Molecular Mechanism for Attractant Signaling to DHMA by E. coli Tsr.

The attractant chemotaxis response of Escherichia coli to norepinephrine requires that it be converted to 3,4-dihydroxymandelic acid (DHMA) by the monoamine oxidase TynA and the aromatic aldehyde dehydrogenase FeaB. DHMA is sensed by the serine chemoreceptor Tsr, and the attractant response requires that at least one subunit of the periplasmic domain of the Tsr homodimer (pTsr) has an intact serine-binding site. DHMA that is generated in vivo by E. coli is expected to be a racemic mixture of the (R) and (S) enantiomers, so it has been unclear whether one or both chiral forms are active. Here, we used a combination of state-of-the-art tools in molecular docking and simulations, including an in-house simulation-based docking protocol, to investigate the binding properties of (R)-DHMA and (S)-DHMA to E. coli pTsr. Our studies computationally predicted that (R)-DHMA should promote a stronger attractant response than (S)-DHMA because of a consistently greater-magnitude piston-like pushdown of the pTsr α-helix 4 toward the membrane upon binding of (R)-DHMA than upon binding of (S)-DHMA. This displacement is caused primarily by interaction of DHMA with Tsr residue Thr156, which has been shown by genetic studies to be critical for the attractant response to L-serine and DHMA. These findings led us to separate the two chiral species and test their effectiveness as chemoattractants. Both the tethered cell and motility migration coefficient assays validated the prediction that (R)-DHMA is a stronger attractant than (S)-DHMA. Our study demonstrates that refined computational docking and simulation studies combined with experiments can be used to investigate situations in which subtle differences between ligands may lead to diverse chemotactic responses.

A Static Microfluidic Device for Investigating the Chemotaxis Response to Stable, Non-linear Gradients.

Microfluidic technology allows fast and precise measurement of chemotaxis responses to both attractant and repellent signals. One of the major drawbacks of current microfluidic chemotaxis assays is the presence of bacterial cells within the concentration gradient flow field, which has the potential for flow effects masking the chemotaxis response. This chapter describes a new microfluidic device for producing stable concentration gradients and measuring the response of cells to the gradient without exposing them to any flow. Unlike other methods described in the literature, this method is capable of producing gradients of any shape, almost instantaneously, allowing the measurement of time-dependent response of cells to a variety of signals.

Conversion of Norepinephrine to 3,4-Dihdroxymandelic Acid in Escherichia coli Requires the QseBC Quorum-Sensing System and the FeaR Transcription Factor.

The detection of norepinephrine (NE) as a chemoattractant by Escherichia coli 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 feaR gene, which encodes a transcription factor that has been shown to be essential for the expression of tynA and feaB, has two predicted QseB-binding sites. One of these sites appears to be in an appropriate position to stimulate transcription from the P1 promoter of the feaR 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 E. coli to convert the host-derived and chemotactically inert NE into the potent bacterial chemoattractant DHMA.IMPORTANCE The chemotaxis of E. coli 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 E. coli (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.

The Norepinephrine Metabolite 3,4-Dihydroxymandelic Acid Is Produced by the Commensal Microbiota and Promotes Chemotaxis and Virulence Gene Expression in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (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 E. coli 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 E. coli, is a chemoattractant for the nonpathogenic E. coli 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 vitro 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 in vivo.

Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.

