Characterization of the active fragments of Spodoptera litura Lebocin-1.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

Elongation factor 1ß' gene from Spodoptera exigua: characterization and function identification through RNA interference.

Elongation factor (EF) is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1ß' from Spodoptera exigua (SeEF-1ß'), its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1ß' mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1ß' mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1ß' dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1ß' was suppressed. The results demonstrate that SeEF-1ß' is a key gene in transcription in S. exigua.

Molecular and functional characterization of a c-type lysozyme from the Asian corn borer, Ostrinia furnacalis.

Some lepidopteran lysozymes have been reported to display activity against Gram-positive and Gram-negative bacteria, in contrast to most lysozymes that are active only against Gram-positive bacteria. OstrinLysC, a c-type lysozyme, was purified from the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae), and shows activity against Gram-positive and Gram-negative bacteria. The NH2-terminal amino acid sequence was determined by Edman degradation and used in a homology cloning strategy. The gene coding for OstrinLysC contains three exons and two introns. The expression profile of the OstrinlysC gene was examined by quantitative real-time PCR. Following injection of the larvae with bacteria, the OstrinlysC gene is strongly up-regulated in immune tissues. Transcripts were also detected in gut tissue. After feeding the larvae with bacteria, OstrinlysC transcripts increased in immune tissues. A very low level of transcript abundance was also detected in gut tissue. These results suggested that the OstrinlysC gene is involved in immune responses. The three dimensional structure of OstrinLysC was predicted. Based on comparison of the 3-D structure of OstrinLysC with that of silkworm lysozyme and chicken lysozyme, we hypothesize that the positive charge-rich surface and the short loop-2, which is close to the cluster of hydrophobic residues, may play important roles in the interaction with the outer membrane of Gram-negative bacterial cell walls.

Bactrocerin-1: a novel inducible antimicrobial peptide from pupae of oriental fruit fly Bactrocera dorsalis Hendel.

A novel antimicrobial peptide, Bactrocerin-1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin-1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin-1 showed very high similarity to the active fragment (46V-65S-NH(2)) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin-1 is a hydrophobic, positively charged, and Lys/Ile/Gly-rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin-1 showed a very broad spectrum of anti-microbial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. Bactrocerin-1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 microM. Analysis of the Helical-wheel projection and the CD spectrum suggested that Bactrocerin-1 contains the amphipathic alpha-helix.