Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

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.

Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections

“…The antagonistic effect of this cation on the candidacidal activity of cationic AMPs is well documented [9,42,82] and has been recently confirmed also in VSF in the case of hepcidin 20 [19]. The antifungal activity of BMAP-28 reported here is consistent with previous studies indicating ability of this peptide to inactivate in vitro a variety of pathogenic fungi grown in planktonic form [7,76], also including a collection of Candida species isolated from clinical samples. In this respect, we show that BMAP-28 is effective against various azole-resistant non-albicans Candida spp.…”

“…The results are reported as the mean ± SD of at least three independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. membrane ergosterol, amphipathic AMPs such as BMAP-28 exert their antimicrobial effects via membrane binding and permeabilization [7,76]. The latter mechanism is faster and somewhat less specific than the azole mechanism and thus, the development of microbial resistance to AMPs is generally less likely to arise.…”

“…In the case of BMAP-28 and LL-37, these differences have resulted in distinct structuring and aggregational properties. BMAP-28 behaves as a random-coil monomer in physiological solution and adopts a helical conformation when interacting with membranes [76] while conversely, LL-37 has a salt-dependent ability to structure and aggregate in saline solution [34]. This distinct behaviour influences the mode of membrane interaction and membrane damage by the two peptides.…”

“…Cell density was assessed by measuring turbidity at 600 nm and was adjusted to obtain the proper inoculum size. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method [76]. Briefly, two-fold serial dilutions of each test agent, either in Sabouraud liquid medium or in VSF, were prepared in 96-well microtiter plates to a final volume of 50 l. A total of 50 l of the adjusted inoculum was added to each well to achieve the final concentration of 5 × 10 4 cells/ml (or 1 × 10 7 cells/ml, when MICs against biofilm-forming concentrations of C. albicans SC5314 were to be determined).…”

“…As microbial adhesion represents a crucial step in biofilm formation and the ability of LL-37 to inhibit the development of bacterial biofilm is well characterized [36,54], we asked ourselves whether LL-37 was also active against biofilm formed by C. albicans. We were further interested in comparison between LL-37 and the bovine ortholog BMAP-28, a cathelicidin peptide [85] highly active against a broad spectrum of pathogens including yeasts and filamentous fungi [7,8,74,76]. BMAP-28 was recently shown to be active against bacterial biofilm formed by cystic fibrosis isolates [59], but its activity against fungal biofilm has not been studied so far.…”