Biology subjects

Katano, H.

Publications and source records attributed to Katano, H..

7 recordsLinked to original sources

bioRxiv2024

Identification of novel wraparound transcripts in JC polyomavirus

JC polyomavirus (JCPyV) is a ubiquitous pathogen causing progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals. Polyomaviruses (PyVs) utilize complex transcriptional regulation mechanisms to express diverse mRNAs from their compact, double-stranded circular DNA genomes. A recent study employed next-generation sequencing (NGS) to provide a detailed transcriptome atlas of PyVs, including BK polyomavirus and simian virus 40. However, detailed information regarding the JCPyV transcriptome remains limited. Here, we conducted a comprehensive analysis combining short-read and long-read NGS methods to unveil the detailed transcriptome atlas of JCPyV. RNA isolated from IMR-32 and 293 cells transfected with the circular JCPyV genome was analyzed by NGS, leading to the discovery of 39 novel viral transcripts in addition to 12 previously annotated ones. Among these novel transcripts, we identified characteristic wraparound transcripts, conserved across PyVs, which are constructed from long primary transcripts generated through continuous, multiple rounds of circular transcription of the viral genome. These wraparound transcripts included both late transcripts harboring leader-to-leader repeated sequences and SuperT transcripts containing multiple LxCxE motifs. Notably, wraparound transcripts, including a SuperT transcript, were also detected in brain tissues from PML patients. Collectively, this study significantly expands our understanding of the JCPyV transcriptome, revealing the expression of wraparound transcripts in PML lesions. These findings provide valuable insights into the molecular basis of JCPyV gene expression and PML pathogenesis, potentially facilitating the development of effective countermeasures against PML. Author SummaryJC polyomavirus (JCPyV) was first identified in 1971 from the brain tissue of a patient with progressive multifocal leukoencephalopathy (PML), a life-threatening neurological disease. Despite over 50 years since the pathogens identification, the prognosis for PML remains extremely poor due to the lack of effective therapeutics. Elucidating the JCPyV transcriptome can expand our understanding of viral gene expression and PML pathogenesis, potentially providing targets for novel therapeutic strategies. Here, we employed a combination of three next-generation sequencing technologies to explore novel viral transcripts, creating a detailed transcriptome atlas of JCPyV. We identified 39 novel viral transcripts in addition to the 12 previously annotated ones in cultured cells transfected with the JCPyV genome. These novel transcripts included wraparound transcripts, which arise from continuous, multiple rounds of circular transcription of the viral genome. Wraparound transcripts were characterized by the presence of tandem repeats of approximately 100 nucleotides. Notably, these characteristic wraparound transcripts were also detected in brain tissues from PML patients. Our study provides new insights into JCPyV gene expression, potentially contributing to the development of effective therapeutics.

microbiology↗

bioRxiv2024

Identification of SPP1-positive macrophages by single-cell spatial analysis in human lung tissues with mycobacterial infection

Tuberculosis and non-tuberculous mycobacterial (NTM) diseases are infections caused by Mycobacterium tuberculosis and non-tuberculous mycobacteria such as the Mycobacterium avium complex, leading to the formation of granulomatous lesions with caseous necrosis in the lungs. Although granulomatous tissues are infiltrated by numerous inflammatory cells, including macrophages, lymphocytes, and neutrophils, the mechanisms underlying granuloma formation caused by mycobacteria remain unclear. In this study, we performed single-cell spatial analysis on lung tissue samples from patients with tuberculosis and NTM diseases to investigate the infiltrating cell populations. We analyzed seven lung lesions and identified individual cell types infiltrating the granulomatous tissue. Based on gene expression profiles, at least four macrophage subtypes were identified. Notably, SPP1-positive macrophages predominantly found infiltrating the granulomatous tissue. Langhans giant cells expressed SPP1, and numerous SPP1-positive macrophages without giant cell morphology were also observed around the granulomas. RNA-seq analysis revealed elevated SPP1 expression in mycobacterium-infected tissues. The SPP1-CD44 signaling pathway was active in SPP1-positive macrophages and their neighboring cells in mycobacterium-infected tissues. SPP1-positive macrophages were also observed around granulomas in other granulomatous diseases, such as granulomatosis with polyangiitis and sarcoidosis. These findings suggest that SPP1-CD44 signaling in SPP1-positive macrophages may play a role in the pathology of granulomatous diseases, including mycobacterial infections. Brief summarySPP1-CD44 signaling in SPP1-positive macrophages may play a role in granuloma formation in mycobacterial and other granulomatous diseases.

