Studies On Functional Genome Of Nosema Bombycis

Microsporidia are an unusual group of eukaryotic, obligate intracellular parasites. They infect a variety of animals, including several commercially important species such as bees, silkworms and salmon. They are common opportunistic pathogens of human in the late stage of AIDS, as well as occasionally infecting immunocompetent individuals, resulting in a number of diseases (e.g., gastroenteritis, encephalitis, and hepatitis). Originally thought to be primitive'amitochondriate' eukaryotes, they are now widely accepted as highly derived relatives of the fungi. As a result of their adaptation to intracellular parasitism, microsporidia show extreme reduction at the molecular, cellular and biochemical levels. In particular, the mitochondria of microsporidia are highly reduced into biochemically and structurally streamlined "mitosomes". Mitosomes have been characterized in the microsporidians Trachipleistophora hominis, Encephalitozoon cuniculi and Antonospora locustae. The mitosome is a morphologically simple compartment bounded by a double membrane. The majority of typical mitochondrial functions are absent. In particular, mitosomes cannot generate ATP by oxidative phosphorylation. Instead, microsporidian mitosomes acquire ATP from the cytosol using nucleotide transporters (NTTs). However, immunolocalization and functional assays in E. cuniculi and T. hominis demonstrated that mitosomes function in the biosynthesis of iron-sulphur clusters. Mitosomes have lost their own genome, all mitosomal precursor proteins must be imported from the cytosol.For Nosema bombycis, there was still poor information reported about its mitosomal proteins. In the present study, based on the genomic data of Nosema bombycis, we firstly identified the mitosomal-related genes via BLAST and HMM search, and compared these genes with other mitosome-containing parasites, then use RT-PCR to investigate the characteristics of transcription. Secondly, we cloned sixteen mitosomal genes for sequencing, and analzyed the conservation and phylogenetic relationship of four mitosomal proteins. Thirdly, we used the yeast expression system to localize16mitosomal proteins. Finally, prokaryotic expression, polyclonal antibodies production, immunoelectron microscopy were carried out to detect the localization of four proteins in the spore of Nosema bombycis.1. Identification of mitosomal genes in the genome of N. bombycisAll the predicted protein sequences of N. bombycis were downloaded from the local database. Mitochondrial protein sequences of yeast and hydrogenosomal proteins sequences of Trichomonas vaginalis were extracted from CBOrg and mitosomal protein sequences of several parasites were downloaded from NCBI. By using BLASTP and Hidden Markov Models,26mitosomal-related genes which encoding21unique mitosomal proteins were identified from the genome of N. bombycis. There are ten proteins involved in iron-sulfur cluster assembly, including Nbfrataxin, NbIsd11, NbNfs, NbIscU, NbmtHsp70, Nbferredoxin, NbFNR, NbGrx5, NbErv1and NbAtm1. Five proteins, NbTom40, NbTom70, NbSam50, NbTim22and NbPam16participate in protein import. Reverse transcription PCR analysis shown that Nbferredoxin, NbTom40and NbTom70can be amplified at24hour post infection (hpi), which suggest they play its role in early stage (meront stage). The transcript of NbSam50, NbGrx5, NbMnsodl, and NbmtHsp70can be detected at72hpi. The others can be detected at96hpi.2. Molecular cloning of mitosomal genes and sequence analysis of four mitosomal proteinsIn this study, we amplified the16mitosomal genes and cloned into pMD19-T vector for sequencing. The sequencing results showed that all genes are consistent with the genomic prediction. We analyzed four mitosome related proteins:(1) NbmtHsp70, the marker of mitosomal identification, possesses the functional amino acids sites:D16, K77, E178, A182, D200, T205, K272,K273and R345. It also contains the two conserved motif GDAW(V/I) and YSPSQI. Compared to yeast homologue, N-terminal transit peptide of mtHsp70is reduced in NbmtHsp70. Phylogenetic analysis showed that NbmtHsp70clusters with mitochondrial Hsp70, which are together derived from a-proteobacteria. It support the hypothesis that Microsporidia is a sister group of fungi.