Comparative Genome-Scale Metabolic Reconstruction and Flux Balance Analysis of Multiple Staphylococcus aureus Genomes Identify Novel Antimicrobial Drug Targets

Mortality due to multidrug-resistant Staphylococcus aureus infection is predicted to surpass that of human immunodeficiency virus/AIDS in the United States. Despite the various treatment options for S. aureus infections, it remains a major hospital- and community-acquired opportunistic pathogen. With the emergence of multidrug-resistant S. aureus strains, there is an urgent need for the discovery of new antimicrobial drug targets in the organism. To this end, we reconstructed the metabolic networks of multidrug-resistant S. aureus strains using genome annotation, functional-pathway analysis, and comparative genomic approaches, followed by flux balance analysis-based in silico single and double gene deletion experiments. We identified 70 single enzymes and 54 pairs of enzymes whose corresponding metabolic reactions are predicted to be unconditionally essential for growth. Of these, 44 single enzymes and 10 enzyme pairs proved to be common to all 13 S. aureus strains, including many that had not been previously identified as being essential for growth by gene deletion experiments in S. aureus. We thus conclude that metabolic reconstruction and in silico analyses of multiple strains of the same bacterial species provide a novel approach for potential antibiotic target identification.Staphylococcus aureus is a major hospital/community-acquired opportunistic pathogen. It causes bacteremia, pneumonia, endocarditis, meningitis, and toxic-shock syndrome in adult humans; skin lesions, impetigo, and abscesses in children; and mastitis in cattle (7, 22, 27). In general, S. aureus infections are treated with β-lactam antibiotics, sulfa drugs, tetracycline, and clindamycin. However, drug-resistant strains, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have emerged from both hospital and community infections in recent years. To date, only one new drug candidate, platensimycin, has been found to be effective against some strains of MRSA and VRSA (30). A recent meta-analysis suggested that mortality due to multidrug-resistant S. aureus in the United States may exceed that from human immunodeficiency virus infections and AIDS (19). This has resulted in a renewed interest in identifying new targets and molecules effective against multidrug-resistant strains of bacteria, and S. aureus in particular.Based on whole-genome sequence comparisons, S. aureus strains can be divided into three divergent groups arising from a common lineage (11). Significant sequence variations between animal and human S. aureus strains have also been identified (15). Though many virulence and drug resistance markers have been studied, the cause of the continuous emergence of multidrug-resistant strains remains elusive, as the resistance phenotype is not attributable to a few studied genes. Combining the data from multilocus sequence typing, microarray analysis, sequence relationships, homologous recombination, and phages of S. aureus, two major groups of clonal strains have been identified (11). A similar conclusion was reached when the S. aureus Newman genome sequence was compared to those of 11 other S. aureus strains (3). These studies not only confirm the clonality of the genome, but also reveal that nearly 20% of the sequence variations are due to prophages and pathogenicity islands.In order to further refine a generic antimicrobial drug target identification scheme (2), we performed metabolic reconstructions of multidrug-resistant and sensitive strains of S. aureus. This was feasible, as the genome sequences of 13 S. aureus strains are now available. They include strain N315 (a MRSA strain), Mu50 (a VRSA strain), JH9 (a vancomycin-nonsusceptible MRSA strain), JH1 (a vancomycin-susceptible, hospital-acquired MRSA strain), COL (a hospital-acquired MRSA strain), 252 (a hospital-acquired MRSA strain), USA 300 (a community-acquired MRSA strain), MW2 (a community-acquired MRSA strain), and RF122 (a bovine mastitis strain).Previous efforts in the metabolic reconstruction and subsequent flux balance analysis (FBA) of a single S. aureus strain (N315) provided valuable but limited insight into the metabolic capabilities of the bacterium (4, 14). Using this strain (20), Becker and Palsson predicted 518 metabolic reactions and 571 metabolites based on a limited set of genes (enzymes) (4). Their study also identified the components of minimal growth medium for S. aureus. Of the six required amino acids, only four (l-alanine, l-arginine, l-proline, and l-glycine) were common to both experimental and computational studies. Glucose (carbon source), phosphate, sulfate, nicotinamide, and thiamine were both experimentally utilized and computationally verified. However, other substrates, such as the nucleosides cytidine and uridine, were predicted not to be required in their metabolic model. A second genome-scale reconstruction of the same strain based on the KEGG ligand database was carried out and yielded 774 metabolic reactions catalyzed by 394 unique enzymes (13). Heinemann et al. (14) also validated their reconstruction using published experimental data and further defined a biomass composition for S. aureus.To reconcile the results of these two previous reconstructions and to address the differences in the metabolic capabilities of various S. aureus strains, we employed comprehensive genomic and metabolic reconstruction methodologies using the ERGO bioinformatics suite (24). This approach enabled us to identify the functional pathways, metabolic reactions, and transport reactions of several sequenced strains of S. aureus. The identified metabolic pathways and their individual reactions were systematically compared with those archived in the KEGG ligand database (17). FBA of such reconstructed metabolic networks have allowed in silico single and double gene deletion experiments, e.g., in Escherichia coli (2, 8, 23, 29). The application of these methods has led us to the identification of single enzymes and synthetic enzyme pairs that are unconditionally required for the growth (biomass production) of all S. aureus strains.     

