In vivo and in vitro assessment of the virulence of Listeria monocytogenes strains

To evaluate whether the in vitro model (invasion and intracellular growth in Caco-2 cells) for determining virulence is a suitable alternative to the in vivo model (50% lethal dose), we compared the levels of virulence obtained with the two models. We tested L. monocytogenes strains isolated from food and clinical samples during three episodes of listeriosis occurring in Italy in the period 1993-1995. We also tested L. monocytogenes strains isolated from food during official control activities. The results obtained from the tested strains varied according to the experimental method adopted: the L. monocytogenes strains featuring the same genetic pattern showed a greater uniformity of response in vivo than in vitro. We can conclude that the in vitro model may be used as an alternative to the animal model to determine Listeria spp pathogenicity, though it cannot distinguish levels of virulence within the L. monocytogenes species.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Inhibitory effects of Leuconostoc mesenteroides 1RM3 isolated from narezushi, a fermented fish with rice, on Listeria monocytogenes infection to Caco-2 cells and A/J mice

Listeria monocytogenes causes listeriosis in humans mainly through consumption of ready-to-eat foods. Immunocompromised persons, the elderly, and pregnant women and their fetuses or newborns are at highest risk for the infection. To isolate probiotic lactic acid bacteria (LAB) with inhibitory effects against L. monocytogenes, we screened for acid and bile resistant LABs from narezushi, a traditional salted and long-fermented fish with cooked rice. Then, inhibitory effects of the selected LABs on L. monocytogenes invasion and infection of human enterocyte Caco-2 cells and Listeria-susceptible A/J mice were determined. From a total of 231 LAB isolates, we selected five acid and bile resistant isolates (four were Lactobacillus plantarum and one was Leuconostoc mesenteroides). Among the five isolates, Ln. mesenteroides (Lnm-1RM3) showed the highest inhibition against L. monocytogenes invasion into Caco-2 cells. In the case of L. monocytogenes orally infected A/J mice, recovery of the pathogen from the spleen was suppressed by drinking water containing 9 log CFU/ml of Lnm-1RM3 cells. The inhibitory effects were also shown by heat-killed Lnm-1RM3 cells. These results suggest that live and also heat-killed Lnm-1RM3 cell intake might prevent L. monocytogenes entero-gastric invasion and infection.     

The InlB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells

InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco-2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells. InlB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non-invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non-invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes , uptake of InlB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.     

轮状病毒LH9株在Vero细胞中的培养条件优化研究

为优化轮状病毒株在Vero细胞上的培养条件,将轮状病毒基因重配株LH9按0.1MOI分别接种于不同规格的细胞培养瓶(100ml、2000ml、3L、15L)。病毒接种采用吸附与未吸附两种方式、病毒收获采取低温冻融后离心与直接离心两种方法,观察分析对病毒滴度的影响。实验中,用CASY细胞计数仪分析活细胞率,病毒接种后逐日观察细胞病变(CPE)并取样,采用细胞半数感染量测定病毒滴度。结果表明,使用不同规格细胞培养瓶经吸附法培养接种、低温破碎法收获的LH9株病毒滴度高,其中以15L立瓶培养滴度最高(6.0～7.0 logCCID50/ml)。     

Use of PCR-restriction fragment length polymorphism of inlA for rapid screening of Listeria monocytogenes strains deficient in the ability to invade Caco-2 cells

A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA. inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA. Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen L. monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L. monocytogenes strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L. monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.     

