Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic Escherichia coli (EHEC). Here we show that nonpathogenic E. coli K-12 grown in the presence of 2 M NE is also attracted to NE. Growth with NE induces transcription of genes encoding the tyramine oxidase, TynA, and the aromatic aldehyde dehydrogenase, FeaB, whose respective activities can, in principle, convert NE to 3,4-dihydroxymandelic acid (DHMA). Our results indicate that the apparent attractant response to NE is in fact chemotaxis to DHMA, which was found to be a strong attractant for E. coli. Only strains of E. coli K-12 that produce TynA and FeaB exhibited an attractant response to NE. We demonstrate that DHMA is sensed by the serine chemoreceptor Tsr and that the chemotaxis response requires an intact serine-binding site. The threshold concentration for detection is <5 nM DHMA, and the response is inhibited at DHMA concentrations above 50 M. Cells producing a heterodimeric Tsr receptor containing only one functional serine-binding site still respond like the wild type to low concentrations of DHMA, but their response persists at higher concentrations. We propose that chemotaxis to DHMA generated from NE by bacteria that have already colonized the intestinal epithelium may recruit E. coli and other enteric bacteria that possess a Tsr-like receptor to preferred sites of infection.T he human gastrointestinal (GI) tract harbors an assortment of bacteria, most of which are harmless or helpful commensals. However, infection of the GI tract by pathogenic bacteria can have devastating consequences. It has been suggested that norepinephrine (NE), the predominant neurotransmitter of the enteric sympathetic nervous system, promotes growth and virulence of enteric bacteria (1) through signaling via adrenergic receptors located either on intestinal epithelial cells (2) or in the bacteria themselves (3, 4). In particular, the bacterial quorum sensor kinase QseC has been implicated in the NE-induced expression of genes whose products are involved in adherence, motility, and pathogenesis (4, 5). However, the concentrations of NE required for effective induction of virulence genes, 50 M in one recent study (6), are higher than those that are expected to occur in the intestinal lumen (7,8). Thus, for NE to activate expression of virulence factors, bacteria would have to navigate to regions of the intestinal epithelium that have locally high concentrations of NE. An obvious candidate for directing such migration is chemotaxis.Chemotaxis in Escherichia coli is well understood at the molecular level. However, the compounds that have been reported as chemoattractants (9) are primarily nutrients: serine and related amino acids, sensed by the chemoreceptor Tsr; aspartate and maltose, sensed by Tar; ribose and galactose, sensed by Trg; and dipeptides and pyrimidines, sensed by Tap. NE has been reported to be an interdomain signaling molecule (5, 10, 11). NE serves as an inducer of v...

We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme selective activators for the recombinant form of human histone deacetylase-8 in the assay system containing Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature is manifested via decrease in the Km value of the enzyme’s substrate and increase in the catalytic turnover rate of the enzyme

Enterohemorrhagic (EHEC) is a commonly occurring foodborne pathogen responsible for numerous multistate outbreaks in the United States. It is known to infect the host gastrointestinal tract, specifically, in locations associated with lymphoid tissue. These niches serve as sources of enteric neurotransmitters, such as epinephrine and norepinephrine, that are known to increase virulence in several pathogens, including enterohemorrhagic The mechanisms that allow pathogens to target these niches are poorly understood. We previously reported that 3,4-dihydroxymandelic acid (DHMA), a metabolite of norepinephrine produced by , is a chemoattractant for the nonpathogenic RP437 strain. Here we report that DHMA is also a chemoattractant for EHEC. In addition, DHMA induces the expression of EHEC virulence genes and increases attachment to intestinal epithelial cells in a QseC-dependent manner. We also show that DHMA is present in murine gut fecal contents and that its production requires the presence of the commensal microbiota. On the basis of its ability to both attract and induce virulence gene expression in EHEC, we propose that DHMA acts as a molecular beacon to target pathogens to their preferred sites of infection.

