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3rd International Symposium on the Advances in Marine Mussel Research, Chioggia, 26-28 August 2019

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3rd International Symposium on the Advances in Marine Mussel Research, Chioggia, 26-28 August 2019

Clusters of nucleotide alignments used for forward primer design. (DOCX 7338 kb)

Success rate of amplicon synthesis in Ranidae species based on used primer. (DOCX 13 kb)

Complete list of frog species obtained from Croatian wild. (DOCX 13 kb)

Antimicrobial peptides (AMPs) are endogenous antibiotics present in all organisms acting directly towards pathogens and also showing immunomodulatory properties. Skin secretions of many tested anuran species have been shown to contain a variety of bioactive peptides that often exhibit antibacterial activity, also against multidrug resistant isolates. We have selectively amplified transcripts likely to encode for AMPs, thus providing their sequences. After RNA extraction from frog skin tissue samples, cDNA synthesis followed by PCR amplification was performed. For this purpose, forward degenerate primers were designed based on highly conserved signal peptide regions from Ranidae, Hylidae and Bombinatoridae together with a reverse primer designed on the poly-A tail of mRNA. Signal peptide regions themselves were derived from sequences already available in DADP database and transcriptome data in SRA database. Resulting amplicons were size-selected and processed by ion semiconductor sequencing, obtaining several thousand sequencing reads which were then assembled into contigs representing nearly full-length AMP-encoding transcripts. Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides from 5 different specimens belonging to 5 different frog species, mostly from Ranidae species. Based on appropriate biophysical properties (e.g. charge, hydrophobicity, amphipathicity) six of the most promising candidates were chosen for chemical synthesis and extensive characterization. All peptides were tested against a panel of Gram-negative and Gram-positive bacteria, as well on tumour cell lines. One peptide, identified in Rana arvalis, proved to be active against both reference ATCC strains and cancer cells, while non-toxic for human MEC-1 cells in a range of concentrations several folds higher then minimal inhibitory concentration (MIC) and IC50 values. Furthermore, the peptide was found to disrupt the bacterial membrane even at sub-MIC concentrations, as observed by flow cytometry and visualised by atomic force microscopy (AFM).

Skin secretions of many tested anuran species have been shown to contain a variety of antimicrobial peptides (AMPs) acting directly towards pathogens, including multidrug resistant isolates, and also showing immunomodulatory properties [1]. We have selectively amplified transcripts likely to encode for AMPs, thus providing their sequences. After RNA extraction from frog skin tissue samples, cDNA synthesis followed by PCR amplification was performed. For this purpose, forward degenerate primers were designed based on highly conserved signal peptide regions [2], together with a reverse primer designed on the poly-A tail of mRNA. Signal peptide regions were derived from sequences deposited in DADP database [3] and transcriptome data in SRA database [4]. Resulting amplicons were size-selected and processed by ion semiconductor sequencing, obtaining several thousand sequencing reads. Those were then assembled into contigs representing nearly full-length AMP-encoding transcripts. Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides from 5 different specimens belonging to 5 different frog species, mostly from Ranidae species. Based on appropriate biophysical properties (e.g. charge, hydrophobicity, amphipathicity) six of the most promising candidates were chosen for chemical synthesis and extensive characterization. All peptides were tested against a panel of Gram-negative and Gram-positive bacteria, as well on tumour cell lines. One peptide, identified in Rana arvalis, proved to be active against both reference ATCC strains and epithelial cancer cell lines, while less toxic for circulating MEC-1 cells in a range of concentrations several fold higher then minimal inhibitory concentration (MIC) and IC50 values. Furthermore, the peptide was found to disrupt the bacterial membrane even at sub-MIC concentrations, as observed by flow cytometry and visualised by atomic force microscopy (AFM). [1] A. Nijnik and R. Hancock, Emerg. Health Threats J. 2, (2009). [2] V. Tessera, F. Guida, D. Juretić, and A. Tossi, FEBS J. 279, 724 (2012). [3] M. Novković, J. Simunić, V. Bojović, A. Tossi, and D. Juretić, Bioinformatics 28, 1406 (2012). [4] Y. Kodama, M. Shumway, and R. Leinonen, Nucleic Acids Res. 40, D54 (2012).

