UbiN, a novel Rhodobacter capsulatus decarboxylative hydroxylase involved in aerobic ubiquinone biosynthesis.

Ubiquinone (UQ) is a lipophilic electron carrier that functions in the respiratory and photosynthetic electron transfer chains of proteobacteria and eukaryotes. Bacterial UQ biosynthesis is well studied in the gammaproteobacterium Escherichia coli, in which most bacterial UQ-biosynthetic enzymes have been identified. However, these enzymes are not always conserved among UQ-containing bacteria. In particular, the alphaproteobacterial UQ biosynthesis pathways contain many uncharacterized steps with unknown features. In this work, we identified in the alphaproteobacterium Rhodobacter capsulatus a new decarboxylative hydroxylase and named it UbiN. Remarkably, the UbiN sequence is more similar to a salicylate hydroxylase than the conventional flavin-containing UQ-biosynthetic monooxygenases. Under aerobic conditions, R. capsulatus ΔubiN mutant cells accumulate 3-decaprenylphenol, which is a UQ-biosynthetic intermediate. In addition, 3-decaprenyl-4-hydroxybenzoic acid, which is the substrate of UQ-biosynthetic decarboxylase UbiD, also accumulates in ΔubiN cells under aerobic conditions. Considering that the R. capsulatus ΔubiD-X double mutant strain (UbiX produces a prenylated FMN required for UbiD) grows as a wild-type strain under aerobic conditions, these results indicate that UbiN catalyzes the aerobic decarboxylative hydroxylation of 3-decaprenyl-4-hydroxybenzoic acid. This is the first example of the involvement of decarboxylative hydroxylation in ubiquinone biosynthesis. This finding suggests that the C1 hydroxylation reaction is, at least in R. capsulatus, the first step among the three hydroxylation steps involved in UQ biosynthesis. Although the C5 hydroxylation reaction is often considered to be the first hydroxylation step in bacterial UQ biosynthesis, it appears that the R. capsulatus pathway is more similar to that found in mammalians.

Development of a prognostic prediction support system for cervical intraepithelial neoplasia using artificial intelligence-based diagnosis.

OBJECTIVE: Human papillomavirus subtypes are predictive indicators of cervical intraepithelial neoplasia (CIN) progression. While colposcopy is also an essential part of cervical cancer prevention, its accuracy and reproducibility are limited because of subjective evaluation. This study aimed to develop an artificial intelligence (AI) algorithm that can accurately detect the optimal lesion associated with prognosis using colposcopic images of CIN2 patients by utilizing objective AI diagnosis. METHODS: We identified colposcopic findings associated with the prognosis of patients with CIN2. We developed a convolutional neural network that can automatically detect the rate of high-grade lesions in the uterovaginal area in 12 segments. We finally evaluated the detection accuracy of our AI algorithm compared with the scores by multiple gynecologic oncologists. RESULTS: High-grade lesion occupancy in the uterovaginal area detected by senior colposcopists was significantly correlated with the prognosis of patients with CIN2. The detection rate for high-grade lesions in 12 segments of the uterovaginal area by the AI system was 62.1% for recall, and the overall correct response rate was 89.7%. Moreover, the percentage of high-grade lesions detected by the AI system was significantly correlated with the rate detected by multiple gynecologic senior oncologists (r=0.61). CONCLUSION: Our novel AI algorithm can accurately determine high-grade lesions associated with prognosis on colposcopic images, and these results provide an insight into the additional utility of colposcopy for the management of patients with CIN2.

Rhoptry kinase protein 39 (ROP39) is a novel factor that recruits host mitochondria to the parasitophorous vacuole of Toxoplasma gondii.

Most intracellular pathogens replicate in a vacuole to avoid the defense system of the host. A few pathogens recruit host mitochondria around those vacuoles, but the molecules responsible for mitochondrial recruitment remain unidentified. It is only in the apicomplexan parasite Toxoplasma gondii, that mitochondrial association factor 1b (MAF1b) has been identified as an association factor for host mitochondria. Here, we show that rhoptry kinase family protein 39 (ROP39) induces host mitochondrial recruitment in T. gondii. We found that the abundance of ROP39 was increased on host mitochondria extracted from human foreskin fibroblasts (HFFs) infected with T. gondii. ROP39 expressed exogenously in HFFs localized on host mitochondria, indicating that it has the potential to bind to host mitochondria without assistance from other parasite factors. Confocal microscopy revealed that ROP39 colocalized with host mitochondria on the membrane of parasitophorous vacuoles, in which the parasites reside. Moreover, we observed about a 10% reduction in the level of mitochondrial association in rop39-knockout parasites compared with a parental strain.

