=141) and L. innocua cells (n=104) indicated that about 15 % of the particles were associated with the cell wall for L. monocytogenes but only 2.4 % for L. innocua. Collectively, these results provide strong evidence that reduced secretion and surface association of LAP in non-pathogenic Listeria probably preclude it from participating in LAP-mediated adhesion to mammalian cells.

    secA2 sequence analysis and secretion of NamA by L. innocua

    We have recently shown that LAP secretion in L. monocytogenes is SecA2-dependent (Burkholder et al., 2009). Since L. innocua had undetectable levels of LAP secretion, we sought to determine whether SecA2 is functional in this species. The secA2 gene from L. innocua (accession no. FJ589744) was cloned and sequenced, and the promoter and ORF were found to be conserved in both species (Supplementary Figs S1 and S2). Amino acid analysis of SecA2 of L. innocua showed 97 % similarity (95 % identity) with SecA2 of L. monocytogenes F4244 (accession no. FJ595243), 10403S (Lenz & Portnoy, 2002), F2365 (Nelson et al., 2004) and EGD (Glaser et al., 2001). Furthermore, Western blot analysis of L. innocua cell wall-associated protein revealed the presence of NamA (Supplementary Fig. S2), a SecA2-dependent protein in the cell wall fraction of L. innocua, suggesting that SecA2 is functional in L. innocua.

    Heterologous expression and binding experiments suggest LAP is unable to reassociate on the surface of L. innocua cells

    We next examined if heterologous expression of LAP of L. monocytogenes in L. innocua could promote LAP-mediated adhesion in L. innocua. In spite of enhanced accumulation of LAP in the cell wall and secreted fractions in recombinant L. innocua (LinnLAPLm) (Fig. 6a⇓), this strain failed to show enhanced adhesion to Caco-2 cells (Fig. 6b⇓), suggesting that secreted LAP may be unable to reassociate on the surface of L. innocua.

    Figure image not available in archive
    Fig. 6.

    (a) Western blot analysis of LAP expression profiles, and (b) adhesion to Caco-2 cells of L. innocua F4248 (wild-type, WT) expressing LAP of L. monocytogenes (LinnLAPLm). Lm, L. monocytogenes F4244; KB208, lap mutant of L. monocytogenes; Linn, L. innocua F4248; LinnLAPLm, L. innocua expressing LAP of L. monocytogenes. Means±sd are plotted in (b).

    Previously, we have shown that reassociation of secreted LAP on the surface of bacterial cells is essential to promote LAP-mediated adhesion in L. monocytogenes (Burkholder et al., 2009). However, in the present study, adhesion of L. innocua to Caco-2 cells was not enhanced by pre-exposure to LAP-containing cell-free supernatant of L. monocytogene, while the lap-mutant KB208 did show enhanced adhesion (Fig. 7⇓). Similarly, adhesion of recombinant L. innocua (LinnLAPLm) exposed to L. monocytogenes LAP preparation was not increased when tested with Caco-2 cells (data not shown). These data, collectively, provide evidence that secreted LAP is unable to reassociate on the surface of L. innocua cells, hence precluding LAP-mediated adhesion in this organism.

    Figure image not available in archive
    Fig. 7.

    Adhesion characteristics of L. innocua (Linn) to Caco-2 cells following exposure to LAP preparation from L. monocytogenes F4244 (Lm). The lap mutant L. monocytogenes KB208 pre-exposed to L. monocytogenes LAP preparation (KB+LmLAP) showed significantly enhanced adhesion while L. innocua pre-exposed to the same preparation (Linn+LmLAP) did not. Values are means±sd of three experiments run in duplicate. Bars labelled with different letters (a, b, c, d) are significantly different at P<0.05.

