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Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence
Published on: December 11, 2012
A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta
1Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Abstract:
Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.
Insights
Researchers identified a new serine proteinase inhibitor, Manduca sexta serpin-2, in insect hemocytes. Its expression increases upon bacterial infection, suggesting a distinct immune role compared to plasma serpins.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Serine proteinase inhibitors (serpins) are crucial in arthropod immunity.
- Manduca sexta serpin-1 is involved in plasma-based immune responses.
- A novel serpin, M. sexta serpin-2, was investigated for its role in insect immunity.
Purpose of the Study:
- To identify and characterize a second serpin gene (serpin-2) in Manduca sexta.
- To determine the expression patterns and cellular localization of M. sexta serpin-2.
- To investigate the functional role of M. sexta serpin-2 in the insect immune response.
Main Methods:
- cDNA cloning and sequencing of M. sexta serpin-2.
- Amino acid sequence comparison with other invertebrate serpins.
- Generation of polyclonal antiserum against recombinant M. sexta serpin-2.
- Western blot, Northern blot, and in situ hybridization analyses.
- Protein purification and enzyme inhibition assays.
Main Results:
- M. sexta serpin-2 cDNA encodes a 381-amino acid protein with 25-40% identity to other invertebrate serpins.
- Antiserum recognized a 43 kDa protein in hemocytes, not plasma.
- Serpin-2 gene expression significantly increased after bacterial challenge.
- In situ hybridization localized serpin-2 mRNA to granular hemocytes in immune-stimulated larvae.
- Purified serpin-2 showed limited inhibitory activity, primarily against human cathepsin G.
Conclusions:
- M. sexta serpin-2 is an intracellular serpin predominantly expressed in hemocytes.
- Its expression is upregulated during bacterial infection, indicating a role in cellular immunity.
- The intracellular localization suggests a function distinct from plasma-based serpins.
