Study On The Effects Of BmIDH And NbNTT1 To Silkworm Energy Metabolism And Nosema Bombycis Proliferation

Microsporidia are obligate eukaryotic intracellular parasites that can infect a variety of invertebrates and vertebrates including human beings.Microsporidia is the important pathogenic pathogen for many animals due to its long infection term but has few prevention and control measures.For the sericulture industry,pebrine caused by Nosema bombycis could cause huge economic losses.Microsporidia does not possess a complete pyruvate dehydrogenase complex and tricarboxylic acid cycle pathway,and are extremely dependent on energy obtained from the host for their replication and proliferation.However,it is still unclear how microsporidia regulates the energy metabolism of the host.This thesis intends to investigate the influence of Nosema bombycis on the host’s energy metabolism,and analyze the changes of energy metabolism and the interaction between microsporidia and the host,which is helpful to elucidate the pathogenesis of pathogens and provide new insight and theoretical support for the treatment and prevention of microsporidia infection.The main results and conclusions of this study are as follows:1.Characteristics analysis of nosema bombycis infectionAfter the oral infection with Nosema bombycis,the body size and weight of fifth instar silkworm larvae were analyzed at 12 hour(h),48 h,72 h and 120 h post-infection.It was found that the larvae of the infected group grew slower than those of the control group at the concentration of 5×10~5 spore per larvae with the infection time increased,and their body weights decreased significantly in 60 h after infection and was extremely significant at 120 hours post infection.Observation of infected cells by transmission electron microscopy revealed that host mitochondria were enriched around microsporidia,suggesting that replication and proliferation of microsporidia was associated with host mitochondria,and statistical changes in the number of mitochondria were significantly increased compared to the control.By detecting the changes of the pyruvate and ATP in the larvae after microsporidium infection,it was found that Nosema bombycis promoted the enhanced metabolism and the accumulation of the metabolic substrate pyruvate in the silkworm and increased the production of ATP in the larvae.2.Transcriptome analysis of changes in host energy metabolic pathwayNosema bombycis enhanced the metabolic level of the host,but the changes of energy metabolic pathways need further analysis.After microsporidia infection in 12 h,24 h,36 h,48 h,60 h,72 h and 120 h,midgut tissue was dissected to extract RNA for transcription library and sequence.Clusters of orthologous groups for eukaryotic complete genomes(KOG)analysis revealed that microsporidian infection leads to the upregulated expression of a large number of host signaling-related genes,while host transcriptional translation and downstream immune defense classes transcription of genes was inhibited.Host carbon metabolism and lipid metabolism genes were significantly upregulated,while host nucleotide metabolism and amino acid metabolism-related genes were significantly downregulated;energy metabolism genes were also partially repressed in the later stages of infection.Further by Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway clustering analysis,the metabolic pathways such as glucose metabolism and tricarboxylic acid cycle showed significant differences at 12 h,24 h,36 h and 48 h post infection,while the differences at 60 h,72 h and 120 h clustered significantly in the glyceride metabolism pathway.It showed that infection by Nosema bombycis promoted host glucose metabolism and lipid degradation,enhanced the ATP synthesis pathway,but inhibited the the synthesis pathway of long chain fatty acids and ATP consumption pathway.Among them,chondroitin sulfate synthase(CSS),glycerol kinase protein(GK),phosphatidylinositol phosphatase SAC1 and ATP-binding cassette transporter ABC transporter were up-regulated.Host galactose kinase(GALK)and hormone-sensitive lipase(HSL)were down-regulated.Vesicular ATP synthase and restorpin interacting proteins were persistently suppressed in the expression changes of host energy metabolism genes;host glucose transporter 1(GLUT 1),isocitrate dehydrogenase(IDH),hexokinase(HK),and thiolase were significantly up-regulated.The changes in key genes of the host energy metabolic pathway were verified by quantitative PCR.ATP and fatty acid assays revealed that microsporidian infection induced ATP accumulation and decreased free fatty acids in host midgut tissues.It was hypothesized that it might promote host glucose uptake,processes of glycolysis and tricarboxylic acid cycle as well as fatty acid degradation,indicating that infection of Nosema bombycis promoted host energy metabolism and enhanced ATP synthesis.3.Effects of BmIDH on host metabolism and microsporidia proliferationThe tricarboxylic acid cycle is one of the most important links of aerobic metabolism in the organism and this study found that the tricarboxylic acid cycle pathway was abnormally changed after infection with Nosema bombycis through the analysis and screening in the previous part.Bombyx Mori isocitrate dehydrogenase(BmIDH)was