Characterization and expression of attacin, an antibacterial protein-encoding gene, from the beet armyworm, Spodoptera exigua (Hübner) (Insecta: Lepidoptera: Noctuidae)

Abstract

To isolate antimicrobial-related genes from the beet armyworm, Spodoptera exigua, we performed GeneFishing, a polymerase chain reaction (PCR)-based differential display technique. An attacin-like complementary DNA (cDNA) including a 3'-untranslated region was identified from among 18 over-expressed genes in microbial-infected larvae. The full-length attacin cDNA from S. exigua cDNA (Seattacin) was cloned using rapid amplification of cDNA ends PCR. The attacin-like cDNA transcript was 765 nucleotides in length, and the predicted polypeptide was 254 amino acids in length with a calculated molecular mass of 27.6 kDa and an isoelectric point of 6.44. The protein sequence of the attacin-like cDNA showed high identity to that of Trichoplusia ni (61.2%). The amino acid sequence identity of Seattacin to the orthologous proteins in Bombyx mori, Manduca sexta, Heliothis virescens, Hlicoverpa armigera, Hyphantria cunea, Hyalophora cecropia, and Drosophila melanogaster was 61.2, 46.1, 44.5, 42.2, 39.5, 45.1, and 24.0%, respectively. To examine possible immune functions of the attacin-like cDNA, its expression was investigated by reverse transcriptase PCR analysis after challenging S. exigua with microorganisms. The attacin-like cDNA was expressed at high levels 12 h post-infection, and its expression was slightly induced 4-8 h post-infection compared to control larvae inoculated with sterile water. Furthermore, induced Seattacin showed biological activity against several bacteria including Escherichia coli DH5α, Pseudomonas cichorii, Bacillus subtilis, and Listeria monocytogenes. These results suggest that the attacin-like cDNA of S. exigua codes for antimicrobial peptides.

Comprehensive Analysis of Gene Expression Profiles of the Beet Armyworm Spodoptera exigua Larvae Challenged with Bacillus thuringiensis Vip3Aa Toxin

“…Up-regulation of AMPs induced by bacterial infection, bacterial feeding (including Bacillus sp), or by Cry toxins, has been previously reported in S. exigua [26-28] and in other lepidopteran [16,30,32,48,49,66-68]. In general, the regulated AMPs exhibited an increase in transcription with time following the same pattern previously described for S. exigua attacin [28].…”

“…In fact, the MAPK p38 pathway is activated to protect the nematode Caenorhabditis elegans or HeLa cells against the Bt Cry5 toxin [22,23,24], and it is activated in Lepidoptera and Diptera after Cry-toxin intoxication [25]. Some studies indicate that S. exigua also responds to bacterial infection through the general mechanisms involved in immune response in insects, producing antimicrobial peptides (AMPs) such as cecropins [26], gloverins [27], or attacins [28], as well as producing “REsponse to PAThogen” (REPAT) proteins in the midgut after Cry1Ca intoxication [29]. …”

Insect antimicrobial peptides and their applications

“…Interestingly, the pro-peptide (P domain) of D. melanogaster attacin-C is longer and proline-rich, which is similar to small proline-rich peptides (see below), and this pro-peptide is active against Gram-negative bacteria (Rabel et al, 2004). Attacins have been identified in many lepidopteran species, including H. cecropia (Hultmark et al, 1983), Heliothis virescens (Ourth et al, 1994), B. mori (Sugiyama et al, 1995), Trichoplusia ni (Kang et al, 1996; Tamez-Guerra et al, 2008), Hyphantria cunea (Kwon et al, 2008), S. cynthia (Kishimoto et al, 2002), Manduca sexta (Rao and Yu, 2010), Helicoverpa armigera (Wang et al, 2010b), and Spodoptera exigua (Bang et al, 2012), as well as in some dipteran species like D. melanogaster (Asling et al, 1995; Dushay et al, 2000) and the Tsetse fly Glossina morsitans (Hao et al, 2001; Wang et al, 2008). …”

“…G. morsitans attacin-A1 is active against E. coli and the protozoan parasite Trypanosoma brucei in vitro (Hu and Aksoy, 2005), H. cunea attacin-B is active against Gram-negative E. coli and Citrobacter freundii as well as the fungus C. albicans (Kwon et al, 2008), S. exigua attacin is active against Gram-negative E. coli and Pseudomonas cichorii , and Gram-positive B. subtilis and Listeria monocytogenes (Bang et al, 2012), and recombinant Drosophila attacin-A is active against E. coli with minor hemolysis against porcine red blood cells (Wang et al, 2010a). Interestingly, the hybrid protein, attacin-coleoptericin, has enhanced antibacterial activity against E. coli , Burkholderia glumae and B. subtilis compared to either attacin or coleoptericin alone (Lee et al, 2013b).…”

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

“…Activated hemocytes kill pathogens through the process of phagocytosis, encapsulation, and nodulation. By contrast, the humoral responses include synthesis of antimicrobial peptides (AMPs) and activation of the phenoloxidase (PO) cascade, and can be activated by several known cascade pathways, such as the NF-kβ pathway, Toll and immune deficiency (Imd) pathway, Janus kinase (JAK)/STAT transcription factors pathway, and enzymatic cascades that regulate melanin formation and clotting [4], [5], [6], [7], [8], [9], [10]. Which of these responses is triggered first upon pathogen invasion of the insect body cavity (hemocoel) is still debated as a matter of contention.…”