ABSTRACT
The interaction of zinc oxide nanoparticles (ZnO NPs) with human haemoglobin (Hb) is studied for the biologically safe application of ZnO NPs in the human body. The Hb corona is formed around the ZnO nanoparticles, directly observed from high-resolution transmission electron microscopy (HRTEM) images. Hb formed 'hard corona' on the surface of ZnO NPs from an exponential association mechanism over a very short duration, as well as unfolding of Hb that occurred over a long lifetime. Dynamic light scattering measurements demonstrated that the ZnO NPs were completely covered by Hb with shell thickness of c. 6 nm that formed a 'hard corona'. Zeta potential measurements represented that the ZnO NPs were fully covered by Hb molecules using an exponential association mechanism. Tryptophans (TRY), as well as heme-porphyrin moieties of Hb, are the major binding sites for ZnO NPs. The nature of the interaction between ZnO NPs and Hb was analysed from the fluorescence quenching of TRYs. Electrostatic interaction, along with the hydrophobic interaction between ZnO NPs and Hb, is responsible for the conformational change in Hb due to increase in the percentage of ß-sheets together with a decrease in α-helices.
Subject(s)
Dynamic Light Scattering , Hemoglobins/chemistry , Nanoparticles/chemistry , Zinc Oxide/chemistry , Humans , Microscopy, Electron, Transmission , Protein Unfolding , Static Electricity , Time FactorsABSTRACT
AIM: To investigate the use of a light scattering sensor, BActerial Rapid Detection using Optical scattering Technology (BARDOT) coupled with a multipathogen selective medium, Salmonella, Escherichia and Listeria (SEL), for concurrent detection of the three major foodborne pathogens in a single assay. METHODS AND RESULTS: BARDOT was used to detect and distinguish the three major pathogens, Salmonella enterica, Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes from food based on colony scatter signature patterns on SEL agar (SELA). Multiple strains of three test pathogens were grown on SELA, and BARDOT was used to generate colony scatter image libraries for inclusive (SEL Library) and exclusive (non-SEL Library) bacterial group. These pathogens were further differentiated using the SEL scatter image library. Raw chicken and hotdog samples were artificially inoculated with pathogens (100 CFU per 25 g each), and enriched in SEL broth at 37°C for 18 h and colonies were grown on SELA for 11-22 h before screening with BARDOT. The BARDOT sensor successfully detected and differentiated Salmonella, STEC and Listeria on SELA with high classification accuracy 92-98%, 91-98% and 83-98% positive predictive values (PPV) respectively; whereas the nontarget strains showed only 0-13% PPV. BARDOT-identified colonies were further confirmed by multiplex PCR targeting inlB gene of L. monocytogenes, stx2 of STEC and sefA of S. enterica serovar Enteritidis. CONCLUSIONS: The results show that BARDOT coupled with SELA can efficiently screen for the presence of three major pathogens simultaneously in a test sample within 29-40 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This innovative SELA-BARDOT detection platform can reduce turnaround time and economic burden on food industries by offering a label-free, noninvasive on-plate multipathogen screening technology for reducing microbial food safety and public health concerns.
Subject(s)
Escherichia coli , Food Microbiology/methods , Listeria monocytogenes , Salmonella enterica , Scattering, Radiation , Animals , Chickens , Escherichia coli/chemistry , Escherichia coli/isolation & purification , Light , Listeria monocytogenes/chemistry , Listeria monocytogenes/isolation & purification , Meat/microbiology , Salmonella enterica/chemistry , Salmonella enterica/isolation & purificationABSTRACT
In this paper, we have used spectroscopic and electron microscopic analysis to monitor the time evolution of the silver nanoparticles (Ag NP)-human hemoglobin (Hb) corona formation and to characterize the interaction of the Ag NPs with Hb. The time constants for surface plasmon resonance binding and reorganization are found to be 9.51 and 118.48 min, respectively. The drop of surface charge and the increase of the hydrodynamic diameter indicated the corona of Hb on the Ag NP surface. The auto correlation function is found to broaden with the increasing time of the corona formation. Surface zeta potential revealed that positively charged Hb interact electrostatically with negatively charged Ag NP surfaces. The change in α helix and ß sheet depends on the corona formation time. The visualization of the Hb corona from HRTEM showed large number of Hb domains aggregate containing essentially Ag NPs and without Ag NPs. Emission study showed the tertiary deformation, energy transfer, nature of interaction and quenching under three different temperatures.
ABSTRACT
AIMS: To investigate the suitability of human Hsp60, a receptor for Listeria adhesion protein (LAP), on paramagnetic beads (PMB) to capture Listeria monocytogenes from food in the presence of other Listeria to facilitate rapid and specific detection of this pathogen. METHODS AND RESULTS: Commercially available streptavidin-coated PMBs were linked with biotinylated Hsp60 (PMB-Hsp60), and the bacterial capture efficiency from pure culture and meat samples was determined. Capture rate was also compared with the monoclonal antibody (MAb)-C11E9-coated beads (PMB-C11E9) and the commercial Dynabeads anti-Listeria. Captured cells were detected and quantified by plating on selective medium, quantitative real-time PCR (qPCR) and a light-scattering sensor. Overall, all ligand-coated beads had similar capture efficiency (varied from 1·8 to 9·2%) for L. monocytogenes under the conditions employed, and the minimum cell number required to achieve such capture was 10³ CFU ml⻹. PMB-Hsp60 had significantly greater capture efficiency for pathogenic Listeria (P < 0·0001) than the nonpathogenic Listeria. In contrast, PMB-C11E9 and Dynabeads anti-Listeria had similar capture efficiency for both. The efficacy of all PMBs to capture L. monocytogenes in the presence of Listeria innocua from food matrices was compared. Although Dynabeads anti-Listeria had the overall best capture efficiency, PMB-Hsp60 was able to selectively capture L. monocytogenes even in the presence of 10-100-fold more L. innocua cells from enriched meat samples. CONCLUSIONS: Data show that the human cell receptor, Hsp60, is suitable for the capture of pathogenic Listeria on PMB in the presence of other Listeria in food. SIGNIFICANCE AND IMPACT OF THE STUDY: As pathogen interaction with host cells is highly specific, host cell receptors could be used as alternate capture molecules on PMB to aid in specific detection of pathogens.
Subject(s)
Chaperonin 60 , Food Microbiology/methods , Listeria monocytogenes/isolation & purification , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Humans , Listeria/isolation & purification , Listeria monocytogenes/immunology , Listeria monocytogenes/metabolism , Meat/microbiology , Sensitivity and Specificity , Streptavidin/metabolismABSTRACT
AIMS: To determine the virulence gene expression of Salmonella Typhimurium in response to sublethal heat stress and determine the adhesion and invasion pattern of heat-stressed Salmonella in Caco-2 intestinal epithelial cells. METHODS AND RESULTS: Transcriptional profiling was employed to capture the virulence gene response of Salm. Typhimurium at 42°C sublethal heat stress. Data indicated an induction of SPI-2 and SPI-5 genes and a repression of SPI-1-encoded genes due to heat stress. Gene expression pattern also showed induced transcription of fimbriae genes and genes present within the stress-associated Rpo regulon. Changes in adhesion and invasion pattern of heat-stressed Salm. Typhimurium were tested in Caco-2 cells. Heat-stressed Salm. Typhimurium showed greater adhesion to Caco-2 cells compared with nonstressed control cells. CONCLUSIONS: Salmonella Typhimurium exposed to sublethal heat stress responds by altered virulence gene expression, which further enhances the adhesion of bacterial cells to intestinal Caco-2 cells. Results indicate a role of physiological stress in Salm. Typhimurium in promoting microbial virulence and host cell vulnerability to infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Studying the Salmonella virulence genes expression in response to sublethal heat stress is crucial for the understanding of the virulence status of Salmonella in temperature-abused foods. Results of this study provide information about the gene response and virulence status of Salmonella pathogenicity factors in response to sublethal heat stress towards host cells.
