EVALUATION OF THE THERAPEUTIC POTENTIAL OF NATURAL ANTIMICROBIAL PEPTIDES FOR HUMAN AND VETERINARY APPLICATION

Innovative antimicrobial drugs effective against emerging and often antibiotic-resistant pathogens are among the most challenging unmet medical needs. Natural antimicrobial peptides (AMPs) hold therapeutic potential as promising novel agents against bacterial and fungal pathogens due to their biological activities including direct killing of invading microbes, modulation of the immune response and low frequency in selecting resistant strains. The aim of the present thesis was to evaluate the functional and mechanistic properties of selected mammalian and fish AMPs in three different settings in view of their possible development as novel anti-infective agents for human and veterinary application. 1) In the first study three structurally diverse bovine peptides have been compared for their capacity to inactivate the yeast-like algae of the genus Prototheca. These microorganisms can cause mastitis in dairy cattle with big economic impact and no cure. The alpha-helical BMAP-28, the proline-rich Bac5 and the cysteine-rich lingual antimicrobial peptide (LAP) have been tested against a P. wickerhamii reference strain and against 12 clinical mastitis isolates in minimum inhibitory concentration (MIC) and time-kill assays. All AMPs were effective in the micromolar range. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular ATP and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at four-fold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. Our results indicate that these three AMPs kill Prototheca with distinct potencies, killing kinetics and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs. 2) In the second study two alpha-helical AMPs, i.e. BMAP-28 and the human cathelicidin LL-37, were tested against Candida isolates from female genital tract infections. Vulvovaginal candidiasis affects approximately 75% of fertile age women with 5-10% incidence of recurrent infection. Importantly, the ability of Candida spp to form stable biofilms often results in serious medical device-related infections. The antifungal activity of these peptides was investigated against a reference C. albicans strain and 24 clinical vaginal isolates of different Candida spp. Under standard experimental conditions, BMAP-28 was highly effective against all Candida isolates (MIC50, 4 µM). Its antifungal effects were also preserved in synthetic vaginal simulated fluid (VSF) at vaginal pH values. The activity of these compounds was further tested against sessile Candida spp. by fluorescence microscopy and XTT reduction assays to quantify the inhibitory effect on biofilm cell viability. BMAP-28 strongly reduced the growth of mature biofilm through a lytic mechanism, whereas LL-37 could only prevent biofilm formation by inhibiting cell adhesion. Interestingly, both BMAP-28 and LL-37 inhibited Candida adhesion to medical grade silicone, suggesting a possible use of these peptides as antimicrobial coatings. 3) The immunomodulatory properties of AMPs have been addressed in the third part of my thesis, in view of developing salmonid AMPs as immunostimulants or vaccine adjuncts to prevent aquaculture infections. For this purpose, I analyzed the effects of BMAP-28 and of the salmonid cathelicidin STF on the respiratory burst and the pro-inflammatory cytokine genes expression in primary head kidney leukocytes from rainbow trout, by using a luminometric method and RT-qPCR analysis, respectively. The oxygen species production (ROS) in Phorbol-myristate-acetate- (PMA) and beta-glucan- stimulated cells was synergistically increased in the presence of micromolar concentrations of each peptide. Moreover, the trout derived STF was able to speed up the respiratory burst kinetics in primary head kidney leukocytes, either stimulated with PMA or with beta-glucan, and was also effective when added alone, though to a lesser extent. Real time PCR studies revealed a synergistic effect on IL-1beta expression following four hour incubation of cells with STF and lipopolysaccharide, whereas no synergy was observed when the cells were incubated with the combination of each peptide with beta-glucan. Each peptide alone did not affect cytokine genes expression and was not toxic to the cells, as assessed by measuring the release of lactate dehydrogenase in the medium. Collectively, the results obtained highlight the antimicrobial and immunomodulatory properties of the peptides under study pointing out the differences in potency, kinetics, and mode of action of each single peptide, thus encouraging further studies in view of the development of these molecules for human and veterinary application.