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The ever-increasing number of chemicals and complex mixtures demands a time-saving and cost-effective platform for environmental risk assessment. However, there is limit promising tool for evaluating the contribution of each component to the total toxicity effects of the mixture. Here, four widely distributed environmental pollutants with different mode-of-actions, i.e., cadmium chloride (Cd), nitrofurazone (NFZ), triclosan (TCS), and tris(2-chloroethyl) phosphate (TCEP), were selected as components of artificial mixture. Integration of leave-one-out method and high-dimensional live cell array system was used to explore relative contribution of each component from the mixture. A quaternary mixture (All_4_chems) and four ternary mixtures (Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP) were investigated by Escherichia coli (E. coli) live cell array system with 90 environmental stress genes modified by green fluorescent protein (GFP) expressing reporter vectors. E. coli cytotoxicity tests demonstrated that TCS has antagonism effect with other three chemicals (Cd, NFZ and TCEP), while it was additive effect in other three binary combinations. A total of 26, 23, 13, 31 and 23 genes were significantly altered with fold-change greater than 2 over the 4 h exposure by All_4_chems, Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP, respectively. Clustering analysis based on time-series gene expression patterns and transcriptional effect level index (TELI) showed that Leave_TCEP has similar profiles with All_4_chems, demonstrating TCEP has the least contribution among four components to the quaternary mixture. Leave_NFZ has the least number of significantly altered genes, implying NFZ has the largest toxicity effect contribution to the quaternary mixture. The relative contribution in different pathways indicated that Cd has the most contribution to the mixture in redox stress, while TCS has the least contribution in DNA stress pathway. Collectively, our results demonstrated the utility of high-dimensional toxicogenomics data and leave-one-out method in prioritizing the relative contribution of each component in mixture.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 mounts specific acid-resistance systems against acid stress, making it more difficult to eradicate in food industry. To date, RNAseq-based analysi...

Purpose. Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of e-polylysine (e-PL). Methodology. The antibacterial activity of e-PL was detected by measuring the minimum inhibitory concentration values as well as the time–kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by β-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology. Key findings. These results demonstrated that e-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of e-PL (16 µg ml−1). In addition, the bacterial membrane was damaged more severely when the concentration of e-PL was increased. Conclusion. The present study investigated the antimicrobial mechanism of e-PL by measuring the content of cytoplasmic β-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that e-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic β-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of e-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

Functional gene fingerprinting of chemicals could be used to understand the direct gene-chemical interaction in the process of toxification from a genome-wide scale. 2, 2-bis (bromomethyl)-1, 3-propanediol (BMP) is a brominated flame retardant with widespread production but with very limited toxicological data. Here the cytotoxicity of BMP was assessed by Escherichia coli ( E. coli ) functional genome-wide knockout mutants screening and the underlying molecular mechanism was investigated. The median inhibition concentration (IC50) of BMP was 1.608 ± 0.078 mg/ml after 24 h exposure. 119 initial, including 66 sensitive and 53 resistant single gene mutants, were identified by a full library screening of BMP at the concentration of IC50. The resistant genes were significantly enriched in nucleobase-containing compound biosynthetic process (GO: 0034654) by gene ontology (GO) biological process analyses, which suggested that the pathway of DNA repair is a critical cellular process in the survival of cells exposed to BMP. Meanwhile, function annotation of all BMP responsive genes suggested the mechanism of BMP was associated with DNA damage, oxidative stress and cellular transmembrane transport process. Many genes were exclusively responsive to BMP comparing with other chemicals that has been assessed by E. coli mutant screening approach, which indicated that BMP has a distinct mode of toxic action. Overall, the functional genomic screening approach presented here provides a great tool to assess the cellular toxicological mechanism of environmental chemicals.

