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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).

Given the current global problem of multidrug resistance in human opportunistic pathogens, antimicrobial peptides (AMPs) show significant potential as novel anti-infective agents1. Notably, they show broad spectrum antibacterial activity and their most common mode of action, namely the disruption of bacterial membrane and/or the formation of pores or lesions, makes it hard for bacteria to gender permanent resistance. The toxicity towards host cells is however a key limitation factor, which imposes the necessity to design artificial AMPs de novo, or redesigning/mutating known natural AMPs to have an increased selectivity index (SI) and reduce side effects. We report four novel Gly and Lys rich peptides which were computationally designed based on a set of training natural peptides with low reported MIC values against E.coli and taking into account a defined set of parameters ; net charge, hydrophobicity, number of Lysines, etc. One additional peptide was designed by creating the specular image of a previously designed sequence. All peptides were named kiadins, from the first three residues in the ‘parent’ peptide sequence1. Susceptibility testing was performed on clinically relevant, drug- resistant bacteria chosen among the ESKAPE pathogens ; carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, 3rd generation cephalosporin-resistant E. coli and Klebsiella pneumoniae, as well as meticillin- resistant Staphylococcus aureus (MRSA). The results show different range of antibacterial activity with respect to Gly substitutions, where the peptides with higher proportion of Gly showed significantly weaker bactericidal effects. The best antibacterial activity was observed for Kiadin-2, which exhibited MIC values from 0.25 to 8 µM, a promising SI values and low cytogenotoxicity on HPBLs, making it a suitable candidate for further development.

E’ noto che, nel tessuto polmonare dei pazienti con fibrosi cistica (FC), P. aeruginosa evolve lentamente, accumulando piccole mutazioni, che accentuano le caratteristiche favorevoli al persistere dell’infezione. Questo fatto, in aggiunta all’evenienza d’infezioni plurime, fa sì che la popolazione di P. aeruginosa, che colonizza un singolo paziente, possa mostrare fenotipi multipli. E’ ipotizzabile che la diversificazione stessa favorisca la persistenza, garantendo un ampio spettro di caratteristiche potenzialmente utili a fronteggiare l’attività dei sistemi di difesa e degli antibiotici. In questo lavoro ci siamo proposti di valutare la diversificazione della popolazione di P. aeruginosa nei confronti della suscettibilità a 5 molecole antibiotiche appartenenti a classi differenti (aztreonam, cefepime, ceftazidime, ciprofloxacina, tobramicina) ed alla catelicidina di origine bovina BMAP-27. I ceppi di P. aeruginosa sono stati isolati ed identificati presso l’Ospedale Bambino Gesù di Roma da 19 pazienti FC con infezione cronica. Ciascun paziente ha fornito da 2 a 4 isolati, provenienti da un unico campione di espettorato e scelti per la diversa morfologia delle colonie. La tipizzazione è stata eseguita mediante macrorestrizione del genoma con SpeI, seguita dalla risoluzione dei frammenti con PFGE. Le MIC sono state valutate con il metodo della microdiluizione in brodo, seguendo le linee guida CLSI. BMAP-27 è stato sintetizzato in fase solida, utilizzando la chimica Fmoc. La tipizzazione degli isolati ha dimostrato che 8/19 pazienti erano colonizzati da un solo ceppo, del quale era possibile distinguere, in base alla morfologia delle colonie, 2 o 3 varianti fenotipiche; 10 pazienti erano infettati da 2 ceppi diversi ed 1 paziente era infettato da tre ceppi. Alcuni ceppi infettavano più pazienti (fino ad un massimo di 7). Lo studio della suscettibilità agli antibiotici ha evidenziato l’ampia diffusione di resistenze e la presenza di differenze significative nelle MIC di ceppi con profilo PFGE diverso coinfettanti lo stesso paziente. In 5 pazienti sono state riscontrate anche variazioni significative di MIC (8-32x) tra varianti fenotipiche dello stesso ceppo, presenti simultaneamente nell’espettorato. Le MIC di BMAP-27 erano comprese tra 2 e 16 μM (MIC90= 8 μM; MIC50= 4 μM) e, rispetto agli antibiotici convenzionali, mostravano una variabilità inferiore, sia tra ceppi diversi che tra varianti fenotipiche dello stesso ceppo (al massimo 4x). Lo studio conferma che, nel polmone del singolo paziente FC, possono essere presenti contemporaneamente più ceppi di P. aeruginosa, e varianti degli stessi, con suscettibilità diverse agli antibiotici. Inoltre, dimostra una minore variabilità delle MIC del peptide BMAP-27.

Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae andmembers of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL-37 and the β-defensin hBD-3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP-29, PG-1 and Bac7(1-35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS-producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors.