N-Heterocyclic dicarboxylic acids: Broad-spectrum inhibitors of metallo-β-lactamases with co-antibacterial effect against antibiotic-resistant bacteria

In an effort to identify novel, broad-spectrum inhibitors against the metallo-β-lactamases (MβLs), several N-heterocyclic derivatives were tested as inhibitors of MβLs CcrA, ImiS, and L1, which are representative enzymes from the distinct MβL subclasses. Three N-heterocyclic dicarboxylic acid derivatives were competitive inhibitors of CcrA and L1, exhibiting K(i) values ?2μM, while only 2,4-thiazolidinedicarboxylic acid (1b) was a competitive inhibitor of ImiS. Two 2-mercapto-1,3,4-thiadiazole derivatives were noncompetitive inhibitors of CcrA and ImiS, exhibiting K(i) values <7μM; however, these same compounds did not inhibit L1. Two 2-mercapto-1,3,4-triazole derivatives were shown not to inhibit any of the tested MβLs. The N-heterocyclic derivatives were tested for antibacterial activity by examining the MIC values for existing antibiotics in the presence/absence of these derivatives. Consistent with the steady-state inhibition data, the inclusion of three N-heterocyclic dicarboxylic acid derivatives resulted in lower MIC values when using Escherichia coli BL21(DE3) cells containing the CcrA or L1 plasmids or Klebsiella pneumoniae (ATCC 700603), while 1b was the only dicarboxylic acid derivative to lower the MIC value of E. coli cells containing the ImiS plasmid. Inclusion of the 2-mercapto-1,3,4-thiadiazole derivatives resulted in lower MIC values for E. coli cells containing ImiS or L1 plasmids; however, these derivatives did not alter the MIC values for K. pneumoniae or E. coli cells containing the L1 plasmid. None of the N-heterocyclic derivatives affected the MIC of two methicillin resistant Staphylococcus aureus (MRSA) strains. Taken together, these studies demonstrate that N-heterocyclic dicarboxylic acids 1a-c and pyridylmercaptothiadiazoles 2a,b are good scaffolds for future broad-spectrum inhibitors of the MβLs.     

Site-selective binding of Zn(II) to metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Extended X-ray absorption fine structure studies of the metallo-β-lactamase L1 from Stenotrophomonas maltophilia containing 1 and 2 equiv of Zn(II) and containing 2 equiv of Zn(II) plus hydrolyzed nitrocefin are presented. The data indicate that the first, catalytically dominant metal ion is bound by L1 at the consensus Zn₁ site. The data further suggest that binding of the first metal helps preorganize the ligands for binding of the second metal ion. The di-Zn enzyme displays a well-defined metal–metal interaction at 3.42 Å. Reaction with the β-lactam antibiotic nitrocefin results in a product-bound species, in which the ring-opened lactam rotates in the active site to present the S1 sulfur atom of nitrocefin to one of the metal ions for coordination. The product bridges the two metal ions, with a concomitant lengthening of the Zn–Zn interaction to 3.62 Å.     

Probing the adaptive response of Escherichia coli to extracellular Zn(II)

The adaptive response of Escherichia coli cells to differing intracellular and extracellular Zn(II) concentrations was evaluated by two-dimensional gel electrophoresis and peptide identifications. Twenty-one Zn(II)-responsive proteins, which were previously not known to be associated with Zn(II), were identified. Most of the proteins were related to cellular metabolism and include membrane transporters and glycolytic and TCA-associated enzymes. The expression levels of no known Zn(II) transporters were identified with these studies. The results of these studies suggest a role of Zn(II) in the expression levels of several E. coli proteins, and the results are discussed in light of recent genomic profiling studies on the adaptive response of E. coli cells to stress by Zn(II) excess.     

Molecular dynamic simulations of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor

The beta-lactam-based antibiotics are among the most prescribed and effective antibacterial agents. Widespread use of these antibiotics, however, has created tremendous pressure for the emergence of resistance mechanisms in bacteria. The most common cause of antibiotic resistance is bacterial production of actamases that efficiently degrade antibiotics. The metallo-beta-lactamases are of particular clinical concern due to their transference between bacterial strains. We used molecular dynamics (MD) simulations to further study the conformational changes that occur due to binding of an inhibitor to the dicanzinc metallo-beta-lactamase from Bacteroides fragilis. Our studies confirm previous findings that the major flap is a major source of plasticity within the active site, therefore its dynamic response should be considered in drug development. However, our results also suggest the need for care in using MD simulations in evaluating loop mobility, both due to relaxation times and to the need to accurately model the zinc active site. Our study also reveals two new robust responses to ligand binding. First, there are specific localized changes in the zinc active site—a local loop flip—due to ligand intercalation that may be critical to the function of this enzyme. Second, inhibitor binding perturbs the dynamics throughout the protein, without otherwise perturbing the enzyme structure. These dynamic perturbations radiate outward from the active site and their existence suggests that long-range communication and dynamics may be important in the activity of this enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mate Discrimination in Invasive Whitefly Species