A novel 2A-peptide-containing plasmid to generate stable Perkinsus marinus cells expressing organelle-targeted genes.

Genetic manipulation techniques for marine protists are not well-established, despite immense efforts. However, Perkinsus marinus is an exception and can be developed as a genetically tractable model organism for related protists. Here, we designed a new plasmid for P. marinus that allows two proteins from a single mRNA to be differently localized using a self-cleaving 2A peptide. This enabled us to establish a stable transfectant expressing a mitochondrially targeted fluorescent protein. The system can be applied to any protein in theory and would make a powerful tool for investigating unique organelles in P. marinus and related dinoflagellates.

Endogenization and excision of human herpesvirus 6 in human genomes.

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.

Molecular and biological analysis revealed genetic diversity and high virulence strain of Toxoplasma gondii in Japan.

Toxoplasma gondii is classified into 16 haplogroups based on a worldwide genotyping study of the parasite. However, only a few isolates from Japan were included in this analysis. To conduct more precise genotyping of T. gondii, we examined the genotypes of Japanese isolates in this study. DNA sequences of 6 loci were determined in 17 Japanese isolates and compared with those of strains of 16 haplogroups. As a result, Japanese isolates were classified into four groups. We investigated the virulence of some Japanese isolates and found a highly virulent strain in mice, comparable to that of RH strain, although this Japanese isolate was sister to strains of haplogroup 2, which show moderate virulence in mice. We further investigated whether this high virulence isolate had different virulence mechanism and strategy to adapt to Japanese host from other strains by comparing the virulence-related genes, ROP5, 18 and the immunomodulatory gene, ROP16 of the isolate with those of archetypical strains (GT1, ME49 and VEG). This analysis indicated the high virulence of the isolate in mice was partly explained by gene sequences of ROP5 and ROP16. These findings lead to the elucidation of biodiversity of T. gondii and have potential to optimize the diagnostic protocol.

A Toxoplasma gondii strain isolated in Okinawa, Japan shows high virulence in Microminipigs.

Toxoplasma gondii strains have been isolated all over the world and their virulence has been examined mainly using laboratory mice. However, T. gondii differs in virulence depending on the host animal species. Therefore, to evaluate the virulence of each strain in domestic animals, it is necessary to examine using not only mice but also the concerned animals. We have shown that TgCatJpOk4, a T. gondii strain recently isolated in Okinawa, Japan, has a high virulence against laboratory mice, comparable to highest virulent RH strain in mice; however, the virulence to domestic animals remains unknown. In this study, we examined the virulence using the Microminipig. After infection, four out of five infected pigs showed severe clinical symptoms: inappetence, hypoactivity and tachypnea. Eventually, three out of the five infected pigs succumbed before the end of the observation. Among the three dead pigs, histological analysis revealed that interstitial pneumonia and spotty necrosis in the liver indicating that the TgCatJpOk4 strain has a high virulence not only in laboratory mice, but in pigs as well.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Puromycin selection for stable transfectants of the oyster-infecting parasite Perkinsus marinus.

Perkinsus marinus is a marine protozoan parasite that infects natural and farmed oysters, attracting attention from researchers in both fisheries and evolutionary biology. The functions of almost all cellular components and organelles are, however, poorly understood even though a draft genome sequence of P. marinus is publicly available. One of the major obstacles for a functional study of the parasite is limited experimental means for genetic manipulation: a transfection method was established in 2008, and the first drug selection system with bleomycin was reported in 2016. We here introduce the second drug-selectable marker for selection of P. marinus transfectants. The parasite growth is efficiently inhibited by puromycin (IC50 = 4.96 µg/mL), and transfection of its resistance gene, puromycin-N-acetyl-transferase (pac), confers resistance to the drug on the parasite. Stable transfectants can be obtained within 2 months by treating with puromycin at 100 µg/mL. Furthermore, combining puromycin and bleomycin treatment can select transfectants co-expressing two marker genes. This dual-transfection method raises the possibility of using co-localization to identify the cellular localization of novel proteins in P. marinus, thereby contributing to the understanding of cellular functions and pathogenesis.

Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5' end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5' end sequence of 28-33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution.

Investigation into the Physiological Significance of the Phytohormone Abscisic Acid in Perkinsus marinus, an Oyster Parasite Harboring a Nonphotosynthetic Plastid.