Modelling pulse wave propagation in the rabbit systemic circulation to assess the effects of altered nitric oxide synthesis

Pulse wave propagation in the mature rabbit systemic circulation was simulated using the one-dimensional equations of blood flow in compliant vessels. A corrosion cast of the rabbit circulation was manufactured to obtain arterial lengths and diameters. Pulse wave speeds and inflow and outflow boundary conditions were derived from in vivo data. Numerical results captured the main features of in vivo pressure and velocity pulse waveforms in the aorta, brachiocephalic artery and central ear artery. This model was used to elucidate haemodynamic mechanisms underlying changes in peripheral pulse waveforms observed in vivo after administering drugs that alter nitric oxide synthesis in the endothelial cells lining blood vessels. According to our model, these changes can be explained by single or combined alterations of blood viscosity, peripheral resistance and compliance, and the elasticity of conduit arteries.     

Allosteric modulation of adenosine receptors

Allosteric modulators for adenosine receptors may have potential therapeutic advantage over orthosteric ligands. Allosteric enhancers at the adenosine A₁ receptor have been linked to antiarrhythmic and antilipolytic activity. They may also have therapeutic potential as analgesics and neuroprotective agents. A₃ allosteric enhancers are postulated to be useful against ischemic conditions or as antitumor agents. In this review, we address recent developments regarding the medicinal chemistry of such compounds. Most efforts have been and are directed toward adenosine A₁ and A₃ receptors, whereas limited or no information is available for A_2A and A_2B receptors. We also discuss some findings, mostly receptor mutation studies, regarding localization of the allosteric binding sites on the receptors.     

Genetic parameters for growth, reproductive and maternal traits in a multibreed meat sheep population

The genetic parameters for growth, reproductive and maternal traits in a multibreed meat sheep population were estimated by applying the Average Information Restricted Maximum Likelihood method to an animal model. Data from a flock supported by the Programa de Melhoramento Genético de Caprinos e Ovinos de Corte (GENECOC) were used. The traits studied included birth weight (BW), weaning weight (WW), slaughter weight (SW), yearling weight (YW), weight gain from birth to weaning (GBW), weight gain from weaning to slaughter (GWS), weight gain from weaning to yearling (GWY), age at first lambing (AFL), lambing interval (LI), gestation length (GL), lambing date (LD - number of days between the start of breeding season and lambing), litter weight at birth (LWB) and litter weight at weaning (LWW). The direct heritabilities were 0.35, 0.81, 0.65, 0.49, 0.20, 0.15 and 0.39 for BW, WW, SW, YW, GBW, GWS and GWY, respectively, and 0.04, 0.06, 0.10, 0.05, 0.15 and 0.11 for AFL, LI, GL, LD, LWB and LWW, respectively. Positive genetic correlations were observed among body weights. In contrast, there was a negative genetic correlation between GBW and GWS (-0.49) and GBW and GWY (-0.56). Positive genetic correlations were observed between AFL and LI, LI and GL, and LWB and LWW. These results indicate a strong maternal influence in this herd and the presence of sufficient genetic variation to allow mass selection for growth traits. Additive effects were of little importance for reproductive traits, and other strategies are necessary to improve the performance of these animals.     

Synthesis and biological evaluation of benzopyran analogues bearing class III antiarrhythmic pharmacophores

We have synthesized a series of compounds combining the hydroxy-benzopyran ring of vitamin E with the methylsulfonylaminophenyl group of class III antiarrhythmic drugs, connected through tertiary amine moieties. Evaluation of the antiarrhythmic and antioxidant activity of the new compounds was carried out on isolated rat heart preparations using the non-recirculating Langendorff mode. The new analogues were present, at 10 microM concentration, during ischemia and reperfusion. Selected compounds were further studied by a conventional microelectrode method in order to get insight into their cellular mode of action. The most active compound, N-[4-[2-[[2-(3,4-dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-yl)ethyl] methylamine]ethyl]phenyl]methanesulfonamide (19a), reduces premature beats, prolongs QT and QRS intervals during ischemia and reperfusion, and reduces MDA content, leading to a fast recovery of the heart. In addition, it exhibits moderate class III antiarrhythmic action.     

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.     

Evidence of lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated by fullerols

An oxidative stress (OS) state is characterized by the generation of Reactive Oxygen Species (ROS) in a biological system above its capacity to counterbalance them [1]. Exposure to OS induces the accumulation of intracellular ROS, which in turn causes cell damage in the form of protein, lipid, and/or DNA oxidations. Such conditions are believed to be linked to numerous diseases or simply to the ageing of tissues. However, the controlled generation of ROS via photosensitizing drugs or photosensitizers (PS) is now widely used to treat various tumors and other infections [2,3]. Here we present a method to track the chemical changes in a cell after exposure to oxidative stress. OS is induced via fullerols, a custom made water soluble derivative of fullerene (C(60)), under visible light illumination. Synchrotron-based Fourier Transform InfraRed Microspectroscopy (S-FTIRM) was used to assess the chemical makeup of single cells after OS exposure. Consequently, a chemical fingerprint of oxidative stress was probed in this study through an increase in the bands linked with lipid peroxidation (carbonyl ester group at 1740 cm(-1)) and protein phosphorylation (asymmetric phosphate stretching at 1240 cm(-1)).