| During the long evolutionary history, the relationships between pathogenic microorganisms and their hosts are very complex. Bacillus bombyseptieus (Bb) is one of the major pathogens of the silkworm, which can cause the silkworm bacterial septicemia by natural infection. Although Bb was identified in 1931, the scientific issues about Bb infection the silkworm such as the molecular pathology and molecular immunology of the silkworm host to Bb infection had never been investigated from now on. Similar conditions can be found about typical fungal pathogen Beauveria bassiana and typical virus pathogen BmNPV infection the silkworm. In order to explore the interaction relationships between pathogens and the silkworm, this study used the biological model of Bb oral infection the host silkworm to investigate the silkworm molecular pathology, its immune response patterns and its pathogenesis. Further, the biological models of silkworm pathogens Beauveria bassiana and BmNPV infection the host silkworm were also used to investigate the host-pathogen interactions at the host genome mRNA level. The main results obtained are showed as follows:(1) Silkworm black-breasted septicemia pathogen Bb is a typical Bacillus, which can produce parasporal crystal and kill the silkworm by oral infection. We cloned a 1.5-kb sequence of its 16S rRNA gene using universal primers. After comparison of the Bb 16S rRNA gene sequence with others in the NCBI database, the resulting phylogenetic tree clearly indicated that Bb belongs to Bacillus with a relatively long branch which might caused by a long-term of independent evolution. The phylogenetic analysis showed that Bb is with closest relationship to Bacillus cereus, close relationship to Bacillus thuringiensis, yet relatively far relationship to Bacillus anthracis and other microbes.(2) Bb oral infection the silkworm can cause the host strong responses which was involved in almost all silkworm tissues. During the infection (3 h,6 h,12 h and 24 h), totally 2,436 genes showed modulation, which is about 17% of all the silkworm genome genes. At the 24 h time point, the host silkworm showed the strongest response. Genome-wide survey of the host genes demonstrated many genes and pathways modulated after the infection. GO analysis of the induced genes indicated that their functions could be divided into 14 categories. KEGG pathway analysis identified that six types of basal metabolic pathway were regulated, including genetic information processing and transcription, carbohydrate metabolism, amino acid and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism.(3) Regardless of virus pathogen BmNPV or fungal pathogen B.bassiana are like Gram-positive bacteria Bb and they all can cause strong host responses. Totally 1804,2436 and 1743 genes showed regulation after BmNPV, Bb and B.bassiana infection respectively during the course of infection. For the silkworm non-pathogen E.coli infection, totally 912 genes showed regulation, which is relatively a smaller number. Searching the identified 665 transcription factors (TFs) of the silkworm genome, there are 68,60,56 and 27 genes were induced by Bb, B.bassiana, BmNPV and E.coli, respectively. Among the genes induced by four microbes, from TFs to total induced genes, BmNPV showed a distinctive induced expression pattern, which showed strong response at the "earlier" time of infection (6 h). However, for the other three microbes'infection, they showed strong responses at the "later" time point of infection (24 h).Notably, a lot of genes encode growth factors, receptors and essential components involved in development signaling pathways such as JH, Wnt, MAPK, P53 and cell cycle were modulated after microbes'infection. Major basic metabolic pathways, including genetic information processing and transcription, nucleic acid metabolism, metabolism of cofactors and vitamins, xenobiotics biodegradation and metabolism, amino acid metabolism and nitrogen metabolism, and carbohydrate metabolism illustrated their modulation. Among them three kinds of pathways that related to metabolism of cofactors and vitamins and amino acid metabolism were common induced after four microbes'infection, including one carbon pool by folate, histidine metabolism and tryptophan metabolism, indicating these pathways are very sensitive after microbes'infection. Totally seven pathways can be identified modulation by pathogens BmNPV, B.bombyseptieus and B.bassiana, including xenobiotics biodegradation and metabolism (benzoate degradation via hydroxylation), carbohydrate metabolism (pyruvate metabolism), nucleotide metabolism (purine metabolism), energy metabolism (nitrogen metabolism), amino acid metabolism (urea cycle and metabolism of amino groups), metabolism of Cofactors and vitamins (pantothenate and CoA biosynthesis and porphyrin and chlorophyll metabolism) indicating these pathways are very sensitive for pathogenic microbes'infection. Among the four induced microbes, BmNPV showed 14 specific pathways, which is much more than B.bombyseptieus (3 pathways), B.bassiana (5 pathways) and E.coli (2 pathways), indicating virus pathogen BmNPV leading to a broader level of host metabolic modulation. Gene expression associated with disease pathogenesis such as cytoskeleton, cuticle proteins, respiratory chain, metalloproteases, proteolytic enzymes coding genes were also modulated at different level. In the molecular immune response field, four kinds of microorganisms induced varying degrees of silkworm innate immune related genes up-regulation, such as signal recognition, signal modulation and effectors.(4) Bb can trigger the host immune responses, including cellular immune response and serine protease cascade melanization response. Real time PCR analysis showed that Bb can induce the silkworm systemic immune response, mainly by the Toll pathway. Anti-microorganism peptides (AMPs), including of Attacin, Lebocin, Enbocin, Gloverin and Moricin families, were upregulated at 24 hours post the infection.(5) Similar to Bacillus thuringiensis (Bt), Bb can also induce a silkworm poisoning-related response. This model has been speculated as follows:after oral infection, Bb interred the ingestion of silkworm. The parasporal crystal produced by Bb could be digested by silkworm midgut proteases. The digested toxins could pass through the peritrophic membrane to bind the aminopeptidase N receptors (APNs) of the midgut epithelial cells to damage them. Bb can go to the silkworm hemolymph from the damaged midgut and finally caused the silkworm toxic septicemia.
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