Mate discrimination could be critical for invasive species that need to locate rare suitable mates and avoid costs associated with misdirected courtships to establish in new environments. Here, we tested whether individuals of two invasive whitefly species in the Bemisia tabaci species complex, commonly known as the B and Q biotypes, could discriminate between potential mates based on their species and sex. Behavioral observations showed that B females were more discriminating than Q females. Males of both species were able to discriminate between mates based on their species and sex, but in general B males discriminated more effectively than Q males. By incorporating these behavioral data into a conceptual model, we show that variation in mating behavior between females of different species was a more significant factor affecting mating than variation between males. These results indicate that mate discrimination could affect interactions between whitefly species and influence a species’ ability to colonize novel environments.     

Characterizing fishing effort and spatial extent of coastal fisheries

Biodiverse coastal zones are often areas of intense fishing pressure due to the high relative density of fishing capacity in these nearshore regions. Although overcapacity is one of the central challenges to fisheries sustainability in coastal zones, accurate estimates of fishing pressure in coastal zones are limited, hampering the assessment of the direct and collateral impacts (e.g., habitat degradation, bycatch) of fishing. We compiled a comprehensive database of fishing effort metrics and the corresponding spatial limits of fisheries and used a spatial analysis program (FEET) to map fishing effort density (measured as boat-meters per km2) in the coastal zones of six ocean regions. We also considered the utility of a number of socioeconomic variables as indicators of fishing pressure at the national level; fishing density increased as a function of population size and decreased as a function of coastline length. Our mapping exercise points to intra and interregional 'hotspots' of coastal fishing pressure. The significant and intuitive relationships we found between fishing density and population size and coastline length may help with coarse regional characterizations of fishing pressure. However, spatially-delimited fishing effort data are needed to accurately map fishing hotspots, i.e., areas of intense fishing activity. We suggest that estimates of fishing effort, not just target catch or yield, serve as a necessary measure of fishing activity, which is a key link to evaluating sustainability and environmental impacts of coastal fisheries.     

Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.     

Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis

Metallo-beta-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a beta-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 +/- 0.2, 1.8 +/- 0.2, and 1.1 +/- 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.     

Cytosine arabinoside rapidly activates Bax-dependent apoptosis and a delayed Bax-independent death pathway in sympathetic neurons

Cytosine arabinoside (ara-C) is a nucleoside analog used in the treatment of hematologic malignancies. One of the major side effects of ara-C chemotherapy is neurotoxicity. In this study, we have further characterized the cell death induced by ara-C in sympathetic neurons. Similar to neurons undergoing trophic factor deprivation-induced apoptosis, ara-C-exposed neurons became hypometabolic before death and upregulated c-myb, c-fos, and Bim. Bax deletion delayed, but did not prevent, ara-C toxicity. Neurons died by apoptosis, indicated by the release of mitochondrial cytochrome-c and caspase-3 activation. p53-deficient neurons demonstrated decreased sensitivity to ara-C, but neither p53 nor multiple p53-regulated genes were induced. Mature neurons showed increased ara-C resistance. These results demonstrate that molecular mechanisms underlying ara-C-induced death are similar to those responsible for trophic factor deprivation-induced apoptosis. However, substantial differences in neuronal death after these two distinct stress stimuli exist since ara-C toxicity, unlike the developmental death, can proceed in the absence of Bax.     

In vivo specificity of EcoRI DNA methyltransferase.

The EcoRI adenine DNA methyltransferase forms part of a bacterial restriction/modification system; the methyltransferase modifies the second adenine within the canonical site GAATTC, thereby preventing the EcoRI endonuclease from cleaving this site. We show that five noncanonical EcoRI sites (TAATTC, CAATTC, GTATTC, GGATTC and GAGTTC) are not methylated in vivo under conditions when the canonical site is methylated. Only when the methyltransferase is overexpressed is partial in vivo methylation of the five sites detected. Our results suggest that the methyltransferase does not protect host DNA against potential endonuclease-mediated cleavage at noncanonical sites. Our related in vitro analysis of the methyltransferase reveals a low level of sequence-discrimination. We propose that the high in vivo specificity may be due to the active removal of methylated sequences by DNA repair enzymes (J. Bacteriology (1987), 169 3243-3250).