The Diversified O-Superfamily in Californiconus californicus Presents a Conotoxin with Antimycobacterial Activity.

Californiconus californicus, previously named Conus californicus, has always been considered a unique species within cone snails, because of its molecular, toxicological and morphological singularities; including the wide range of its diet, since it is capable of preying indifferently on fish, snails, octopus, shrimps, and worms. We report here a new cysteine pattern conotoxin assigned to the O1-superfamily capable of inhibiting the growth of Mycobacterium tuberculosis (Mtb). The conotoxin was tested on a pathogen reference strain (H37Rv) and multidrug-resistant strains, having an inhibition effect on growth with a minimal inhibitory concentration (MIC) range of 3.52⁻0.22 µM, similar concentrations to drugs used in clinics. The peptide was purified from the venom using reverse phase high-performance liquid chromatography (RP-HPLC), a partial sequence was constructed by Edman degradation, completed by RACE and confirmed with venom gland transcriptome. The 32-mer peptide containing eight cysteine residues was named O1_cal29b, according to the current nomenclature for this type of molecule. Moreover, transcriptomic analysis of O-superfamily toxins present in the venom gland of the snail allowed us to assign several signal peptides to O2 and O3 superfamilies not described before in C. californicus, with new conotoxins frameworks.

Community ecology across bacteria, archaea and microbial eukaryotes in the sediment and seawater of coastal Puerto Nuevo, Baja California.

Microbial communities control numerous biogeochemical processes critical for ecosystem function and health. Most analyses of coastal microbial communities focus on the characterization of bacteria present in either sediment or seawater, with fewer studies characterizing both sediment and seawater together at a given site, and even fewer studies including information about non-bacterial microbial communities. As a result, knowledge about the ecological patterns of microbial biodiversity across domains and habitats in coastal communities is limited-despite the fact that archaea, bacteria, and microbial eukaryotes are present and known to interact in coastal habitats. To better understand microbial biodiversity patterns in coastal ecosystems, we characterized sediment and seawater microbial communities for three sites along the coastline of Puerto Nuevo, Baja California, Mexico using both 16S and 18S rRNA gene amplicon sequencing. We found that sediment hosted approximately 500-fold more operational taxonomic units (OTUs) for bacteria, archaea, and microbial eukaryotes than seawater (p < 0.001). Distinct phyla were found in sediment versus seawater samples. Of the top ten most abundant classes, Cytophagia (bacterial) and Chromadorea (eukaryal) were specific to the sediment environment, whereas Cyanobacteria and Bacteroidia (bacterial) and Chlorophyceae (eukaryal) were specific to the seawater environment. A total of 47 unique genera were observed to comprise the core taxa community across environment types and sites. No archaeal taxa were observed as part of either the abundant or core taxa. No significant differences were observed for sediment community composition across domains or between sites. For seawater, the bacterial and archaeal community composition was statistically different for the Major Outlet site (p < 0.05), the site closest to a residential area, and the eukaryal community composition was statistically different between all sites (p < 0.05). Our findings highlight the distinct patterns and spatial heterogeneity in microbial communities of a coastal region in Baja California, Mexico.

Antimycobacterial Activity: A New Pharmacological Target for Conotoxins Found in the First Reported Conotoxin from Conasprella ximenes.

Mycobacterium tuberculosis is the etiological agent of tuberculosis, an airborne infectious disease that is a leading cause of human morbidity and mortality worldwide. We report here the first conotoxin that is able to inhibit the growth of M. tuberculosis at a concentration similar to that of two other drugs that are currently used in clinics. Furthermore, it is also the first conopeptide that has been isolated from the venom of Conasprella ximenes. The venom gland transcriptome of C. ximenes was sequenced to construct a database with 24,284 non-redundant transcripts. The conopeptide was purified from the venom using reverse phase high performance liquid chromatography (RP-HPLC) and was analyzed using electrospray ionization-mass spectrometry (ESI-MS/MS). No automatic identification above the identity threshold with 1% of the false discovery rate was obtained; however, a 10-amino-acid sequence tag, manually extracted from the MS/MS spectra, allowed for the identification of a conotoxin in the transcriptome database. Electron transfer higher energy collision dissociation (EThcD) fragmentation of the native conotoxin confirmed the N-terminal sequence (1-14), while LC-MS/MS analysis of the tryptic digest of the reduced and S-alkylated conotoxin confirmed the C-terminal region (15-36). The expected and experimental molecular masses corresponded, within sub-ppm mass error. The 37-mer peptide (MW 4109.69 Da), containing eight cysteine residues, was named I1_xm11a, according to the current nomenclature for this type of molecule.

In Silico Identification of Protein Disulfide Isomerase Gene Families in the De Novo Assembled Transcriptomes of Four Different Species of the Genus Conus.

Small peptides isolated from the venom of the marine snails belonging to the genus Conus have been largely studied because of their therapeutic value. These peptides can be classified in two groups. The largest one is composed by peptides rich in disulfide bonds, and referred to as conotoxins. Despite the importance of conotoxins given their pharmacology value, little is known about the protein disulfide isomerase (PDI) enzymes that are required to catalyze their correct folding. To discover the PDIs that may participate in the folding and structural maturation of conotoxins, the transcriptomes of the venom duct of four different species of Conus from the peninsula of Baja California (Mexico) were assembled. Complementary DNA (cDNA) libraries were constructed for each species and sequenced using a Genome Analyzer Illumina platform. The raw RNA-seq data was converted into transcript sequences using Trinity, a de novo assembler that allows the grouping of reads into contigs without a reference genome. An N50 value of 605 was established as a reference for future assemblies of Conus transcriptomes using this software. Transdecoder was used to extract likely coding sequences from Trinity transcripts, and PDI-specific sequence motif "APWCGHCK" was used to capture potential PDIs. An in silico analysis was performed to characterize the group of PDI protein sequences encoded by the duct-transcriptome of each species. The computational approach entailed a structural homology characterization, based on the presence of functional Thioredoxin-like domains. Four different PDI families were characterized, which are constituted by a total of 41 different gene sequences. The sequences had an average of 65% identity with other PDIs. Using MODELLER 9.14, the homology-based three-dimensional structure prediction of a subset of the sequences reported, showed the expected thioredoxin fold which was confirmed by a "simulated annealing" method.