Subject(s)
Gene Expression Regulation, Bacterial , Hot Temperature , Salmonella typhimurium/genetics , Salmonella typhimurium/pathogenicity , Virulence Factors/genetics , Bacterial Adhesion/physiology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Caco-2 Cells , Genomic Islands/genetics , Humans , Salmonella Infections/microbiology , Salmonella Infections/pathology , Salmonella typhimurium/metabolism , Stress, Physiological/genetics , Transcriptome , Virulence Factors/metabolismABSTRACT
AIM: To develop antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food products. METHODS AND RESULTS: Aptamer, a single-stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer-A8, specific for internalin A, an invasive protein of L. monocytogenes, was used in the fibre-optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti-Listeria antibody, P66, was immobilized on streptavidin-coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647-conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10(3) CFU ml(-1). This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10(2) CFU 25 g(-1) ) ready-to-eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. CONCLUSION: Based on the data presented in this study, the antibody-aptamer functionalized fibre-optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates feasibility and novel application of aptamer on fibre-optic biosensor platform for the sensitive detection of L. monocytogenes from food products.
Subject(s)
Antibodies, Bacterial , Aptamers, Nucleotide , Biosensing Techniques/methods , Food Microbiology , Listeria monocytogenes/isolation & purification , Meat Products/microbiology , Bacterial Proteins/analysis , Fiber Optic Technology , Food Analysis/methods , Listeria monocytogenes/immunologyABSTRACT
AIM: To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. METHODS AND RESULTS: Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria-Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37 degrees C but not at 4 degrees C. Transmission electron microscopy, enzyme-linked immunosorbent assay, and mRNA analysis further supported these observations. CONCLUSION: Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. SIGNIFICANCE AND IMPACT OF THE STUDY: As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody-based detection of L. monocytogenes.
Subject(s)
Bacterial Proteins/genetics , Food Microbiology , Listeria monocytogenes/metabolism , Membrane Proteins/genetics , Bacteriological Techniques , Blotting, Western/methods , Culture Media , Enzyme-Linked Immunosorbent Assay/methods , Listeria monocytogenes/classification , Microscopy, Electron, Transmission , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Serotyping , Species Specificity , TemperatureABSTRACT
AIMS: To study the reaction patterns of selected antibodies to Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes cells exposed to various environmental stresses. METHODS AND RESULTS: Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes cells subjected to different environmental stress of temperatures (4 and 45 degrees C), NaCl (5.5%), oxidative stress (15 mmol(-1) H2O2), acidic pH (5.5) and ethanol (5%) for 3 h (short-term stress) or for 5 days (long-term stress) were analysed by ELISA and Western blotting. The ELISA results indicated that most stresses caused 12-16% reductions in reaction for anti-E. coli O157:H7 and 20-48% reductions for anti-Salmonella polyclonal antibodies during short-term stress, whereas the most stresses exhibited enhanced reaction (44-100% increase) with the anti-L. monocytogenes polyclonal antibody. During long-term stress exposure to combined stress conditions of pH 5.5, 3.5% NaCl at 12 degrees C or at 4 degrees C, antibody reactions to the three pathogens were highly variable with the combined stress at 4 degrees C showing the most reductions (8-40%). Likewise, there were about 18-59% reductions in antibody reactions with pathogens when cultured in hotdog samples with the combined stress conditions. Western blot analyses of crude cell surface antigens from both short- and long-term stressed cells revealed that the changes in antibody reactions observed in ELISA were either because of repression, expression or possible denaturation of antigens on the surface of cells. CONCLUSIONS: Overall, the antibody reactions were significantly reduced in pathogens exposed to both short- and long-term environmental stresses in culture medium or in meat sample because of expression, repression or denaturation of specific antigens in cells. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to ensure the reliable detection of foodborne pathogens using antibody-based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first as the physiological activities in cells are often altered in response to a stress.