微载体高密度培养Vero细胞的研究

微载体是动物细胞高密度培养的有效手段。首先在硅化的方瓶中对Cytodex 1、Cy-todex 3、Biosilon、Bellco Glass Microcarrier、CT-1、CT-3、MC-1、CT-28种国产和进口微载体进行了比较和筛选。确定以Biosilon作为Vero细胞高密度培养的首选微载体。用500mlWheaton搅拌瓶探索影响Vero细胞高密度培养的条件,表明50～60mg/ml的微载体浓度、1～2×10⁶/ml的细胞接种密度、适当的通气(95％O_2+5％CO₂)对该细胞的高密度培养具有重要意义。在200ml培养体积的Wheaton搅拌瓶中,微载体浓度为50～60mg/ml,细胞接种密度为9.24×10⁵/ml,搅拌速度为65～85r/min,经25d培养,Vero细胞密度可达2.34×10⁷/ml,表明50～60mg/ml的微载体浓度对培养细胞没有毒性。接着在1.5L CelliGen生物反应器中进行培养,细胞接种密度为4.98×10⁵/ml,培养体积为1.2L,日灌流量从0.20L逐渐加大到3.65L,经22d连接灌流培养,最终细胞密度可达2.05×10⁷/ml。     

Select Listeria monocytogenes subtypes commonly found in foods carry distinct nonsense mutations in inlA, leading to expression of truncated and secreted internalin A, and are associated with a reduced invasion phenotype for human intestinal epithelial cells

The surface protein internalin A (InlA) contributes to the invasion of human intestinal epithelial cells by Listeria monocytogenes. Screening of L. monocytogenes strains isolated from human clinical cases (n=46), foods (n=118), and healthy animals (n=58) in the United States revealed mutations in inlA leading to premature stop codons (PMSCs) in L. monocytogenes ribotypes DUP-1052A and DUP-16635A (PMSC mutation type 1), DUP-1025A and DUP-1031A (PMSC mutation type 2), and DUP-1046B and DUP-1062A (PMSC mutation type 3). While all DUP-1046B, DUP-1062A, DUP-16635A, and DUP-1031A isolates (n=76) contained inlA PMSCs, ribotypes DUP-1052A and DUP-1025A (n=72) contained isolates with and without inlA PMSCs. Western immunoblotting showed that all three inlA PMSCs result in the production of truncated and secreted InlA. Searches of the Pathogen Tracker database, which contains subtype and source information for more than 5,000 L. monocytogenes isolates, revealed that the six ribotypes shown to contain isolates with inlA PMSCs were overall more commonly isolated from foods than from human listeriosis cases. L. monocytogenes strains carrying inlA PMSCs also showed significantly (P=0.0004) reduced invasion of Caco-2 cells compared to isolates with homologous 3' inlA sequences without PMSCs. Invasion assays with an isogenic PMSC mutant further supported the observation that inlA PMSCs lead to reduced invasion of Caco-2 cells. Our data show that specific L. monocytogenes subtypes which are common among U.S. food isolates but rare among human listeriosis isolates carry inlA mutations that are associated with, and possibly at least partially responsible for, an attenuated invasion phenotype.     

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion

Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.     

A role for ActA in epithelial cell invasion by Listeria monocytogenes

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes . An in frame Δ actA mutant was constructed in a hyperinvasive strain of prfA * genotype, in which all genes of the PrfA-dependent virulence regulon, including actA , are highly expressed in vitro . Loss of ActA production in prfA * bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the Δ actA prfA * mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA * bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro ) or prfA *Δ actA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA * strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.     

Intracellular growth of Listeria monocytogenes insertional mutant deprived of protein p60

The study of the mutant strain described here demonstrates that several characteristics contribute to maximal virulence of pathogenic strains of L. monocytogenes. The invasion levels of L. monocytogenes JB1115, a p60-deficient strain, were the same as for the parent strain L. monocytogenes 1043S in J774 macrophage-like cells. The invasion level of Listeria strains in Int407 cells was 100 times lower than in J774 cells. In epithelial Int407 cells, the time of division of p60- strain L. monocytogenes JB1115 was 43% slower than for the parent strain. In this study, two lisosomotrophic agents, ammonium chloride and chlorquinoline were tested in experimental L. monocytogenes 1043S and p60-deprived mutant JB1115 infection in both cell lines. The presence of ammonium chloride increased the level of infection (calculated as number of gentamicin-resistant cells) of both Listeria strains, but in the case of infection by p60 mutant, the increased amount of ammonium chloride showed only a minimal effect on the number of isolated bacteria. In both cell lines treated with chlorquinoline we observed a decrease in the number of viable intracellular bacteria isolated from infected monolayers. Our observation of parental and mutated strains of Listeria showed that phospholipase activity also depends on the presence of p60 protein. Mutated strain showed 31.46% reduction of PI-PLC activity measured in normal growth conditions. Protein p60 plays a role not only in listeriolysin O mediated haemolytic activity but full phospholipase C activity is also dependent on the presence of the Iap protein.     

