Biology subjects

Watanabe, A.

Publications and source records attributed to Watanabe, A..

43 records · Page 3Linked to original sources

bioRxiv2020

Comparative genome and transcriptome analyses revealing interspecies variations in the expression of fungal biosynthetic gene clusters

Filamentous fungi produce various bioactive compounds that are biosynthesized by a set of proteins encoded in biosynthetic gene clusters (BGCs). For an unknown reason, large parts of the BGCs are transcriptionally silent under laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional regulation of fungal secondary metabolism is not fully understood from an evolutionary viewpoint. To address this issue, we conducted comparative genomic and transcriptomic analyses using five closely related species of the Aspergillus section Fumigati: Aspergillus fumigatus, Aspergillus lentulus, Aspergillus udagawae, Aspergillus pseudoviridinutans, and Neosartorya fischeri. From their genomes, 298 secondary metabolite (SM) core genes were identified, with 27.4% to 41.5% being unique to a species. Compared with the species-specific genes, a set of section-conserved SM core genes was expressed at a higher rate and greater magnitude, suggesting that their expression tendency is correlated with the BGC distribution pattern. However, the section-conserved BGCs showed diverse expression patterns across the Fumigati species. Thus, not all common BGCs across species appear to be regulated in an identical manner. A consensus motif was sought in the promoter region of each gene in the 15 section-conserved BGCs among the Fumigati species. A conserved motif was detected in only two BGCs including the gli cluster. The comparative transcriptomic and in silico analyses provided insights into how the fungal SM gene cluster diversified at a transcriptional level, in addition to genomic rearrangements and cluster gains and losses. This information increases our understanding of the evolutionary processes associated with fungal secondary metabolism. Author summaryFilamentous fungi provide a wide variety of bioactive compounds that contribute to public health. The ability of filamentous fungi to produce bioactive compounds has been underestimated, and fungal resources can be developed into new drugs. However, most biosynthetic genes encoding bioactive compounds are not expressed under laboratory conditions, which hampers the use of fungi in drug discovery. The mechanisms underlying silent metabolite production are poorly understood. Here, we attempted to show the diversity in fungal transcriptional regulation from an evolutionary viewpoint. To meet this goal, the secondary metabolisms, at genomic and transcriptomic levels, of the most phylogenetically closely related species in Aspergillus section Fumigati were compared. The conserved biosynthetic gene clusters across five Aspergillus species were identified. The expression levels of the well-conserved gene clusters tended to be more active than the species-specific, which were not well-conserved, gene clusters. Despite highly conserved genetic properties across the species, the expression patterns of the well-conserved gene clusters were diverse. These findings suggest an evolutionary diversification at the transcriptional level, in addition to genomic rearrangements and gains and losses, of the biosynthetic gene clusters. This study provides a foundation for understanding fungal secondary metabolism and the potential to produce diverse fungal-based chemicals.

genomics↗

bioRxiv2020

NRF1 and NRF3 complementarily maintain a basal proteasome activity in cancer cells through CPEB3-mediated translational repression

Proteasomes are protease complexes essential for cellular homeostasis, and their activity is crucial for cancer cell growth. However, the mechanism of how proteasome activity is maintained in cancer cells has remained unclear. The CNC family transcription factor NRF1 induces the expression of almost all proteasome-related genes under proteasome inhibition. NRF1 and its phylogenetically closest homolog NRF3 are both highly expressed in several types of cancers, such as colorectal cancer. Herein, we demonstrate that NRF1 and NRF3 complementarily maintain basal proteasome activity in cancer cells. A double knockdown of NRF1 and NRF3 impaired the basal proteasome activity in cancer cells and the cancer cell resistance to a proteasome inhibitor anticancer drug bortezomib by significantly reducing basal expression of seven proteasome-related genes, including PSMB3, PSMB7, PSMC2, PSMD3, PSMG2, PSMG3, and POMP. Interestingly, the molecular basis behind these cellular consequences was that NRF3 repressed NRF1 translation by the gene induction of translational regulator CPEB3, which binds to NRF1-3'UTR and decreases polysome formation on NRF1 mRNA. Consistent results were obtained from clinical analysis, wherein patients with cancer having higher CPEB3/NRF3-expressing tumors exhibit poor prognosis. These results provide the novel regulatory mechanism of basal proteasome activity in cancer cells through an NRF3-CPEB3-NRF1 translational repression axis.