    Analysis of enzyme activity of LAP from different Listeria species

    Since the SPR-sensor binding studies demonstrated that purified LAP of all the Listeria species interacts with the mammalian cell receptor Hsp60, we examined enzyme activity of all LAPs to determine if the protein could still aid in bacterial growth and physiology. We tested crude rLAP preparations for both ADH and ALDH activities. The ADH activity of rLAP of all Listeria species ranged from 8.65±3.47 to 16.64±2.51 mU (mg protein)−1 and was significantly higher than the ADH activity of the parental E. coli BL21(DE3) strain (P=0.0015). The ALDH activity of the LAP preparations ranged from 165.3±109 to 314.6±30.55 mU mg−1, and these values were higher than the ALDH activity of E. coli BL21(DE3) (Table 3⇓). These data clearly indicate that LAP of both pathogenic and non-pathogenic Listeria species is active and possesses bifunctional ADH and ALDH activities, capable of catalysing both alcohol and acetaldehyde as substrate for energy during growth.

    Table 3.

    ADH and ALDH activity of recombinant LAP from Listeria species

    Means±sd are shown. Values in a column labelled with A or B were significantly different as analysed by Tukey's test at P<0.05.

    DISCUSSION

    Adherence of L. monocytogenes to the host cell surface is an important event during infection, and involves a number of cell surface proteins, including internalins, Ami and FbpA (Bierne & Cossart, 2007). We previously reported the significance of LAP as a pathogenic factor in L. monocytogenes, promoting bacterial adhesion to intestinal epithelial cells by interacting with mammalian receptor Hsp60 (Jaradat et al., 2003; Pandiripally et al., 1999; Wampler et al., 2004). LAP homologues are present in non-pathogenic Listeria species, and thus it was speculated that LAP may serve as a general colonization/adhesion factor in Listeria. To address this hypothesis, we sought to determine the role of LAP as an adhesion/colonization factor in both pathogenic and non-pathogenic Listeria species. The lap genes from all Listeria species showed 98 % similarity in nucleotide sequence (Fig. 1⇑), suggesting that LAP may possess a similar function in all Listeria spp. Previously, LAP from L. monocytogenes was identified as an alcohol acetaldehyde dehydrogenase homologue, whose adhesion property was confirmed in E. coli expressing recombinant LAP (Kim et al., 2006). In Entamoeba histolytica, this enzyme (designated EhADH2; 97 kDa) is a major adhesion factor only in pathogenic strains that interacts with eukaryotic fibronectin (Espinosa et al., 2001; Torian et al., 1990; Yang et al., 1994). A database search further revealed lap to possess 63–75 % sequence similarities with alcohol acetaldehyde dehydrogenases from other bacteria (Staphylococcus aureus, Streptococcus agalactiae, Clostridium acetobutylicum, Pasteurella multocida and E. coli), although the function has not been elucidated in these organisms.

    We examined whether LAP from different Listeria spp. would display adhesion properties similar to L. monocytogenes (Kim et al., 2006). In a competitive adhesion assay, exogenous Hsp60 blocked 47–68 % of adhesion to Caco-2 cells only for the pathogenic species L. monocytogenes and L. ivanovii, while no significant changes were exhibited in the adhesion of the non-pathogenic species L. innocua, L. welshimeri, L. seeligeri and L. grayi (Fig. 2⇑). Furthermore, Hsp60 also caused about 50 % reduction in adhesion in inlAB mutant strains (Supplementary Fig. S1), suggesting that LAP-mediated adhesion occurs independent of internalin, which is consistent with our previous report (Burkholder et al., 2009). When interaction of purified recombinant LAPs from both pathogenic and non-pathogenic Listeria species with human Hsp60 was examined, all showed equivalent binding affinity to Hsp60 (Table 3⇑) and the dissociation constant values (KD) were within the range for biological molecules (Pazos et al., 2004). It is possible that the non-pathogens have a very low level of LAP on their surface, and hence exogenous Hsp60 has no effect on LAP-mediated adhesion in these species, which was demonstrated by a competitive adhesion assay (Fig. 2a⇑). Flow cytometry and ELISA analysis of Listeria cells labelled with either anti-LAP mAb or purified Hsp60 also confirmed that non-pathogenic Listeria cells indeed have significantly lower levels of surface-associated LAP than the pathogenic Listeria (Fig. 4⇑). These findings were further verified here by Western blotting (Fig. 5a⇑) and transmission immunoelectron microscopy (Fig. 5b⇑), and previously on a biochip sensor, where a Hsp60-coated biochip selectively detected only pathogenic Listeria species, with reduced or no interaction with non-pathogenic species (Koo et al., 2009). Although there is no apparent difference in lap transcript levels for L. monocytogenes and L. innocua strains, the observed differences in protein amounts could be due to either the mRNA not being translated into proteins or a post-transcriptional mechanism preventing protein translation (Johansson et al., 2002; Pelech, 2004).

