Characterization of Prototheca CYP51/ERG11 as a possible target for therapeutic drugs.

Prototheca spp. are achlorophyllous algae, ubiquitous in nature. An increasing number of human and animal cases of Prototheca infection (protothecosis) are reported, and antifungal azoles, which inhibit sterol 14α-demethylase (CYP51/ERG11) involved in ergosterol biosynthesis, have empirically been used for the treatment of protothecosis. Although Prototheca, like fungi, has ergosterol in the cell membrane, efficacy of the antifungal azoles in the treatment of protothecosis is controversial. For investigating the interaction of azole drugs with Prototheca CYP51/ERG11, the CYP51/ERG11 genomic genes of four strains of P. wickerhamii and one strain each of P. cutis and P. miyajii were isolated and characterized in this study. Compared with the CYP51/ERG11 gene of chlorophyllous Auxenochlorella Protothecoides, it is possible that ProtothecaCYP51/ERG11 gene, whose exon-intron structure appeared to be species-specific, lost introns associated with the loss of photosynthetic activity. Analysis of the deduced amino acid sequences revealed that Prototheca CYP51/ERG11 and fungal CYP51/ERG11 are phylogenetically distant from each other although their overall structures are similar. Our basic in silico studies predicted that antifungal azoles could bind to the catalytic pocket of Prototheca CYP51/ERG11. It was also suggested that amino acid residues away from the catalytic pocket might affect the drug susceptibility. The results of this study may provide useful insights into the phylogenetic taxonomy of Prototheca spp. in relationship to the CYP51/ERG11 structure and development of novel therapeutic drugs for the treatment of protothecosis. LAY SUMMARY: Cases of infection by microalgae of Prototheca species are increasing. However, effective treatment has not been established yet. In this study, gene and structure of Prototheca's CYP51/ERG11, an enzyme which might serve as a target for therapeutic drugs, were characterized for the first time.

Protothecosis in Dogs and Cats-New Research Directions.

Protothecosis refers to disease of humans and animals caused by infection with fungus-like, colourless microalgae of the genus Prototheca. Although protothecosis remains an uncommon infection, increasing numbers of human and animal cases are being diagnosed worldwide. This review summarises major new findings in basic science (sequencing analyses of sterol 14α-demethylase (CYP51/ERG11) genes and organelle genomes of Prototheca wickerhamii) to elucidate taxonomic features of this pathogen. Furthermore, this review updates and summarises the clinical features, diagnosis and treatment of protothecosis in dogs and cats. This content of this review is based on information presented at the medical phycology symposium held in the 20th Congress of the International Society for Human and Animal Mycology ( https://www.isham.org/ ).

Molecular Characterization of Prototheca strains isolated in China revealed the first cases of protothecosis associated with Prototheca zopfii genotype 1.

In this study, six strains of Prototheca isolated in China from human patients diagnosed as protothecosis and cows with mastitis were characterized by polymerase chain reaction (PCR) and nucleotide sequencing of the ribosomal RNA gene (rDNA) and by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The results indicated that three strains isolated from the human patients were P. zopfii genotype 1, revealing the first cases of human protothecosis associated with P. zopfii genotype 1. The remaining three strains were shown to be P. zopfii genotype 2. Interestingly, one strain isolated from the cerebrospinal fluid of the human patient appeared to have both of the genotype 1- and 2-specific alleles in the small subunit (SSU) rDNA although it was classified by MALDI-TOF MS as genotype 2. For genotyping of certain strains of P. zopfii, it may be necessary to comprehensively evaluate the diversity in the SSU rDNA sequences and the MALDI-TOF MS results.

Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd.

Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis.

Species of the genus Prototheca are achlorophyllous algae and ubiquitous in nature, and so far, six species have been listed in this genus: Prototheca wickerhamii, Prototheca zopfii, Prototheca blaschkeae, Prototheca cutis, Prototheca stagnora and Prototheca ulmea. A strain of the genus Prototheca, IFM 53848T, was isolated in Japan from a patient with systemic protothecosis and had been designated P. wickerhamii. Our previous study, by using PCR analysis, revealed that its SSU rRNA gene (rDNA) was distinctively larger than that of P. wickerhamii and other species of the genus Prototheca. In this study, molecular analysis showed that the exceptionally large SSU rDNA of IFM 53848T contains four group I introns. The morphology of IFM 53848T was indistinguishable from those of P. wickerhamii or P. cutis, and phylogenetic analyses, based on the sequences of the SSU rDNA exons and the D1/D2 region of the large subunit rDNA, indicated that IFM 53848T was closely related to P. cutis. On the other hand, unlike P. cutis, IFM 53848T failed to assimilate fructose or lysine and grew well at higher temperatures of up to 42 °C. In addition, the nucleotide sequence of the ribosomal internal transcribed spacer and the matrix assisted laser desorption ionization time-of-flight mass spectrometry profile of IFM 53848T were clearly distinct from those of P. cutis. The results strongly suggest that IFM 53848T represents a novel species, and so the seventh member of the genus Prototheca, which we have named Prototheca miyajii sp. nov. The unique characteristics of the strain may provide useful insights into the systematic taxonomy of the genus Prototheca.

