Biology subjects

Sawai, K.

Publications and source records attributed to Sawai, K..

5 recordsLinked to original sources

bioRxiv2026

Transposon insertion sequencing identifies genetic determinants of intrinsic rifamycin resistance in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to many antimicrobial agents, including rifampicin, a frontline anti-tuberculosis drug, severely limiting treatment options. Here, we used transposon insertion sequencing (Tn-Seq) to perform a genomewide screen to identify genes required for intrinsic rifampicin resistance in M. abscessus. We uncovered candidate genes that confer intrinsic resistance to the rifamycin-class antibiotics, rifampicin and rifabutin. The genes we identified included previously reported genes such as arr, helR, and MAB_2807. By comparing our results with the rifampicin intrinsic resistance gene in Mycobacterium tuberculosis, we found that the mechanisms underlying rifampicin intrinsic resistance were distinct between the two species. The contribution of seven representative candidate genes to rifampicin resistance was confirmed by characterizing targeted gene deletion or transposon insertion mutants. Among these determinants, MAB_2807 was identified as a major efflux-based contributor to rifamycin resistance, and its disruption increased intracellular rifampicin accumulation. In addition to known resistance determinants, Tn-Seq revealed contributions from many genes involved in cell envelope processes to rifamycin resistance. Guided by this genetic signature, we evaluated the combined effects of cell wall-targeting antimicrobial agents with rifamycins. We found that rifamycins displayed selective synergistic interactions with specific {beta}-lactam antibiotics. Interestingly, we found that rifamycin exposure altered cell envelope ultrastructure and increased the accumulation of the peptidoglycan precursor UDP-N-acetylglucosamine, suggesting that rifampicin perturbs envelope-associated metabolic homeostasis. These findings highlight rifamycin-induced vulnerable cellular processes that may inform rational combination strategies. ImportanceRifamycins are cornerstone antibiotics for the treatment of mycobacterial infections, yet they are ineffective against Mycobacterium abscessus. Although several key resistance mechanisms have been described, the full genetic basis of intrinsic rifamycin resistance remains to be elucidated. Here, we used transposon insertion sequencing to systematically identify genes that contribute to intrinsic rifamycin resistance in M. abscessus. Because recent studies have shown that chemical modification of rifamycins can overcome specific intrinsic resistance mechanisms, a comprehensive understanding of these determinants could facilitate the development of next-generation rifamycins with improved activity against M. abscessus. In addition, we unexpectedly found that rifamycin exposure alters cell envelope structure, the intracellular levels of a key metabolic intermediate in peptidoglycan biosynthesis, and susceptibility to certain {beta}-lactams in M. abscessus. These findings may help guide the rational development of combination therapeutic strategies with next-generation rifamycins.

microbiology↗

bioRxiv2026

Stable isotope-assisted computational mass spectrometry reveals root-specific alkaloids in Glycyrrhiza species

Licorice (Glycyrrhiza) is a medicinal plant widely used in approximately 70% of traditional Japanese Kampo formulations and is known to produce a wide array of specialized metabolites with diverse pharmacological properties. Although hundreds of metabolites have been reported, the overall chemical diversity of Glycyrrhiza remains poorly characterized. Here, using mass spectrometry data obtained from fully 13C-labeled leaves and roots of Glycyrrhiza uralensis and Glycyrrhiza glabra, we determined the carbon number, followed by molecular formula and substructure prediction in combination with MS/MS similarity-based molecular networking. After excluding redundant ions, including isotopic peaks, adducts, and in-source fragments, we extracted 3,060 unique metabolite features with assigned carbon numbers. Among these, substructure information was assigned to 1,015 features (33%) across the four plant tissues, revealing the tissue-specific metabolome profiles. Furthermore, we discovered five previously unreported alkaloids, homopipecolic acid-conjugated flavonoids, in the roots of G. uralensis and G. glabra, and Glycine max, another member of the Fabaceae family. Two of these structures were validated using nuclear magnetic resonance spectroscopy. We further proposed a biosynthetic route involving a spontaneous reaction between 1-piperideine and malonyl glycoside substrates and confirmed the formation of the conjugated product using authentic standards.

biochemistry↗

bioRxiv2022

Unique genomic sequences in novel Mycobacterium avium subsp. hominissuis lineage enabled fine scale tracing the transmission route during pig movement

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct. HighlightsO_LIVariable number of tandem repeat analysis of Mycobacterium avium subsp. hominissuis (MAH) isolated from pigs (n=50) were conducted. C_LIO_LIDraft genome sequences of MAH (n=30) and genome analysis were conducted. C_LIO_LIPig MAHs were genetically far from East Asian human isolates and close to those of Western countries. C_LIO_LINovel MAH lineage which were transmitted farms by pig movement was found. C_LIO_LIHuman MAH isolates influenced the evolution of pig isolates. C_LI

microbiology↗

bioRxiv2021

Inhibitory effect of an anti-prokineticin-1 antibody on liver metastasis in mice injected with human colorectal cancer cell lines

Controlling hematogenous metastases is an effective treatment strategy for colorectal cancer. Multidisciplinary treatment for colorectal cancer has made great strides, and molecularly-targeted drugs have greatly improved the prognosis of patients. However, currently accepted molecularly- targeted therapeutic agents require concomitant use with anticancer agents. Thus, new molecularly-targeted drugs need to be developed. The prokineticin family of angiogenic factors has the potential of becoming target molecules. Among them, prokineticin-1 (PROK1) is involved in the promotion of angiogenesis, tumor growth, and liver metastases in colorectal cancer. We manufactured our own anti-PROK1 antibody and verified its effect in inhibiting liver metastases and prolonging survival. The method involved creating liver metastasis model mice using human colorectal cancer cell lines. These mice were divided into anti-PROK1 antibody administration and control groups. Mice were treated intraperitoneally with antibodies or phosphate-buffered saline (control) every 3 days. The number of liver metastatic lesions and survival time of each group were compared. The number of metastatic lesions decreased, and survival time was significantly prolonged in the antibody-treated group. Furthermore, using microarray and immunostaining in both groups, we confirmed the effect of administering the anti-PROK1 antibody on the oxidation, reduction, and apoptotic processes, and cell division of tumors, and that alterations were suppressed in 72.1% of the genes examined. The expression of transforming growth factor-{beta} (TGF-{beta}), a tumor suppressor gene, was increased. The increased expression of TGF-{beta} via PROK1 antibody administration may suppress the cancer cell proliferation ability, leading to liver metastasis suppression and prolonging the survival time of mice.

cancer biology↗

bioRxiv2021

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. Genome analysis on MAH of human isolates has been proceeding, however, those of pigs are limited despite its potential source of infection to human. In the current study, we obtained 30 draft genome sequences of MAH of pigs reared in Japan. The 30 draft genomes consisted of 4,848,678 - 5,620,788 bp length, 4,652 - 5,388 coding genes and 46 - 75 (Med: 47) tRNAs. All isolates had restriction modification associated genes and 185 - 222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

microbiology↗