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Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms

Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of... Introduction: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb ) microorganisms possess a 54- kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. Findings: The analysis revealed that two of six strains possessed a clb gene cluster. These clb strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. Conclusion: These results support the observations that the clb gene cluster is widely distributed in nature and clb E. coli having genotoxic potencies is not rare among microorganisms. Keywords: Colibactin, Genotyping, Genotoxicity Introduction DNA double-strand breaks and interstrand cross-links in Colibactin is a small genotoxic molecule produced by En- human cell lines and in animals infected with clb E. coli terobacteriaceae, including certain Escherichia coli (E. coli) strains, resulting in generation of gene mutations [1]. The strains harbored in the human gut, and is involved in the clb E. coli stimulates growth of colon tumors under etiology of colorectal cancer. The colibactin-producing conditions of host inflammation, and is found with in- (clb ) microorganisms possess a 54-kilobase genomic island creased frequency in inflammatory bowel disease, familial (clb gene cluster) encoding polyketide synthases (PKSs), adenomatous polyposis, and colorectal cancer patients [2, nonribosomal peptide synthetases (NRPSs), and PKS-NRPS 3]. We previously reported that E. coli strains isolated from hybrid megasynthetases [1]. Nougayrede et al. observed a Japanese colorectal cancer patient produced colibactin and showed genotoxicity in in vitro assays [4, 5]. However, the chemical structure of the genotoxin, the molecular * Correspondence: kawanisi@riast.osakafu-u.ac.jp mechanism of its mutagenesis/carcinogenesis, and distribu- Graduate School of Science and Radiation Research Center, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8570, tion of the clb gene cluster among microorganisms have Japan not been fully clarified yet. Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kawanishi et al. Genes and Environment (2020) 42:12 Page 2 of 6 Table 1 Summary of genotyping and genotoxicity analyses at the microbe division of the RIKEN BioResource Re- Strain clb Gene cluster Genotoxicity (MN test) search Center (Tsukuba, Japan), which is participating in JCM1246 –– the National BioResource Project of the MEXT, Japan. E. coli Nissle 1917 strain was obtained from Mutaflor, JCM1649T –– Ardeypharm, GmbH. (Herdecke, Germany), and used as JCM5263 + + a a clb strain [1]. The host tester strain E. coli ZA227 JCM5491 + + used in the umu test was kindly supplied by Dr. Mie JCM18426 –– Watanabe-Akanuma (Institute of Environmental Toxi- JCM20114 –– cology, Tokyo, Japan). PCR analysis and electrophoresis Nissle 1917 + + for genotyping of the clb gene cluster was carried out Genotyping data are also confirmed in ref. [4] b with the oligonucleotide primers, as previously re- Genomic data are also from ref. [1] ported [4]. For genome analysis with next-generation The present study aimed to assess the distribution of sequencing, the E. coli genomic DNA was purified the clb gene cluster among E. coli strains randomly with MonoFas DNA Purification Kit V (GL Sciences chosen from the Japan Collection of Microorganisms, In., Tokyo, Japan). Library construction and paired- with genotyping of the gene cluster. To evaluate the as- end sequencing were carried out using the Miseq sociation between presence of the cluster and genotoxi- (Illumina Inc., San Diego, CA, U.S.A) with the Miseq city, we examined the genotoxicity/clastogenicity of reagent kits v2 (300 cycles). The raw sequence data these E. coli strains in rodent cells using the in vitro mi- were mapped by the HISAT2 program (ver. 2.1.0, cronucleus (MN) test. Using the umu test, DNA damage Johns Hopkins University, Baltimore, MD, U.S.A) to in a bacterial tester strain treated with crude extracts of the genome of Nissle1917 (GCA_000714595.1) as a the E. coli was also evaluated. reference sequence. The mapped files were converted to bam files by using SAMtools (ver1.9, http://www. Materials and methods