BACKGROUND: Methionyl-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the Escherichia coli methionine aminopeptidase (MetAP) catalyzed reaction, and is routinely used for screening compounds to identify potential antibiotic agents. In pursuit of screening the enzyme's inhibitors, we observed that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequestration of the substrate by HP-ß-CD. METHODS: The mechanistic path for the HP-ß-CD mediated inhibition of MetAP was probed by performing a detailed account of steady-state kinetics, ligand binding, X-ray crystallographic, and molecular modeling studies. RESULTS: X-ray crystallographic data of the ß-cyclodextrin-substrate (ß-CD-MetAMC) complex reveal that while the AMC moiety of the substrate is confined within the CD cavity, the methionine moiety protrudes outward. The steady-state kinetic data for inhibition of MetAP by HP-ß-CD-MetAMC conform to a model mechanism in which the substrate is "bridged" between HP-ß-CD and the enzyme's active-site pocket, forming HP-ß-CD-MetAMC-MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-ß-CD-bound MetAMC substrate does not reach within the proximity of the enzyme's catalytic metal center, and thus the substrate fails to undergo cleavage. CONCLUSIONS: The data presented herein suggests that the bridging of the substrate between the enzyme and HP-ß-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity. GENERAL SIGNIFICANCE: Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-ß-CD mediated delivery of drugs under pathophysiological conditions.

Chemotaxis of Escherichia coli to norepinephrine (NE) requires conversion of NE to 3,4-dihydroxymandelic acid.

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 µM 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 µM. 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.

Probing the metal ion selectivity in methionine aminopeptidase via changes in the luminescence properties of the enzyme bound europium ion.

We report herein, for the first time, that Europium ion (Eu(3+)) binds to the "apo" form of Escherichia coli methionine aminopeptidase (EcMetAP), and such binding results in the activation of the enzyme as well as enhancement in the luminescence intensity of the metal ion. Due to competitive displacement of the enzyme-bound Eu(3+) by different metal ions, we could determine the binding affinities of both "activating" and "non-activating" metal ions for the enzyme via fluorescence spectroscopy. The experimental data revealed that among all metal ions, Fe(2+) exhibited the highest binding affinity for the enzyme, supporting the notion that it serves as the physiological metal ion for the enzyme. However, the enzyme-metal binding data did not adhere to the Irving-William series. On accounting for the binding affinity vis a vis the catalytic efficiency of the enzyme for different metal ions, it appears evident that that the "coordination states" and the relative softness" of metal ions are the major determinants in facilitating the EcMetAP catalyzed reaction.

Histone deacetylase activators: N-acetylthioureas serve as highly potent and isozyme selective activators for human histone deacetylase-8 on a fluorescent substrate.

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 K(m) value of the enzyme's substrate and increase in the catalytic turnover rate of the enzyme.

Synthesis of barbiturate-based methionine aminopeptidase-1 inhibitors.

The syntheses of a new class of barbiturate-based inhibitors for human and Escherichia Coli methionine aminopeptidase-1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency.

Alternative Modes of Binding of Recombinant Human Histone Deacetylase 8 to Colloidal Gold Nanoparticles.

Histone deacetylases are intimately involved in the transcriptional regulation of genes, and they are high priority drug targets for cancer therapy. Due to prevalence of several sulfhydryl groups on the surface of histone deacetylase 8, we explored the possibility of its binding to colloidal gold nanoparticles by determining its potentials to inhibit the flocculation as well as retaining the enzyme activity. It was observed that although both these processes conformed to the binding affinity of the gold-histone deacetylase 8 conjugate as being equal to 15-20 nM, only 30% of the nanoparticle-bound enzyme exhibited the enzymatic activity. In the light of the structural features of histone deacetylase 8, we propose that the enzyme interacts with the gold nanoparticles via the surface exposed thiol groups, and such interaction occurs in two alternative modes. Whereas the enzyme bound via mode-1 is catalytically inactive (presumably due to the orientation of the enzyme's active site toward the gold nanoparticle surface), and it prevents the flocculation of the nanoparticles, the enzyme bound via mode-2 shows the full catalytic activity (as its active site is believed to be oriented away from the nanoparticle surface). Although the histone deacetylase 8 bound to AuNP via mode-2 exhibits the same inhibitory potency against Trichostatin A as the free enzyme, the former is more susceptible to thermal denaturation. The potential of potent interaction between gold nanoparticles and histone deacetylase 8 via alternative modes may find diagnostic and/or therapeutic applications for different forms of cancers.