Studies On The Mitochondrial-related Genes From Nosema Bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites.They can infect a wide variety of animals ranging from invertebrates to vertebrates,and they are the common fateful pathogens for sericulture,fishery and shrimp farms.Since the first microsporidia,Nosema bombycis,has been discovered in the middle of the nineteenth century,thus far,more than 1 300 microsporidia species belonging to 160 genera have been reported,from which 14 species belonging to 8 genera can lead to several syndromes in immunocompetent hosts of human and cause opportunistic infections in acquired immunodeficiency syndrome(AIDS) patients. Microsporidia draws great interest to scientists because they are closely related to human health. Microsporidia were even thought to be "primitive" eukaryotes without microspordial but are now widely recognized as being fungi that have undergone reductive evolution as so to appear to be primitive organisms.And now,a highly reduced microspordial organelle has been identified, so-called "mitosome".In general,the structure,composition and function in energy transfer of microspordia may play an important role in the process of microsporidia infecting.Thus microspordia has been a focus of research in microsporidian pathology.14 microspordial relative genes were reported in microsporidia which have also been demonstrated to involve in the metabolism of mitosome.However,most of those genes greatly reduced and the function and action mechanism of them in the microsporidia were unknown yet.N. bombycis is known as a pathogen of silkworm pebrine,which usually prevails in sericulture.The prevention and treatment of pebrine is still an important and difficult topic for sericulture.Now,the complete genomic sequence of N.bombycis has been finished in our labs.By comparative with genome datas and EST datas between N.bombycis and other species,such as human and yeast,we identified the candidate of mitochondrial-related genes.Among candidate genes,we have cloned some important genes,analyzed and discussed their location and function in the Nosema bombycis.Some major research results were obtained as following:1 Bioinformatics analysis of mitochondrial-related gene of microsporidia N.bombycisAfter comparison of total genome datas of N.bombycis and mitochondrial protein library,we obtained mitochondrial-related genes from N.bombycis,the potential function of mitochondrial-related genes of N.bombycis was predicted by analysis of function and structure.We further finished the cloning,expressing and location of mitochondrial-related genes of N. bombycis.We obtained 171 mitochondrial-related proteins include frataxin,nifu,nifs and ferredoxin FeS cluster assembly system,Mn-SOD,thioredoxin reductase for antioxidation way, mtHsp70,Tim22/17and mtTom70 for the folding of protease and proteins except the related protein for oxidative phosphorylation and electron transport chain.N.bombycis have mitochondrial type ABC Transporter same as other insect,nevertheless,the number of copy is distinctly less to Encephalitozoon cuniculi,Cryptosporidium parvum,human and yeast,but closer to the Antonospora locustae.Even if N.bombycis exsisted partily mitochondrial related proteins coded by genome,it have no the function of electron transport and production of ATP.We have found a series genes involved in Fe-S cluser,that might will provide us clues to the function of mitochondrial related gene about N.bombycis by the comparation of Fe-S cluser in other microspordia species.Collinearity was been known as one of the major characteristic of microspordia genome.We have found that some mitochondrial-related genes are not have collinearity,such as ATP/ADP carrier protein,ADP/ATP carrier protein,nifu,MnSOD,ABC transporter microspordial type,while some have better relation of coilinearity such as frataxin,nifs,Adrenodoxin(ferredoxin),mtHsp70, Tim22/17,microspordiall Glycerol-3-phosphate dehydrogenase,PDH E1α,PDH E1β.Several event of insert,invert and loss of frataxin gene have occurred among different related species,for instance adrenodoxin and mitochondrial glycerol-3-phosphate dehydrogenase found neither in yeast nor in microspordia,which maybe result from the transposon(NBO0016-0045,NBO0016-0056, NBO0084-0025) near the loci.This suggested that most of mitochondrial-related genes sequences in microspordia were steady and conservative.The differences of codon bias between mitochondrial-related genes or genome reveals that the strength of natural select depend on nucleotide composition.Comparative of mitochondrial-related genes,genome of N.bombycis and E.cuniculi,the result had shown that the selection strength of genome of N.bombycis appears to be very poor,whose baise code mainly result from nucleotide composition.Analysis of codon bias of mitochondrial-related genes of N.bombycis suggested that mitochondrial-related genes of N.bombycis appear to very poor,only been influced by natural selection strength,whose codon bias is mainly about compositon of nucleotide the same as that the mitochondrial-related gene nuclear coding.In order to study of the phylogenetic evolution of N.bombycis,multiple alignment were done by using amino acid sequences of frataxin,nifs and nifu from fungal,prokaryote,protozoa,together with the gene of N.bombycis.The result indicated that mitochondrial-related genes of N.bombycis, Encephalitozoon cuniculi and Antonospora locusta fall in one clade,close to typical evolution relation of high eukaryote,junior to protozoa,and is far from prokaryote.It can be supposed that the mitochondrial type frataxin protein of high eukaryote and microspordia is most similar,indicating microspordia may have a mitochondrial-like organelle and posses a part of mitochondrial function. Besides,the phylogenetic tree has shown that N.bombycis is closer to protozoa which also proved that microspordia is the sister group of fungi.We have constructed the pathway of 171 mitochondrial-related homologous genes of N. bombycis include Fe-S cluster assembly system,protein