Subject(s)
Antibodies, Bacterial/analysis , Bacteria/isolation & purification , Environment , Food Microbiology , Bacteria/classification , Blotting, Western/methods , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli O157/immunology , Escherichia coli O157/isolation & purification , Humans , Listeria monocytogenes/immunology , Listeria monocytogenes/isolation & purification , Meat Products/microbiology , Oxidative Stress/physiology , Salmonella enteritidis/immunology , Salmonella enteritidis/isolation & purificationABSTRACT
AIMS: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments. METHODS AND RESULTS: The reaction patterns of antibodies to L. monocytogenes held in stressful environments for a short duration (3 h) or grown for extended periods (16-72 h) were investigated. During both short or prolonged exposure to stress environments of high temperature (45 degrees C) and NaCl (>1.5%, w/v), reactions of whole cells of L. monocytogenes to antibodies were severely affected as determined by ELISA and by the reduced expression of the antibody-reactive 66 kDa antigen in the Western blot assay. Conversely, cold (4-15 degrees C) or acid (pH 2-3) stress environments had very little effect on antigen expression or antibody reaction. Additionally, heat-killed cells showed reduced reactions to these antibodies when compared with unheated cells. Artificially created stress environments in hotdog slurry also affected the antigen expression in L. monocytogenes. Immunoelectron microscopy revealed that the antibody-reactive antigens were uniformly present on the surface of the cells. Morphological characteristics following growth in stressed environments revealed that heat stress at 45 degrees C caused L. monocytogenes cells to be elongated and to form clumps; whereas, osmotic stress (5.5% NaCl, w/v) caused filamentous appearance with multiple septa along the length of the cell. CONCLUSIONS: These results indicated that MAb C11E9 or EM-7G1 could detect L. monocytogenes from cold or acid-stress environments; however, they may show weaker reactions with heat or osmotically stressed cells or cells grown at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria in food are routinely subjected to various stresses, induced by cold, heat, salt or acid during processing and storage. Whether stresses would modify the expression of cellular antigens of L. monocytogenes is of a great concern for immunodetections in food products.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Bacterial/analysis , Listeria monocytogenes/immunology , Antigens, Surface/analysis , Blotting, Western/methods , Cold Temperature , Culture Media , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/analysis , Food Microbiology , Hot Temperature , Hydrogen-Ion Concentration , Microscopy, Electron , Sodium Chloride , Time FactorsABSTRACT
AIMS: To assess the presence of virulence genes in environmental and foodborne Escherichia coli isolates using the TaqMan PCR system. METHODS AND RESULTS: Three TaqMan pathogen detection kits called O157:H7, StxI and StxII were used to investigate the presence of virulence genes in Escherichia coli isolates. All 54 foodborne E. coli O157:H7 isolates showed expected results using these kits. Ninety (15%) of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TaqMan PCR amplification products from these 90 isolates were analysed by agarose gel electrophoresis, and 90% (81 of 90) of the environmental samples contained the expected PCR product. Sixty-six of these 90 were chosen for serotyping tests and only 35% (23 of 66) showed agglutination with both anti-O157 and anti-H7 antibodies. Further ribotyping of 16 sero-positive isolates in an automated Riboprinter did not identify these to be O157:H7. Multiplex PCR with primers for eaeA, stxI and stxII genes was used to confirm the TaqMan results in 10 selected environmental isolates. CONCLUSIONS: All three TaqMan pathogen detection kits were useful for virulence gene analysis of prescreened foodborne O157:H7 isolates, while the O157:H7-specific kit may not be suitable for virulence gene analysis of environmental E. coli isolates, because of high false positive identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to rapidly identify the presence of pathogenic E. coli in food or environmental samples is essential to avert outbreaks. These results are of importance to microbiologists seeking to use TaqMan PCR to rapidly identify pathogenic E. coli in environmental samples. Furthermore, serotyping may not be a reliable method for identification of O157:H7 strains.
