Nosema Bombycis Can Regulate Host Cell Metabolism And Apoptosis By Utilizing Self-derived Hexokinase NbHXK And Host-derived Hexokinase BmHXK

Microsporidia is a class of obligate intracellular eukaryotic microorganisms.They are widely found in nature and can infect a wide variety of animals ranging from invertebrates such as insects to vertebrates like human.Nosema bombycis was the first microsporidian recognized by humans and was able to infect silkworm and cause Pébrine disease,resulting in significant economic losses and a serious threat to the sustainable development of the sericulture industry in various countries.Therefore,an in-depth and detailed study of the molecular mechanisms of N.bombycis infection and proliferation not only has important theoretical value,but also lays the foundation for the prevention and treatment of microsporidiosis.For the intracellular parasitism microsporidia,the function of secreted proteins is extremely important,and many biological processes require the help of secretory proteins,such as the formation of microsporidian spore wall,the regulation of host physiological metabolism,and the defense of host cell's immune system.Therefore,the identification of secreted proteins has very important significance not only for the understanding of its proliferation mechanism,but also for the prevention and control of microsporidia.Since microsporidia cannot be genetically edited using existing techniques,in recent years,the research on N.bombycis has mainly focused on the function analysis of its infection device.However,studies on secretory proteins that has played a role in the proliferation of microsporidia in host cells are rarely reported.In view of this,secreteory proteins in the whole genome of N.bombycis were predicted and analyzed by microsporidia database(Microsporidia DB)and silkworm pathogen database(Silk Path DB),and the N.bombycis hexokinase Nb HXK was screened out.Nb HXK is a metabolic enzyme of the glycolytic pathway but has a signal peptide sequence.We hypothesize that this is likely related to N.bombycis obligate intracellularparasitism as well as the proliferation pattern which highly dependent on host cell material and energy supply.In order to further explore the function of the N.bombycis hexokinase Nb HXK,we conducted the following studies and obtained the following results:1.N.bombycis is able to secrete hexokinase Nb HXKThe N.bombycis hexokinase Nb HXK gene is 1287 bp in length and encodes a protein containing 428 amino acid residues.Domain analysis revealed that amino acids1-25 of the Nb HXK are predicted signal peptide sequences,it also contains two conserved hexokinase domains,separately located in amino acids 29-228 and 235-424.Phylogenetic analysis revealed that Nb HXK was classified into the microsporidian hexokinase and N.bombycis was closely related to Nosema ceranae and Nosema apis.The signal peptide sequence of Nb HXK gene was cloned into the yeast signal peptide trapping vector p SUC2T7M13 ORI,and then transformed into the invertase-deficient yeast strain YTK12.Compared with the positive control and the negative control,the signal peptide sequence of Nb HXK was able to induce the secretion of the target protein invertase,and promoted the growth of transformed yeast on YPRAA medium.At the same time,the signal peptide sequences of Al HXK gene and Na HXK gene can achieve the same secretion effect,indicating that most of the microsporidia may have evolved a hexokinase gene with a signal peptide sequence.This is closely related to its intracellular parasitic lifestyle.2.Infection with N.bombycis can upregulate the expression of host Bm HXK and Bm GLUTAfter mulberry leaves coated with N.bombycis(1×106grains per silkworm)were added to the fifth instar silkworm of Dazao line,the midgut tissues of silkworm from the N.bombycis infection group and the fresh water control group were taken out and collected at different time points,seperately.After extracting the m RNA,a q RT-PCR study was performed.By detecting the expression level of Nb SSU,it was found that the copy number of N.bombycis began to exponentially increase in the midgut tissue cells of the silkworm after infection for 24 h.However,by detecting the expression level of Nb HXK,it was found that there are two distinct up-regulated peaks after 6-12 h and48-72 h,which are likely related to the proliferation cycle of N.bombycis in host cells.The expression levels of Bm HXK and Bm GLUT were significantly up-regulated in the silkworm midgut cells after N.bombycis infection.The expression level of Bm HXK wasup-regulated about 25 times at 48 h after infection,and the expression of Bm GLUT was increased about 8 times at 18 h after