pathology↗

bioRxiv2023

Prolonged Kaposi's Sarcoma-associated Herpesvirus vIL-6 Exposure Enhances Inflammatory Responses by Epigenetic Reprogramming

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and possesses greater risks of having other complications, which include malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol-Linked Alkylation for the Metabolic Sequencing and Cleavage Under Target & Release Using Nuclease analysis, we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNAPII with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged NF-KB p65 binding after the lipopolysaccharide stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitor, OTX015, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals. Author summaryCombined and continuous cytokine stimulation triggers transcription reprogramming and is often used for specific tissue development. Continuous vIL-6 exposure occurs in KSHV-infected patients and leads to inflammatory cytokine storm with high mortality. However, possible epigenetic reprogramming by the vIL-6 and its association with pathogenesis remain unclear. Here we demonstrate the establishment of a new chromatin landscape mediated by BRD4 through prolonged vIL-6 exposure which contributes to more robust and rapid transcription and increased cytokines production. Inhibition of BRD4 suppressed this inflammatory response. Our results indicate that targeting the epigenetic effect of viral cytokines may lead to novel therapies for KSHV-induced inflammatory cytokine storms.

microbiology↗

bioRxiv2022

Hypoxic conditions promote Candida glabrata colonization in the intestinal tract and EPA6 plays a significant role in hypoxic adhesion to intestinal cells

Candida glabrata, a fungal pathogen colonizing mucocutaneous membranes and indwelling medical devices, is associated with invasive infections, especially in immunocompromised individuals. Candidiasis could be of endogenous and exogenous origins. Endogenous infections are considered to derive from the invasion of Candida species colonizing the digestive mucosa. Investigations of the gut-to-bloodstream translocation mechanisms of Candida species remain limited, although environmental oxygen levels have been recently suggested to alter the human fungal pathogen phenotypes. Moreover, human fungal pathogens, including Candida, colonizing or invading less oxygenated tissues encounter altered oxygen circumstances. Therefore, phenotype investigation under hypoxic conditions could provide valuable novel insights into the host-pathogen interaction mechanisms. This study aimed to elucidate the adhesion capabilities and mechanisms of C. glabrata depending on various oxygen levels. We performed C. glabrata adhesion assays to Caco-2 cells under aerobic, microaerobic (5 vol% oxygen), and anaerobic conditions, conducted RNA-seq to identify candidate genes functioning on hypoxic adhesion. We then generated deletants of these genes and evaluated both their adhesion to Caco-2 cells under anaerobic conditions and their colonization ability in the hypoxic intestinal tract in a mouse model. We observed significant differences in Caco-2 cell adhesion in response to different oxygen levels. Under hypoxic conditions, the C. glabrata adhesion capability increased and the expression levels of seven adhesion-related genes were up-regulated. Among these mutants, the adhesion capability of epa6{Delta} decreased the most. The epa6{Delta} mutant exhibited significantly lower intestinal colonization in mice than the wild-type. To the best of our knowledge, this study first describes the hypoxic adjustment of C. glabrata to intestinal cell adhesion, in which EPA6 plays the most significant role. If Epa6p function could be inhibited, it may contribute to reducing endogenous infection. Phenotype investigation under hypoxic conditions could provide valuable novel insights into the host-pathogen interaction mechanisms. Author SummaryCandida glabrata is the second most common pathogen of Candida infections (i.e., candidiasis), colonizing mucocutaneous membranes, indwelling medical devices, thereby causing bloodstream- and medical device-related infections and often leading to high morbidity and mortality. Candidiasis could be of endogenous and exogenous origins. Endogenous infections are considered to derive from the invasion of Candida species colonizing the digestive mucosa. Investigations of the gut-to-bloodstream colonizing and translocation mechanisms of Candida species remain limited. Interestingly, recent studies suggest that environmental oxygen levels could alter the human fungal pathogen phenotypes. This study thus focused on the relationship between the colonization and adhesion capability of C. glabrata in the gastrointestinal tract depending on the environmental oxygen level to address the underlying mechanisms. Our results suggest that anaerobic conditions promote C. glabrata adhesion and EPA6 plays a significant role in hypoxic adhesion, opening new perspectives in various affiliated fields and related research domains. If Epa6p function could be inhibited, it may contribute to control the colonization in the gut and following translocation. C. glabrata is known to be low-susceptible to azole antifungals. A novel antifungal agent type, such as one targeting these adhesive molecules, should thus be considered and further related studies would be necessary.

