Contribution to Carbapenem Resistance and Fitness Cost of DcuS/DcuR, RcsC/RcsB, and YehU/YehT Two-Component Systems in CTX-M-15-Producing Escherichia coli.

Alteration in two-component systems (TCSs), which are signal transduction pathways in prokaryotes, can result in antibiotic resistance. Recently, it has been shown that the overexpression, using a multicopy cloning vector, of the dcuR, rcsB, and yehT genes, which code for the response regulator (RR) part of TCSs, enhanced the minimal inhibitory concentrations (MICs) of carbapenems in Escherichia coli K-12 derivative KAM3. Herein, the contribution to carbapenem resistance of the DcuS/DcuR, RcsC/RcsB, and YehU/YehT TCSs was assessed in E. coli K-12 derivative BW25113 (A phylogroup) and 536 (B2 phylogroup) recipient strains in combination with extended-spectrum ß-lactamase that exhibit a weak carbapenemase activity. The genes encoding both the sensor kinase (SK) and the RR, on the one hand, and the genes encoding the SK only, on the other hand, of these regulating pathways were disrupted. Subsequently, the mutants and their parental strains were transformed by a recombinant plasmid encoding the CTX-M-15 gene, before testing their susceptibility to carbapenems and their fitness. Results showed a trade-off between enhanced MICs for ertapenem, which remained above the clinical resistance breakpoint, and decreased growth rate, specifically for the 536 strain SK mutants. In conclusion, mutations in dcuS/dcuR, rcsC/rcsB, and yehU/yehT genes may be a pivotal first-step event in the development of carbapenem resistance.

Global assessment of its network dynamics reveals that the kinase Plk1 inhibits the phosphatase PP6 to promote Aurora A activity.

Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. The subcellular localization and substrate interactions of Plk1 are tightly controlled and require its binding to phosphorylated residues. To identify phosphorylation-dependent interactions within the Plk1 network in human mitotic cells, we performed quantitative proteomics on HeLa cells cultured with kinase inhibitors or expressing a Plk1 mutant that was deficient in phosphorylation-dependent substrate binding. We found that many interactions were abolished upon kinase inhibition; however, a subset was protected from phosphatase opposition or was unopposed, resulting in persistent interaction of the substrate with Plk1. This subset includes phosphoprotein phosphatase 6 (PP6), whose activity toward Aurora kinase A (Aurora A) was inhibited by Plk1. Our data suggest that this Plk1-PP6 interaction generates a feedback loop that coordinates and reinforces the activities of Plk1 and Aurora A during mitotic entry and is terminated by the degradation of Plk1 during mitotic exit. Thus, we have identified a mechanism for the previously puzzling observation of the Plk1-dependent regulation of Aurora A.

Rapid detection of protein phosphatase activity using Zn(II)-coordinated gold nanosensors based on His-tagged phosphopeptides.

We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution. In contrast, unphosphorylated peptides and His6-deficient phosphopeptides did not change the color of the AuNP solution. As a result, protein phosphatase 1 (PP1) activity and its inhibition were easily quantified with high sensitivity by determining the extinction ratio (E520/E700) of colloidal AuNPs. Most importantly, this method was capable of detecting protein phosphatase 2A (PP2A) activity in immunoprecipitated plant extracts. Because PPs play pivotal roles in mediating diverse signal transduction pathways as primary effectors of protein dephosphorylation, we anticipate that our method will be applied as a rapid format method to analyze the activities of various PPs and their inhibition.

First transcriptomic analysis of the economically important parasitic nematode, Trichostrongylus colubriformis, using a next-generation sequencing approach.