    We recently showed that LAP secretion and its reassociation with the cell wall are essential for adhesion of L. monocytogenes to Caco-2 cells (Burkholder et al., 2009). The adhesion potential of L. monocytogenes AKB-103 (ΔsecA2), which is deficient for LAP secretion, is similar to that of the non-pathogenic L. innocua, which indicates that either low expression of LAP or an inefficient secretory system is responsible for the reduced level of adherence in this non-pathogen. However, cloning and sequence analysis of the secA2 gene and protein secretion analysis for a SecA2-dependent protein, NamA, revealed that a functional secA2 is present in L. innocua (Supplementary Fig. S3), consistent with previous observations made with three different strains of L. monocytogenes 10403S (Lenz et al., 2003), EGD (Machata et al., 2005) and F4244 (Burkholder et al., 2009). These data provide strong evidence that the SecA2 system is functional and imply that possible inadequate secretion of LAP due to a lower level of expression by L. innocua or a lack of reassociation of LAP on the surface of cells is most likely responsible for reduced adhesion to Caco-2 cells.

    Although LAP accumulation in the cell wall and in the supernatant was slightly increased in the recombinant L. innocua strain expressing lap of L. monocytogenes, this strain still did not show enhanced adhesion (Fig. 6⇑), suggesting that the secreted LAP is probably unable to reassociate on the surface of cells. Previously, we demonstrated that the virulence potential of LAP is mediated by the secreted form of the protein and subsequent reassociation on the surface of pathogenic L. monocytogenes (Burkholder et al., 2009). The strongest evidence for such surface reassociation came from an experiment in which LAP-mediated adhesion was restored in the L. monocytogenes lap mutant strain, KB208, following exposure to a LAP preparation from L. monocytogenes wild-type. In contrast, in this study, the LAP (from L. monocytogenes)-mediated adhesion could not be restored in L. innocua, suggesting that this bacterium possibly lacks factor(s) that facilitate reassociation of LAP; this is currently under investigation in our laboratory.

    Surface association is a common phenomenon for many virulence proteins with or without a cell wall anchoring domain that are involved in adhesion or invasion to mammalian cells (Pancholi & Chhatwal, 2003; Scott & Barnett, 2006). For example, InlA with its LPXTG motif forms a cross-link with the sortase substrate, while InlB with its GW (glycine tryptophan) modules interacts with lipoteichoic acid in the cell wall (Bierne & Cossart, 2007). Metabolic or housekeeping enzymes that serve as adhesion/invasion factors are often referred to as ‘anchorless adhesins’ (Pancholi & Chhatwal, 2003; Scott & Barnett, 2006) and the mechanism by which they interact with the cell wall is not well understood. Glyceraldehyde-3-phosphate dehydrogenase, present on the cell surface in group III streptococci (Seifert et al., 2003) and C. albicans (Gil-Navarro et al., 1997) serves as a virulence factor. Similarly, alcohol acetaldehyde dehydrogenase found on the surface of only pathogenic strains of E. histolytica, not in non-pathogenic strains, serves as an adhesion factor (Torian et al., 1990). The secreted glyoxalate enzyme malate synthase in M. tuberculosis reassociates on the surface and allows bacteria to adhere to host laminin and fibronectin (Kinhikar et al., 2006). Similarly, secreted LAP has been shown to reassociate on the surface of L. monocytogenes cells prior to binding to mammalian cells (Burkholder et al., 2009). In this study, our preliminary data provide evidence that secreted LAP is unable to reassociate on the surface of non-pathogenic Listeria species, thus preventing LAP-mediated adhesion to mammalian cells.