Desiccation Related Proteins (DRPs), firstly described in desiccation tolerant plants, are present also in several unrelated bacterial and algal groups. Although their role is still unknown, their presence seems to confer an increased desiccation tolerance in some extremophile bacteria, including the Deinococcus/Thermus phylum. In this study we describe our findings on the DRPs present in the transcriptome of Trebouxia gelatinosa, a member of the most common lichen-forming genus of green algae. Thirteen sequences were classified as DRPs: they are characterized by a c.170 aa long ferritin-like domain (PF13668), followed by one or two domains of unknown function. This gene family has undergone relevant expansion in T. gelatinosa: in the majority of green algae DRPs are present in few copies or they are completely absent. Further, their diversification finds no parallelism in other desiccation tolerant organisms investigated so far. Bayesian phylogenetic inference pointed out that DRPs of green algae are unlikely to be orthologous to those found in Embryophyta. Conversely, they share an unexpected sequence similarity to DRPs found in bacteria. This result led us to consider a bacterial origin for Trebouxia DRP genes, which may have ancestrally been acquired by horizontal gene transfer (HGT) from lichen-associated bacteria.

This is the open peer reviewers comments and recommendations regarding the submitted GigaScience article and/or dataset.

Scatter plots representing paired comparisons of gene expression profiles between tissues. (PDF 1316Â kb)

Multiple sequence alignment of the consensus of the 17-mer repeated units present in the byssal cuticle proteins of Mytilisepta virgata, Septifer biforcatus and Bathymodiolus thermophilus. (PDF 122Â kb)

The esterase activity of carbonic anhydrase (CA) was investigated in mussels sampled at 24 locations along the Croatian coast of Adriatic Sea. The gills were the target tissue because the respiratory, ionic transport and pH regulatory enzyme function of CA and its potential usage as biomarkers of environmental pollution was the main topic. Total esterase activity was measured in cytosolic fraction by colorimetric end-point reaction using p-nitrophenyl acetate as enzyme substrate. CA activity was estimated by the same enzymatic reaction using acetazolamide as a specific CA inhibitor. The results of total esterase activities in winter (March; all sites average value 0.137±0.057) were lower than determined for summer season (August; 0.153±0.036) at almost all investigated locations. CA activities determined in gills of mussel sampled in winter ranged from 1.75% to 24.65% of total esterase activities and in summer samples were lower (0.83 to 13.45%). Although recent research showed potential application of CA activity in bioassay and biomarker in pollution studies, further research is needed. Here we report a short-term simple colorimetric microplate method which can be applied for analyses of large numbers of samples. Furthermore in this study we characterized full length coding sequence (cDNA) of M. galloprovincialis carbonic anhydrase II (CAII). The CAII cDNA (with the 5’ and 3’ untranslated regions) is 1317 bp long. The putative open reading frame encodes a polypeptide of 256 amino acids, with a theoretical pI/Mw 5.87/28.416 kDa and conserved domains (active site and zinc binding site).

The transcriptome of Trebouxia gelatinosa, belonging to one of the most common genus of lichen photobionts, gave an interesting overview of the mechanisms that underlay the desiccation tolerance in this species, regarding structure, physiology and biochemistry. The analysis of the annotated transcripts of both the dehydrated and rehydrated cultured alga revealed interesting and peculiar features of this poikilohydric organism. In particular the presence of a large number of desiccation-related proteins (DRPs) was highlighted, most of them affected by at least one of the two treatments. The DRP family has been first described in the resurrection plants, then also in other plants and in green algae. In T. gelatinosa 13 sequences are classified as desiccation-related proteins. These identified sequences, clearly pertaining to the same multigenic family, are usually characterized by a c.170 a long ferritinlike domain (PF13668), followed by a C-terminal region of variable length without known annotated domains. Nine out of the 13 annotated transcripts were significantly responsive to dehydration and/or rehydration by either being up- or down-regulated. Because the number of DRP genes predicted in the analysed genomes of vascular plants is generally low - ranging from 0 to 5 - this gene family seems to have undergone an expansion in T. gelatinosa. Although the exact role of DRPs in the dehydration/rehydration processes is still unclear, their massive response, both in terms of gene number and fold change, to the hydric status of T. gelatinosa points out that they are prominent players in drought tolerance not only in resurrection plants but also in lichen photobionts. Our study aims at expanding the knowledge of the DRP family in rebouxia, in order to understand the role of these proteins and their expansion in desiccation tolerance and in relation to lichen symbiosis.