microbiology↗

bioRxiv2021

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure. Author SummarySARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

microbiology↗

bioRxiv2021

The peripheral T cell population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus 2

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that began in December 2019. Lymphopenia is a common feature in severe cases of COVID-19; however, the role of T cell responses during infection is unclear. Here, we inoculated six cynomolgus monkeys, divided into two groups according to the CD3+ T cell population in peripheral blood, with two clinical isolates of SARS-CoV-2: one of East Asian lineage and one of European lineage. After initial infection with the isolate of East Asian lineage, all three monkeys in the CD3+ low group showed clinical symptoms, including loss of appetite, lethargy, and transient severe anemia with/without short-term fever, within 14 days post-infection (p.i.). By contrast, all three monkeys in the CD3+ high group showed mild clinical symptoms such as mild fever and loss of appetite within 4 days p.i. and then recovered. After a second inoculation with the isolate of European lineage, three of four animals in both groups showed mild clinical symptoms but recovered quickly. Hematological, immunological, and serological tests suggested that the CD3+ high and low groups mounted different immune responses during the initial and second infection stages. In both groups, anti-viral and innate immune responses were activated during the early phase of infection and re-infection. However, in the CD3+ low group, inflammatory responses, such as increased production of monocytes and neutrophils, were stronger than those in the CD3+ high group, leading to more severe immunopathology and failure to eliminate the virus. Taken together, the data suggest that the peripheral T lymphocyte population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with SARS-CoV-2. Author summarySARS-CoV-2 infection causes an illness with clinical manifestations that vary from asymptomatic or mild to severe; examples include severe pneumonia and acute respiratory distress syndrome. Lymphopenia, which is common in severe COVID-19 cases, is characterized by markedly reduced numbers of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells. Here, we showed that cynomolgus monkeys selected according to the T cell populations in peripheral blood have different outcomes after experimental infection with SARS-CoV-2. These findings will increase our understanding of disease pathogenesis and may facilitate the development of animal models for vaccine evaluation.

pathology↗

bioRxiv2020

Suberoyl bis-hydroxamic acid reactivates Kaposis sarcoma-associated herpesvirus through histone acetylation and induces apoptosis in lymphoma cells

Kaposis sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposis sarcoma as well as primary effusion lymphoma (PEL), an aggressive B-cell neoplasm which mostly arises in immunocompromised individuals. At present, there is no specific treatment available for PEL and its prognosis is poor. Lytic replication of KSHV is also associated with a subset of multicentric Castleman diseases. In this study, we found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation in PEL cells in a dose-dependent manner. Next-generation sequencing analysis showed that more than 40% of all transcripts expressed in SBHA-treated PEL cells originated from the KSHV genome compared with less than 1% in untreated cells. Chromatin immunoprecipitation assays demonstrated that SBHA induced histone acetylation targeting the promoter region of the KSHV replication and transcription activator gene. However, there was no significant change in methylation status of the promoter region of this gene. In addition to its effect of KSHV reactivation, this study revealed that SBHA induces apoptosis in PEL cells in a dose-dependent manner, inducing cleavage of caspases and expression of proapoptotic factors, including Bim and Bax. These findings suggest that SBHA reactivates KSHV from latency and induces apoptosis through the mitochondrial pathway in PEL cells. Therefore, SBHA can be considered a new tool for induction of KSHV reactivation, and could provide a novel therapeutic strategy against PEL. ImportanceKaposis sarcoma and primary effusion lymphoma cells are latently infected with Kaposis sarcoma-associated herpesvirus (KSHV), whereas KSHV replication is frequently observed in multicentric Castleman disease. Although KSHV replication can be induced by some chemical reagents (e.g. 12-O-tetradecanoylphorbol-13-acetate), the mechanism of KSHV replication is not fully understood. We found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation with high efficiency, through histone acetylation in the promoter of the replication and transcription activator gene, compared with 12-O-tetradecanoylphorbol-13-acetate. SBHA also induced apoptosis through the mitochondrial pathway in KSHV-infected cells, with a lower EC50 than measured for viral reactivation. SBHA could be used in a highly efficient replication system for KSHV in vitro, and as a tool to reveal the mechanism of replication and pathogenesis of KSHV. The ability of SBHA to induce apoptosis at lower levels than needed to stimulate KSHV reactivation, indicates its therapeutic potential.

microbiology↗