    The lack of LAP-mediated virulence in non-pathogens is possibly due to evolutionary adaptation to the environment (Freitag et al., 2009; Jacob & Monod, 1961). Pathogens are required to maintain and exhibit enhanced LAP expression for adhesion to host cells, while non-pathogens are not. Thus they are possibly adapted to maintain LAP in its cellular environment as a minimum so as to perform its metabolic function. As suggested earlier, unlike other virulence factors, expression of metabolic enzyme is generally not under the control of any regulatory mechanism (Pancholi & Chhatwal, 2003). Therefore, we speculate that higher accumulation of LAP in pathogens (or reduced level in non-pathogens) and reassociation on the surface of pathogens is a natural adaptation to dynamically changing environments in a context-dependent manner (Bennett et al., 2008).

    Since LAP is both a housekeeping enzyme and an adhesion factor, we determined whether the protein would still maintain its enzymic activity to participate in metabolic processes. LAPs from all Listeria spp. tested were bifunctional and displayed both ADH and ALDH activities (Table 3⇑). Thus, it is conceivable that enzymic activity does not contribute to the virulence potential of the pathogenic Listeria species. Adhesion of LAP to Hsp60 is a critical attribute in pathogenic Listeria species. It is possible that LAP–Hsp60 interaction may facilitate infection by promoting uptake of bacteria into the intestinal epithelial cells or by promoting unknown downstream signalling events in the eukaryotic cells (Henderson et al., 2006), areas which are now a focus of our intense investigation. The finding that LAP is an active metabolic enzyme with additional adhesion properties adds to the growing list of housekeeping enzymes which serve as pathogenic factors not only in Listeria (Bierne & Cossart, 2007) but also in other micro-organisms (Pancholi & Chhatwal, 2003).

    In summary, we have shown that the lap sequences from both pathogenic and non-pathogenic Listeria species are 98 % similar, yet LAP does not serve as an adhesion factor in non-pathogenic Listeria cells. Interestingly, purified LAP from pathogenic or non-pathogenic Listeria species exhibited strong interaction with the mammalian cell receptor Hsp60. Despite showing strong interaction with Hsp60 in vitro, LAP from non-pathogenic Listeria species does not participate in adhesion to host cells in a cell culture model. Our observations that LAP secretion and reassociation are inadequate on the surface of non-pathogenic Listeria species lead us to hypothesize that pathogens express high levels of surface-associated LAP to sustain a parasitic life cycle in the host.

    Acknowledgments

    Part of this research was supported through a cooperative agreement with the Agricultural Research Service of the US Department of Agriculture, project number 1935-42000-035, the Center for Food Safety Engineering at Purdue University, and Purdue Faculty Scholar Funding. Our sincere thanks to Debby Sherman for assistance with the electron microscopy, Vishu Nanduri for the adhesion experiment, and Professor Michael G. Johnson and Dr Abhijit Mukhopadhyay for critical review of the manuscript.