Subject(s)
Bacterial Typing Techniques/methods , Environmental Microbiology , Escherichia coli/classification , Escherichia coli/pathogenicity , Genes, Bacterial , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli O157/classification , Escherichia coli O157/genetics , Escherichia coli O157/pathogenicity , Food Microbiology , Humans , Polymerase Chain Reaction/methods , Reagent Kits, Diagnostic , Ribotyping/methods , Virulence/geneticsABSTRACT
Immunity to Listeria monocytogenes is largely mediated by T lymphocytes. Recently, B lymphocytes or their secreted products are implicated to provide immunity against L. monocytogenes infection. To understand whether L. monocytogenes can infect and kill B cells as a possible strategy to initiate an infection, we examined the effects of L. monocytogenes on a human B lymphoma (Ramos RA-1) and mouse primary B cells in vitro. L. monocytogenes infection resulted in significantly (pSubject(s)
Apoptosis
, B-Lymphocytes/microbiology
, Listeria monocytogenes/physiology
, Listeriosis/microbiology
, Animals
, B-Lymphocytes/immunology
, B-Lymphocytes/ultrastructure
, Bacterial Adhesion
, Cell Line, Transformed
, Cells, Cultured
, Cytotoxicity, Immunologic
, Flow Cytometry
, Humans
, Listeria/immunology
, Listeria/physiology
, Listeria monocytogenes/immunology
, Listeriosis/immunology
, Mice
, Mice, Inbred BALB C
, Microscopy, Electron, Scanning
, Microscopy, Fluorescence
, Tumor Cells, Cultured
ABSTRACT
Thirty Listeria monocytogenes isolates from human patients and foods originated from two different geographic locations without any epidemiological relations were analyzed for their genotypic and phenotypic virulence gene expressions and genetic relatedness. All strains contained virulence genes, inlA, inlB, actA, hlyA, plcA and plcB, with expected product size in PCR assay except for the actA gene. Some strains produced actA gene product of 268 and others 385 bp. Phenotypically, all were hemolytic but showed variable expressions of phospholipase activity. Ribotyping classified isolates into 12 different groups based on the similarity to DuPont Identification numbers (DID), which consisted primarily of clinical or food isolates or both. Cluster analysis also indicated possible existence of clones of L. monocytogenes that are found in food or human hosts or are evenly distributed between these two. Two isolates (F1 from food and CHL1250 from patient) had unique ribotype patterns that were not previously reported in the RiboPrinter database. This study indicates distribution of diverse L. monocytogenes strains in clinical and food environments. The isolates showed 92-99% genetic homogeneity, in spite of their origins from two different geographic locations and environments.
Subject(s)
Listeria monocytogenes/genetics , Virulence/genetics , Animals , Food Microbiology , Genotype , Humans , Listeria monocytogenes/classification , Meat Products , Phenotype , Phylogeny , Polymerase Chain Reaction , RibotypingABSTRACT
The Vero cell assay presently used for virulence testing of verotoxigenic Escherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was developed that measures endogenous lactate dehydrogenase (LDH) release from Vero or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp-2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strain also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cells. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell lines. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli showing positive LDH values. Similarly, RiboPrinter analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations from VTEC revealed severe lysis, vacuole formation and death in Vero cells and multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric cytotoxicity assay described here can provide quantitative data for determining the virulence potential of verotoxigenic E. coli in 12-16 h.
Subject(s)
Escherichia coli Infections/microbiology , Escherichia coli/classification , Escherichia coli/pathogenicity , L-Lactate Dehydrogenase/metabolism , Shiga Toxins/biosynthesis , Animals , Bacteriological Techniques , Cattle , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli O157/classification , Escherichia coli O157/genetics , Escherichia coli O157/metabolism , Escherichia coli O157/pathogenicity , Humans , Polymerase Chain Reaction , Ribotyping , Shiga Toxin 1/genetics , Shiga Toxin 1/toxicity , Shiga Toxin 2/genetics , Shiga Toxin 2/toxicity , Shiga Toxins/genetics , Vero Cells , VirulenceABSTRACT
The major form of autosomal dominant polycystic kidney disease (ADPKD) results from mutation of a gene (PKD1) of unknown function that is essential for the later stages of renal tubular differentiation. In this report, we describe a novel cell culture system for studying how PKD1 regulates this process. We show that expression of human PKD1 in MDCK cells slows their growth and protects them from programmed cell death. MDCK cells expressing PKD1 also spontaneously form branching tubules while control cells form simple cysts. Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic diseases. Our study suggests that PKD1 may function to regulate both pathways, allowing cells to enter a differentiation pathway that results in tubule formation.
Subject(s)
Apoptosis , Kidney Tubules/cytology , Proteins/metabolism , Animals , Cell Division , Cell Line , Collagen/metabolism , Dogs , Humans , Kidney Tubules/metabolism , Membrane Proteins/metabolism , Phenotype , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/pathology , Proteins/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TRPP Cation ChannelsABSTRACT
The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2 mM) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mM gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA+ hlyA+) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.