molecular biology↗

bioRxiv2019

Biosynthesis of β-(1->5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus

The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise -(1[->]2)-/-(1[->]6)-mannan and {beta}-(1[->]5)-/{beta}-(1[->]6)-galactofuranosyl chains. We previously revealed that GfsA is a {beta}-galactofuranoside {beta}-(1[->]5)-galactofuranosyltransferase involved in the biosynthesis of {beta}-(1[->]5)-galactofuranosyl chains. Here, we clarified the entire biosynthesis of {beta}-(1[->]5)-galactofuranosyl chains in A. fumigatgus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are {beta}-galactofuranoside {beta}-(1[->]5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize {beta}-(1[->]5)-galactofuranosyl oligomers up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from the strains {Delta}gfsB, {Delta}gfsC, {Delta}gfsAC, and {Delta}gfsABC revealed that GfsA and GfsC synthesized all {beta}-(1[->]5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans, and GfsB exhibited limited function in A. fumigatus. The loss of {beta}-(1[->]5)-galactofuranosyl residues decreased the hyphal growth rate and conidia formation ability as well as increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immune-compromised mice.\n\nIMPORTANCE{beta}-(1[->]5)-galactofuranosyl residues are widely distributed in the subphylum Pezisomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of {beta}-(1[->]5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for their understanding. In this study, we show that GfsA and GfsC are responsible for the biosynthesis of all {beta}-(1[->]5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicates that {beta}-(1[->]5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of {beta}-(1[->]5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the subphylum Pezisomycotina.

microbiology↗

bioRxiv2019

Structural basis for two-way communication between dynein and microtubules

The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain of dynein via an a-helical coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulated MTBD either in high-affinity or low-affinity states by introducing a disulfide bond between the coiled-coils and analyzed the resulting structures by NMRand cryo-EM. In the MT-unbound state, the affinity changes of MTBD were achieved by sliding of the N-terminal -helix by one half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, cryo-EM analysis showed that MT binding induced further sliding of the N-terminal -helix even without the disulfide bond, which suggests the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.

biophysics↗

bioRxiv2019

An alternative nisin A resistance mechanism affects virulence in Staphylococcus aureus

Nisin A is a bacteriocin produced by Lactococcus lactis and is widely used as a food preservative. Staphylococcus aureus has the BraRS-VraDE system providing resistance against low concentrations of nisin A. The BraRS is one of a two-component system that senses nisin A by BraS and finally induces the expression of ABC transporter VraDE by phosphorylated BraR. Previously, we isolated a highly nisin A resistant strain with increased VraDE expression due to a mutation of braRS. In this study, we isolated a BraRS-VraDE-independent, nisin A resistant mutant from S. aureus MW2. These mutants, designated SAN2 (S. aureus nisin resistant) and SAN469, had a mutation in pmtR (MW1875) which encodes a transcriptional regulator responsible for the expression of the pmtA-D operon. As a result, this mutant exhibited a high level of constitutive production of PmtA-D, a transporter responsible for the export of phenol soluble modulin (PSM). We also obtained two pmtA-D overexpressing, nisin A resistant mutants which contained a point mutation in pmtR from other S. aureus strains.\n\nCharacterization of the mutants revealed that they have a decreased susceptibility to human beta defensin-3 and LL37, which are innate immune factors. Additionally, these mutants showed higher hemolytic activity than the MW2 original strain. Furthermore, in a mouse bacteremia model, the SAN2 strain exhibited a lower survival rate than the MW2 original strain. These results indicate that the over expression of pmtA-D due to the pmtR mutation is an alternative nisin A resistance, which also affects virulence in S. aureus.\n\nAuthor SummaryRecently, the emergence of antibiotic resistant bacteria such as MRSA, MDRP and CRE have brought serious problems for chemotherapy in the world. In addition, many antibacterial agents such as disinfectants and food additives are widely used. Therefore, it raises the possibility that bacteria are becoming resistant to all antibacterial agents. In this study, we investigated whether S. aureus become resistant against nisin A, one of the food additives. Finally, we isolated nisin A highly resistant S. aureus strains. Among these strains, we identified that one strain designated as SAN2 showed nisin A resistance by the overproduction of Pmts which were involved in the secretion of virulence factors called PSMs. We identified a mutation of pmtR gene encoding a regulator for pmt genes. SAN2 strain showed the decreased susceptibility to human antimicrobial peptides and the increased hemolytic activity. Finally, SAN2 showed higher lethal activity in mouse bacteremia model. Our study provides new insights into that S. aureus may cause resistance against various antibacterial food additives, together with the altering the virulence.