    References

    1. Autret, N., Raynaud, C., Dubail, I., Berche, P. & Charbit, A. (2003). Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence. Infect Immun 71, 4463–4471.
    2. Bakardjiev, A. I., Theriot, J. A. & Portnoy, D. A. (2006). Listeria monocytogenes traffics from maternal organs to the placenta and back. PLoS Pathog 2, e66.
    3. Bennett, M. R., Pang, W. L., Ostroff, N. A., Baumgartner, B. L., Nayak, S., Tsimring, L. S. & Hasty, J. (2008). Metabolic gene regulation in a dynamically changing environment. Nature 454, 1119–1122.
    4. Bergmann, S., Rohde, M., Chhatwal, G. S. & Hammerschmidt, S. (2001). α-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol Microbiol 40, 1273–1287.
    5. Bhunia, A. K. & Johnson, M. G. (1992). Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen. Appl Environ Microbiol 58, 1924–1929.
    6. Bhunia, A. K., Ball, P. H., Fuad, A. T., Kurz, B. W., Emerson, J. W. & Johnson, M. G. (1991). Development and characterization of a monoclonal antibody specific for Listeria monocytogenes and Listeria innocua. Infect Immun 59, 3176–3184.
    7. Bierne, H. & Cossart, P. (2007). Listeria monocytogenes surface proteins: from genome predictions to function. Microbiol Mol Biol Rev 71, 377–397.
    8. Blandino, A., Caro, I. & Cantero, D. (1997). Comparative study of alcohol dehydrogenase activity in flor yeast extracts. Biotechnol Lett 19, 651–654.
    9. Braun, L., Dramsi, S., Dehoux, P., Bierne, H., Lindahl, G. & Cossart, P. (1997). InIB: an invasion protein of Listeria monocytogenes with a novel type of surface association. Mol Microbiol 25, 285–294.
    10. Bubert, A., Kuhn, M., Goebel, W. & Kohler, S. (1992). Structural and functional properties of the p60 proteins from different Listeria species. J Bacteriol 174, 8166–8171.
    11. Bubert, A., Hein, I., Rauch, M., Lehner, A., Yoon, B., Goebel, W. & Wagner, M. (1999). Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl Environ Microbiol 65, 4688–4692.
    12. Bueno, V. F., Banerjee, P., Banada, P. P., de Mesquita, A. J., Lemes-Marques, E. G. & Bhunia, A. K. (2010). Characterization of Listeria monocytogenes isolates of food and human origins from Brazil using molecular typing procedures and in vitro cell culture assays. Int J Environ Health Res 20, 43–59.
    13. Burkholder, K. M., Kim, K.-P., Mishra, K. K., Medina, S., Hahm, B.-K., Kim, H. & Bhunia, A. K. (2009). Expression of LAP, a SecA2-dependent secretory protein, is induced under anaerobic environment. Microbes Infect 11, 859–867.
    14. Chhatwal, G. S. (2002). Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors. Trends Microbiol 10, 205–208.
    15. Drevets, D. A. & Bronze, M. S. (2008). Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion. FEMS Immunol Med Microbiol 53, 151–165.
    16. Espinosa, A., Yan, L., Zhang, Z., Foster, L., Clark, D., Li, E. & Stanley, S. L., Jr (2001). The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) protein is necessary for amebic growth and survival and requires an intact C-terminal domain for both alcohol dehydrogenase and acetaldehyde dehydrogenase activity. J Biol Chem 276, 20136–20143.
    17. Freitag, N. E., Port, G. C. & Miner, M. D. (2009). Listeria monocytogenes from saprophyte to intracellular pathogen. Nat Rev Microbiol 7, 623–628.
    18. Fujimoto, S. & Ike, Y. (2001). pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis. Appl Environ Microbiol 67, 1262–1267.
    19. Gahan, C. G. & Hill, C. (2005). Gastrointestinal phase of Listeria monocytogenes infection. J Appl Microbiol 98, 1345–1353.
    20. Gaillard, J. L., Berche, P., Frehel, C., Gouin, E. & Cossart, P. (1991). Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell 65, 1127–1141.
    21. Gil-Navarro, I., Gil, M. L., Casanova, M., O'Connor, J. E., Martinez, J. P. & Gozalbo, D. (1997). The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen. J Bacteriol 179, 4992–4999.