Background/Purpose Carcinoembryonic antigen (CEA) is an attractive target for immunotherapy because it is expressed minimally in normal tissue, but is overexpressed in a wide variety of malignant epithelial tissues. Lactic acid bacteria (LABs), widely used in food processes, are attractive candidates for oral vaccination. Thus, we examined whether LABs could be used as a live vaccine vector to deliver CEA antigen. Methods CEA was cloned into an Escherichia coli / Lactococcus lactis shuttle vector pSEC:LEISS under the control of a nisin promoter. For displaying the CEA on the cell surface of the L. lactis strain, the anchor motif LcsB from the S-layer protein of Lactobacillus crispatus was fused with CEA. Intracellular and cell surface expression of the CEA–LcsB fusion was confirmed by western blot analysis. Results Significantly higher levels of CEA-specific secretory immunoglobulin A in the sera of mice were observed upon oral administration of strain cultures containing the CEA–LcsB fused protein. In addition, the CEA–LcsB antigen group showed a higher spleen index compared to the CEA antigen alone or negative control, demonstrating that surface-displayed CEA antigen could induce a higher immune response. Conclusion These results provided the first evidence for displaying CEA antigen on the cell surfaces of LABs as oral vaccines against cancer or infectious diseases.

Background Foodborne diseases caused by foodborne pathogens have increasingly become a worldwide public health concern. Due to potential harmful effects of synthetic chemicals, there is a pressure for adoption of natural alternatives to obtain microbial safety of food. Tea tree oil (TTO) exhibited a wide range of pharmacological actions attribute to the broad spectrum activities. However, to the best of our knowledge, no systematic research on the mode of antibacterial actions of TTO against Listeria monocytogenes (L. monocytogenes) and Escherichia coli (E. coli) in vitro models have been conducted so far. Results The present investigation reported on the antimicrobial activities of TTO and examined its possible antimicrobial mode of action against L. monocytogenes and E. coli. Results showed that the susceptibility of L. monocytogenes were excellent with the lower minimal inhibitory concentration (MIC) values and larger inhibition zones. TTO changed the integrity of the membrane, as evidenced by the release of 260 nm absorbing intracellular materials and the alteration of membrane potential. The results of flow cytometry showed that TTO caused bacterial membrane permeabilization in a dose-dependent manner. The remarkable cellular morphological changes in bacteria caused by TTO were observed using the scanning electron microscope, indicating cell damage. In addition, antimicrobial preserving properties of TTO were evaluated by time-kill assay after its incorporation in cucumber juice, the results showed TTO successfully inhibited L. monocytogenes and E. coli development, at room temperature and in refrigerator (25 °C and 4 °C) respectively, demonstrating it had good preservative activities in food system. Conclusions These findings suggested that TTO exhibited good antimicrobial effect against food-borne pathogens and could be potentially used in food industries as a food preservative.

The knowledge of gene-chemical interaction can be used to derive toxicological mechanism of chemical pollutants, therefore, it might be useful to discriminate chemicals with different mechanisms. In this study, three narcotic chemicals (4-chlorophenol (4-CP), 3, 4-dichloroaniline (DCA) and 2, 2, 2-trichloroethanol (TCE)) and three specific acting chemicals (triclosan (TCS), clarithromycin (CLARY), sulfamethoxazole (SMX)) were assessed by Escherichia coli ( E. coli ) genome-wide knockout screening. 66, 97, 88, 144, 198 and 180 initial robust hits were identified by exposure to 4-CP, DCA, TCE, TCS, CLARY and SMX with two replicates at the concentration of IC50, respectively. The average fold change values of responsive mutants to the three narcotic chemicals were smaller than the three specific acting chemicals. The common gene ontology (GO) term of biological process enriched by the three narcotic chemicals was “response to external stimulus” (GO: 0009605). Other GO terms like “lipopolysaccharide biosynthetic process” (induced by 4-CP) and “purine nucleotide biosynthetic process” (induced by DCA) were also influenced by the narcotic chemicals. The toxic target of three known specific acting chemicals could be validated by GSEA of responsive genes. Four genes ( flhC, fliN, fliH and flhD ) might serve as potential biomarkers to distinguish narcotic chemicals and specific acting chemicals. The E. coli functional genomic approach presented here has shown great potential not only for the molecular mechanistic screening of chemicals, rather it can discriminate chemicals based on their mode-of-action.

