Microbiological survey and genomic analysis of Cronobacter sakazakii strains isolated from US households and retail foods.

Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

Cyclic-disulfide-based prodrugs for cytosol-specific drug delivery.

The cytosolic conversion of therapeutically relevant nucleosides into bioactive triphosphates is often hampered by the inefficiency of the first kinase-mediated step. Nucleoside monophosphate prodrugs can be used to bypass this limitation. Herein we describe a novel cyclic-disulfide class of nucleoside monophosphate prodrugs with a cytosol-specific, reductive release trigger. The key event, a charge-dissipating reduction-triggered cyclodeesterification leads to robust cytosolic production of the cyclic 3',5'-monophosphate for downstream enzymatic processing. The antiviral competence of the platform was demonstrated with an O-benzyl-1,2-dithiane-4,5-diol ester of 2'-C-methyluridine-3',5'-phosphate. Both in vitro and in vivo comparison with the clinically efficacious ProTide prodrug of 2'-deoxy-2'-α-fluoro-ß-C-methyluridine is provided. The cytosolic specificity of the release allows for a wide range of potential applications, from tissue-targeted drug delivery to intracellular imaging.

Application of a Rat Liver Drug Bioactivation Transcriptional Response Assay Early in Drug Development That Informs Chemically Reactive Metabolite Formation and Potential for Drug-induced Liver Injury.

Drug-induced liver injury is a major reason for drug candidate attrition from development, denied commercialization, market withdrawal, and restricted prescribing of pharmaceuticals. The metabolic bioactivation of drugs to chemically reactive metabolites (CRMs) contribute to liver-associated adverse drug reactions in humans that often goes undetected in conventional animal toxicology studies. A challenge for pharmaceutical drug discovery has been reliably selecting drug candidates with a low liability of forming CRM and reduced drug-induced liver injury potential, at projected therapeutic doses, without falsely restricting the development of safe drugs. We have developed an in vivo rat liver transcriptional signature biomarker reflecting the cellular response to drug bioactivation. Measurement of transcriptional activation of integrated nuclear factor erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1) electrophilic stress, and nuclear factor erythroid 2-related factor 1 (NRF1) proteasomal endoplasmic reticulum (ER) stress responses, is described for discerning estimated clinical doses of drugs with potential for bioactivation-mediated hepatotoxicity. The approach was established using well benchmarked CRM forming test agents from our company. This was subsequently tested using curated lists of commercial drugs and internal compounds, anchored in the clinical experience with human hepatotoxicity, while agnostic to mechanism. Based on results with 116 compounds in short-term rat studies, with consideration of the maximum recommended daily clinical dose, this CRM mechanism-based approach yielded 32% sensitivity and 92% specificity for discriminating safe from hepatotoxic drugs. The approach adds new information for guiding early candidate selection and informs structure activity relationships (SAR) thus enabling lead optimization and mechanistic problem solving. Additional refinement of the model is ongoing. Case examples are provided describing the strengths and limitations of the approach.

The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation.

Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal "Neh3" domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity.

The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress.

The induction of many antioxidant and phase II drug-metabolizing enzymes by phenolic antioxidants and electrophilic compounds is regulated at the transcriptional level. The response to these compounds is mediated by the cis-acting antioxidant response element (ARE) found in the promoter of the encoding genes. The transcription factor NF-E2-related factor 2, or Nrf2, has emerged as the central protein that binds to the ARE to activate gene transcription. Data from many studies indicate that Nrf2 is constitutively and ubiquitously expressed in a number of tissues and cell lines and is thus responsible for the low-level expression of its target genes observed under physiological conditions. However, in cells exposed to oxidative stress, Nrf2 activity is increased, further driving the transcriptional activation of genes whose expression is essential to control cellular redox homeostasis. Recent studies suggest that the activation of Nrf2 involves a coordinated process and is regulated at multiple levels. Nrf2 activity is believed to be repressed through the binding of the cytoskeleton-associated protein Keap1, and its activation involves mechanisms that interfere with this interaction. Activation of Nrf2 has also been demonstrated to be dependent on mechanisms that mediate its stabilization. In this review, the mechanisms controlling this activation process as reported in recent studies will be examined and discussed, with particular emphasis on those affecting Nrf2 stability at the molecular level.

Prevalence and counts of Salmonella and enterohemorrhagic Escherichia coli in raw, shelled runner peanuts.