    22. Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P. & other authors (2001). Comparative genomics of Listeria species. Science 294, 849–852.
    23. Gupta, S., Mat-Jan, F., Latifi, M. & Clark, D. P. (2000). Acetaldehyde dehydrogenase activity of the AdhE protein of Escherichia coli is inhibited by intermediates in ubiquinone synthesis. FEMS Microbiol Lett 182, 51–55.
    24. Hamon, M., Bierne, H. & Cossart, P. (2006). Listeria monocytogenes: a multifaceted model. Nat Rev Microbiol 4, 423–434.
    25. Henderson, B., Allan, E. & Coates, A. R. (2006). Stress wars: the direct role of host and bacterial molecular chaperones in bacterial infection. Infect Immun 74, 3693–3706.
    26. Holmes, A. R., McNab, R., Millsap, K. W., Rohde, M., Hammerschmidt, S., Mawdsley, J. L. & Jenkinson, H. F. (2001). The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence. Mol Microbiol 41, 1395–1408.
    27. Jacob, F. & Monod, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3, 318–356.
    28. Jaradat, Z. W., Wampler, J. W. & Bhunia, A. W. (2003). A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines. Med Microbiol Immunol 192, 85–91.
    29. Jelski, W., Chrostek, L., Szmitkowski, M. & Markiewicz, W. (2006). The activity of class I, II, III and IV alcohol dehydrogenase isoenzymes and aldehyde dehydrogenase in breast cancer. Clin Exp Med 6, 89–93.
    30. Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M. & Cossart, P. (2002). An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551–561.
    31. Jonquieres, R., Bierne, H., Fiedler, F., Gounon, P. & Cossart, P. (1999). Interaction between the protein InIB of Listeria monocytogenes and lipoteichoic acid: a novel mechanism of protein association at the surface of Gram-positive bacteria. Mol Microbiol 34, 902–914.
    32. Kim, K.-P. (2004). Genetic identification and characterization of Listeria adhesion protein, an alcohol acetaldehyde dehydrogenase homologue in Listeria monocytogenes. PhD thesis, Department of Food Science, Purdue University.
    33. Kim, K. P., Jagadeesan, B., Burkholder, K. M., Jaradat, Z. W., Wampler, J. L., Lathrop, A. A., Morgan, M. T. & Bhunia, A. K. (2006). Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor. FEMS Microbiol Lett 256, 324–332.
    34. Kinhikar, A. G., Vargas, D., Li, H., Mahaffey, S. B., Hinds, L., Belisle, J. T. & Laal, S. (2006). Mycobacterium tuberculosis malate synthase is a laminin-binding adhesin. Mol Microbiol 60, 999–1013.
    35. Koo, O. K., Jeong, D. W., Lee, J. M., Kim, M. J., Lee, J. H., Chang, H. C., Kim, J. H. & Lee, H. J. (2005). Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Leuconostoc mesenteroides isolated from kimchi. Biotechnol Lett 27, 505–510.
    36. Koo, O. K., Liu, Y., Shuaib, S., Bhattacharya, S., Ladisch, M. R., Bashir, R. & Bhunia, A. K. (2009). Targeted capture of pathogenic bacteria using a mammalian cell receptor coupled with dielectrophoresis on a biochip. Anal Chem 81, 3094–3101.
    37. Lathrop, A. A., Jaradat, Z. W., Haley, T. & Bhunia, A. K. (2003). Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor. J Immunol Methods 281, 119–128.
    38. Lecuit, M., Nelson, D. M., Smith, S. D., Khun, H., Huerre, M., Vacher-Lavenu, M. C., Gordon, J. I. & Cossart, P. (2004). Targeting and crossing of the human maternofetal barrier by Listeria monocytogenes: role of internalin interaction with trophoblast E-cadherin. Proc Natl Acad Sci U S A 101, 6152–6157.
    39. Lenz, L. L. & Portnoy, D. A. (2002). Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype. Mol Microbiol 45, 1043–1056.
    40. Lenz, L. L., Mohammadi, S., Geissler, A. & Portnoy, D. A. (2003). SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc Natl Acad Sci U S A 100, 12432–12437.