(2)NbNfs possesses the typical conserved regions implicated in cysteine desulfurase activity but does not contain a recognizable transit peptide targeting to mitochondrion. Phylogenetic analysis showed that N. bombycis cysteine desulfurase clusters with fungal mitochondrial cysteine desulfurases, which belong to group I derived from α-proteobacteria.(3) Tom40, the main component of the translocase of the outer mitochondrial membrane in eukaryotes, although reduced in size, NbTom40canform a β-barrel structure composed of19β-strands. Phylogenetic analysis shows that NbTom40forms a clade with Tom40sequences from other species, distinct from a related clade of voltage-dependent anion channels (VDACs). The NbTom40contains a β-signal motif, among amion acid residues, a polar residue is substituted by glycine.(4) NbNTT1, a nucleotide transporter is similar to ADP/ATP translocases from plastids and bacterial parasites, composed of12β-strands. Sequence alignment showed that NbNTTl harbors four conserved amino acids:K61, E158, D292and K446. The sequence identity is29%between EcNTT3and NbNTT1.3. Localization of mitosomal proteins in Saccharomyces cerevisiaeAnalyzing the cleavable N-terminal presequence by Mitopred, MitPred, Mitoprot, Predotar and PSORT Ⅱ indicated that most of mitosomal matrix proteins of N. bombycis lost the cleavable N-terminal transit peptide. Only four mitosomal proteins, Nbfrataxin, NbPDHEla, NbErvl and NbG3PDH, are equipped with a putative short N-terminal transit peptide and a conserved arginine residue at the-2position relative to the cleavage site.In order to assess the localization of mitosomal proteins, we express the GFP fusion protein in S. cerevisiae, the full length of16mitosomal genes except NbG3PDH were cloned into the expression vector pUG35, and then transformed into the yeast strain CEN.PK2. Positive recombinant yeast was screened on the SC-Ura plate, subsequently induced in the SC-Ura-Met for expression, and the yeast cells were stained with the mitochondrial probe Mitotracker Red. The result showed that NbTom40, NbTom70and NbSam50, specifically target green fluorescent protein to the mitochondria of S. cerevisiae. Our results suggested that NbTom40, NbTom70and NbSam50function as a part of the core mitosomal protein import apparatus. In addition, two proteins taking part in iron-sulfur assembly, NbNfs and Nbferredoxin also localize to the mitochondrion, which suggested that NbNfs and Nbferredoxin may be targeted to the mitosome of N. bombycis depending on the internal targeting signals instead of transit peptide, and the mitosome in N. bombycis may retain the function of iron sulfur assembly.4. Immunolocalization of mitosomal proteins in the spore of N. bombycis In this chapter, we constructed seven prokaryotic expression vector: NbIscU-pColdI, NbmtHsp70-pET30a, NbTom40-pET30a, NbTom70-pET30a, NbSam50-pET30a, NbNfs-pET30a and NbNTTl-△TM--DHFR-pET30a. These recombinant vectors were transformed into E. coli Rosetta strain, and induced by1mM IPTG. Six fusion proteins, rmtHsp70, rNbNTT1-△TM-DHFR, rNbIscU, rNbNfs, rNbTom40and rNbTom70expressed in E. coli. Among them, four fusion proteins (rmtHsp70, rNbNTT1-△TM-DHFR, rNbIscU and rNbNfs) are expressed as inclusion bodies. Using Ni-NTA affinity chromatography, rmtHsp70, rNbNTTI-△TM-DHFR, rNbIscU and rNbNfs were purifiedand used to immunize Kunming mice for preparing polyclonal antibody, respectively. Western blotting analysis showed that NbNfs, NbIscU and NbNTTl were expressed in mature spores of N. bombycis. However, Western blotting analysis showed that the polyclonal antibody of NbmtHsp70can recognize two type of Hsp70. Indirect fluorescent assay indicated NbNTTl may localize on the cytoplasm membrane of spores, and was involved in energy import. Colloidal gold immunolocalization displayed that NbNfs and NbIscU was mainly distributed in cytoplasm of the spore. Some gold particles of NbmtHsp70are clustered together, which may indicate the position of mitosome in the spore of N. bombycis.In general, we identified a set of mitosome-related genes in N. bombycis, and classified them according to their function. Expression pattern of sixteen genes were analyzed with RT-PCR and yeast localization, we found five proteins could be targeted to yeast mitochondrion. Four proteins immunolocalization were achieved and we found the mitosome distribution pattern in spore of N. bombycis. Our works provided more information of mitosome and paved a way for functional genomics of mitosome of N. bombycis.