The syntheses of a new class of barbiturate-based inhibitors for human and E. Coli Methionine Aminopeptidase -1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency. KeywordsType-1 MetAP inhibitors; barbiturates Cellular protein synthesis starts with an N-terminal methionine in the eukaryotic cells or with a formylmethionine in the prokaryotic cells. Removal of the methionine residue is essential for proper folding, post-translational modifications and translocation of the synthesized proteins. 1 The metalloenzymes methionine aminopeptidases (MetAPs) catalyze the hydrolytic removal of the N-terminal methionine residue from the newly synthesized proteins. 1 While the prokaryotic cells contain only the type-1 enzyme, eukaryotic cells contain both the type-1 and -2 enzymes in the cytosol. 2 Deletion of the MetAP is shown to be lethal in various bacteria. 2Since the discovery that the irreversible MetAP-2 inhibitor fumagillin prevents angiogenesis, both reversible and irreversible inhibitors for this enzyme have been extensively studied as potential anti-angiogenesis and anti-cancer agents. 3 Selective inhibition of MetAP-1 in bacteria (or in parasites) over the human enzymes has been shown to be a promising approach in designing new antibacterial 4 and antimalarial 5 agents. The role of MetAP-1 in human cell cycle progression has been elucidated recently. 6 Selective inhibition of the human MetAP-1 (over MetAP-2) led to the arrest of cell division and induction of apoptosis in leukemia cells. 6In contrast to MetAP-2, there are only a few reports in the literature on inhibition of human MetAP-1. Peptide hydroxamic acids are reported to be competitive inhibitors for human MetAP-1 with moderate potency. 7 Substituted pyridines were identified as another class of human MetAP-1 inhibitors using high-throughput screening of 175,000 small organic molecules. 3b Ovalicin, the highly-potent, non-competitive, natural product inhibitor for *Corresponding authors. Tel: +1 701 231 7888; fax: +1 701 231 8331; e-mail addresses: sanku.mallik@ndsu.edu; dk.srivastava@ndsu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Under physiological conditions, the metal ions in the active site of MetAPs are not firmly established. The active site binds to two divalent transition metal ions and the residues responsible for binding to the metal ions are conserved. 4 One of these two bound metal ions undergoes a rapid exchange with free metal ions in solution. 4 Since Co (II) ions activate all MetAPs, usually the inhibitors...

The detection of norepinephrine (NE) as a chemoattractant by strain K-12 requires the combined action of the TynA monoamine oxidase and the FeaB aromatic aldehyde dehydrogenase. The role of these enzymes is to convert NE into 3,4-dihydroxymandelic acid (DHMA), which is a potent chemoattractant sensed by the Tsr chemoreceptor. These two enzymes must be induced by prior exposure to NE, and cells that are exposed to NE for the first time initially show minimal chemotaxis toward it. The induction of TynA and FeaB requires the QseC quorum-sensing histidine kinase, and the signaling cascade requires new protein synthesis. Here, we demonstrate that the cognate response regulator for QseC, the transcription factor QseB, is also required for induction. The related quorum-sensing kinase QseE appears not to be part of the signaling pathway, but its cognate response regulator, QseF, which is also a substrate for phosphotransfer from QseC, plays a nonessential role. The promoter of the gene, which encodes a transcription factor that has been shown to be essential for the expression of and, has two predicted QseB-binding sites. One of these sites appears to be in an appropriate position to stimulate transcription from the P promoter of the gene. This study unites two well-known pathways: one for expression of genes regulated by catecholamines (QseBC) and one for expression of genes required for metabolism of aromatic amines (FeaR, TynA, and FeaB). This cross talk allows to convert the host-derived and chemotactically inert NE into the potent bacterial chemoattractant DHMA. The chemotaxis of K-12 to norepinephrine (NE) requires the conversion of NE to 3,4-dihydroxymandleic acid (DHMA), and DHMA is both an attractant and inducer of virulence gene expression for a pathogenic enterohemorrhagic (EHEC) strain. The induction of virulence by DHMA and NE requires QseC. The results described here show that the cognate response regulator for QseC, QseB, is also required for conversion of NE into DHMA. Production of DHMA requires induction of a pathway involved in the metabolism of aromatic amines. Thus, the QseBC sensory system provides a direct link between virulence and chemotaxis, suggesting that chemotaxis to host signaling molecules may require that those molecules are first metabolized by bacterial enzymes to generate the actual chemoattractant.