    41. Machata, S., Hain, T., Rohde, M. & Chakraborty, T. (2005). Simultaneous deficiency of both MurA and p60 proteins generates a rough phenotype in Listeria monocytogenes. J Bacteriol 187, 8385–8394.
    42. Membrillo-Hernandez, J., Echave, P., Cabiscol, E., Tamarit, J., Ros, J. & Lin, E. C. (2000). Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. Genetic and biochemical studies of the mutant proteins. J Biol Chem 275, 33869–33875.
    43. Nelson, K. E., Fouts, D. E., Mongodin, E. F., Ravel, J., DeBoy, R. T., Kolonay, J. F., Rasko, D. A., Angiuoli, S. V., Gill, S. R. & other authors (2004). Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res 32, 2386–2395.
    44. Pancholi, V. & Chhatwal, G. S. (2003). Housekeeping enzymes as virulence factors for pathogens. Int J Med Microbiol 293, 391–401.
    45. Pandiripally, V. K., Westbrook, D. G., Sunki, G. R. & Bhunia, A. K. (1999). Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2. J Med Microbiol 48, 117–124.
    46. Pazos, M. J., Alfonso, A., Vieytes, M. R., Yasumoto, T., Vieites, J. M. & Botana, L. M. (2004). Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions. Anal Biochem 335, 112–118.
    47. Pelech, S. (2004). Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions. Curr Pharm Biotechnol 5, 69–77.
    48. Pentecost, M., Otto, G., Theriot, J. A. & Amieva, M. R. (2006). Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion. PLoS Pathog 2, e3.
    49. Pilgrim, S., Kolb-Maurer, A., Gentschev, I., Goebel, W. & Kuhn, M. (2003). Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility. Infect Immun 71, 3473–3484.
    50. Sabet, C., Toledo-Arana, A., Personnic, N., Lecuit, M., Dubrac, S., Poupel, O., Gouin, E., Nahori, M. A., Cossart, P. & Bierne, H. (2008). The Listeria monocytogenes virulence factor InlJ is specifically expressed in vivo and behaves as an adhesin. Infect Immun 76, 1368–1378.
    51. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    52. Schaumburg, J., Diekmann, O., Hagendorff, P., Bergmann, S., Rohde, M., Hammerschmidt, S., Jansch, L., Wehland, J. & Karst, U. (2004). The cell wall subproteome of Listeria monocytogenes. Proteomics 4, 2991–3006.
    53. Schubert, W. D., Urbanke, C., Ziehm, T., Beier, V., Machner, M. P., Domann, E., Wehland, J., Chakraborty, T. & Heinz, D. W. (2002). Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin. Cell 111, 825–836.
    54. Scott, J. R. & Barnett, T. C. (2006). Surface proteins of gram-positive bacteria and how they get there. Annu Rev Microbiol 60, 397–423.
    55. Seifert, K. N., McArthur, W. P., Bleiweis, A. S. & Brady, L. J. (2003). Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization, enzymatic activity, and protein-protein interactions. Can J Microbiol 49, 350–356.
    56. Torian, B. E., Reed, S. L., Flores, B. M., Creely, C. M., Coward, J. E., Vial, K. & Stamm, W. E. (1990). The 96-kilodalton antigen as an integral membrane protein in pathogenic Entamoeba histolytica: potential differences in pathogenic and nonpathogenic isolates. Infect Immun 58, 753–760.
    57. Vazquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J. & Kreft, J. (2001). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14, 584–640.
    58. Wampler, J. L., Kim, K. P., Jaradat, Z. & Bhunia, A. K. (2004). Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells. Infect Immun 72, 931–936.
    59. Waseem, A., Yaqoob, M. & Nabi, A. (2006). Flow-injection determination of thyroxine using immobilized enzyme with tris (2,2′-bipyridyl)ruthenium(III) chemiluminescence detection. Anal Sci 22, 1095–1098.
    60. Yang, W., Li, E., Kairong, T. & Stanley, S. L., Jr (1994). Entamoeba histolytica has an alcohol dehydrogenase homologous to the multifunctional adhE gene product of Escherichia coli. Mol Biochem Parasitol 64, 253–260.