infection.Studies at the cellular level also revealed that N.bombycis infection caused the up-regulation of Bm HXK expression in two silkworm cell lines Bm N-SWU1 and Bm E,respectively.The above results indicate that N.bombycis can upregulate the expression of Bm HXK and Bm GLUT in host cells during its proliferation.3.Both Nb HXK and Bm HXK can promote the proliferation of N.bombycisSubcellular localization identification revealed that Bm HXK distributed at both nucleus and cytoplasm,but mainly located in the cytoplasm and accumulating around the cell membrane.Nb HXK is only located in the cytoplasm of the host and is evenly distributed.After co-transformation of Nb HXK and Bm HXK in silkworm cells,PBS starvation for 6 h,it was found that the localization of Bm HXK changed,the cytoplasm localization was reduced,and the nuclear localization was significantly increased,while Nb HXK could not respond to starvation and was uniformly distributed in the silkworm cytoplasm.Through the overexpression of Nb HXK and Bm HXK in silkworm cells,knockout of Bm HXK,recovery of Nb HXK after knockout of Bm HXK,then infected N.bombycis and detected its proliferation,we found that Nb HXK and Bm HXK were able to promote the proliferation of N.bombycis.Treatment of N.bombycis-infected silkworm cells with the hexokinase inhibitor 2-DG also revealed the same results.Hexokinase inhibitors inhibit the proliferation of N.bombycis.The above studies indicate that Nb HXK and Bm HXK have different subcellular localization characteristics and can both promote the proliferation of N.bombycis.4.Nb HXK and Bm HXK interact with Bm VDAC,respectivelyStudies on human HK-II(hexokinase-II)have revealed that HK-II can interact with VDAC(voltage-dependent anion channel)to facilitate the rapid acquisition of energy molecule ATP for phosphorylation of glucose to produce glucose-6-phosphate.Therefore,we cloned a voltage-dependant anion channel gene from Bombyx mori(Bm VDAC)and constructed a gene expression vector with a HA tag.By immunoprecipitation,bimolecule fluorescence complementation and fluorescence co-localization experiments,it was verified that Bm HXK and Nb HXK can interact with Bm VDAC,respectively.The results showed that the hexokinase Bm HXK in the silkworm could interact with Bm VDAC,which is consistent with the results in otherspecies.Surprisingly,it was also found that the N.bombycis hexokinase Nb HXK also interacts with Bm VDAC.Interaction with Bm VDAC may be the molecular basis for the function of secretory hexokinase Nb HXK in host cells.5.Nb HXK and Bm HXK can enhance host cell metabolism and inhibit host cell apoptosisThis study further explored the function of the two hexokinases Nb HXK and Bm HXK in the metabolism and apoptosis of silkworm cells.Detection of glucose content in cell culture solution showed that Nb HXK and Bm HXK could promote glucose uptake in silkworm cells.Detection of intracellular ATP content showed that Nb HXK and Bm HXK could increase intracellular ATP level.MTS assay showed that overexpression of Nb HXK and Bm HXK can promote the proliferation of silkworm cells.Reactive oxygen detection shows that both Nb HXK and Bm HXK can reduce the level of reactive oxygen species in the silkworm cells.Flow cytometry analysis after hydrogen peroxide induced apoptosis showed that Bm HXK can reduce the apoptosis rate of silkworm cells and the apoptosis rate of silkworm cells was significantly increased while Bm HXK knocked out.The activities of Caspase 3/7 and Caspase 9were also detected after hydrogen peroxide induced apoptosis.It was found that overexpression of Nb HXK and Bm HXK can inhibit host cell apoptosis to some extent.The above studies indicate that Nb HXK and Bm HXK can enhance the energy metabolism of host cells,and inhibit the apoptosis of host cells through the interaction with Bm VDAC,together to promote the proliferation of N.bombycis.In summary,this study found that N.bombycis is able to secrete hexokinase Nb HXK into host cytoplasm,and up-regulate the expression of Bm HXK and Bm GLUT in the host cell,together to enhance the glycolysis of the host cell and promote the production of more ATP and intermediate metabolites.At the same time,the interaction between Bm VDAC and two hexokinases inhibited the apoptosis of silkworm cells to some extent,and thus ensured and promoted the proliferation process of N.bombycis in silkworm cells.This study reveals the molecular mechanism of N.bombycis in regulating metabolism and apoptosis of silkworm cells,not only lays the foundation for the prevention and control of Pébrine disease,but also provides new ideas and targets for the prevention and treatment of human infection microsporidiosis.