Drug Target for Pathogenic Amoebae

Drug marking for Pathogenic AmoebaeHorizontal Gene ravish of a Chlamydial transfer RNA-Guanine Transglycosylase Gene to Specific Algal and Protozoan Lineages A Putative Drug Target for Pathogenic AmoebaeAbstracttRNA- guanine transglycosylases atomic number 18 gear up in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of specific tRNAs. They are also cognise to modulate virulence in bacteria such as Shigella flexneri, which has prompted the reading of drugs that chasten the kick the bucket of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotes ( algae and protozoa) which are more comparable to their bacterial counterparts than previously characterized eucaryotic tRNA-guanine transglycosylases. In silico compendium of these bacterial-like tRNA-guanine transglycosylasesrevealed thatthe majority are predicted to be targeted to mitochondria, although some are likely to come in to chloroplasts, t he secretory pathway or the cytosol. We provide evidence demonstrating that the constituent convert theseenzymes was acquired by these eukaryotic lineages via horizontal gene transfer which from the Chlamydiae. Given that the S. flexneri tRNA-guanine transglycosylase skunk be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentiallybe targeted in a similar look in pathogenic amoebae that make these enzymes such as Acanthamoeba castellanii.Keywords mitochondria, tRNA-guanine transglycosylase, queuine tRNA-ribosyltransferase, horizontal gene transfer, tRNA, queuosine, ChlamydiaeAbbreviationsTGTase tRNA-guanine transglycosylaseE-TGTase Eukaryotic tRNA-guanine transglycosylaseB-TGTase Bacterial tRNA-guanine transglycosylaseBL-TGTase Bacterial-like tRNA-guanine transglycosylaseHGT Horizontal gene transferIntroduction secondary registration of tRNAshas been implicated in tRNA structure, aminoacyl tRNA synthetase interaction andinfluencin g codon-anticodon basepairing1. The function of the adaption will depend on itstype and the position of the modified base. For example, most(prenominal) bases that are modified within the anticodon loop (positions 34-36) of tRNAsare important for accurate displacement by facilitating interactions with their cognate codons in mRNAs 1. One such modification that influences codon-anticodon basepairingis the internalisation of queuine within the anticodon loop.Queuosine is a modified guanosine analogue fix in tRNAs from all three domains of life.Despite its wide phylogenetic distribution, queuosine is only found in a select group of tRNAs (tRNAHis, tRNAAsp, tRNATyr and tRNAAsn) 2.Reduced incorporation of queuosine in these tRNAs alters their codon recognition skill and has been linked to various cancers 3,4.tRNA-guanine transglycosylasesQueuosine modification of tRNA is mediated by tRNA-guanine transglycosylases (TGTases)(also know as queuine tRNA-ribosyltransferases). TGTases cat alyze this modification via base exchange where the guanine at position 34 of the tRNA is post-transcriptionally removed and substituted with queuine or a queuine predecessor 5.Eukaryotes are not capable of de novo queuine synthesis but acquire it finished diet or their gastrointestinal microbiota 6.After its encyclopedism, the eukaryotic TGTase (E-TGTase) mediates the replacement of guanine with queuine in the anticodon loop. In contrast, queuosine modification of bacterial tRNA is more complex. Prokaryotesuse GTP-cyclohydrolase-like enzymes tosynthesizea queuine precursor(e.g. preQ1) from GTP. The bacterial TGTase (B-TGTase) whence mediates the base exchange with guanine to incorporate preQ1, unlike E-TGTases that use queuine itself as the substrate.This incorporatedpreQ1 is then modified by S-adenosylmethionine tRNA ribosyltransferase to epoxyQ, which is further modified to manikin queuosine 6.In addition to tRNA modification, B-TGTasesplay a role in regulating the font of bacterial genes.TGTase mutants (vacC) in the bacterium Shigella flexneri exhibit reduced expression of the virG and ipaBCDgenes, which encode virulence factors that facilitate the spread and invasion of the pathogen 7. This is a result ofthe VacCTGTase beingrequired to restrict a single base in virF mRNA, which encodes the transcriptional activator ofvirG and ipaBCD8.Thus, B-TGTases can modify substrates otherthan tRNA and are important mediators of bacterial virulence. As a result, B-TGTases lay down served as a targetfor the growing of drugs that interfere with their function 9.Here we report a in the buff group of TGTases in eukaryotes that display significantly greater resemblance to B-TGTases than E-TGTases. We hereby refer to these proteins as bacterial-like TGTases (BL-TGTases).In silico analysis identified 25 BL-TGTases in obvious protozoan and algal lineages and the reason for their similarity to B-TGTases is explored in this article.Variation in the subcellular loca lization of bacterial-like tRNA-guanine transglycosylasesTo investigate the putative subcellular localization of BL-TGTases, three bioinformatic programs were utilized Mitoprot 10, Predotar 11 and Target P 12. The putativelocalizationfor each BL-TGTase was backup manedby predictions from at least two of the three programs.Most BL-TGTases throw N-terminal mitochondrial targeting signals (Table 1), suggesting a role in modification of mitochondrial tRNAs.Interestingly, the BL-TGTases from Ostreococcus lucimarinus and Chondrus crispus were predicted to coiffure to mitochondria with matchless program (Predotar) but to the plastid with another (Target P). While it is possible that these two proteins whitethorn localize to some(prenominal) organelles, further experimentation is required to elucidate