Dissemination of the Flavivirus Subgenomic Replicon Genome and Viral Proteins by Extracellular Vesicles.

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.

Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis.

The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and the transconjugant strains. Sequence analysis revealed a conjugative multidrug resistance plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a ß-lactamase gene. The 7.1 kb element and the previously reported element Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that these elements generate a circular copy of one specific strand without leaving an empty site on the donor molecule, in contrast to the movement of integron gene cassettes or ICE/IMEs discovered to date. These elements are termed SEs (strand-biased circularizing integrative elements): SE-6945 (the 7.1 kb element) and SE-6283 (Tn6283). The copy number and location of SE-6945 in the chromosome affected the antibiotic resistance levels of the transconjugants. SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that SEs are involved in the spread of AMR genes among marine bacteria.

Phenotypic Characteristics of Prototheca Species Occurring in Humans and Animals.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and also occur in animal intestines; occasionally, infections in humans and animals are observed. In this study, we conducted tests of assimilative abilities and thermotolerance in comparison with morphological characteristics of six opportunistic species (Prototheca blaschkeae, Prototheca bovis, Prototheca ciferrii, Prototheca cutis, Prototheca miyajii, and Prototheca wickerhamii) along with Prototheca paracutis. Five of the seven species could be differentiated by physiological characteristics, but P. wickerhamii and P. cutis had identical profiles. Of the cattle-associated species, only P. bovis was able to grow at 42°C. Both type strains of P. cutis and P. miyajii were most susceptible to ravuconazole compared with the other azoles.

Application of the Luminescence Syncytium Induction Assay to Identify Chemical Compounds That Inhibit Bovine Leukemia Virus Replication.

Bovine leukemia virus (BLV) infection causes endemic bovine leukemia and lymphoma, resulting in lower carcass weight and reduced milk production by the infected cattle, leading to economic losses. Without effective measures for treatment and prevention, high rates of BLV infection can cause problems worldwide. BLV research is limited by the lack of a model system to assay infection. To overcome this, we previously developed the luminescence syncytium induction assay (LuSIA), a highly sensitive and objectively quantifiable method for visualizing BLV infectivity. In this study, we applied LuSIA for the high-throughput screening of drugs that could inhibit BLV infection. We screened 625 compounds from a chemical library using LuSIA and identified two that markedly inhibited BLV replication. We then tested the chemical derivatives of those two compounds and identified BSI-625 and -679 as potent inhibitors of BLV replication with low cytotoxicity. Interestingly, BSI-625 and -679 appeared to inhibit different steps of the BLV lifecycle. Thus, LuSIA was applied to successfully identify inhibitors of BLV replication and may be useful for the development of anti-BLV drugs.

A chemical compound for controlled expression of nmt1-driven gene in the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is a useful model organism for studying a variety of eukaryotic cellular events such as the cell cycle control mechanisms. For inducible expression of exogenous genes in S. pombe, vectors carrying the nmt1 (no message in thiamine 1) promoter are most commonly used. Although nmt1 is a potent promoter, its transcription activity is drastically repressed in the presence of a low concentration of thiamine. Therefore, a combination of thiamine and nmt1 promoter is convenient for regulating gene expression in an all-or-none fashion. However, it has been difficult to adjust the nmt1 promoter activity in a controlled manner. Here we describe a chemical compound, designated as YAM2, whose repressive activity on the nmt1 promoter has a wider linear range than thiamine. Expression of exogenous proteins, such as human immunodeficiency virus type 1 Vpr and jellyfish green fluorescent protein, driven by the nmt1 promoter is gradually repressed by YAM2 in a dose-dependent manner. YAM2 does not exhibit a detectable level of cytotoxicity at a concentration required to fully repress the nmt1 promoter. The compound may serve as a useful tool for controlled expression of the nmt1-driven gene in S. pombe.

Development of biliary stent applying the antibacterial activity of silver: A literature review.

