No abstract

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate 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 fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP 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.

OriginalAntimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3Highlights 38• Salmonid cathelicidin-derived peptides show medium-sensitive antimicrobial activity 39• They are not cytotoxic to fish cells and promote proliferation of trout fibroblasts 40• Peptide STF(1-37) potentiates phagocytosis and respiratory burst in trout leukocytes 41• STF(1-37) and β-glucan act synergistically to immunostimulate trout leukocytes 42Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role 44 in the first line of defense against infections. They have been identified in several salmonid species, 45 where the putative mature peptides are unusually long and rich in serine and glycine residues, often 46 arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. 47Fragments of 24 to 40 residues, spanning specific motifs and conserved sequences in grayling or 48 brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial 49 activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as 50 laboratory reference strains. They were not active in complete medium, but showed varying potency 51 and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only 52 under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated 53 bacteria. However, circular dichroism analyses indicated that they did not significantly adopt 54 ordered conformations in membrane-like environments. The peptides were not hemolytic or 55 cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the 56 fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic 57 activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional 58 interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The 59 three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL 60 simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further 61 analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased 62 intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the 63 immunostimulant β-glucan, it synergistically potentiated both phagocytic up...