Proline-rich antimicrobial peptides (PrAMPs) internalize into susceptible bacteria using specific transporters and interfere with protein synthesis and folding. To date, mammalian PrAMPs have so far been identified only in artiodactyls. Since cetaceans are co-phyletic with artiodactyls, we mined the genome of the bottlenose dolphin Tursiops truncatus, leading to the identification of two PrAMPs, Tur1A and Tur1B. Tur1A, which is orthologous to the bovine PrAMP Bac7, is internalized into Escherichia coli, without damaging the membranes, using the inner membrane transporters SbmA and YjiL/MdM. Furthermore, like Bac7, Tur1A also inhibits bacterial protein synthesis by binding to the ribosome and blocking the transition from the initiation to the elongation phase. By contrast, Tur1B is a poor inhibitor of protein synthesis and may utilize another mechanism of action. An X-ray structure of Tur1A bound within the ribosomal exit tunnel provides a basis to develop these peptides as novel antimicrobial agents.

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis, and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH-1 and CATH-2, have been identified and characterized in teleosts (ray-finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath-1 and cath-2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700-fold induction of cath-2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up-regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.

Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1–35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1–15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

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