ABSTRACT
Lactobacillus acidophilus are Gram-positive bacteria distributed in diverse environments, and as a component of the normal microbiota of gastrointestinal and urogenital tract, they are relevant for human beings. Classified as lactic acid bacteria, due to the production of lactic acid, Lactobacillus can also produce antimicrobial peptides (AMPs), which is a compound synthesized by all forms of life aiming for protecting themselves from threats and to increase their competitivity to survive in a specific environment. AMPs are molecules capable of inhibiting the growth of microorganisms and, due to the indiscriminate use of conventional antibiotics and the emergence of multi-resistant bacteria, they have become an alternative, not only for treating multi-resistant infections, but also for the identification of probiotic products and food conservation. Considering the rampant rise of bacterial resistance to classical antimicrobials, the present study aimed to isolate and characterize AMPs from L. acidophilus extracts. Lactobacillus acid extract was pre-fractionated on disposable cartridges, followed by a high-performance liquid chromatography (HPLC). The collected fractions were evaluated in a liquid growth inhibition assay allowing to identify eight fractions with antimicrobial activity, and one of them showed antimicrobial activity against Candida albicans and, for this reason, was further characterized by mass spectrometry (MS). A peptide with a molecular mass of 1788.01 Da, showing the primary sequence NEPTHLLKAFSKAGFQ, as determined by MS, was named as Doderlin. Interestingly, antimicrobial molecules isolated from L. acidophilus have already been described previously, but few reports describe AMPs effective against C. albicans as the one reported here. We show here that this newly discovered molecule has a biological property with potential to be used in pharmaceutical and food companies, in the fight against contamination and/or for treating infections caused by microorganisms, respectively.
ABSTRACT
Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the ß-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 µg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 µg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 µg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 µg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.
