Involvement of JNK and caspase activation in hoiamide A-induced neurotoxicity in neocortical neurons.

The frequent occurrence of Moorea producens (formerly Lyngbya majuscula) blooms has been associated with adverse effects on human health. Hoiamide A is a structurally unique cyclic depsipeptide isolated from an assemblage of the marine cyanobacteria M. producens and Phormidium gracile. We examined the influence of hoiamide A on neurite outgrowth in neocortical neurons and found that it suppressed neurite outgrowth with an IC50 value of 4.89 nM. Further study demonstrated that hoiamide A stimulated lactic acid dehydrogenase (LDH) efflux, nuclear condensation and caspase-3 activity with EC50 values of 3.66, 2.55 and 4.33 nM, respectively. These data indicated that hoiamide A triggered a unique neuronal death profile that involves both necrotic and apoptotic mechanisms. The similar potencies and similar time-response relationships between LDH efflux and caspase-3 activation/nuclear condensation suggested that both necrosis and apoptosis may derive from interaction with a common molecular target. The broad-spectrum caspase inhibitor, Z-VAD-FMK completely inhibited hoiamide A-induced neurotoxicity. Additionally, hoiamide A stimulated JNK phosphorylation, and a JNK inhibitor attenuated hoiamide A-induced neurotoxicity. Collectively, these data demonstrate that hoiamide A-induced neuronal death requires both JNK and caspase signaling pathways. The potent neurotoxicity and unique neuronal cell death profile of hoiamide A represents a novel neurotoxic chemotype from marine cyanobacteria.

On being the right size: the impact of population size and stochastic effects on the evolution of drug resistance in hospitals and the community.

The evolution of drug resistant bacteria is a severe public health problem, both in hospitals and in the community. Currently, some countries aim at concentrating highly specialized services in large hospitals in order to improve patient outcomes. Emergent resistant strains often originate in health care facilities, but it is unknown to what extent hospital size affects resistance evolution and the resulting spillover of hospital-associated pathogens to the community. We used two published datasets from the US and Ireland to investigate the effects of hospital size and controlled for several confounders such as antimicrobial usage, sampling frequency, mortality, disinfection and length of stay. The proportion of patients acquiring both sensitive and resistant infections in a hospital strongly correlated with hospital size. Moreover, we observe the same pattern for both the percentage of resistant infections and the increase of hospital-acquired infections over time. One interpretation of this pattern is that chance effects in small hospitals impede the spread of drug-resistance. To investigate to what extent the size distribution of hospitals can directly affect the prevalence of antibiotic resistance, we use a stochastic epidemiological model describing the spread of drug resistance in a hospital setting as well as the interaction between one or several hospitals and the community. We show that the level of drug resistance typically increases with population size: In small hospitals chance effects cause large fluctuations in pathogen population size or even extinctions, both of which impede the acquisition and spread of drug resistance. Finally, we show that indirect transmission via environmental reservoirs can reduce the effect of hospital size because the slow turnover in the environment can prevent extinction of resistant strains. This implies that reducing environmental transmission is especially important in small hospitals, because such a reduction not only reduces overall transmission but might also facilitate the extinction of resistant strains. Overall, our study shows that the distribution of hospital sizes is a crucial factor for the spread of drug resistance.

Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D'.

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A' (compounds 1a/1b), as well as two new derivatives, BAC-D/D' (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey's method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host's environment.IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.

Identification of compounds exhibiting inhibitory activity toward the Pseudomonas tolaasii toxin tolaasin I using in silico docking calculations, NMR binding assays, and in vitro hemolytic activity assays.

Using in silico docking calculations, NMR analysis of target-ligand binding, and hemolytic activity assays, we searched a 30,000-compound library for an effective inhibitor of tolaasin I, a Pseudomonas tolaasii toxin that causes virulent infection in mushrooms. Of more than 30,000 compounds screened in silico, two compounds were selected. One of these compounds, sorbitololeic acid, bound to tolaasin I and inhibited its hemolytic activity in vitro. Therefore, sorbitololeic acid can be a potential inhibitor of tolaasin I.

Characteristics of inositol phosphorylceramide synthase and effects of aureobasidin A on growth and pathogenicity of Botrytis cinerea.

Inositol phosphorylceramide (IPC) synthase is the key enzyme with highly conserved sequences, which is involved in fungal sphingolipid biosynthesis. The antibiotic aureobasidin A (AbA) induces the death of fungi through inhibiting IPC synthase activity. The mutations of AUR1 gene coding IPC synthase in fungi and protozoa causes a resistance to AbA. However, the mechanism of AbA resistance is still elusive. In this paper, we generated two mutants of Botrytis cinerea with AbA-resistance, BcAUR1a and BcAUR1b, through UV irradiation. BcAUR1a lost an intron and BcAUR1b had three amino acid mutations (L197P, F288S and T323A) in the AUR1 gene. AbA strongly inhibits the activity of IPC synthase in wild-type B. cinerea, which leads to distinct changes in cell morphology, including the delay in conidial germination, excessive branching near the tip of the germ tube and mycelium, and the inhibition of the mycelium growth. Further, AbA prevents the infection of wild-type B. cinerea in tomato fruits via reducing oxalic acid secretion and the activity of cellulase and pectinase. On the contrary, AbA has no effect on the growth and pathogenicity of the two mutants. Although both mutants show a similar AbA resistance, the molecular mechanisms might be different between the two mutants.

Characterization of cereulide synthetase, a toxin-producing macromolecular machine.

Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.

Effects of the mycotoxin destruxin A on Locusta migratoria visceral muscles.

Destruxins, a family of cyclic peptides, are produced by various species of entomopathogenic fungi. These peptides have been shown to influence calcium-dependent processes in insect cell lines and tissues, such as skeletal muscles. To better understand the mechanism of action of these peptide toxins on insect muscular tissues, we have evaluated the effects of destruxin A on the contractions of oviducts and hindgut of Locusta migratoria. In oviducts, destruxin A increased the frequency of spontaneous contractions and induced a dose-dependent tonic contraction; the EC(50) for lower lateral and upper lateral oviducts was 0.7 microM and 8.7 microM, respectively. In hindgut, destruxin A also caused an increase in the frequency of spontaneous contractions; the EC(50) was 3.2 microM. The action of destruxin A was abolished in Ca(2+)-free saline or when the Ca(2+) channel blocker CoCl(2) was added to the incubation saline. Likewise, the presence of 50 microM nifedipine or 100 microM verapamil in the medium reduced the magnitude of destruxin A's effect, particularly in hindgut. The depolarization of muscle membranes by 100 mM K(+) saline prevented the action of destruxin A. Preincubation of lower lateral oviducts in the intracellular Ca(2+) antagonist TMB-8 did not have any effect on destruxin A action; however, preincubation in the calmodulin inhibitor trifluoperazine greatly reduced the effect of destruxin A. Taken together, these results show that destruxin A has an excitatory effect on contractions of insect visceral muscles of L. migratoria. Destruxin A-induced contractions appear to be dependent on extracellular, but not on intracellularly-released Ca(2+), which suggest that this peptide toxin might be acting on insect visceral muscle by facilitating an influx of extracellular Ca(2+).

Identification of a specific inhibitor of the dishevelled PDZ domain.

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.