Three major outbreaks of salmonellosis linked to consumption of peanut butter during the last 6 years have underscored the need to investigate the potential sources of Salmonella contamination in the production process flow. We conducted a study to determine the prevalence and levels of Salmonella in raw peanuts. Composite samples (1,500 g, n = 8) of raw, shelled runner peanuts representing the crop years 2009, 2010, and 2011 were drawn from 10,162 retained 22-kg lot samples of raw peanuts that were negative for aflatoxin. Subsamples (350 g) were analyzed for the presence of Salmonella and enterohemorrhagic Escherichia coli. Salmonella was found in 68 (0.67%) of 10,162 samples. The highest prevalence rate (P < 0.05) was for 2009 (1.35%) compared with 2010 (0.36%) and 2011 (0.14%). Among four runner peanut market grades (Jumbo, Medium, No. 1, and Splits), Splits had the highest prevalence (1.46%; P < 0.05). There was no difference (P > 0.05) in the prevalence by region (Eastern versus Western). Salmonella counts in positive samples (most-probable-number [MPN] method) averaged 1.05 (range, 0.74 to 5.25) MPN per 350 g. Enterohemorrhagic E. coli was found in only three samples (0.030%). Typing of Salmonella isolates showed that the same strains found in Jumbo and Splits peanuts in 2009 were also isolated from Splits in 2011. Similarly, strains isolated in 2009 were also isolated in 2010 from different peanut grades. These results indicated the persistence of environmental sources throughout the years. For five samples, multiple isolates were obtained from the same sample that had different pulsed-field gel electrophoresis types. This multistrain contamination was primarily observed in Splits peanuts, in which the integrity of the kernel is usually compromised. The information from the study can be used to develop quantitative microbial risk assessments models.

The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress.

A major mechanism in the cellular defense against oxidative or electrophilic stress is activation of the Nrf2-antioxidant response element signaling pathway, which controls the expression of genes whose protein products are involved in the detoxication and elimination of reactive oxidants and electrophilic agents through conjugative reactions and by enhancing cellular antioxidant capacity. At the molecular level, however, the regulatory mechanisms involved in mediating Nrf2 activation are not fully understood. It is well established that Nrf2 activity is controlled, in part, by the cytosolic protein Keap1, but the nature of this pathway and the mechanisms by which Keap1 acts to repress Nrf2 activity remain to be fully characterized and are the topics of discussion in this minireview. In addition, a possible role of the Nrf2-antioxidant response element transcriptional pathway in neuroprotection will also be discussed.

A mutation of Keap1 found in breast cancer impairs its ability to repress Nrf2 activity.

Keap1 is the substrate recognition module of a Cullin 3-based E3 ubiquitin ligase. Its primary role is to catalyze the ubiquitylation of the Nrf2 transcription factor. Oxidative stress blocks the E3 ligase activity of Keap1 which stabilizes Nrf2 allowing it to drive the expression of certain antioxidant and drug metabolizing enzymes. A recent study identified a mutation in the Keap1 gene (Keap1C23Y) that is present in breast cancer. Using reporter gene assays we show that Keap1C23Y is impaired in its ability to repress Nrf2 dependent transcription. Unlike wild-type Keap1, we found that Keap1C23Y failed to stimulate the degradation of Nrf2. Co-immunopreciptation experiments showed that Keap1C23Y retains its ability to interact with Nrf2 and Cullin 3. In contrast, we found that Keap1C23Y could not efficiently promote the ubiquitylation of Nrf2, suggesting that its intrinsic biological activity might have been compromised. These results revealed an unexpected role for the N-terminal region of Keap1 in regulating its E3 ligase activity. Importantly, our findings suggest that a paradox exists whereby Nrf2 activity is beneficial in non-malignant cells but in cancer cells it may provide a selective advantage for clonal expansion.

Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1.