htslib.org), and the read coverages were generated by E. coli strains and genotyping StringTie (ver1.3.5, Johns Hopkins University) and the Six E. coli strains (Escherichia coli (Migula 1895) Castel- heatmap was constructed using the CIMminer pro- lani and Chalmers 1919) were randomly chosen and gram (National Cancer Institute, Bethesda, MD. purchased from the Japan Collection of Microorganisms U.S.A). + − + − Fig. 1 Typical gel images of amplicons from genomic DNA of a clb and a clb strain. Genomic DNA of JCM5263 (clb ) and JCM20114 (clb ) were analyzed. The clb genes and expected sizes of their amplicons (bp) in PCR are as follows: clbA, 613; clbB, 555; clbC, 503, clbD, 431; clbF, 465; clbG, 599; clbH, 693; clbI, 643; clbJ, 544; clbK, 690; clbL, 401; clbM, 592; clbN, 581; clbO, 438; clbP, 464; clbQ, 430 Kawanishi et al. Genes and Environment (2020) 42:12 Page 3 of 6 Fig. 2 The read coverage of the clb genes in genomic DNA of E. coli strains determined by Illumina MiSeq. The color represents the read coverage of the indicated clb gene in the indicated strains Infection and in vitro micronucleus test described [5]. Briefly, E. coli cells were harvested from Bacterial infection to Chinese hamster ovary (CHO) 10 mL of overnight culture in LB media (O.D. = 1.7–2) AA8 cells and the MN test were carried out as previ- by centrifugation, and extracts of E. coli were prepared ously described [5]. Briefly, the CHO cells (4 × 10 cells/ with 1 mL of BugBuster protein extraction reagent dish) were seeded in ϕ60 mm plastic cell culture dishes (Novagen, Merck Millipore Co., Tokyo, Japan). The cell 1 day before the infection procedure. The bacteria were lysates were collected by centrifugation at 16,000×g for cultured until OD = 0.5 at 37 °C in Infection Medium 20 min at 4 °C. The umu assay using ZA227/pSK1002 (IM) (RPMI1640 medium (Nacalai Tesque., Kyoto, tester strain was conducted as previously reported [6, 7]. Japan) + 25 mM HEPES, 5% fetal bovine serum (FBS, ZA227 is derived from E. coli K-12, which dose not Sigma-Aldrich, MO USA)). The infection was carried posses the clb gene cluster [1]. The tester strain in 1 mL out with 3 mL of IM containing E. coli at the indicated of the TGA medium and 20 μL of the extracts from the multiplicity of infection (MOI) (number of bacteria per clb E. coli strains were incubated for 3 h at 37 °C. As a cell at the onset of infection). After being treated with solvent and positive controls, 20 μL of BugBuster solu- bacteria for 4 h, the CHO cells were cultured for a fur- tion and 10 μLof1 μg/mL 4-nitroquinoline 1-oxide (4- ther 20 h in cell culture medium supplemented with NQO) (Nacalai Tesque) were used, respectively. 200 μg/mL gentamicin (Nacalai Tesque). The MN test was then performed, and the number of cells with MN Results and discussion was recorded based on the observation of 1000 inter- Genotyping phase cells. Relative cell growth was calculated using the First, we assessed the presence of the clb genes, i.e., 16 formula: clb genes (clbA-clbD, clbF-clbQ) by detecting each amplicon after PCR with specific primer sets to the Relative cell growth ¼ðÞ number of treated cells genes. As a positive control strain, we analyzed the known clb strain Nissle 1917, which is a commensal ðÞ number of non−treated cells strain also widely used as a probiotic treatment for intes- tinal disorders [1]. In genomic DNA from JCM5263, umu test JCM5491 and Nissle 1917, we observed amplicons corre- The DNA damaging potency of bacterial cell extracts sponding to all 16 clb genes (Table 1 and Fig. 1). How- was estimated using the umu test, as previously ever, some or all of the amplicons corresponding to the Kawanishi et al. Genes and Environment (2020) 42:12 Page 4 of 6 Fig. 3 Micronuclei formation in CHO AA8 cells infected with clb E. coli. Relative cell growth and mean values of MN frequencies at least 1000 cells are shown. In the graph, MOI = 0 represents the vehicle control (treatment with IM). Horizontal red lines in MN graphs indicate MN frequencies two fold higher than those of each vehicle control. N.A. indicates data are not available due to the high cytotoxicity Fig. 4 Dose-dependent induction of micronuclei in CHO AA8 cells infected with clb E. coli. Mean ± SD values of at least three independent experiments are shown. MOI = 0 represents the vehicle control. * indicates p < 0.05 and ** indicates p < 0.01 (versus that of MOI = 0) according to the t-test Kawanishi et al. Genes and Environment (2020) 42:12 Page 5 of 6 Fig. 5 Induction of SOS response (umuC gene) by E. coli extracts in umu test. Relative LacZ activity to the clb strain JCM1649T. Mean values of duplicated determinations are shown. 