Results of previous studies have indicated that 6-HO-BDE-47, the addition of the hydroxyl (HO) group to the backbone of BDE-47, significantly increased the toxicity of the chemical compared to its postulated precursor analogues, BDE-47 and 6-MeO-BDE-47. However, whether such a result is conserved across polybrominated diphenyl ether (PBDE) congeners was unknown. Here, cytotoxicity of 32 PBDE analogues (17 HO-PBDEs and 15 MeO-PBDEs) was further tested and the underlying molecular mechanism was investigated. A total of 14 of the 17 HO-PBDEs inhibited growth of Escherichia coli during 4 or 24 h durations of exposure, but none of the MeO-PBDEs was cytotoxic at the concentrations tested. 6-HO-BDE-47 and 2-HO-BDE-28 were most potent with 4 h median effect concentrations (EC50) of 12.13 and 6.25 mg/L, respectively, which trended to be lesser with a longer exposure time (24 h). Expression of 30 modulated and validated genes by 6-HO-BDE-47 in a previous study was also observed after exposure to other HO-PBDE analogues. For instance, uhpT was upregulated by 13 HO-PBDEs, and three rRNA operons (rrnA, rrnB, and rrnC) were downregulated by 8 HO-PBDEs. These unanimous responses suggested a potential common molecular signaling modulated by HO-PBDEs. To explore new information on mechanisms of action, this work was extended by testing the increased susceptibility of 182 mutations of transcriptional factors (TFs) and 22 mutations as genes modulated by 6-HO-BDE-47 after exposure to 6-HO-BDE-47 at the 4 h IC50 concentration. Although a unanimous upregulation of uhpT was observed after exposure to HO-PBDEs, no significant shift in sensitivity was observed in uhpT-defective mutants. The 54 genes, selected by cut-offs of 0.35 and 0.65, were determined to be responsible for "organic acid/oxoacid/carboxylic acid metabolic process" pathways, which supported a previous finding.

Triphenyltin chloride (TPTC), which has been extensively used in industry and agriculture, can occur at concentrations in the environment sufficient to be toxic. Here, potency of TPTC to modulate genes in a library containing 1,820 modified green fluorescent protein (GFP)-expressing promoter reporter vectors constructed from Escherichia coli K12 strains was determined. Exposure to TPTC resulted in 22 (fold change > 2) or 71 (fold change > 1.5) differentially expressed genes. The no observed transcriptional effect (NOTEC) and median transcriptional effect concentrations (TEC50) were determined to be 0.036 and 0.45 mg/L in E. coli. These responses were 1,230 and 97 times more sensitive than the acute median effect concentration (EC50) required to inhibit growth of cells, which demonstrated that this live cell array represents a sensitive method to assess toxic potency of chemicals. The 71 differentially expressed genes could be classified into seven functional groups. Of all the altered genes, three groups which encoded for catalytic enzymes, regulatory proteins, and structural proteins accounted for 28 %, 18 %, and 14 % of all altered genes, respectively. The pattern of differential expression observed during this study was used to elucidate the mechanism of toxicity of TPTC. To determine potential relationships among genes that were changed greater than 2.0-fold by exposure to TPTC, a correlation network analysis was constructed, and four genes were related to aroH, which is the primary target for metabolic regulation of aromatic biosynthesis by feedback inhibition in bacteria. The genes rnC, cld, and glgS were selected as potential biomarkers for TPTC, since their expression was more than 2.0-fold greater after exposure to TPTC.

Recently some novel alternative flame retardants (NFRs), which have been widely applied to meet demands for mandated flame retardation of products, have been detected in various matrices of the environment. However, knowledge on toxic effects and associated molecular mechanisms of these chemicals was limited. Here, toxic mechanisms of action of six NFRs, bis (2-ethylhexyl) phosphate (BEHP), chlorendic acid (Het acid), 2,2-bis (bromomethyl)-1,3-propanediol (BMP), tris (2-butoxyethyl) phosphate (TBEP), triethyl phosphate (TEP), tributyl phosphate (TBP) were investigated by use of a library containing ∼1820 modified green fluorescent protein (GFP) expressing promoter reporter vectors constructed from Escherichia coli K12(E.coli). BEHP, Het acid, BMP, TBEP, TEP, TBP inhibited growth of E. coli with 4 h 10%-inhibition concentrations of 53.0-3102.3 μM. A total of 119, 44, 26, 131, 62, 103 genes out of 336 genes selected during preliminary screening were significantly altered with fold-changes greater than 1.5 by BEHP, Het acid, BMP, TBEP, TEP and TBP, respectively. GO analyses of responsive genes suggested that RNA and primary metabolism process were involved in molecular mechanisms of toxicity. Chemical clustering based on expression of 62 multi-responsive genes showed that BEHP, TBP and TBEP were grouped together, which is consistent with similarity of their chemical structures, especially for BEHP and TBP. Clustering by molecular descriptors and molecular activity by use of the multivariate classification system ToxCast was consistent with that by profiles of multi-responsive genes. The results of this study demonstrated the utility of the E. coli, whole-cell assay for determining mechanisms of toxic action of chemicals.

