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論文(リポジトリ) |
Sato, Yukuto ; Hermawan, Idam ; Kakita, Tetsuya ; Okano, Sho ; Imai, Hideyuki ; Nagai, Hiroto ; Kimura, Ryosuke ; Yamashiro, Tetsu ; Kajita, Tadashi ; Toma, Claudia
概要:
Background Leptospirosis, a zoonosis caused by species in the spirochete genus Leptospira, is endemic to the Yaeyama reg
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ion in Okinawa, subtropical Japan. Species of the P1 subclade “virulent” group, within the genus Leptospira, are the main etiological agents of leptospirosis in Okinawa. However, their environmental persistence is poorly understood. This study used a combination of bacterial isolation and environmental DNA (eDNA) metabarcoding methods to understand the eco-epidemiology of leptospirosis in this endemic region. Findings Polymerase chain reaction (PCR) characterized twelve human clinical L. interrogans isolates belonging to the P1 subclade “virulent” subgroup and 11 environmental soil isolates of the P1subclade “low virulent” subgroup (genetically related to L. kmetyi, n = 1; L. alstonii, n = 4; L. barantonii, n = 6) from the Yaeyama region targeting four virulence-related genes (lipL32, ligA, ligB and lpxD1). Clinical isolates were PCR positive for at least three targeted genes, while all environmental isolates were positive only for lipL32. Analysis of infected renal epithelial cells with selected clinical and environmental strains, revealed the disassembly of cell-cell junctions for the Hebdomadis clinical strain serogroup. Comparison of leptospiral eDNA during winter and summer identified operational taxonomic units corresponding to the species isolated from soil samples (L. kmetyi and L. barantonii) and additional P2 subclade species (L. licerasiae, L. wolffii-related, among others) that were not detected by soil cultivation. Total Leptospira read counts were higher in summer than in winter and the analysis of leptospiral/animal eDNA relationship suggested Rattus spp. as a potential reservoir animal. Conclusion Our study demonstrated high environmental Leptospira diversity in the Yaeyama region, particularly during summer, when most of the leptospirosis cases are reported. In addition, several Leptospira species with pathogenic potential were identified that have not yet been reported in Yaeyama; however, the environmental persistence of P1 subclade species previously isolated from human clinical cases in this region was absent, suggesting the need of further methodology development and surveillance.
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2.
論文(リポジトリ) |
Sebastián, Isabel ; Okura, Nobuhiko ; Humbel, M. Bruno ; Xu, Jun ; Hermawan, Idam ; Matsuura, Chiaki ; Hall, Malgorzata ; Takayama, Chitoshi ; Yamashiro, Tetsu ; Nakamura, Shuichi ; Toma, Claudia
概要:
Bacterial pathogens have evolved multiple strategies to disassemble epithelial cell apical junctional complexes (AJCs) and infect epithelial cells. Leptospirosis is a widespread zoonotic infection, mainly caused by Leptospira
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interrogans, and its dissemination across host cell barriers is essential for its pathogenesis. However, the mechanism of bacterial dissemination across epithelial cell barriers remains poorly characterised. In this study, we analysed the interaction of L. interrogans with renal proximal tubule epithelial cells (RPTECs) and found that at 24 hr post-infection, L. interrogans remain in close contact with the plasma membrane of the RPTEC by extracellularly adhering or crawling. Leptospira interrogans cleaved E-cadherin and induced its endocytosis with release of the soluble N-terminal fragment into the extracellular medium. Concomitantly, a gradual decrease in transepithelial electrical resistance (TEER), mislocalisation of AJC proteins (occludin, claudin-10, ZO-1, and cingulin) and cytoskeletal rearrangement were observed. Inhibition of clathrin-mediated E-cadherin endocytosis prevented the decrease in TEER. We showed that disassembly of AJCs in epithelial cells and transmigration of bacteria through the paracellular route are important for the dissemination of L. interrogans in the host.
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3.