BACKGROUND: Endoscopic transpapillary stenting is commonly performed in patients with obstructive jaundice caused by a biliary stricture. Although the plastic stent (PS) is widely used for biliary drainage because of the low-cost and easy procedure, patency is short after placement in the bile duct because of the small diameter. Dysfunction of PS is primarily caused by biliary sludge that forms as a result of bacterial adhesion and subsequent biofilm formation on the inner surface of the stent. It is well known that silver ions have excellent antibacterial activity against a wide range of microorganisms. OBJECTIVE: This review provides an overview and perspective of the significance of silver-coated biliary stents. METHODS: We collected literature regarding silver-coated biliary stents, reviewed the current research/development status and discussed their possible usefulness. RESULTS: To date, several in vivo/vitro studies evaluated the patency of silver-blended or silver-coated biliary stents. These studies suggested that the silver coating on a PS was likely to prolong the patency period. CONCLUSION: The development of biliary stents using silver is expected to prolong stent patency and prevent frequent stent replacement.

Effects of the Japanese Encephalitis Virus Genotype V-Derived Sub-Viral Particles on the Immunogenicity of the Vaccine Characterized by a Novel Virus-Like Particle-Based Assay.

Japanese encephalitis virus (JEV) is classified into five genotypes labelled I through V. Although the genotype V (GV) JEV was originally found and had apparently been limited in Malaysia for more than 50 years, its emergence in Korea and China has recently been reported. Therefore, the GV JEV might be spreading over new geographical regions as a cause of potential public health problems. However, it is unknown whether the currently available JEV vaccines are effective against the emerging GV strains. To investigate this issue, a novel virus-like particle-based neutralizing assay was developed in this study. By using this assay, the inactivated JEV vaccine used in Japan and the recombinant sub-viral particles (SVPs) bearing the E protein of the GV Muar strain were characterized for the immunogenicity against the GV JEV. Although the inactivated vaccine alone failed to elicit a detectable level of neutralizing antibodies against the GV JEV, the vaccine added with the Muar-derived SVPs induced relatively high titers of neutralizing antibodies, associated with the efficient Th1 immune responses, against the GV JEV. The results indicate that addition of the GV JEV-derived antigens may be useful for developing the vaccine that is universally effective against JEV including the emerging GV strains.

A Short Peptide Derived from the ZorO Toxin Functions as an Effective Antimicrobial.

Antimicrobial peptides are potential molecules for the development of novel antibiotic agents. The ZorO toxin of a type I toxin-antitoxin system in Escherichia coli O157:H7 is composed of 29 amino acids and its endogenous expression inhibits E. coli growth. However, little is known about its inhibitory mechanism. In this study, we demonstrate that the ZorO localized in the inner membrane affects the plasma membrane integrity and potential when expressed in E. coli cells, which triggers the production of cytotoxic hydroxyl radicals. We further show that five internal amino acids (Ala-Leu-Leu-Arg-Leu; ALLRL) of ZorO are necessary for its toxicity. This result prompted us to address the potential of the synthetic ALLRL peptide as an antimicrobial. Exogenously-added ALLRL peptide to Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, and a fungus, Candida albicans, trigger cell membrane damage and exhibit growth defect, while having no effect on Gram-negative bacterium, E. coli. The ALLRL peptide retains its activity under the physiological salt concentrations, which is in contrast to natural antimicrobial peptides. Importantly, this peptide has no toxicity against mammalian cells. Taken together, an effective and short peptide, ALLRL, would be an attractive antimicrobial to Gram-positive bacteria and C. albicans.

Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-stimulatory Signal Domain Exhibits High Functional Potency.

Chimeric antigen receptor (CAR) is a hybrid molecule consisting of an antigen-binding domain and a signal transduction domain. The artificial T cells expressing CAR (CAR-T cells) are expected to be a useful tool for treatment of various diseases, such as cancer. The addition of a co-stimulatory signal domain (CSSD) to CAR is shown to be critical for modulating CAR-T cell activities. However, the interplay among types of CSSDs, effector functions, and characteristics of CAR-T cells is largely unknown. To elucidate the interplay, we analyzed effector functions, differentiation to memory T cell subsets, exhaustion, and energy metabolism of the CAR-T cells with different CSSDs. Comparing to the CAR-T cells bearing a CD28- or 4-1BB-derived CSSD, which are currently used for CAR-T cell development, we found that the CAR-T cells with a herpes virus entry mediator (HVEM)-derived CSSD exhibited enhanced effector functions and efficient and balanced differentiation to both central and effector memory subsets, associated with an elevated energy metabolism and a reduced level of exhaustion. Thus, we developed the CAR-T cells bearing the CSSD derived from HVEM with high functional potency. The HVEM-derived CSSD may be useful for developing effective CAR-T cells.

Case report of cutaneous protothecosis caused by Prototheca wickerhamii designated as genotype 2 and current status of human protothecosis in Japan.