Cytotoxicity of 6-HO-BDE-47 and its two analogues, BDE-47 and 6-MeO-BDE-47, and the associated molecular mechanisms were assessed by use of a live cell reporter assay system which contains a library of 1820 modified green fluorescent protein (GFP) expressing promoter reporter vectors constructed from E. coli K12 strains. 6-HO-BDE-47 inhibited growth of E. coli with a 4 h median effect concentration (EC50) of 22.52 ± 2.20 mg/L, but neither BDE-47 nor 6-MeO-BDE-47 were cytotoxic. Thus, 6-HO-BDE-47 might serve as an antibiotic in some living organisms. Exposure to 6-HO-BDE-47 resulted in 65 (fold change >2) or 129 (fold change >1.5) genes being differentially expressed. The no observed transcriptional effect concentration (NOTEC) and median transcriptional effect concentration (TEC50) based on transcriptional end points, of 6-HO-BDE-47 were 0.0438 and 0.580 mg/L, respectively. The transcriptional responses were 514- and 39-fold more sensitive than the acute EC50 to inhibit cell growth. Most of the genes that were differentially expressed in response to 6-HO-BDE-47 were not modulated by BDE-47 or 6-MeO-BDE-47. These results suggest that cytotoxicity of 6-HO-BDE-47 to E. coli was via a mechanism that was different from that of either BDE-47 or 6-MeO-BDE-47. Gene expression associated with metabolic pathways was more responsive to 6-HO-BDE-47, which suggests that this pathway might be the primary target of this compound.

Diseases associated with porcine circovirus type 2 (PCV2) infections are becoming a major problem for the swine industry worldwide. The capsid protein (Cap) of PCV2 is an antigen important for both early diagnosis and development of vaccines. In this study, Lactococcus lactis was used as vehicle to deliver the PCV2 antigen in an attempt to develop oral vaccine. A cap gene with a deleted nuclear localization signal sequence (dcap) was cloned into an Escherichia coli/L. lactis shuttle vector pSEC: LEISS under the control of a nisin promoter. Intracellular and extracellular expression of the dCap was confirmed by Western blot analysis. Significantly higher levels of PCV2-specific IgG in the sera of mice were observed upon oral administration of strain cultures expressing the PCV2 antigen. These results suggest that it is feasible to use L. lactis as an antigen delivery vehicle for developing oral vaccines against PCV2 infection.

Surface water samples constantly receive a vast mixture of micropollutants mainly originating from wastewater treatment plants (WWTPs). High-throughput live cell arrays provide a promising method for the characterization of the effects of chemicals and the associated molecular mechanisms. In the present study, this test system was evaluated for the first time for the characterization of a set of typical surface water extracts receiving effluent from WWTPs. The extracts containing complex mixtures of micropollutants were analyzed for the expression of 90 stress responsive genes in the Escherichia coli reporter gene assay. The most affected pathways and the genes most sensitive to surface water samples suggested prominent stress-responsive pathways for wastewater-impacted surface water, such as oxidative stress, DNA damage, and drug resistance. Samples strongly affecting particular pathways were identified by statistical analysis of gene expression. Transcription data were correlated with contamination data from chemical screening and percentages of wastewater in the samples. Samples with particular effects and outstanding chemical composition were analyzed. For these samples, hypotheses on the alteration of the transcription of genes involved in drug resistance and DNA repair attributable to the presence of pharmaceuticals were drawn.

