Putative Antimicrobial Peptide Research Articles

Production and characterization of a novel antimicrobial peptide HKABF by Pichia pastoris

HKABF, Hippocampus kuda antibacterial factor, is a putative antimicrobial peptide identified from the cDNA library of the brooding pouch of H. kuda Bleeker. The putative mature peptide, fused with a 6His-tag, an anionic peptide (SAPLNPDNID) and to an enterokinase cleavage site was expressed in Pichia pastoris. The mature HKABF peptide, released by enterokinase cleavage, showed antimicrobial activity against Staphylococcus aureus and Staphylococcus saprophyticus at low concentrations (0.5–2.5 μg/ml). The recombinant mature peptide was heat-stable and had low hemolytic activity. Circular dichroism (CD) spectroscopy data indicates its classification as a CSαβ-type antimicrobial peptide. This study serves as an impetus for the production and further research of this antimicrobial peptide.

Identification of immune inducible genes from the velvet worm Epiperipatus biolleyi (Onychophora)

Onychophora are the next relatives of Arthropoda and, hence, represent an important taxon to unravel relationships among Insecta, Crustacea, Arachnida, and Myriapoda. Here, we screened for immune inducible genes from the onychophoran Epiperipatus biolleyi (Peripatidae) by injecting crude bacterial LPS and applying the suppression subtractive hybridization technique. Our analysis of 288 cDNAs resulted in identification of 36 novel genes in E. biolleyi whose potential homologues from other animals are known to mediate immune-related signaling (e.g. mitogen-activated protein kinase kinase 1 and immunoglobulin enhancer binding protein), to be involved in cellular processes (e.g. perilipin and myosin light chain), or to act as immune effector molecules (e.g. lysosomal beta-galactosidase, a putative antimicrobial peptide and a potential thiolester containing protein). Comparisons with homologous genes from other animals including the two most favored ecdysozoan model organisms of innate immunity research, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, provide further insights into the origin and evolution of Arthropoda immunity.

The impact of membrane lipid composition on antimicrobial function of an α-helical peptide

VP1, a putative α-helical antimicrobial peptide (α-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500 μM. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract ( circa 4.5 mN m −1) but transient pressure changes in corresponding E. coli monolayers ( circa 1.0 mN m −1), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 (Δ G Mix < 0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide (Δ G Mix > 0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of α-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.

Evolution of the Primate Cathelicidin: CORRELATION BETWEEN STRUCTURAL VARIATIONS AND ANTIMICROBIAL ACTIVITY

Cathelicidin genes homologous to the human CAMP gene, coding for the host defense peptide LL-37, have been sequenced and analyzed in 20 primate species, including Great Apes, hylobatidae, cercopithecidae, callithricidae, and cebidae. The region corresponding to the putative mature antimicrobial peptide is subject to a strong selective pressure for variation, with evidence for positive selection throughout the phylogenetic tree relating the peptides, which favors alterations in the charge while little affecting overall hydrophobicity or amphipathicity. Selected peptides were chemically synthesized and characterized, and two distinct types of behavior were observed. Macaque and leaf-eating monkey RL-37 peptides, like other helical antimicrobial peptides found in insect, frog, and mammalian species, were unstructured in bulk solution and had a potent, salt and medium independent antimicrobial activity in vitro, which may be the principal function also in vivo. Human LL-37 and the orangutan, hylobates, and callithrix homologues instead showed a salt-dependent structuring and likely aggregation in bulk solution that affected antimicrobial activity and its medium dependence. The two types of peptides differ also in their interaction with host cells. The evolution of these peptides has thus resulted in distinct mechanisms of action that affect the direct antimicrobial activity and may also modulate accessory antimicrobial functions due to interactions with host cells.

An antimicrobial peptide of the earthworm Pheretima tschiliensis: cDNA cloning, expression and immunolocalization.

A cDNA encoding a putative antimicrobial peptide (named PP-1) was obtained using a rapid amplification of cDNA ends from the Asian earthworm, Pheretima tschiliensis. PP-1 showed 77.6% homology with the antimicrobial peptide lumbricin I isolated from the earthworm Lumbricus rubellus. PP-1 lacked an obvious signal peptide sequence. RT-PCR analysis demonstrated that this gene was expressed mainly in the body wall. PP-1 was expressed in Escherichia coli as a fusion protein with a maltoze-binding protein. A polyclonal antiserum was raised in mice using this recombinant fusion protein as antigen. Immunohistochemical studies showed that PP-1 was only in the mucus of the epidermis.

Biological characterization of a novel mammalian antimicrobial peptide

A putative antimicrobial peptide of 34 residues was recently deduced from a bovine cathelicidin gene sequence and named BMAP-34. A peptide based on the deduced sequence was chemically synthesized and used to study the localization, structure and biological activities of BMAP-34. A Western blot analysis using antibodies raised to the synthetic peptide showed that BMAP-34 is stored as proform in the cytoplasmic granules of bovine neutrophils. CD spectroscopy indicates that the peptide assumes an amphipathic α-helical conformation, as also predicted by secondary structure analysis. The peptide exerts a broad spectrum antimicrobial activity against both Gram-negative and Gram-positive organisms, and is not active against eukaryotic cells. When tested on Escherichia coli ML-35, the kinetics of bacterial killing and of inner membrane permeabilization are slower than those observed for other α-helical peptides derived from cathelicidins.

Structural organization of the bovine cathelicidin gene family and identification of a novel member

Cathelicidins are a group of myeloid antimicrobial peptide precursors found in a variety of mammalian species. Transcripts of this family show a highly conserved 5′ region corresponding to the 5′ untranslated region, signal peptide and propiece, and diverse 3′ regions encoding structurally varied C-terminal sequences that correspond to mature antimicrobial peptides after proteolytic release from the precursors. To establish the size of the bovine gene family encoding these proteins, λ genomic clones were isolated by screening a bovine library with a probe based on the conserved cDNA region of bovine members. Restriction mapping, hybridization studies and sequence analysis identified 11 distinct genes that based on the intergenic distances of contiguous genes appear to be in close physical proximity. Among these, a novel gene encoding the precursor of a putative α-helical antimicrobial peptide was recognized and sequenced. The novel gene appears to be expressed in bovine bone marrow myeloid cells, spleen and testis.