Abstract
In contrast to vertebrate immune systems, invertebrates lack an adaptive response and rely solely on innate immunity in which antimicrobial peptides (AMPs) play an essential role. Most of them are membrane active molecules that are typically unstructured in solution and adopt secondary/tertiary structures upon binding to phospholipid bilayers. This work presents the first characterization of a constitutive AMP from the hemolymph of an Opiliones order animal: the harvestman Acutisoma longipes. This peptide was named longipin. It presents 18 aminoacid residues (SGYLPGKEYVYKYKGKVF) and a positive net charge at neutral pH. No similarity with other AMPs was observed. However, high sequence similarity with heme-lipoproteins from ticks suggested that longipin might be a protein fragment. The synthetic peptide showed enhanced antifungal activity against Candida guilliermondii and C. tropicalis yeasts (MIC: 3.8–7.5 μM) and did not interfered with VERO cells line viability at all concentrations tested (200–0.1 μM). This selectivity against microbial cells is related to the highest affinity of longipin for anionic charged vesicles (POPG:POPC) compared to zwitterionic ones (POPC), once microbial plasma membrane are generally more negatively charged compared to mammalian cells membrane. Dye leakage from carboxyfluorescein-loaded POPG:POPC vesicles suggested that longipin is a membrane active antimicrobial peptide and FT-IR spectroscopy showed that the peptide chain is mainly unstructured in solution or in the presence of POPC vesicles. However, upon binding to POPG:POPC vesicles, the FT-IR spectrum showed bands related to β-sheet and amyloid-like fibril conformations in agreement with thioflavin-T binding assays, indicating that longipin is an amyloid antimicrobial peptide.
Highlights
The invertebrate immune system lacks a specific response against invading microorganisms because it is unable to produce antibodies that can recognize these pathogens
The fractions with antimicrobial activity were revaluated against M. luteus
We presented the purification, characterization, spectrum of biological activity and vesicle interaction studies of a new antimicrobial peptide isolated from the hemolymph of the harvestman Acutisoma longipes
Summary
The invertebrate immune system lacks a specific response against invading microorganisms because it is unable to produce antibodies that can recognize these pathogens. Different from the vertebrates, which possess an adaptive response, their defense mechanisms against invading agents rely solely on the innate immunity [1]. Cellular and humoral reactions are part of invertebrate innate immunity and act in concert to combat invading agents. The clotting cascade and the action of antimicrobial peptides (AMPs) are part of the humoral response. In addition to avoiding hemolymph leakage, clotting can physically trap microorganisms, thereby favoring the action of antimicrobial molecules such as AMPs that are involved in direct microbial killing [2, 3]. AMPs play an important role in invertebrate innate immunity
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