4-NQO as a positive control of DNA damaging agent (incubated for 3 h at 37 °C) 16 genes were not detected in JCM1649T, JCM1246, treated with JCM5491 and JCM20114 at MOI = 100 JCM18426 and JCM20114. The presence or absence of were 2.6% (data not shown) and 24%, respectively. the clb gene cluster in the strains was also confirmed JCM5491 and JCM20114 were hemolysin-positive with next-generation sequencing of the bacterial gen- strains (data not shown), therefore, their high cyto- omic DNA (Fig. 2). We concluded that three among toxicity might be involved in hemolysin. Since the seven strains, i.e., six strains randomly chosen from the MN test cannot be performed under such highly- Japan Collection of Microorganisms and the positive cytotoxic conditions, we tried lower-MOI treatments control strain Nissle 1917, harbored the clb gene cluster and found that at MOI = 6.25, JCM5491 induced MN (Table 1). It has been reported that 20.8% of healthy with frequency 2.5-fold greater than that at MOI = 0 people who have neither inflammatory bowel disease (Fig. 3). We concluded that clb JCM5263, JCM5491 nor colorectal cancer as well as 66.7% of colorectal can- and Nissle 1917 are MN-induction positive strains cer patients harbor clb E. coli [8]. Furthermore, this (Table 1). We also confirmed that infections with gene cluster is found not only in E. coli but also in Kleb- JCM5263 and Nissle 1917 resulted in dose- siella pneumoniae, Enterobacter aerogenes and Citrobac- dependent MN-inductions (Fig. 4). ter koseri [8, 9]. The bacterial clb gene cluster seems to Since DNA damage is known to induce MN [10], be well-distributed in nature. we examined the extracts of clb E. coli (JCM5263 and Nissle 1917) for induction of an SOS response in In vitro genotoxicity analysis the umu test. The extracts were prepared using Next, for screening of their genotoxicities, the MN- BugBuster reagent, which disrupts the cell walls and inducing activity of the E. coli strains was examined liberates the cytosol. Increased SOS responses were using the CHO AA8 cell line, since the test is a con- observed in the extracts of clb from both JCM5263 venient and reliable for evaluating genotoxicity [10]. and Nissle 1917 compared with that of clb As showninFig. 3, the degree of induction varied JCM1649T (Fig. 5). The relative SOS-induction levels among the strains. In the present study, we deter- by the extracts of both JCM5263 and Nissle 1917 mined that E. coli induces MN-frequency at least were 1.5 times higher than that of JCM1649T. The twofold compared with MOI = 0 as an MN-induction induction level by the positive control agent 4-NQO positive strain. Evidently, JCM5263 and Nissle 1917 (1.0 μg/mL) was 4.3-fold that by JCM1649T. These re- were MN-induction positive strains, that is, infection sults indicate that the clb E. coli extracts have weak of both strains at MOI = 100 induced MN with fre- potency for SOS induction. The clbSgene encodes a quency 2.5- to 7-fold greater than that at MOI = 0. resistance protein blocking the genotoxicity of coli- The level of cytotoxicity also varied. Infection of bactin and ClbS protein functions as an antidote for JCM5491 and JCM20114 led to high cytotoxic effects colibactin-autotoxicity in clb E. coli [11]. Presumably, in CHO cells. The relative growths of CHO cells the presence of ClbS protein in the extracts in the Kawanishi et al. Genes and Environment (2020) 42:12 Page 6 of 6 present study attenuated their DNA-damaging Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kyoto, Japan. potency. Received: 24 January 2020 Accepted: 17 February 2020 Conclusion Genotyping analysis revealed that two of six E. coli References strains randomly chosen from the Japan Collection of 1. Nougayrede JP, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, Gottschalk G, Buchrieser C, Hacker J, Dobrindt U, Oswald E. Escherichia coli induces DNA Microorganisms possessed a clb gene cluster. The clb double-strand breaks in eukaryotic cells. Science. 