Transcriptome Landscape Of Midgut Immunity And Functional Study Of Peptidoglycan Recognition Protein-S5 In Bombyx Mori

Part I: Regulation of Bombyx mori midgut transcriptome by bacteria. Innate immunity is conserved from insect to human and functions as the first line of the host defense against invasive microbes. Most of pathogens in insects are from feeding. The gut can provide physical and physiological barriers to prevent gut microbes breaking through gut and permeating into hemolymph. Production of ROS and AMPs in gut of Drosophila melanogaster is efficient to protect insects from invasive pathogens infection and maintain the hemeostasis of symbiotic bacteria. As a kind of economic insect in lepidopteran, Bombyx mori is little known about the battles between midgut immune system and invasive bacteria. Via the study of the gut immune system in Bombyx mori, it is expected to discover the mechanism that how the gut immune system of lepidopteran works which may be used for pest biological control in the future.In this study, we sequenced the transcriptome of the midgut in Bombyx mori infected with B. bombyseptieus and Y. pseudotuberculosis by feeding. Via the mortality assay, we found that larval silkworms are more susceptible to B. bombyseptieus than to Y. pseudotuberculosis at the early stage of infection. However, in the late stage the larvae are more susceptible to Y. pseudotuberculosis than to B. bombyseptieus. After analysis of hierarchical clustering, venn diagram, Gene Ontology(GO) and KEGG, we also found the transcriptomes of midgut in silkworm infected by those two kind of bacteria are regulated in different patterns, Gram-positive bacteria B. bombyseptieus is capable to induced stronger responses than the Gram-negative bacteria Y. pseudotuberculosis after oral infection in silkworm midgut, bacterial invasion is able to result in more energy consumption for infected silkworms to eliminate the invading bacteria, ROS play important roles in eliminating invading bacteria at early stage and AMPs are responsible for eliminating invading bacteria at late stage in silkworm midgut. Additionally, we found that Toll pathway plays some roles in silkworm midgut immune system.Part II: Functional study of peptidoglycan recognition protein-S5 in Bombyx mori. Recognition of invading microbes as non-self is the first step of immune responses. Peptidoglycan recognition proteins(PGRPs), a family of pattern recognition proteins, play key roles in the innate immune system against invading pathogens and parasites in vertebrates and invertebrates. Peptidoglycans(PGs), which are conserved in most bacteria as an essential component of cell wall, are recognized by PGRPs as pathogen-associated molecular pattern(PAMP). Twelve PGRPs have been identified in the silkworm, Bombyx mori, through bioinformatics analysis. In this study, we expressed Bombyx mori PGRP-S5 and its mutant PGRP-S5_C177S, and functional studies showed that PGRP-S5 is able to bind to PGs, is an amidase with a key enzyme site C177 which is not required for binding to PGs, and is a antibacterial protein depended on its amidase activity. Additionally, PGRP-S5 is also involved in the prophenoloxidase activation pathway and processing of PG by PGRP-S5 is necessary for formation of the pathway initiation complex. More importantly, we found that PGRP-S5 negatively regulates antimicrobial peptides generation in an amidase-dependent manner, likely through the IMD pathway. Thus, silkworm PGRP-S5 acts as a sensor, a modulator, and an effector in the silkworm humoral immune system.