their subcellular locations. The BL-TGTase from the diatom Phaeodactylum tricornutum was predicted to localize to the endoplasmic reticulum (ER) of the secretory pathway, indicating it ma ymodify other substrates in this organelle.Bacterial-like tRNA-guanine transglycosylase genes originated from a Chlamydial gene acquired via horizontal gene transferWhile the localization of BL-TGTases varied, all 25 of the proteins exhibited higher levels of amino acid similarity to B-TGTases despite their existence in eukaryotes. A Bayesian analysis of phylogeny using MrBayes 13 withBL-TGTases,B-TGTases and E-TGTasesconfirmedthis similarity(Figure 1).The BL-TGTases were most similar to TGTases from members of the Chlamydiae.In fact, the Chlamydial TGTases were more similar to BL-TGTases than other B-TGTases. Given that Chlamydiaeare bacteria, the topographic anatomy of the tree in the present study is incongruent with the universal tree of life. Instead, this topology is consistent with a horizontal gene transfer (HGT) event. That is, the genes encoding BL-TGTases originated from a Chlamydial TGTase-encoding gene that was acquired via prokaryote-to-eukaryote HGT.In addition to th e strong statistical support for the BL-TGTase-Chlamydial TGTase sister group, there are several other factors that support this notion. The Chlamydiaeare known to be major contributors of genes to several eukaryotic genomes via HGT 14,15. This includes genes encoding tRNA modification enzymes such as the Chlamydial tRNA guanine methyltransferases found in protozoa, diatoms and algae16,17 and Chlamydial tRNA genes in vascular plants 18.Similarly to the present study, sister groups were detect between the Chlamydial and the horizontally acquired eukaryotic genes in these cases. Lastly, the majority of eukaryotic lineages in which we identified BL-TGTases have previouslybeen reported to possess HGT-derived genes acquired from the Chlamydiae16,19. Thus, the notion that BL-TGTases resulted from the acquisition of a B-TGTase from the Chlamydiaevia HGT in eukaryotes is highlyplausible.Indirect acquisition of a Chlamydial tRNA-guanine transglycosylase in protozoa via anon-Chlamydialbacter ial intermediateInterestingly, a B-TGTase sequence from the -proteobacterium Candidatus Babela massiliensisclustered with the BL-TGTases of protistsrather than the B-TGTases (Figure 1). Although the protozoan BL-TGTases displayed similarity to Chlamydial B-TGTases, the possibility of a HGT event from Ca. B. massiliensisto protistswas present. SinceChlamydiae and -proteobacteria are not about related, the phylogeny of their B-TGTases was investigated. Interestingly, the Ca. B. massiliensisTGTase clustered with the Chlamydial TGTase clade rather than other -proteobacterial (Pelobacter, Geobacter, Myxococcus, Desulfobulbus) B-TGTases (Figure 2).Ca.B. massiliensisand members of the Chlamydiae are found as obligate intracellular symbionts of protists such as Acanthamoeba, Dictyosteliumand Naegleria 20,21. The presence of both of these bacteria within the one eukaryotic cell would provide the example conditions for HGT between them. Therefore, it is likely that at least two independent HGT events have occurred1) The Chlamydiae donated a TGTase-encoding gene to an ancestral Ca.B. massiliensisspecies and 2) Ca.B. massiliensisthen donated this gene to theAmoebozoa and Heterolobosea.How the BL-TGTase genes were acquired in the algal lineages bear ons to be elucidated, but may have occurred via additional HGT events (either prokaryote-to-eukaryote or eukaryote-to-eukaryote).Bacterial-like tRNA-guanine transglycosylases as drug targets for pathogenic amoebaeIn addition to their role in queuosine modification of tRNAs, TGTases are important for S. flexneri virulence 7,8.As a result, studies have focused on the development of TGTase inhibitors that specifically target the S. flexneri B-TGTase to treat shigellosis,while the E-TGTases of the human host remain unaffected. Some of these inhibitors,such as lin-benzoguanine,function by occupying the binding site for preQ122,23.While most eukaryotic species that possess BL-TGTases are non-pathogenic, we identified a BL-TGTase in Acanthamoeba castellanii, the causative element of amoebic keratitis and encephalitis.Naegleria gruberi, whichalso has a BL-TGTase, is non-pathogenic, but is closely related to Naegleria fowleri, the etiologic means of primary amoebic meningoencephalitis, which may possess an unidentified BL-TGTase. Given the development of B-TGTase inhibitors has already been achieved, the BL-TGTasesin pathogenic eukaryotes could also potentially be targeted with the same drugs. Alternatively, advanced inhibitors could be developed following resolution of the BL-TGTase crystal structure. To confirm BL-TGTases as a putative drug target future research should cause to characterize these proteins and determine if they have retained their prokaryotic functions and mechanism of action. conclude remarksIn this report, we have described a group of TGTases in algae and protozoa (BL-TGTases). Theseproteins are predicted to localize to various subcellular locations including mitochondria, chloroplasts, the ER and the cytosol, depending on the organism. Lastly, we showed that via threefold HGT events, BL-TGTases were originallyfrom bacteria of the Chlamydiae lineage. The bacterial origin of these proteins could be exploited in the development of drugs similar to those synthesized for the S. flexneri B-TGTase. Research into the identification and synthesis of BL-TGTase inhibitors may provide a novel treatment for infectious diseases which are caused by pathogenic amoebae that possess these proteins.