An 85-year-old Japanese woman presented with infiltrative erythema and ulceration on the extensor surface of her right forearm. Direct microscopic examination demonstrated spherical and morula-like sporangia, while histopathology revealed numerous microorganisms with a mulberry-like appearance in the dermis. Staining of the microorganisms also showed mulberry-like sporangia that resembled the spokes of a wheel. The isolated yeast-like microorganism had been identified as Prototheca wickerhamii genotype 2 in another independent study on the basis of its morphological, biochemical and genetic analysis. This case of protothecosis was recorded in Kyushu, Japan, and oral treatment with itraconazole 200 mg/day for 2 months was effective. Herein, we also summarize and analyze 39 cases of human protothecosis reported in Japan since the first record in 1983.

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate.

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

Multiple antiviral activities of the antimalarial and anti-hepatitis C drug candidates N-89 and N-251.

The chemically synthesized endoperoxide compound N-89 and its derivative N-251 were shown to have potent antimalarial activity. We previously demonstrated that N-89 and N-251 potently inhibited the RNA replication of hepatitis C virus (HCV), which belongs to the Flaviviridae family. Since antimalarial and anti-HCV mechanisms have not been clarified, we were interested whether N-89 and N-251 possessed the activity against viruses other than HCV. In this study, we examined the effects of N-89 and N-251 on other flaviviruses (dengue virus and Japanese encephalitis virus) and hepatitis viruses (hepatitis B virus and hepatitis E virus). Our findings revealed that N-89 and N-251 moderately inhibited the RNA replication of Japanese encephalitis virus and hepatitis E virus, although we could not detect those anti-dengue virus activities. We also observed that N-89 and N-251 moderately inhibited the replication of hepatitis B virus at the step after viral translation. These results suggest the possibility that N-89 and N-251 act on some common host factor(s) that are necessary for viral replications, rather than the possibility that N-89 and N-251 directly act on the viral proteins except for HCV. We describe a new type of antiviral reagents, N-89 and N-251, which are applicable to multiple different viruses.

HIV-1 Vpr induces G2 cell cycle arrest in fission yeast associated with Rad24/14-3-3-dependent, Chk1/Cds1-independent Wee1 upregulation.

Viral protein R (Vpr), an accessory protein of human immunodeficiency virus type 1 (HIV-1), induces the G2 cell cycle arrest in fission yeast for which host factors, such as Wee1 and Rad24, are required. Catalyzing the inhibitory phosphorylation of Cdc2, Wee1 is known to serve as a major regulator of G2/M transition in the eukaryotic cell cycle. It has been reported that the G2 checkpoint induced by DNA damage or incomplete DNA replication is associated with phosphorylation and upregulation of Wee1 for which Chk1 and Cds1 kinase is required. In this study, we demonstrate that the G2 arrest induced by HIV-1 Vpr in fission yeast is also associated with increase in the phosphorylation and amount of Wee1, but in a Chk1/Cds1-independent manner. Rad24 and human 14-3-3 appear to contribute to Vpr-induced G2 arrest by elevating the level of Wee1 expression. It appears that Vpr could cause the G2 arrest through a mechanism similar to, but distinct from, the physiological G2 checkpoint controls. The results may provide useful insights into the mechanism by which HIV-1 Vpr causes the G2 arrest in eukaryotic cells. Vpr may also serve as a useful molecular tool for exploring novel cell cycle control mechanisms.

[Studies on HIV/AIDS using the fission yeast Schizosaccharomyces pombe].

Human immunodeficiency virus (HIV) is a causative agent of acquired immunodeficiency syndrome (AIDS) and a member of Retrovirus family. The name of "retrovirus" is said to be derived from "reverse-transcribing oncogenic virus." Living up to its name, retrovirus has contributed to oncology, especially in the field of cancer pathogenesis. Retrovirus research has led to discovery of a number of oncogenes as well. Since the discovery of HIV in 1983, however, retrovirus has also been considered as an important etiologic agent that could incapacitate the cells involved in immune responses. As of the end of 2004, the number of people living with HIV/AIDS is estimated to be as large as 40 million. Every year, nearly 5 million people are newly infected with HIV, and 3 million people die of AIDS in the world, mainly in Africa and southeastern and southern Asia. Despite extensive studies, detailed mechanisms of HIV pathogenesis are still unclear, and efforts are being made to clarify functions of various HIV proteins and identify the cellular factors that could interact with the HIV proteins. One of the HIV accessory proteins, Vpr, causes the host cell cycle arrest at G2 phase, which may play an important pathogenic role in AIDS induction. Exploiting the fission yeast Schizosaccharomyces pombe useful for cell cycle studies, I've been trying to elucidate the mechanism by which Vpr induces the G2 arrest as presented in this review.

Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene.

Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V.