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.

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.

Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities.

Characterization of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan is the key component forming the backbone of bacterial cell wall. It can be recognized by a group of pattern recognition receptors, known as peptidoglycan recognition proteins (PGRPs) in insects and higher animals. PGRPs may serve as immune receptors or N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). Here, we report the characterization of a short PGRP, PGRP-S1, from the oriental armyworm, Mythimna separata. MsePGRP-S1 cDNA encodes a protein of 197 amino acids (aa) with a PGRP domain of about 150 aa. MsePGRP-S1 was expressed in several tissues of naïve larvae, including hemocytes, midgut, fat body and epidermis. Bacterial challenges caused variable changes in different tissues at the mRNA level. The recombinant protein bound strongly to Staphylococcus aureus and purified peptidoglycans from Staphylococcus aureus and Bacillus subtilis. It can inhibit the growth of gram-negative and gram-positive bacteria by disrupting bacterial surface. It can degrade peptidoglycans from Escherichia coli and Staphylococcus aureus. Taken together, these data demonstrate that M. separata PGRP-S1 is involved in defending against bacteria.

Molecular cloning and analysis of PGRP-L1 and IMD from silkworm Bombyx mori.

Peptidoglycan is one of the major components of bacterial cell wall. The innate immune system of insects utilizes a group of peptidoglycan recognition proteins (PGRPs) for the recognition of specific peptidoglycans and activating immune signaling pathways. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are two important signaling molecules of the IMD pathway. Here we cloned and characterized PGRP-L1 and IMD from the domesticated silkworm Bombyx mori (BmPGRP-L1 and BmIMD). BmPGRP-L1 gene consists of five exons that encodes a polypeptide of 304 amino acids with a transmembrane region and an extracellular PGRP domain. The PGRP domain lacks key residues for the amidase activity. BmIMD cDNA encodes a polypeptide of 250 amino acids with a death domain. BmPGRP-L1 and BmIMD were expressed in various tissues and induced by bacterial challenges. In addition, in vivo blocking of the PGRP domain by the antiserum or purified antibody significantly reduced the expression of some antimicrobial peptide genes. The extracellular region of BmPGRP-L1 bound to diaminopimelic acid-type and lysine-type peptidoglycans. Overexpression of full-length BmIMD in Drosophila Schneider 2 cells significantly induced three antimicrobial peptide genes. These results suggest that BmPGRP-L1 and BmIMD may be players in the IMD pathway of B. mori. This study provides a foundation for further studies on the functions of silkworm IMD pathway.

Immune functions of insect ßGRPs and their potential application.

Insects rely completely on the innate immune system to sense the foreign bodies and to mount the immune responses. Germ-line encoded pattern recognition receptors play crucial roles in recognizing pathogen-associated molecular patterns. Among them, ß-1,3-glucan recognition proteins (ßGRPs) and gram-negative bacteria-binding proteins (GNBPs) belong to the same pattern recognition receptor family, which can recognize ß-1,3-glucans. Typical insect ßGRPs are comprised of a tandem carbohydrate-binding module in the N-terminal and a glucanase-like domain in the C-terminal. The former can recognize triple-helical ß-1,3-glucans, whereas the latter, which normally lacks the enzymatic activity, can recruit adapter proteins to initiate the protease cascade. According to studies, insect ßGRPs possess at least three types of functions. Firstly, some ßGRPs cooperate with peptidoglycan recognition proteins to recognize the lysine-type peptidoglycans upstream of the Toll pathway. Secondly, some directly recognize fungal ß-1,3-glucans to activate the Toll pathway and melanization. Thirdly, some form the 'attack complexes' with other immune effectors to promote the antifungal defenses. The current review will focus on the discovery of insect ßGRPs, functions of some well-characterized members, structure-function studies and their potential application.

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.

A single-CRD C-type lectin is important for bacterial clearance in the silkworm.

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca(2+)-dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 (CTL-S3), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus. More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens.