Regulation of Antimicrobial Peptide Genes in the Tobacco Hornworm Manduca sexta
The innate immune system, also known as natural or non-specific immune system, is conserved from insects to humans. The insect innate immune system is composed of humoral and cellular components. Insect humoral responses mainly include synthesis of antimicrobial peptides (AMPs) and activation of the prophenoloxidase (PPO) system. Expression of AMPs is regulated by two signaling pathways, the Toll-Spätzle (Spz) and immune deficiency (IMD) pathways, in Drosophila melanogaster. My research has been focusing on pattern recognition receptors (PRRs) and regulation of AMP genes in the tobacco hornworm, Manduca sexta. In chapter 2, I showed direct interaction between M. sexta MsToll and MsSpz-C108 by Co-immunoprecipitation (Co-IP), and demonstrated that co-expression of MsToll and MsSpz-C108 can activate AMP gene promoters in S2 cells by dual luciferase assays. My results confirm a Toll-Spz pathway in an insect other than Drosophila. In chapter 3, a Toll-ML (MD2 (myeloid differentiation protein 2) - related lipid-recognition)-LPS signaling pathway was identified in M. sexta. The Co-IP assay showed that MsToll, MsML-1 and lipopolysaccharide (LPS) could form a receptor complex. More importantly, I showed that co-expression of MsToll and MsML-1 could up-regulate iv AMP genes activated by LPS. My results for the first time showed that a Toll-ML-LPS signaling pathway is conserved from insects to humans. In chapter 4, I identified Relish short isoforms and Dorsal in M. sexta. Dorsal and Relish belong to the nuclear factor-κB (NF-κB)/Rel family. I showed that M. sexta Dorsal and Rel2 (Relish) isoforms could regulate AMP genes differently, and Dorsal and Rel2 could form heterodimers, which negatively regulated AMP genes. This is a novel finding about NF-κB factors in regulation of AMP gene expression. Characterization of a new member (βGRP3) of the β-1, 3-glucan recognition proteins in M. sexta was presented in chapter 5. I discovered novel functions of M. sexta βGRP3 in the βGRP family proteins: (1) calcium-dependent agglutination of bacterial cells, and (2) antibacterial (bacteriostatic) activity. In Chapter 6, I characterized function of M. sexta gloverin. I showed that M. sexta gloverin can bind to different microbial cell wall components and has activity against different microorganisms. My results suggested that gloverin’s broad spectrum of activity may be related to its binding ability to microbial cell wall components. In Chapter 7, I summarized my discoveries in insect innate immune signaling pathways and proposed future research directions. My research has contributed to a better understanding of innate immunity in insects and vertebrates, regulation of gene expression by NF-κB factors, and evolution of the innate immune signaling pathways.
Table of Contents
General introduction of insect innate immune signaling pathways -- A toll-spatzle pathway in the tobacco hornworm, manduca sexta -- A toll-ML-LPS pathway in the tobacco hornworm, manduca sexta -- Dorsal-REL2 heterodimers negatively regulate antimicrobial peptide expression in manduca sexta -- Characterization of a novel manduca sexta beta-1,3-glucan recognition protein-3 (BGRP3) with multiple functions -- Manduca sexta gloverin microbial components and is active against bacteria and fungi -- Research summary and future direction