Some organisms have retained plastids even after they have lost the ability to photosynthesize. Several studies of nonphotosynthetic plastids in apicomplexan parasites have shown that the isopentenyl pyrophosphate biosynthesis pathway in the organelle is essential for their survival. A phytohormone, abscisic acid, one of several compounds biosynthesized from isopentenyl pyrophosphate, regulates the parasite cell cycle. Thus, it is possible that the phytohormone is universally crucial, even in nonphotosynthetic plastids. Here, we examined this possibility using the oyster parasite Perkinsus marinus, which is a plastid-harboring cousin of apicomplexan parasites and has independently lost photosynthetic ability. Fluridone, an inhibitor of abscisic acid biosynthesis, blocked parasite growth and induced cell clustering. Nevertheless, abscisic acid and its intermediate carotenoids did not affect parasite growth or rescue the parasite from inhibition. Moreover, abscisic acid was not detected from the parasite using liquid chromatography mass spectrometry. Our findings show that abscisic acid does not play any significant roles in P. marinus.

Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus.

Perkinsus species are notorious unicellular marine parasites that infect commercially important mollusk species including clams and oysters. Recent accumulation of molecular information will greatly facilitate the understanding of Perkinsus biology and development of strategies to control infection. However, the limited availability of methods for genetic manipulation has hindered molecular-based studies of the parasites. In particular, the lack of a drug selection system requires manual isolation of fluorescent cells under a microscope to establish transfected cell lines. Here, we introduce a drug selection system using a glycopeptide antibiotic, bleomycin, and a vector containing the resistance gene Sh-ble. Perkinsus marinus is sensitive to bleomycin, and 100µg/ml of this drug completely blocks its proliferation. Concomitant expression of Sh-ble enables us to specifically select transfected cells in the presence of the drug. We believe that this system provides new opportunities for functional analyses of this parasite.

Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes.

Drug resistance compromises control of malaria. Here, we show that resistance to a commonly used antimalarial medication, atovaquone, is apparently unable to spread. Atovaquone pressure selects parasites with mutations in cytochrome b, a respiratory protein with low but essential activity in the mammalian blood phase of the parasite life cycle. Resistance mutations rescue parasites from the drug but later prove lethal in the mosquito phase, where parasites require full respiration. Unable to respire efficiently, resistant parasites fail to complete mosquito development, arresting their life cycle. Because cytochrome b is encoded by the maternally inherited parasite mitochondrion, even outcrossing with wild-type strains cannot facilitate spread of resistance. Lack of transmission suggests that resistance will be unable to spread in the field, greatly enhancing the utility of atovaquone in malaria control.

Identification of centromere regions in chromosomes of a unicellular red alga, Cyanidioschyzon merolae.

To investigate the evolution of centromere architecture in plant cells, it is important to identify centromere regions of primitive algae, such as Cyanidioschyzon merolae. In a previous genome project, in silico analysis predicted an AT-rich region in each chromosome as putative centromere regions. Here, we identified a centromere position in each chromosome by ChIP-on-chip analysis using an anti-CENP-A antibody. The identified centromeres were of the regional type, about 2-3 kb in length and contained no consensus or repeat elements. Centromeres in primitive eukaryotic plant cells may have originated from these regional type centromeres.

Direct evidence for the atovaquone action on the Plasmodium cytochrome bc1 complex.

Atovaquone, a coenzyme Q analogue has been indicated to specifically target the cytochrome bc1 complex of the mitochondrial respiratory chain in the malarial parasite and other protozoan. Various mutations in the quinone binding site of the cytochrome b gene of Plasmodium spp. such as M133I, L144S, L271V, K272R, Y268C, Y268S, Y268N, and V284F are suggesting to associate with resistance to atovaquone. There is no direct evidence of relation between the mutations and resistance to atovaquone in Plasmodium parasite that has been available. Technical difficulties in isolating active assayable mitochondria in the malarial parasite hinder us to obtain direct biochemical evidence to support the relation between the mutations and drug resistance. The establishment of a mitochondrial isolation method for the malaria parasite has allowed us to test the degree of resistance of Plasmodium berghei isolates to atovaquone directly. We have tested the activity of dihydroorotate (DHO)-cytochrome c reductase in various P. berghei atovaquone resistant clones in the presence of a wide concentration range of atovaquone. Our results show the IC(50) of P. berghei atovaquone resistant clones is much higher (1.5 up to 40 nM) in comparison to the atovaquone sensitive clones (0.132-0.465 nM). The highest IC(50) was revealed in clones carrying Y268C and Y268N mutations (which play an important role in atovaquone resistance in Plasmodium falciparum), with an approximately 100-fold increase. The findings indicate the importance of the mutation in the quinone binding site of the cytochrome b gene and that provide a direct evidence for the atovaquone inhibitory mechanism in the cytochrome bc1 complex of the parasite.

Phylogenomic analysis of "red" genes from two divergent species of the "green" secondary phototrophs, the chlorarachniophytes, suggests multiple horizontal gene transfers from the red lineage before the divergence of extant chlorarachniophytes.