Trichostrongylus colubriformis (Strongylida), a small intestinal nematode of small ruminants, is a major cause of production and economic losses in many countries. The aims of the present study were to define the transcriptome of the adult stage of T. colubriformis, using 454 sequencing technology and bioinformatic analyses, and to predict the main pathways that key groups of molecules are linked to in this nematode. A total of 21,259 contigs were assembled from the sequence data produced from a normalized cDNA library; 7876 of these contigs had known orthologues in the free-living nematode Caenorhabditis elegans, and encoded, amongst others, proteins with 'transthyretin-like' (8.8%), 'RNA recognition' (8.4%) and 'metridin-like ShK toxin' (7.6%) motifs. Bioinformatic analyses inferred that relatively high proportions of the C. elegans homologues are involved in biological pathways linked to 'peptidases' (4%), 'ribosome' (3.6%) and 'oxidative phosphorylation' (3%). Highly represented were peptides predicted to be associated with the nervous system, digestion of host proteins or inhibition of host proteases. Probabilistic functional gene networking of the complement of C. elegans orthologues (n=2126) assigned significance to particular subsets of molecules, such as protein kinases and serine/threonine phosphatases. The present study represents the first, comprehensive insight into the transcriptome of adult T. colubriformis, which provides a foundation for fundamental studies of the molecular biology and biochemistry of this parasitic nematode as well as prospects for identifying targets for novel nematocides. Future investigations should focus on comparing the transcriptomes of different developmental stages, both genders and various tissues of this parasitic nematode for the prediction of essential genes/gene products that are specific to nematodes.

Drosophila notal bristle as a novel assessment tool for pathogenic study of Tau toxicity and screening of therapeutic compounds.

To elucidate the Tau gain-of-toxicity functional mechanism and to search for potential treatments, we overexpressed human Tau variants (hTau) in the dorsal mesothorax (notum) of Drosophila. Overexpression of Tau variants caused loss of notal bristles, and the phenotype was used for evaluating toxicity of ectopic Tau. The bristle loss phenotype was found to be highly associated with the toxicity of hyperphosphoryled Tau in flies. We have shown that the bristle loss phenotype can be rescued either by reducing Glycogen synthase kinase 3beta (GSK3beta)/Shaggy (Sgg) activity or overexpressing Bbeta2 regulatory subunits of PP2A. Elevated expression of the Drosophila Bbeta2 homolog, Twins (Tws), also alleviated neuritic dystrophy of the dorsal arborization (da) neuron caused by Tau aggregation. Additionally, lowering endogenous Tau dosage was beneficial as it ameliorated the bristle loss phenotype. Finally, the bristle loss phenotype was used to evaluate the efficacy of potential therapeutic compounds. The GSK3beta inhibitor, alsterpaullone, was found to suppress toxicity of Tau in a concentration-dependent manner. The notum of Drosophila, thus, provides a new tool and insights into Tau-induced toxicity. It could also potentially assist in screening new drugs for possible therapeutic intervention.

Sigma A recognition sites in the Bacillus subtilis genome.

A hidden Markov model of sigma(A) RNA polymerase cofactor recognition sites in Bacillus subtilis, containing either the common or the extended -10 motifs, has been constructed based on experimentally verified sigma(A) recognition sites. This work suggests that more information exists at the initiation site of transcription in both types of promoters than previously thought. When tested on the entire B. subtilis genome, the model predicts that approximately half of the sigma(A) recognition sites are of the extended type. Some of the response-regulator aspartate phosphatases were among the predictions of promoters containing extended sites. The expression of rapA and rapB was confirmed by site-directed mutagenesis to depend on the extended -10 region.

Cell behaviour as a dynamic attractor in the intracellular signalling system.

We present a model of the cell signalling network based on the generic properties of interactions between protein kinases (PKs) and protein phosphatases (PPs) inside cells. The model is designed to examine the global properties and intrinsic dynamics of the phosphorylation system. A genetic algorithm (GA) is used to evolve populations of "cells". The GA selects cells and ranks them based on an analysis of the dynamics of the proteins within the networks from a series of different random starting conditions. The fittest cells are taken to be those which can generate a variety of different "behaviours" from a series of different initial conditions. During the GA, intracellular protein interactions evolve via mutation and an analogue of domain shuffling between protein types that is thought to occur during biological evolution. The dynamics of the simulated networks are presented and we discuss the hypothesis that changes in the behaviour of a cell may be interpretable as a switch between attractor basins in the intracellular signalling network.

Energetic cost of activation processes during contraction of swine arterial smooth muscle.