transportation and folding system, glycerophosphate shuttle system,antioxidation way and so on.Aithrough the copies numbers of relative gene between N.bombycis and Encephalitozoon cuniculi microspordiai varies,the major structure and function is same.2 Cloning,expressing and sequences analysis of mitochondrial-related protein ofN.bombycisWe focused 3 mitochondrial-related proteins:frataxin,nifu,ADP/ATP carrier protein by eukaryote express and RT-PCR.Frataxin gene is 306 bp.There are 3α-spiral which is loaded in N-terminal,center and C-terminal of protein,highly conservative in secondary structure and 1β-fold whose folding loaction also very conservative.In that,we have supposed that these structure is essential in the function of protein.Frataxin of microspordia is lack ofα-spiral with the location of 52-65 by compared of eukaryote,is lack ofα-spiral with the location of 40-45,61-66 by compared of yeast,can be guessed thatα-spiral with the location of 61-66 is related to protein location in cytoplasm,α-spiral with the location of 40-45 is related to protein location in microspordia.This conclusion also proved that the protein from microspordia is different from the typical eukaryote,the former lacks of loaction information maybe interpret reduction of loaction information or very different in nature.There are no mitochondrial targeting sequence by prediction analysis of Nbfra using PRORT program or MITOPROTII.Nbfra have a Arg in the location of 100 is same to CyaY analogues of plant,yeast and bacterial.Also,human frataxin is been found a His residues binding with iron in this Ioaction.Nbnifu gene which coded 140 amino acid residues is shorter to the nifu of typical eukaryote and Encephalitozoon cuniculi.The nifu gene is relative conservative in its structure,but have differences between N-terminal and C-terminal,which is highly similarity to the protein of E.cuniculi(78% identity at amino acid level).Nifu is lack of any transmembrane structure or signal peptide,and only has one conservative domain Nifu-N.The microspordia mitochondrial protein might be different from of typical eukaryote mitochondrial proteins,or the former is lack of location sigal,which caused highly reduction of location sigai or very different.There are no mitochondriai location sequence by prediction analysis of Nbfra using PRORT program or MITOPROTII.Nbadp/atp is 674bp which coded 224 amino acid,and its pI value is 9.15.In this study,we have found 3 transmembrane area by SMART software as typical membrane proteins.Compared with other homologue gene,Nbadp/atp is incomplete,lacking of most sequence information,and have high homology with ADP/ATP carrier proteinl of Encephalitozoon cuniculi.We predicated that ADP /ATP carrier protein located in mitochondrial membrane,transfers the ATP synthesised from mitochondrion to cytoplasm,transfers ADP back to mitochondrion.Frataxin,nifu,ADP/ATP carrier protein also have the activity of tanscription but poorer express activity.Frataxin exists in the total protein of N.bombycis and without a proprotein by western blotting analysis.3 Study on the location of mitochondrial-related gene encoding protein of N.bombycis To verify the expressing specificity and subcellular location characteristics features,IFA and immune electron microscopy were been used.It is obvious clear of nuclear field and ball-shaped mitochondria in the cell by MitoTracke Red CMXRos dying.Most of spores possessed red flurescence and differentiated clearly,nuclear field by DAPI dying and MitoTracke Red CMXRos is different.The cell location by fluorescent antibody and MitoTracker Red CMXRos dying is overlapped partialy,which can explained both mitochondria-like organelle and mitochondrial proteins coding by genome are exsist in N.bombycis.However,the function of these protein remains to be further researched.The results were shown that 8 mitochondrial-related proteins binding to spore of N.bombycis, is not overlapping with DAPI dying,and have no cross reaction with host cell of N.bombycis.The antibody signals of frataxin,ferredoxin,tim22/17,ATP/ADP carrier protein exsist in cytoplasm near the spore wall,have ball-shaped fluorscence sigai which might be michondrial relict organelle-mitosome,with 6 point-shaped signals in a spore.Nifs and nifu were just locating in the cytoplasm.MtTom70 and ADP/ATP carrier protein that possess continuous line-shaped flurescence sigal,can be guessd in the spore membrane.Location analysis of mitochondriai-related genes of frataxin,nifs,ferredoxin and mtTom70 proteins were done by immuno-gold labeling.It is clear to see that inner structure of spores of N. bombycis,such as polar tube,posterior vacuole,nucleus,spore wall and so on.Two to four Colloidal particle gold particles often cluster together in frataxin and ferredoxin which may be a mitosome. Nifs diffused in spore cytoplasm freedomly while mtTom70 along with spore wall.4 A preliminary analysis of mitochondrial-related proteins of microsporidia N.bombycisFirstly,organelles of N.bombycis were seperated by using percoll density gradient centrifugation and sucrose density gradient centrifugation methods.The results showed that two methods could not enrich and purify organelle.Then co-immunoprecipitation were used to obtain more mitochondrial-related proteins by polyclonal antibody,negative blood as a control.Cytoplasm protein was prepared from spores of N. bombycis by frost thawing method.Eleven proteins effected on frataxin,two were unkown proteins. One unknown protein associated with scaffolding protein nifu was found by analysis of MALDI-TOF-MS cutting gel method.The conjecture was that the eight proteins associated with TIM22/17 were four unknown proteins and three hypothesis proteins.One hypothesis protein effected on ATP/ADP carrier protein.Four proteins associated with nifs,in which one was unkown protein and two were hypothetical proteins.Two proteins were connected with ferredoxin and three proteins with tom70.Only one protein was related with adp/atp carrier protein.Some proteins had a signal peptide or transmembrane domain.