Subject(s)
Dithiothreitol , Listeria monocytogenes/physiology , Animals , Cattle , Chickens , Culture Media , Humans , Listeria monocytogenes/growth & development , Listeria monocytogenes/isolation & purification , Listeria monocytogenes/metabolism , Meat , Mice , Microscopy, Electron, Scanning , Serum Albumin, Bovine , Tumor Cells, CulturedABSTRACT
The human kidney is composed of roughly 1.2-million renal tubules that must maintain their tubular structure to function properly. In autosomal dominant polycystic kidney disease (ADPKD) cysts develop from renal tubules and enlarge independently, in a process that ultimately causes renal failure in 50% of affected individuals. Mutations in either PKD1 or PKD2 are associated with ADPKD but the function of these genes is unknown. PKD1 is thought to encode a membrane protein, polycystin-1, involved in cell-cell or cell-matrix interactions, whereas the PKD2 gene product, polycystin-2, is thought to be a channel protein. Here we show that polycystin-1 and -2 interact to produce new calcium-permeable non-selective cation currents. Neither polycystin-1 nor -2 alone is capable of producing currents. Moreover, disease-associated mutant forms of either polycystin protein that are incapable of heterodimerization do not result in new channel activity. We also show that polycystin-2 is localized in the cell in the absence of polycystin-1, but is translocated to the plasma membrane in its presence. Thus, polycystin-1 and -2 co-assemble at the plasma membrane to produce a new channel and to regulate renal tubular morphology and function.
Subject(s)
Calcium Channels/metabolism , Kidney Tubules/metabolism , Membrane Proteins/metabolism , Polycystic Kidney, Autosomal Dominant/metabolism , Proteins/metabolism , Animals , CHO Cells , Calcium/metabolism , Calcium Signaling , Cations/metabolism , Cell Membrane/metabolism , Cricetinae , Electrophysiology , Humans , Membrane Proteins/genetics , Molecular Sequence Data , Mutation , Polycystic Kidney, Autosomal Dominant/genetics , Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , TRPP Cation Channels , TransfectionABSTRACT
Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117-124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42 degrees C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42 degrees C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25 degrees C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37 degrees C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.
Subject(s)
Bacterial Adhesion , Bacterial Outer Membrane Proteins/biosynthesis , Listeria monocytogenes/growth & development , Listeria monocytogenes/metabolism , Blotting, Western , Caco-2 Cells/microbiology , Colony Count, Microbial , Enzyme-Linked Immunosorbent Assay , Humans , Listeria monocytogenes/genetics , Listeria monocytogenes/pathogenicity , TemperatureABSTRACT
Adhesion of Listeria monocytogenes to intestinal endothelial cells is an important initial event in the pathogenesis of infection which is not well understood. The suggestion has been made that some proteins, including internalin and actin polymerisation protein (ActA), and carbohydrate molecules mediate, at least in part, the adhesion of listeria to certain cultured mammalian cells. This study investigated the role of a L. monocytogenes cell-surface protein of 104 kDa (p104) in adhesion to human intestinal enterocyte-like Caco-2 cell lines by transposon (Tn916) mutagenesis and a p104-specific monoclonal antibody (MAb-H7). Genotypic and phenotypic characteristics of Tn916-transformed L. monocytogenes strains, AAMU530 and AAMU572, revealed that these strains did not express p104, and the transposon had been inserted at a single locus in the structural gene. Strains AAMU530 and AAMU572 yielded only 10 and 6.3% adhesion to Caco-2 cells. Coating of L. monocytogenes and L. innocua wild-type strains with MAb-H7 reduced adhesion to Caco-2 cells from 100% to 50 and 45%, respectively, whereas on isotype control MAb EM-7G1 had no effect. Western blot analysis with MAb-H7 indicated that p104 is present in all Listeria spp. except in L. grayi. Furthermore, p104 is also present in internalin (BUG8) and ActA (LUT12) deficient strains, suggesting that p104 is indeed different from internalin or ActA proteins. Cytotoxicity analysis of strains AAMU530 and AAMU572 demonstrated that these strains, although haemolytic and phospholipase-positive, were avirulent when tested with a hybridoma B-lymphocyte cell line. Loss of virulence could be attributed to the interruption of adhesion of mutant strains to the hybridoma cell line. These results strongly suggest that p104 is an adhesion factor in L. monocytogenes and possibly in other Listeria species and is involved in adhesion to intestinal cells.