論文(リポジトリ) |
Gamage, Chandika D. ; Sato, Yukuto ; Kimura, Ryosuke ; Yamashiro, Tetsu ; Toma, Claudia
概要:
Background:Leptospirosis is one of the most significant zoonoses across the world not only because of its impact on huma
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n and animal health but also because of the economic and social impact on agrarian communities. Leptospirosis is endemic in Sri Lanka where paddy farming activities, the use of draught animals in agriculture, and peridomestic animals in urban and rural areas play important roles in maintaining the infection cycle of pathogenic Leptospira, especially concerning animals as a potential reservoir. In this study, an environmental DNA (eDNA) metabarcoding methodology was applied in two different agro-ecological regions of Sri Lanka to understand the eco-epidemiology of leptospirosis.\nMethodology/Principal findings:Irrigation water samples were collected in Kandy District (wet zone mid-country region 2) and Girandurukotte, Badulla District (intermediate zone low-country region 2); and analysed for the presence of pathogenic Leptospira, associated microbiome and the potential reservoir animals. Briefly, we generated PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. The analysis of eDNA showed different environmental microbiomes in both regions and a higher diversity of Leptospira species circulating in Kandy than in Girandurukotte. Moreover, the number of sequence reads of pathogenic Leptospira species associated with clinical cases such as L. interrogans was higher in Kandy than in Girandurukotte. Kandy also showed more animal species associated with pathogenic bacterial species than Girandurukotte. Finally, several pathogenic bacterial species including Arcobacter cryaerophilus, responsible for abortion in animals, was shown to be associated with pathogenic Leptospira.\nConclusions/Significance:Leptospirosis has been considered to be endemic in wet regions, consistently, leptospiral sequences were detected strongly in Kandy. The great Leptospira species diversity in Kandy observed in this study shows that the etiological agents of leptospirosis in Sri Lanka might be underestimated. Furthermore, our eDNA metabarcoding can be used to discriminate bacterial and animal species diversity in different regions and to explore environmental microbiomes to identify other associated bacterial pathogens in the environment.
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4.
論文(リポジトリ) |
Sato, Yukuto ; Mizuyama, Masaru ; Sato, Megumi ; Minamoto, Toshifumi ; Kimura, Ryosuke ; Toma, Claudia
概要:
Leptospires, which cause the zoonotic disease leptospirosis, persist in soil and aqueous environments. Several factors,
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including rainfall, the presence of reservoir animals, and various abiotic and biotic components interact to influence leptospiral survival, persistence, and pathogenicity in the environment. However, how these factors modulate the risk of infection is poorly understood. Here we developed an approach using environmental DNA (eDNA) metabarcoding for detecting the microbiome, vertebrates, and pathogenic Leptospira in aquatic samples. Specifically, we combined 4 sets of primers to generate PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. Using our method to analyze the eDNA of leptospirosis-endemic areas in northern Okinawa, Japan, we found that the microbiota in each river shifted over time. Operating taxonomic units corresponding to pathogenic L. alstonii, L. kmetyi, and L. interrogans were detected in association with 12 nonpathogenic bacterial species. In addition, the frequencies of 11 of these species correlated with the amount of rainfall. Furthermore, 10 vertebrate species, including Sus scrofa, Pteropus dasymallus, and Cynops ensicauda, showed high correlation with leptospiral eDNA detection. Our eDNA metabarcoding method is a powerful tool for understanding the environmental phase of Leptospira and predicting human infection risk.
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5.
論文(リポジトリ) |
Toma, Claudia ; Murray, Gerald L. ; Nohara, Toshitsugu ; Mizuyama, Masaru ; Koizumi, Nobuo ; Adler, Ben ; Suzuki, Toshihiko
概要:
Leptospira interrogans is responsible for the zoonotic disease leptospirosis. The pathogenic mechanisms of this spirocha
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ete remain poorly understood; however, virulence has been correlated with increased phagocytic uptake and survival within macrophages. Leptospiral outer membrane proteins are thought to be responsible for persistence in vivo via interaction with specific host components. In this study, we analysed the transcriptional profile of a virulent strain and its culture‐attenuated derivative strain to identify bacterial factors that may be involved in pathogenesis. Two outer membrane proteins, LMB216 and LigB (leptospiral immunoglobulin‐like protein B) were downregulated more than 10‐fold in the culture‐attenuated strain. We show that both proteins play a role in leptospiral uptake by macrophages and that LMB216, as well as LigB, enhances the binding of leptospires to fibronectin. Taken together, our results indicate that LMB216 plays a role in pathogen interaction with host molecule/s, which may contribute to pathogenesis of leptospirosis.
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6.
論文(リポジトリ) |
Suzuki, Toshihiko ; Franchi, Luigi ; Toma, Claudia ; Ashida, Hiroshi ; Ogawa, Michinaga ; Yoshikawa, Yuko ; Mimuro, Hitomi ; Inohara, Naohiro ; Sasakawa, Chihiro ; Nunez, Gabriel
概要:
Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but th
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e precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1β processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial–host interactions.
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