Microbiome associated with the tetrodotoxin-bearing anuran Brachycephalus pitanga.

The genus Brachycephalus includes small species of aposematic anurans known as microendemic, occurring in the mountains of the Atlantic Forest. Brachycephalus ephippium, B. nodoterga and B. pernix have been reported to contain the neurotoxin tetrodotoxin in skin and viscera. The biological conservation of several Brachycephalus species is currently threatened by climate change, deforestation, and the pandemic caused by the fungus Batrachochytrium dendrobatidis (Bd). Despite the well-known importance of amphibians' associated bacteria in the defensive role against pathogens, there is still a poor understanding of amphibian microbiome composition. The present study investigated the composition of B. pitanga microbial community and the presence of TTX in the host and in cultures of bacterial isolates, using a combination of metagenomics, bacterial culture isolation, mass spectrometry and metabolomic analyses. Results of culture-dependent and -independent analyses characterized the microbial communities associated with the skin and viscera of B. pitanga. Mass spectrometry analysis indicated the presence of TTX in host tissues, while bacterial production of TTX was not observed under the experimental conditions used in this investigation. This is the first report confirming the occurrence of TTX in B. pitanga.

Muricauda brasiliensis sp. nov., isolated from a mat-forming cyanobacterial culture.

Strain K001 was isolated from a cyanobacterial culture derived from Abrolhos, a reef bank microbial mat (South Atlantic Ocean-Brazil). Cells of K001 are Gram stain-negative, catalase and oxidase-positive, non-motile, rod-shaped, and with or without appendages. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain K001 belongs to the genus Muricauda. The highest strain K001 16S rRNA gene identity, ANI, and dDDH, respectively, are with M. aquimarina (98.90%, 79.23, 21.60%), M. ruestringensis (98.20%, 80.82, 23.40%), and M. lutimaris (97.86%, 79.23, 22.70%). The strain grows at 15-37 °C and between 0.5 and 10% NaCl. The major fatty acids of strain K001 are iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). The polar lipids are represented by phosphatidylethanolamine, three unidentified aminolipids, and three unidentified polar lipids. The major respiratory quinone is MK-6. The G+C content of the DNA of strain K001 is 41.62 mol%. Based on polyphasic analysis of strain K001, it was identified as a novel representative of the genus Muricauda and was named Muricauda brasiliensis sp. nov. The type strain is K001 (=CBMAI 2315T = CBAS 752T).

New tetrodotoxin analogs in Brazilian pufferfishes tissues and microbiome.

While tetrodotoxin (TTX) is commonly found in pufferfish tissues, it is unclear if bacterial symbionts isolated from pufferfish tissues can produce TTX. In this investigation, UPLC qTOF-MS/MS analysis of tissue extracts obtained from Sphoeroides spengleri and Canthigaster figuereidoi identified TTX in their composition, indicating their consumption is unsafe. UPLC qTOF-MS/MS analysis coupled with Molecular Networking indicated new TTX analogs (methyl-TTX, TTX-acetate, hydroxypropyl-TTX and glycerol-TTX). Bacterial extracts from sixteen strains revealed a compound with a [M+H]+ ion at m/z 320.1088, identical to TTX. However, TTX itself was not detected in these cultures by UPLC-MS/MS. Neurotoxicity of Vibrio A665 purified fraction 2 (with precursor [M+H]+ ion at m/z 320.1088) was significant in human neural stem cells (hNSCs), but the Nav blockage activity was not confirmed by the veratridine/ouabain essays, indicating a possible difference in the mechanism of action between the bacterium A665 purified fraction 2 and TTX. Vibrios symbionts of pufferfish point out involving in the production of TTX precursors.

UHPLC-MS and MALDI-MS study of aluminum phthalocyanine chloride and development of a bioanalytical method for its quantification in nanoemulsions and biological matrices.

Metal phthalocyanines are promising components in photodynamic therapy. Aluminum phthalocyanine chloride (AlClPc) has been used to treat oral cancer in mice, human carious tissue, lung cancer cells and other conditions. To overcome the high hydrophobicity of AlClPc, phthalocyanine is often encapsulated in nanoformulations. Despite increased usage, little is known about the pharmacokinetics and biodistribution of AlClPc. The aim of this study was the development and validation of a UHPLC-MS method for the determination of AlClPc in solution after extraction from nanoformulations and biological matrices such as plasma and tissue. The described method has been assayed as to selectivity, linearity, limits of detection and quantification, precision and recovery. The present study is the first to describe the behavior of AlClPc in biological matrices with mass spectrometry as well as the first to describe the chromatographic behavior of AlClPc contaminants. Molecular mass analysis identified dechlorination of AlClPc by both LC/MS and MALDI-MS and an adduct formation in LC/MS. The parameters observed indicated that the method has applicability and robustness for use in biodistribution studies.

The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases.

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0-10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0-8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

Anti-proliferative and cytotoxic activity of pentadactylin isolated from Leptodactylus labyrinthicus on melanoma cells.

Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.