Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus.

Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1-NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure-activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

The promise of copper ionophores as antimicrobials.

Antibiotic-resistant microbe-mediated deaths are a major worldwide health issue. Unfortunately, due to microbial adaptation to develop resistance, some antibiotics are nullified early in their usage, and worse, resistance is detected before they can even be prescribed. Copper's toxicity since antiquity against microbes at the host-pathogen interface offers a fascinating weapon to fight antimicrobial resistance. Here, we briefly review why copper is so effective, how drugs that work with copper are effective antimicrobials, and how compounds such as these could reinvigorate investment in antimicrobial development.

Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against Streptococcal species.

Despite the availability of several vaccines against multiple disease-causing strains of Streptococcus pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. N,N-dimethyldithiocarbamate (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including S. pneumoniae. Here, DMDCs efficacy against Streptococcal pathogens Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus anginosus was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives' effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against S. pneumoniae TIGR4 and tested in bactericidal and animal models. Two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an in vivo murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.

Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors.

Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniaecop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs.IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.

αM-Conotoxin MIIIJ Blocks Nicotinic Acetylcholine Receptors at Neuromuscular Junctions of Frog and Fish.

We report the discovery and functional characterization of αM-Conotoxin MIIIJ, a peptide from the venom of the fish-hunting cone snail Conus magus. Injections of αM-MIIIJ induced paralysis in goldfish (Carassius auratus) but not mice. Intracellular recording from skeletal muscles of fish (C. auratus) and frog (Xenopus laevis) revealed that αM-MIIIJ inhibited postsynaptic nicotinic acetylcholine receptors (nAChRs) with an IC50 of ~0.1 µM. With comparable potency, αM-MIIIJ reversibly blocked ACh-gated currents (IACh) of voltage-clamped X. laevis oocytes exogenously expressing nAChRs cloned from zebrafish (Danio rerio) muscle. αM-MIIIJ also protected against slowly-reversible block of IACh by α-bungarotoxin (α-BgTX, a snake neurotoxin) and α-conotoxin EI (α-EI, from Conus ermineus another fish hunter) that competitively block nAChRs at the ACh binding site. Furthermore, assessment by fluorescence microscopy showed that αM-MIIIJ inhibited the binding of fluorescently-tagged α-BgTX at neuromuscular junctions of X. laevis,C. auratus, and D. rerio. (Note, we observed that αM-MIIIJ can block adult mouse and human muscle nAChRs exogenously expressed in X. laevis oocytes, but with IC50s ~100-times higher than those of zebrafish nAChRs.) Taken together, these results indicate that αM-MIIIJ inhibits muscle nAChRs and furthermore apparently does so by interfering with the binding of ACh to its receptor. Comparative alignments with homologous sequences identified in other fish hunters revealed that αM-MIIIJ defines a new class of muscle nAChR inhibitors from cone snails.

Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins.

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.