induced 10-25 fold up-regulation over multiple time points post Nosema bombycis infection.BmIDH was detected by quantitative real-time polymerase chain reaction(q RT-PCR)in cells infected with Nosema bombycis and up-regulated 20-140-fold compared with control cells.The BmIDH protein has a nicotinamide adenine dinucleotide phosphate(NADP)-dependent binding site,a conserved structural domain,and possibly an oligomerized functional domain.Overexpressing BmIDH localizes to the cytoplasm in Bm N-SWU1 cells and was co-localized with mitochondria.To further investigate the effect of BmIDH on host energy metabolism,overexpression of BmIDH detected the accumulation of cellular ATP and promoted the uptake of glucose from the environment.MTS assay revealed that overexpression of BmIDH enhanced the conversion of cells to MTS and promoted cellular metabolic viability,which in turn promoted the proliferation rate of Nosema bombycis in the host cells.BmIDH was knocked down by CRISPR/Cas9 gene editing,and a significant reduction in ATP production was detected,while the ability of microsporidia to proliferate from infection was somewhat restored,but the effect was weaker than that of controls,indicating that the pathway of intracellular ATP production after microsporidia infection is dependent on BmIDH.Microsporidia proliferation was also significantly inhibited,indicating that BmIDH Protein is important for the proliferation of microsporidia.Knocking down BmIDH slightly reduced the host’s ability to absorb glucose,and the difference was not significant compared to the control,indicating that host glucose uptake was not dependent on BmIDH.The inhibition of glucose transport by GLUT1 through the glucose transporter inhibitor BAY-876 indirectly inhibited the glycolytic pathway of the treated cells,weakened cellular energy metabolism,and resulted in a decrease in intracellular ATP content,indicating that the host relied on GLUT1 for glucose cis-concentration transmembrane transport during microsporidial infection,and the inhibition of GLUT1activity could ultimately inhibit the proliferation of microsporidia.BmIDH and GLUT1,as important host factors,have the potential to be molecular targets against microsporidia.4.Functional study and interaction analysis of NbNTT1The up-regulation of host energy metabolism genes induced by Nosema bombycis provides a material basis for microsporidia to acquire ATP,and further studies on how microsporidia utilize host energy will help to better understand the interaction process between pathogen and host.Microsporidia could transport the ATP by transporters.The Nucleotide transporter(NTT),NBO＿251gi001,was selected by transcriptome analysis to be consistently up-regulated in microsporidia infestation and proliferation,and has a high expression profile and is associated with ATP transport.The Nosema bombycis nucleotide transporter(NbNTT1)protein has a signal peptide sequence and four transmembrane domains as well as a typical ATP translocation domain,which is highly conserved at functional domain positions.In homologous evolutionary relationships,it is more closely related to the Papilio xuthus and the Nosema cerana.It is located on the side of the host cell membrane,partly in the cytoplasm,and its structure contains signal peptides related to the transmembrane domain.The expression of NbNTT1 can promote the silkworm cells produce more ATP and has the ability to promote the proliferation of microsporidia.In order to identify whether the function of NbNTT1 is closely related to host BmIDH,by knocking down BmIDH after overexpressing NbNTT1,it has a certain restorative ability on host ATP synthesis,which is speculated that the transport of ATP by NbNTT1 during infection decreases the concentration of ATP in host cells and cannot meet the cellular ATP requirement,thus inducing the host to accelerate ATP production.It also indicates that NbNTT1 may have the ability to promote host ATP production independent of BmIDH,which is presumably related to the NbNTT1interacting proteins.NbNTT1 was screened by co-immunoprecipitation assay to interact with four silkworm proteins and three microsporidian proteins during infection.Through immunofluorescence co-localization and co-immunoprecipitation assay,it was proved that NbNTT1 interacts with Bombyx mori Heat Shock Protein Bm HSP70,Bombyx mori Adenine Nucleotide Transporter Bm ANT and Nosema bombycis protein Nb Actin.Host interaction protein Bm HSP70 of NbNTT1 was highly upregulated after microsporidia infection,and Bm ANT was inhibited after infection.The overexpression of NbNTT1showed the same trend.Presumably,Bm HSP70 enhances the level of glycolysis and ATP production in the presence of NTT1,and Bm HSP70 may be involved in NTT1-mediated ATP transport as a co-molecular partner.Host ANT mediates mitochondrial energy transport,and the interaction between NbNTT1with Bm ANT presumably inhibit Bm ANT’s ATP transport ability.These studies support the establishment of an energy interaction model between microsporidia and the host,which is conducive to a clearer understanding of the regulation of host energy metabolism during microsporidian infection and provides a theoretical basis for the creation of anti-microsporidian molecular target materials.