1. The objective of this study was to partition the increase in ATP consumption during contraction of swine carotid arterial smooth muscle estimated from suprabasal oxygen consumption (suprabasal JO2) and lactate release (Jlactate) into a component associated with cross-bridge cycling (JX) and one reflecting activation (JA). 2. Two experimental approaches-varying length under constant activation, and varying activation at a long length (1.8 times the optimal length for force development (Lo)) where force generation is minimal-revealed a linear dependence of JO2 and activation energy (JA) on cross-bridge phosphorylation. Protocols inducing a large increase in myosin regulatory light chain (MRLC) phosphorylation at 1.8 Lo resulted in significant elevations of JO2 and marked reductions in the economy of force maintenance. Our evidence suggests that this is primarily due to the increased cost of cross-bridge phosphorylation. 3. The extrapolated estimate of JA during maximal K(+)-induced depolarization made by varying length was 16%, while at 1.8 Lo it was 33% of the suprabasal JO2 at Lo. Calculated activation energies ranged from 17 to 45% of the suprabasal JO2 at Lo and from 72 to 87% of the suprabasal JO2 at 1.8 Lo under stimulation conditions that varied steady-state MRLC phosphorylation from 15 to 50%. 4. The results suggest that the kinetics of cross-bridge phosphorylation-dephosphorylation can rival those of cross-bridge cycling during isometric contractions in swine arterial smooth muscle.

Overexpression and purification of human calcineurin alpha from Escherichia coli and assessment of catalytic functions of residues surrounding the binuclear metal center.

Calcineurin is an important signal-transducing enzyme in many cell types including T lymphocytes and is a common target for the immunosuppressants cyclosporin A and FK506. The crystal structures of both calcineurin [Griffith et al. (1995) Cell 82, 507-522; Kissinger et al. (1995) Nature 378, 641-644] and a related enzyme, protein phosphatase-1 [Goldberg et al. (1995) Nature 376, 745-753], revealed that this class of serine/threonine phosphatases contain in their putative active sites a binuclear metal center formed by an Asn, two Asp, and three His residues. In addition, one His and two Arg residues lie in close vicinity of the binuclear metal centers. The importance of the binuclear metal center and its surrounding residues in catalysis by calcineurin has not been investigated experimentally. Herein, we report an efficient bacterial expression and purification system for human calcineurin alpha. Using this system, a systematic alanine-scan mutagenesis on the residues surrounding the putative active site was performed. It was found that an intact binuclear metal center is essential for the catalytic activity of the enzyme. In addition, His151, Arg122, and Arg254 also exhibited either a loss or a dramatic decrease in catalytic activity upon mutation into alanines. Interestingly, the Arg254Ala mutant retained a small but significant amount of catalytic activity toward the small substrate p-nitrophenyl phosphate, but is completely inactive toward a phosphopeptide substrate, suggesting that this arginine may be involved in the binding of phosphoprotein substrates as well as in catalysis. As all the residues in the putative active site are conserved between different eukaryotic serine/threonine phosphatases, these results should apply to all members of this family of protein phosphatases.

A molecular basis for different interactions of marine toxins with protein phosphatase-1. Molecular models for bound motuporin, microcystins, okadaic acid, and calyculin A.

The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and -2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and -LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.

A dynamical model of the distributed interaction of intracellular signals.

The behaviour of cells is coordinated by a large array of different types of intracellular protein kinases, protein phosphatases and second messenger molecules. Current experimental evidence suggests that these kinases, phosphatases and other messengers form a vast complicated interconnected signalling network inside the cell. A generic model of this signalling network is presented which is specifically designed to examine the global properties that can be expected from a model based on the available knowledge of these molecular interactions. Different protein types are represented as nodes in a network that interact via a connection matrix. During interactions, idealised kinases and phosphatases activate and deactivate other protein types by altering the level of phosphorylation of their regulatory sites. The occupancy of regulatory sites on protein kinases and phosphatases in turn determines their activity. Monte Carlo simulations are carried out on ensembles of networks. Steady states and periodic behaviour are observed in these networks. We discuss the potential of this type of model for understanding cell behaviour.