Increasing production and applications of nano zinc oxide particles (nano-ZnO) enhances the probability of its exposure in occupational and environmental settings, but toxicity studies are still limited. Taking the free Zn ion (Zn(2+)) as a control, cytotoxicity of a commercially available nano-ZnO was assessed with a 6-h exposure in Escherichia coli (E. coli). The fitted dose-cytotoxicity curve for ZnCl2 was significantly sharper than that from nano-ZnO. Then, a genome-wide gene expression profile following exposure to nano-ZnO was conducted by use of a live cell reporter assay system with library of 1820 modified green fluorescent protein (GFP)-expressing promoter reporter vectors constructed from E. coli K12 strains, which resulted in 387 significantly altered genes in bacterial (p 0.001). These altered genes were enriched into ten biological processing and two cell components (p 0.05) terms through statistical hypergeometric testing, strongly suggesting that exposure to nano-ZnO would result a great disturbance on the functional gene product synthesis processing, such as translation, gene expression, RNA modification, and structural constituent of ribosome. The pattern of expression of 37 genes altered by nano-ZnO (fold change2) was different from the profile following exposure to 6 mg/L of free zinc ion. The result indicates that these two Zn forms might cause toxicity to bacterial in different modes of action. Our results underscore the importance of understanding the adverse effects elicited by nano-ZnO after entering aquatic environment.

Background Pathway alterations reflected as changes in gene expression regulation and gene interaction can result from cellular exposure to toxicants. Such information is often used to elucidate toxicological modes of action. From a risk assessment perspective, alterations in biological pathways are a rich resource for setting toxicant thresholds, which may be more sensitive and mechanism-informed than traditional toxicity endpoints. Here we developed a novel differential networks (DNs) approach to connect pathway perturbation with toxicity threshold setting. Methods Our DNs approach consists of 6 steps: time-series gene expression data collection, identification of altered genes, gene interaction network reconstruction, differential edge inference, mapping of genes with differential edges to pathways, and establishment of causal relationships between chemical concentration and perturbed pathways. A one-sample Gaussian process model and a linear regression model were used to identify genes that exhibited significant profile changes across an entire time course and between treatments, respectively. Interaction networks of differentially expressed (DE) genes were reconstructed for different treatments using a state space model and then compared to infer differential edges/interactions. DE genes possessing differential edges were mapped to biological pathways in databases such as KEGG pathways. Results Using the DNs approach, we analyzed a time-series Escherichia coli live cell gene expression dataset consisting of 4 treatments (control, 10, 100, 1000 mg/L naphthenic acids, NAs) and 18 time points. Through comparison of reconstructed networks and construction of differential networks, 80 genes were identified as DE genes with a significant number of differential edges, and 22 KEGG pathways were altered in a concentration-dependent manner. Some of these pathways were perturbed to a degree as high as 70% even at the lowest exposure concentration, implying a high sensitivity of our DNs approach. Conclusions Findings from this proof-of-concept study suggest that our approach has a great potential in providing a novel and sensitive tool for threshold setting in chemical risk assessment. In future work, we plan to analyze more time-series datasets with a full spectrum of concentrations and sufficient replications per treatment. The pathway alteration-derived thresholds will also be compared with those derived from apical endpoints such as cell growth rate.

Mixtures of naphthenic acids (NAs), which include cyclopentyl and cyclohexyl carboxylic acids, have been suggested to be toxic components in oils spills, effluents from the petrochemical industry and in oil sands process waters (OSPW). The present study demonstrated, for the first time, an application of a high throughput live bacterial cell array in a genome-scale investigation of the toxic mechanisms of environmental chemicals, a commercial NAs technical mixture extracted from crude oil. Real time gene profiling of time- and concentration- dependent responses of live cells exposed to NAs for three hours was conducted using a library of 1800 fluorescent transcriptional reporters for Escherichia coli (E. coli) growing in 384-well plates. The response patterns obtained after exposure to NAs suggested that the primary cellular responses were up-regulation of genes in the pentose phosphate pathway, involved in the molecular function of NADP or NADPH binding, and down-regulation of the ATP-binding cassette (ABC) transporter complex. Transcriptional networks that were significantly modulated by NAs included those that were regulated by transcriptional factors such as CRP-, RecA-, and GadE. Down-regulation of the SOS response pathway suggested that DNA damage might not be the direct result of NAs within the first three hours of exposure. However, CRP-dependent genes modulated by exposure to NAs indicated that the cellular level of cyclic AMP was altered immediately upon exposure of cells to NAs. Furthermore, the linear range of the concentration-response curve of the selected gene reporters encompassed a range of concentrations between 10 and 1000 mg NAs/L, which covers concentrations typically observed in the environment and makes this assay system ideal for the detection of environmental NAs.