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.

Molecular Characterization of a Mitochondrial Manganese Superoxide Dismutase From Chilo suppressalis (Lepidoptera: Crambidae).

In insects, superoxide dismutases (SODs) play a critical role in the scavenging of harmful reactive oxygen species (ROS) and protecting against oxidative stress induced by various environmental stresses. The Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is an economically important insect pest of rice crops. In this study, a mitochondrial manganese SOD (Cs-mMnSOD) gene was characterized in C. suppressalis. The deduced Cs-mMnSOD protein has typical highly conserved features of mitochondrial manganese SODs, including four manganese binding residues, the signature DVWEHAYY peptide, and a mitochondrial-targeting sequence at the N-terminus. Transcription of Cs-mMnSOD was detectable at all developmental stages, but highest in pupae. Furthermore, the mRNA level of Cs-mMnSOD was strongly upregulated (more than twofold increase) following exposure to low and high temperatures (4, 30 and 35°C), insecticides (chlorpyrifos and chlorantraniliprole), and chemical reagents (cumene hydroperoxide, paraquat, H2O2 and CdCl2), but slightly elevated (less than twofold increase) in response to 8°C, abamectin and CuSO4. Additionally, the Cs-mMnSOD transcription results were consistent with the enzymatic activity data of the protein product. Purified recombinant Cs-mMnSOD protein expressed in Escherichia coli displayed SOD activity and thermostability. Furthermore, E. coli cells overexpressing Cs-mMnSOD exhibited long-term resistance to the oxidative inducers cumene hydroperoxide and paraquat. Our findings indicate that Cs-mMnSOD plays an important role in protecting C. suppressalis against oxidative damage.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura.

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.