Nrf2 regulates the expression of genes encoding antioxidant proteins involved in cellular redox homeostasis. Previous studies have suggested that activation of Nrf2 is mediated by mechanisms promoting its dissociation from Keap1, a cytosolic repressor that acts to sequester the transcription factor in the cytoplasm. As a short-lived protein, Nrf2 is also activated by mechanisms leading to its stabilization in cells under stress, and recent evidence indicates that Keap1 has an active role in the control of its stability. In this report, using immunocytochemistry, cell fractionation, and chromatin immunoprecipitation analyses, we found that Nrf2 is primarily a nuclear protein and that it is expressed and recruited to the chromatin constitutively to drive basal gene expression. Furthermore, we found evidence indicating that Keap1 may repress Nrf2 activity by transiently shuttling into the nucleus to promote its ubiquitylation. The data suggested that the steady-state level of Nrf2 is maintained by a dynamic pathway that balances its constitutive expression with a Keap1-regulated degradation process downstream of its role as a transcriptional activator. We suggest that the stabilization of Nrf2 in cells under stress represents the central regulatory response mediated by mechanisms that interfere with its interaction with Keap1, leading to the induction of antioxidant enzymes important to maintain cellular redox homeostasis.

Regulatory mechanisms controlling gene expression mediated by the antioxidant response element.

The expression of genes encoding antioxidative and Phase II detoxification enzymes is induced in cells exposed to electrophilic compounds and phenolic antioxidants. Induction of these enzymes is regulated at the transcriptional level and is mediated by a specific enhancer, the antioxidant response element or ARE, found in the promoter of the enzyme's gene. The transcription factor Nrf2 has been implicated as the central protein that interacts with the ARE to activate gene transcription constitutively or in response to an oxidative stress signal. This review focuses on the molecular mechanisms whereby the transcriptional activation mediated by the interaction between the ARE and NF-E2-related factor 2 (Nrf2) is regulated. Recent studies suggest that the sequence context of the ARE, the nature of the chemical inducers, and the cell type are important for determining the activity of the enhancer in a particular gene.

CYP1A1 induction by pyridine and its metabolites in HepG2 cells.

Pyridine and its metabolites have been shown in previous studies to induce cytochrome P4501A1 (CYP1A1) expression in vivo in the rat and in vitro in cultured human lung explants. In this study, we assessed the role of the metabolites in CYP1A1 induction by the parent compound. This was accomplished by comparing pyridine, 2-hydroxypyridine, 3-hydroxypyridine, pyridine N-oxide, and N-methylpyridinium in terms of the induction of CYP1A1 mRNA, CYP1A1 catalytic activity, and a xenobiotic response element-directed chloramphenicol acetyltransferase reporter gene, using HepG2 cells as the experimental system. We also assessed the effect of expression of the pyridine-metabolizing enzyme cytochrome P4502E1 on CYP1A1 induction by the parent pyridine. Only 2-hydroxypyridine significantly induced the CYP1A1 mRNA expression and CYP1A1-preferential activity ethoxyresorufin O-deethylase in wild-type HepG2 cells. Similarly, only 2-hydroxypyridine induced the expression of a xenobiotic response element-directed reporter gene in transfected HepG2 cells. Pyridine elevated CYP1A1 mRNA abundance 4.6-fold in HepG2 cells transfected with a human CYP2E1 expression vector relative to the abundance of the transcript in empty vector-transfected (control) HepG2 cells; the elevation was inhibited by the CYP2E1 inhibitor dimethyl sulfoxide. The results indicate that CYP1A1 induction by pyridine is mediated largely by metabolites, the formation of which may be catalyzed by CYP2E1.

Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.

Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response.

Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome.

Nrf2 (NF-E2-related factor 2) is a central transcription factor involved in the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes via the antioxidant response element. Nrf2 has previously been found to undergo nuclear translocation by a phosphorylation-dependent mechanism mediated by protein kinase C in HepG2 cells treated with tert-butylhydroquinone, beta-naphthoflavone, or 12-O-tetradecanoylphorbol-13-acetate. In the present report, we have found that the levels of Nrf2 were increased in cells treated with tert-butylhydroquinone or beta-naphthoflavone by a post-transcriptional mechanism. Treatment of HepG2 cells with cycloheximide resulted in the loss of Nrf2 within 30 min. By contrast, treatment with the proteasome inhibitors (lactacystin or MG-132) caused an accumulation of Nrf2 as well as an induction of reporter gene activity in cells transfected with the GSTA2 antioxidant response element-chloramphenicol acetyl transferase construct. Similarly, the protein phosphatase inhibitor okadaic acid also caused an accumulation of Nrf2, whereas the reverse effects were observed with PD 98059 and U 0126, two compounds that block the activation of the MAPK/ERK signaling cascade. These data suggest that Nrf2 is degraded by the ubiquitin-dependent pathway and that phosphorylation of Nrf2 leads to an increase in its stability and subsequent transactivation activity.