The plastids of chlorarachniophytes were derived from an ancestral green alga via secondary endosymbiosis. Thus, genes from the "green" lineage via secondary endosymbiotic gene transfer (EGT) are expected in the nuclear genomes of the Chlorarachniophyta. However, several recent studies have revealed the presence of "red" genes in their nuclear genomes. To elucidate the origin of such "red" genes in chlorarachniophyte nuclear genomes, we carried out exhaustive single-gene phylogenetic analyses, including two operational taxonomic units (OTUs) that represent two divergent sister lineages of the Chlorarachniophyta, Amorphochlora amoeboformis ( = Lotharella amoeboformis; based on RNA sequences newly determined here) and Bigelowiella natans (based on the published genome sequence). We identified 10 genes of cyanobacterial origin, phylogenetic analysis of which showed the chlorarachniophytes to branch with the red lineage (red algae and/or red algal secondary or tertiary plastid-containing eukaryotes). Of the 10 genes, 7 demonstrated robust monophyly of the two chlorarachniophyte OTUs. Thus, the common ancestor of the extant chlorarachniophytes likely experienced multiple horizontal gene transfers from the red lineage. Because 4 of the 10 genes are obviously photosynthesis- and/or plastid-related, and almost all of the eukaryotic OTUs in the 10 trees possess plastids, such red genes most likely originated directly from photosynthetic eukaryotes. This situation could be explained by a possible cryptic endosymbiosis of a red algal plastid before the secondary endosymbiosis of the green algal plastid, or a long-term feeding on a single (or multiple closely related) red algal plastid-containing eukaryote(s) after the green secondary endosymbiosis.

The search for the missing link: a relic plastid in Perkinsus?

Perkinsus marinus (Phylum Perkinsozoa) is a protozoan parasite that has devastated natural and farmed oyster populations in the USA, significantly affecting the shellfish industry and the estuarine environment. The other two genera in the phylum, Parvilucifera and Rastrimonas, are parasites of microeukaryotes. The Perkinsozoa occupies a key position at the base of the dinoflagellate branch, close to its divergence from the Apicomplexa, a clade that includes parasitic protista, many harbouring a relic plastid. Thus, as a taxon that has also evolved toward parasitism, the Perkinsozoa has attracted the attention of biologists interested in the evolution of this organelle, both in its ultrastructure and the conservation, loss or transfer of its genes. A review of the recent literature reveals mounting evidence in support of the presence of a relic plastid in P. marinus, including the presence of multimembrane structures, characteristic metabolic pathways and proteins with a bipartite N-terminal extension. Further, these findings raise intriguing questions regarding the potential functions and unique adaptation of the putative plastid and/or plastid genes in the Perkinsozoa. In this review we analyse the above-mentioned evidence and evaluate the potential future directions and expected benefits of addressing such questions. Given the rapidly expanding molecular/genetic resources and methodological toolbox for Perkinsus spp., these organisms should complement the currently established models for investigating plastid evolution within the Chromalveolata.

Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata).

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria.

Cyanobacterial contribution to the genomes of the plastid-lacking protists.

BACKGROUND: Eukaryotic genes with cyanobacterial ancestry in plastid-lacking protists have been regarded as important evolutionary markers implicating the presence of plastids in the early evolution of eukaryotes. Although recent genomic surveys demonstrated the presence of cyanobacterial and algal ancestry genes in the genomes of plastid-lacking protists, comparative analyses on the origin and distribution of those genes are still limited. RESULTS: We identified 12 gene families with cyanobacterial ancestry in the genomes of a taxonomically wide range of plastid-lacking eukaryotes (Phytophthora [Chromalveolata], Naegleria [Excavata], Dictyostelium [Amoebozoa], Saccharomyces and Monosiga [Opisthokonta]) using a novel phylogenetic pipeline. The eukaryotic gene clades with cyanobacterial ancestry were mostly composed of genes from bikonts (Archaeplastida, Chromalveolata, Rhizaria and Excavata). We failed to find genes with cyanobacterial ancestry in Saccharomyces and Dictyostelium, except for a photorespiratory enzyme conserved among fungi. Meanwhile, we found several Monosiga genes with cyanobacterial ancestry, which were unrelated to other Opisthokonta genes. CONCLUSION: Our data demonstrate that a considerable number of genes with cyanobacterial ancestry have contributed to the genome composition of the plastid-lacking protists, especially bikonts. The origins of those genes might be due to lateral gene transfer events, or an ancient primary or secondary endosymbiosis before the diversification of bikonts. Our data also show that all genes identified in this study constitute multi-gene families with punctate distribution among eukaryotes, suggesting that the transferred genes could have survived through rounds of gene family expansion and differential reduction.