2006;313:848–51. JCM5263, JCM5491 and Nissle 1917 (as clb control 2. Cuevas-Ramos G, Petit CR, Marcq I, Boury M, Oswald E, Nougayrede JP. strain) exhibited MN induction in CHO cells. The cell Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. Proc Natl Acad Sci U S A. 2010;107:11537–42. extracts of JCM5263 and Nissle 1917 also had DNA- 3. Vizcaino MI, Crawford JM. The colibactin warhead crosslinks DNA. Nat damaging potency in a bacterial umu test. These results Chem. 2015;7:411–7. support the observations that clb gene clusters are 4. Hirayama Y, Tsunematsu Y, Yoshikawa Y, Tamafune R, Matsuzaki N, Iwashita Y, Ohnishi I, Tanioka F, Sato M, Miyoshi N, Mutoh M, Ishikawa H, Sugimura widely distributed in nature and that clb E. coli, which H, Wakabayashi K, Watanabe K. Activity-based probe for screening of high- has genotoxic potency, is not rare among colibactin producers from clinical samples. Org Lett. 2019;21:4490–4. microorganisms. 5. Kawanishi M, Hisatomi Y, Oda Y, Shimohara C, Tsunematsu Y, Sato M, Hirayama Y, Miyoshi N, Iwashita Y, Yoshikawa Y, Yagi T, Sugimura H, Abbreviations Wakabayashi K, Watanabe K. In vitro genotoxicity analyses of E. coli 4-NQO: 4-nitroquinoline 1-oxide; CHO: Chinese hamster ovary; producing colibactin isolated from a Japanese colorectal cancer patient. J clb : colibactin-producing; MN: micronucleus; MOI: multiplicity of infection Toxicol Sci. 2019;44(12):871–6. 6. Oda Y. Development and progress for three decades in umu test systems. Acknowledgements Genes Environ. 2016;38:24. Not applicable. 7. Oda Y, Nakamura S, Oki I, Kato T, Shinagawa H. Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens. Mutat Res. 1985;147:219–29. Authors’ contribution 8. Arthur JC, Perez-Chanona E, Muhlbauer M, Tomkovich S, Uronis JM, Fan TJ, MK, K. Wakabayashi and K. Watanabe designed the study. MK, Y Hisatomi Campbell BJ, Abujamel T, Dogan B, Rogers AB, Rhodes JM, Stintzi A, and CS performed micronucleus assays with mammalian cells. YO carried out Simpson KW, Hansen JJ, Keku TO, Fodor AA, Jobin C. Intestinal inflammation umu tests. YT, MS, Y Hirayama, NM and YY conducted genotyping. HS, targets cancer-inducing activity of the microbiota. Science. 2012;338:120–3. YI, MM, HI and TY critically discussed the study. MK wrote the manuscript. All 9. Putze J, Hennequin C, Nougayrede JP, Zhang W, Homburg S, Karch H, authors read and approved the final manuscript. Bringer MA, Fayolle C, Carniel E, Rabsch W, Oelschlaeger TA, Oswald E, Forestier C, Hacker J, Dobrindt U. Genetic structure and distribution of the Authors’ information colibactin genomic island among members of the family Enterobacteriaceae. Not applicable. Infect Immun. 2009;77:4696–703. 10. Hayashi M. The micronucleus test -most widely used in vivo genotoxicity Funding test. Genes Environ. 2016;38:18. This study was supported by Grants-in-Aid for Scientific Research (Grant 11. Bossuet-Greif N, Dubois D, Petit C, Tronnet S, Martin P, Bonnet R, Oswald E, Number: 17 K08841) from the Japan Society for the Promotion of Science Nougayrede JP. Escherichia coli ClbS is a colibactin resistance protein. Mol (JSPS) to Y.Y. and the Development of Innovative Research on Cancer Thera- Microbiol. 2016;99:897–908. peutics from Japan Agency for Medical Research and Development (AMED) (K. Watanabe, 16ck0106243h0001), Innovative Areas from MEXT, Japan (K. Watanabe, 16H06449), the Takeda Science Foundation (K. Watanabe), the In- Publisher’sNote stitution of Fermentation at Osaka (K. Watanabe), the Princess Takamatsu Springer Nature remains neutral with regard to jurisdictional claims in Cancer Research Fund (K. Watanabe, 16–24825), and the Yakult Bio-Science published maps and institutional affiliations. Foundation (K. Watanabe). Availability of data and materials Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details Graduate School of Science and Radiation Research Center, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8570, Japan. Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan. Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan. Department of Tumor Pathology, Hamamatsu University School of Medicine, Shizuoka, Japan. School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Tokyo, Japan. Division of Prevention, Center for Public Health Sciences, National Cancer Center, Tokyo, Japan. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes and Environment Springer Journals

Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms

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Abstract

Introduction: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb ) microorganisms possess a 54- kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. Findings: The analysis revealed that two of six strains possessed a clb gene cluster. These clb strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. Conclusion: These results support the observations that the clb gene cluster is widely distributed in nature and clb E. coli having genotoxic potencies is not rare among microorganisms. Keywords: Colibactin, Genotyping, Genotoxicity Introduction DNA double-strand breaks and interstrand cross-links in Colibactin is a small genotoxic molecule produced by En- human cell lines and in animals infected with clb E. coli terobacteriaceae, including certain Escherichia coli (E. coli) strains, resulting in generation of gene mutations [1]. The strains harbored in the human gut, and is involved in the clb E. coli stimulates growth of colon tumors under etiology of colorectal cancer. The colibactin-producing conditions of host inflammation, and is found with in- (clb ) microorganisms possess a 54-kilobase genomic island creased frequency in inflammatory bowel disease, familial (clb gene cluster) encoding polyketide synthases (PKSs), adenomatous polyposis, and colorectal cancer patients [2, nonribosomal peptide synthetases (NRPSs), and PKS-NRPS 3]. We previously reported that E. coli strains isolated from hybrid megasynthetases [1]. Nougayrede et al. observed a Japanese colorectal cancer patient produced colibactin and showed genotoxicity in in vitro assays [4, 5]. However, the chemical structure of the genotoxin, the molecular * Correspondence: kawanisi@riast.osakafu-u.ac.jp mechanism of its mutagenesis/carcinogenesis, and distribu- Graduate School of Science and Radiation Research Center, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8570, tion of the clb gene cluster among microorganisms have Japan not been fully clarified yet. Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kawanishi et al. Genes and Environment (2020) 42:12 Page 2 of 6 Table 1 Summary of genotyping and genotoxicity analyses at the microbe division of the RIKEN BioResource Re- Strain clb Gene cluster Genotoxicity (MN test) search Center (Tsukuba, Japan), which is participating in JCM1246 –– the National BioResource Project of the MEXT, Japan. E. coli Nissle 1917 strain was obtained from Mutaflor, JCM1649T –– Ardeypharm, GmbH. (Herdecke, Germany), and used as JCM5263 + + a a clb strain [1]. The host tester strain E. coli ZA227 JCM5491 + + used in the umu test was kindly supplied by Dr. Mie JCM18426 –– Watanabe-Akanuma (Institute of Environmental Toxi- JCM20114 –– cology, Tokyo, Japan). PCR analysis and electrophoresis Nissle 1917 + + for genotyping of the clb gene cluster was carried out Genotyping data are also confirmed in ref. [4] b with the oligonucleotide primers, as previously re- Genomic data are also from ref. [1] ported [4]. For genome analysis with next-generation The present study aimed to assess the distribution of sequencing, the E. coli genomic DNA was purified the clb gene cluster among E. coli strains randomly with MonoFas DNA Purification Kit V (GL Sciences chosen from the Japan Collection of Microorganisms, In., Tokyo, Japan). Library construction and paired- with genotyping of the gene cluster. To evaluate the as- end sequencing were carried out using the Miseq sociation between presence of the cluster and genotoxi- (Illumina Inc., San Diego, CA, U.S.A) with the Miseq city, we examined the genotoxicity/clastogenicity of reagent kits v2 (300 cycles). The raw sequence data these E. coli strains in rodent cells using the in vitro mi- were mapped by the HISAT2 program (ver. 2.1.0, cronucleus (MN) test. Using the umu test, DNA damage Johns Hopkins University, Baltimore, MD, U.S.A) to in a bacterial tester strain treated with crude extracts