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β-Lactamases (BLs) able to hydrolyze β-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC β-lactamase (K i = 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant β-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-β-lactamases (MBL). A multiligand set of boronic acid (BA) β-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant β-lactamases., This work was supported by the OPTObacteria project within the 7FP (Grant agreement no: 286998; www.optobacteria.eu), and by the National Institute of Health (Grant no: GM63815). JB was supported by the Spanish Network for Research in Infectious Pathology (REIPI) RD12/0015/0012, funded by Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, and co-financed by the European Development Regional Fund “A way to achieve Europe” ERDF.

Background Multi-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: ( i ) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR–TS), ( ii ) the primary off-targets: human DHFR and TS, and ( iii ) the secondary on-target: human folate receptor β , a folate/antifolate transporter. Methods We computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFR–TS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. Results Our analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures. Conclusions The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents. General significance Apart from the target-based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress β-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through β-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.

Protein expression changes induced in thioglycolate-elicited peritoneal murine macrophages (M Phi) by infection with type III Group B Streptococcus (GBS) are described. Proteins from control M Phi and M Phi incubated 2 h with live or heat-inactivated GBS were separated by 2-DE. Proteins whose expression was significantly different in infected M Phi, as compared with control cells, were identified by MS/MS analysis. Changes in the expression level of proteins involved in both positive and negative modulation of phagocytic functions, stress response and cell death were induced in M Phi by GBS infection. In particular, expression of enzymes playing a key role in production of reactive oxygen species was lowered in GBS-infected M Phi. Significant alterations in the expression of some metabolic enzymes were also observed, most of the glycolytic and of the pentose-cycle enzymes being down-regulated in M Phi infected with live GBS. Finally, evidence was obtained that GBS infection affects the expression of enzymes or enzyme subunits involved in ATP synthesis and in adenine nucleotides interconversion processes.

We investigated the adsorption and catalytic activity of myoglobin (Mb) immobilized on colloidal particles of zirconia covalently grafted with phosphoric (ZrO2-P) and benzenephosphonic acid (ZrO2-BP). The maximum adsorption was reached after 1 h of contact and was greater on a hydrophilic support, ZrO2-P, compared to a hydrophobic support, ZrO2-BP. The equilibrium isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the surface of the nanoparticles. The nanostructured biocomposites are active in the oxidation of 2-methoxyphenol (guaiacol) by hydrogen peroxide. The oxidation catalyzed by immobilized Mb followed a Michaelis-Menten kinetics, similar to that observed in the oxidation by free Mb. Furthermore, the catalytic efficiency is similar to that of free Mb and higher than that of "large-size" biocatalysts (with sizes larger than 1 mum). In the latter case, the kinetic parameters, k(cat) and K(M), indicate that this is mostly due to an increased affinity of the nano-biocomposite for the substrate. The activity of the nano-biocomposites decreases slightly as the amount of adsorbed protein increases. This is mainly due to the formation of a nonordered monolayer, which reduces the accessibility of the substrate to the active center.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress β-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through β-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.

The adsorption of myoglobin (Mb) onto phosphate grafted-zirconia (ZrO2-P) nanoparticles was studied in terms of conformational studies and thermal stability, determined by circular dichroism (CD), differential scanning calorimetry (DSC), and atomic force microscopy (AFM). The changes in protein structure have been correlated with the catalytic activity of free and adsorbed Mb. CD and DSC studies indicate marked rearrangements in Mb structure upon adsorption onto phosphate-grafted zirconia nanoparticles. These structural rearrangements of Mb could be responsible for the loss of catalytic activity observed for the adsorbed Mb. In particular, the conformational changes due to the adsorption process induced a reduction of kcat and KM. AFM measurements indicate that the interaction with the grafted-zirconia nanoparticles also affects the morphology of the bound protein, inducing the nucleation of prefibrillar-like aggregates.

Non-ionic water-in-oil (w/o) microemulsions of pentaoxyethylenenonylphenyl ether (Igepal-CO520)/cyclohexane/water at 25 °C have been used as reaction vessels to obtain zirconium hydrogen phosphate nanoparticles.