Diverse geno- and phenotypes of persistent Listeria monocytogenes isolates from fermented meat sausage production facilities in Portugal

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

Adhesion,invasion, and translocation characteristics of Listeria monocytogenes serotypes in Caco-2 cell and mouse models

Adhesion is a crucial first step in Listeria monocytogenes pathogenesis. In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model. Adhesion characteristics of all 13 serotypes of L. monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles. The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R(2)) was only 0.1236. In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles. However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R(2) = 0.3371). Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model.     

In vitro and in vivo invasiveness of different pulsed-field gel electrophoresis types of Listeria monocytogenes

The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food. Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types. Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study. PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food. Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of L. monocytogenes commonly found in food are less invasive than others to Caco-2 cells. In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed. In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10(9) L. monocytogenes cells were performed for isolates of PFGE types 1, 2, 5, and 15. After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Recurrent and sporadic Listeria monocytogenes contamination in alheiras represents considerable diversity, including virulence-attenuated isolates

Microbiological characterization of alheiras, traditional smoked meat sausages produced in northern Portugal, had previously shown that more than 60% of the lots analyzed were contaminated with Listeria monocytogenes at levels higher than 100 CFU/g. In order to better understand L. monocytogenes contamination patterns in alheiras, we characterized 128 L. monocytogenes isolates from alheiras using a variety of subtyping techniques (i.e., molecular serotyping; arsenic, cadmium, and tetracycline resistance typing; and pulsed-field gel electrophoresis [PFGE]). Subtyping of isolates from products collected on two separate dates provided evidence for the persistence of specific L. monocytogenes PFGE types in the production and distribution chains of alheiras from four different processors. A subset of 21 isolates was further characterized using ribotyping and Caco-2 cell invasion assays to evaluate the pathogenic potential of L. monocytogenes present in alheiras. Caco-2 invasion assays revealed seven isolates with invasion efficiencies that were less than 20% of that of the control strain 10403S. All seven isolates had premature stop codons in inlA that represented three distinct mutations, which had previously been observed in isolates from the United States or France. Our findings indicate the need for a comprehensive approach to control L. monocytogenes in alheiras, including strategies to reduce persistence. The presence of considerable diversity in invasion phenotypes among L. monocytogenes strains present in alheiras, including the presence of subtypes likely to be virulence attenuated, may provide an opportunity to initially focus control strategies on the subtypes most likely to cause human disease.     

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

Listeria adhesion protein (LAP) is an important adhesion factor in Listeria monocytogenes and interacts with its cognate receptor, mammalian heat shock protein 60 (Hsp60). The genetic identity of LAP was determined to be alcohol acetaldehyde dehydrogenase (Aad). A recombinant Escherichia coli strain expressing aad confirmed the involvement of Aad in adhesion to Caco-2 cells. Binding kinetics (ka) of recombinant LAP (rLAP) to Hsp60 was examined in a surface plasmon resonance sensor and was determined to be 5.35 x 10(8) M(-1) s(-1) and it was equivalent to the binding of anti-Hsp60 antibody (ka = 2.15 x 10(9) M(-1) s(-1)) to Hsp60. In contrast, Internalin B, an adhesion/invasion protein from L. monocytogenes, used as a control, had binding kinetics (ka) of only 2.9 x 10(6) M(-1) s(-1). The KD value of rLAP was 1.68 x 10(-8) M, which was significantly lower than Internalin B (KD = 6.5 x 10(-4) M). These results suggest that Hsp60 has significantly higher avidity for anti-Hsp60 antibody and LAP than Internalin B. In summary, LAP is identified as an alcohol acetaldehyde dehydrogenase and binding of recombinant E. coli to Caco-2 cells or rLAP to Hsp60 protein was found to be highly specific.