Development of Technology for Analyzing Symbiotic Microbial Genome Data

Intestinal bacteriome (Intestinal flora): A wide variety of microorganisms live in our bodies. In recent years, the relationship between the intestinal microbiota (intestinal flora) and health and disease has become a matter of great interest. The number of genes in the intestinal microflora is approximately 10 times greater than the number of human genes, yielding 20 million genes. Recent studies have shown that an imbalance in the human gut microbiome can affect not only intestinal diseases such as colitis, but also neurodegenerative diseases and psychiatric disorders such as depression. An important research agenda for the next 10 years will be to accumulate meta-genomic data that comprehensively reads DNA sequences from the intestinal flora, and to link these meta-genomic data to predict, prevent, and treat health conditions and diseases. In this research, we are developing algorithms for detecting dysbiosis in the bacterial flora in pathway units and artificial intelligence to interpret the results in relation to human health conditions and diseases.

Revealing mechanism behind fecal microbiota transplantation: The bacterium Clostridioides difficile (C. difficile) is an opportunistic but drug-resistant bacterium that is present in very small numbers in healthy people. When strong antibiotics are used for some treatment, various bacteria die in the gut, but C. difficile survives. C. difficile then produces toxins that cause inflammation in the intestines, sometimes resulting in severe enteritis. In recent years, highly toxic C. difficile has emerged in Europe and the United States, and the disease is attracting a great deal of attention. According to the U.S. CDC, C. difficile infection is one of the five major threats that require the most attention. Although antimicrobial agents such as vancomycin and metronidazole are used for treatment, the disease is often recurrent and refractory, especially in Western countries, resulting in tens of thousands of deaths per year in the United States. In recent years, fecal transplantation therapy*1, in which feces from a healthy person is injected into the intestinal tract using an endoscope, etc., is very effective for recurrent C. difficile-associated enteritis, and is being practiced. However, how fecal transplant therapy affects recurrent C. difficile-associated enteritis and leads to improvement of the disease has not been fully elucidated. We extracted gut bacterial genomes and gut viral genomes from fecal samples of 9 patients with recurrent C. difficile-associated enteritis who responded to fecal transplantation therapy and their donors, and performed whole genome sequencing of each. Using our research group's proprietary technology, the analysis pipeline for intestinal bacteria and viruses (Cell Host Microbe, 2020), we analyzed their compositional proportions and infection relationships. As a result, we showed for the first time in the world that fecal transplantation therapy can not only dramatically change the compositional ratio and infection relationship of disturbed intestinal bacteria and viruses, but also restore the function of intestinal microflora (Gastroenterology, 2021).

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Intestinal virome: Along with bacteria, a wide variety of viruses exist in the human intestinal tract, many of which are not viruses that infect our cells, such as coronaviruses, but rather bacteriophages that infect bacteria. Bacteriophages affect the bacterial flora by infecting bacteria, multiplying themselves, and destroying them (lysis), but a comprehensive method to analyze bacteriophages has not yet been developed. We have succeeded in developing a protocol for comprehensive analysis of bacteriophages using a next-generation sequencer in collaboration with Professor Satoshi Uematsu. In addition, we are also developing data analysis technology to analyze bacteriophage composition using the sequencing data. Using these technologies, we are developing computational technologies to elucidate the relationship between bacteriophages and bacteria (host-parasite associations) by simultaneously analyzing the bacteriome and virome data from human feces.

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Next-generation phage therapy for multi-agent resistant bacteria: Lysogenic phages, which insert their own genome into the bacterial genome, are not supposed to be used in phage therapy. The Shigatoxin in Shigella dysenteriae and Verotoxin in E. coli O157 were both transmitted by phages, which can cause such dangerous gene transmission. However, it is very difficult to investigate the host-parasite associations of lytic phages that do not involve genomic insertions, and the CRISPR region only reveals the history of past infections, which may have already acquired resistance. Therefore, we are developing a novel phage therapy that uses phage-derived enzymes to lyse target pathogenic bacteria without using phage itself. Since this novel phage therapy uses only enzymes, various enzymes of lysogenic phages, which could not be used in the past, can be used in this therapy. We have sequenced the enzyme endolysin, which is capable of killing C. difficile, the causative bacteria of pseudomembranous colitis, from metagenomic data. Furthermore, we have actually synthesized endolysins from the identified sequences, and have even conducted infection experiments in mice. Mice were infected with C. difficile and developed severe intestinal inflammation and almost died, but C. difficile was effectively eradicated in mice that were introduced to our endolysin after infection, and death was virtually non-existent.