Subject(s)
Bacterial Adhesion/physiology , Bacterial Outer Membrane Proteins/physiology , Caco-2 Cells/microbiology , Listeria monocytogenes/cytology , Bacterial Outer Membrane Proteins/genetics , Blotting, Southern , Blotting, Western , Conjugation, Genetic , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic , Humans , Hybridomas , Listeria monocytogenes/genetics , Mutagenesis, Insertional , Polymerase Chain ReactionABSTRACT
The endothelial expression of adhesion molecules by proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been suggested to contribute to the initiation of atherosclerotic plaque formation. Since lactosylceramide (LacCer) accumulates in large quantities in human atherosclerotic plaque, we have explored its role in TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and their consequent adhesion to polymorphonuclear leukocytes (PMNs). We found that TNF-alpha increased LacCer synthesis by way of stimulating the activity of UDP-galactose:glucosylceramide beta(1-->4)-galactosyltransferase in a time-dependent fashion. The TNF-alpha-induced expression of ICAM-1 was abrogated by D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of UDP-galactose:glucosylceramide beta(1-->4)-galactosyltransferase. However, the addition of LacCer reversed the D-PDMP effect on TNF-alpha-induced ICAM-1 expression in human umbilical vein endothelial cells. Northern hybridization analysis of mRNA levels and enzyme-linked immunosorbent assays revealed that LacCer (5 microM) specifically stimulated ICAM-1 at both the transcriptional and translational levels. This was accompanied by the adhesion of PMNs, which was visualized by confocal microscopy. Further studies revealed that LacCer stimulated the endogenous generation of superoxide radicals (O-2) about 5-fold compared with the control by specifically activating plasma membrane-associated NADPH-dependent oxidase. This phenomenon was blocked by the antioxidant N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, and the NADPH oxidase inhibitor, diphenylene iodonium. Overexpression of endogeneous CuZn-superoxide dismutase via an adenoviral vector carrying cDNA for CuZn-superoxide dismutase, also inhibited LacCer-induced ICAM-1 expression in endothelial cells. In sum, our findings suggest that LacCer may play the role of a lipid second messenger in TNF-alpha-induced pathogenesis by activating an oxidant-sensitive transcriptional pathway that leads to the adhesion of PMNs to endothelial cells.
Subject(s)
Antigens, CD , Endothelium, Vascular/physiology , Gene Expression Regulation , Intercellular Adhesion Molecule-1/genetics , Lactosylceramides/physiology , Neutrophils/physiology , Transcription, Genetic , Tumor Necrosis Factor-alpha/pharmacology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cells, Cultured , E-Selectin/biosynthesis , E-Selectin/genetics , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Kinetics , Lactosylceramides/pharmacology , Morpholines/pharmacology , Neutrophils/drug effects , RNA, Messenger/genetics , Time Factors , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/physiology , Umbilical Veins , Vascular Cell Adhesion Molecule-1/biosynthesis , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
Depending on the growth medium used for enrichment of bacterial cells prior to assay, the monoclonal antibody (MAb) EM-6E11 recognizing Listeria genus-specific epitope on 43 and 94 to 97 kDa cell-surface antigens (A. K. Bhunia and M. G. Johnson, Appl. Environ. Microbiol. 58:1924-1929, 1992) exhibited extensive variability in the detection of Listeria species. MAb EM-6E11 strongly detected live cells of all Listeria species and all serotypes of L. monocytogenes by ELISA when cells were grown in nonselective brain heart infusion (BHI) broth, in selective Listeria enrichment broth (LEB), or in Listeria repair broth (LRB). In contrast, EM-6E11 detected only four of the thirteen serotypes of L. monocytogenes (serotypes 1/2c, 3b, 4ab, and 7) when cells were grown in the UVM1 formulation of Listeria enrichment broth (UVM1) or Fraser broth (FRB). This MAb failed to react with live cells of four other Listeria species, including L. ivanovii, L. welshimeri, L. grayi, and L. murrayi cells grown in UVM1 or FRB. Heating of Listeria cells at 100 degrees C for 20 min, irrespective of the enrichment media used, led to large losses of MAb EM-6E11 reactivity in ELISA, suggesting that the specific cell-surface epitopes involved may not be heat stable. Our results confirm that MAb EM-6E11 is suitable for detection of live cells but not heat-killed cells of Listeria spp. and can be used in conjunction with an enrichment step in BHI, LEB, or LRB but not in UVM1 or FRB.