microbiology↗

bioRxiv2019

Investigation of betaine as a novel psychotherapeutic for schizophrenia

Betaine is known to act against various biological stresses and its levels were reported to be decreased in schizophrenia patients. Using Chdh (a gene for betaine synthesis)-deficient mice and betaine-supplemented inbred mice, we assessed the role of betaine in psychiatric pathophysiology, and its potential as a novel psychotherapeutic, by leveraging metabolomics, behavioral-, transcriptomics and DNA methylation analyses. The Chdh-deficient mice revealed remnants of psychiatric behaviors along with schizophrenia-related molecular perturbations. Betaine supplementation elicited genetic background-dependent improvement in cognitive performance, and suppressed methamphetamine (MAP)-induced behavioral sensitization. Furthermore, betaine rectified the altered antioxidative and proinflammatory responses induced by MAP and in vitro phencyclidine treatments. Notably, betaine levels were decreased in the postmortem brains from schizophrenia, and a coexisting elevated carbonyl stress, a form of oxidative stress, demarcated a subset of schizophrenia with \"betaine deficit-oxidative stress pathology\". We revealed the decrease of betaine levels in glyoxylase 1 (GLO1)-deficient hiPSCs, which shows elevated carbonyl stress, and the efficacy of betaine in alleviating it, thus supporting a causal link between betaine and oxidative stress conditions. Furthermore, a CHDH variant, rs35518479, was identified as a cis-expression quantitative trait locus (QTL) for CHDH expression in postmortem brains from schizophrenia, allowing genotype-based stratification of schizophrenia patients for betaine efficacy. In conclusion, the present study underscores the potential benefit of betaine in a subset of schizophrenia.

neuroscience↗

bioRxiv2019

Isolation of lactic acid bacteria capable of reducing environmental alkyl and fatty acid hydroperoxides, and the effect of their oral administration on oxidative-stressed nematodes and rats

Reinforcement of hydroperoxide-eliminating activity in the intestines and colon should prevent associated diseases. We previously isolated a lactic acid bacterium, Pediococcus pentosaceus Be1, that facilitates a 2-electron reduction of hydrogen peroxide to water. In this study, we successfully isolated an alternative lactic acid bacterium, Lactobacillus plantarum P1-2, that can efficiently reduce environmental alkyl hydroperoxides and fatty acid hydroperoxides to their corresponding hydroxy derivatives through a 2-electron reduction. Each strain exhibited a wide concentration range with regard to the environmental reducing activity for each hydroperoxide. Given this, the two lactic acid bacteria were orally administered to the oxygen-sensitive short-lived nematode mutant, and this resulted in a significant expansion of its lifespan. This observation suggests that P. pentosaceus Be1 and L. plantarum P1-2 inhibit internal oxidative stress. To determine the specific organs involved in this response, we performed a similar experiment in rats, involving induced lipid peroxidation by iron-overloading. We observed that only L. plantarum P1-2 inhibited colonic mucosa lipid peroxidation in rats with induced oxidative stress.

microbiology↗