of the genome of Nissle1917 (GCA_000714595.1) as a the E. coli was also evaluated. reference sequence. The mapped files were converted to bam files by using SAMtools (ver1.9, http://www. Materials and methods htslib.org), and the read coverages were generated by E. coli strains and genotyping StringTie (ver1.3.5, Johns Hopkins University) and the Six E. coli strains (Escherichia coli (Migula 1895) Castel- heatmap was constructed using the CIMminer pro- lani and Chalmers 1919) were randomly chosen and gram (National Cancer Institute, Bethesda, MD. purchased from the Japan Collection of Microorganisms U.S.A). + − + − Fig. 1 Typical gel images of amplicons from genomic DNA of a clb and a clb strain. Genomic DNA of JCM5263 (clb ) and JCM20114 (clb ) were analyzed. The clb genes and expected sizes of their amplicons (bp) in PCR are as follows: clbA, 613; clbB, 555; clbC, 503, clbD, 431; clbF, 465; clbG, 599; clbH, 693; clbI, 643; clbJ, 544; clbK, 690; clbL, 401; clbM, 592; clbN, 581; clbO, 438; clbP, 464; clbQ, 430 Kawanishi et al. Genes and Environment (2020) 42:12 Page 3 of 6 Fig. 2 The read coverage of the clb genes in genomic DNA of E. coli strains determined by Illumina MiSeq. The color represents the read coverage of the indicated clb gene in the indicated strains Infection and in vitro micronucleus test described [5]. Briefly, E. coli cells were harvested from Bacterial infection to Chinese hamster ovary (CHO) 10 mL of overnight culture in LB media (O.D. = 1.7–2) AA8 cells and the MN test were carried out as previ- by centrifugation, and extracts of E. coli were prepared ously described [5]. Briefly, the CHO cells (4 × 10 cells/ with 1 mL of BugBuster protein extraction reagent dish) were seeded in ϕ60 mm plastic cell culture dishes (Novagen, Merck Millipore Co., Tokyo, Japan). The cell 1 day before the infection procedure. The bacteria were lysates were collected by centrifugation at 16,000×g for cultured until OD = 0.5 at 37 °C in Infection Medium 20 min at 4 °C. The umu assay using ZA227/pSK1002 (IM) (RPMI1640 medium (Nacalai Tesque., Kyoto, tester strain was conducted as previously reported [6, 7]. Japan) + 25 mM HEPES, 5% fetal bovine serum (FBS, ZA227 is derived from E. coli K-12, which dose not Sigma-Aldrich, MO USA)). The infection was carried posses the clb gene cluster [1]. The tester strain in 1 mL out with 3 mL of IM containing E. coli at the indicated of the TGA medium and 20 μL of the extracts from the multiplicity of infection (MOI) (number of bacteria per clb E. coli strains were incubated for 3 h at 37 °C. As a cell at the onset of infection). After being treated with solvent and positive controls, 20 μL of BugBuster solu- bacteria for 4 h, the CHO cells were cultured for a fur- tion and 10 μLof1 μg/mL 4-nitroquinoline 1-oxide (4- ther 20 h in cell culture medium supplemented with NQO) (Nacalai Tesque) were used, respectively. 200 μg/mL gentamicin (Nacalai Tesque). The MN test was then performed, and the number of cells with MN Results and discussion was recorded based on the observation of 1000 inter- Genotyping phase cells. Relative cell growth was calculated using the First, we assessed the presence of the clb genes, i.e., 16 formula: clb genes (clbA-clbD, clbF-clbQ) by detecting each amplicon after PCR with specific primer sets to the Relative cell growth ¼ðÞ number of treated cells genes. As a positive control strain, we analyzed the known clb strain Nissle 1917, which is a commensal ðÞ number of non−treated cells strain also widely used as a probiotic treatment for intes- tinal disorders [1]. In genomic DNA from JCM5263, umu test JCM5491 and Nissle 1917, we observed amplicons corre- The DNA damaging potency of bacterial cell extracts sponding to all 16 clb genes (Table 1 and Fig. 1). How- was estimated using the umu test, as previously ever, some or all of the amplicons corresponding to the Kawanishi et al. Genes and Environment (2020) 42:12 Page 4 of 6 Fig. 3 Micronuclei formation in CHO AA8 cells infected with clb E. coli. Relative cell growth and mean values of MN frequencies at least 1000 cells are shown. In the graph, MOI = 0 represents the vehicle control (treatment with IM). Horizontal red lines in MN graphs indicate MN frequencies two fold higher than those of each vehicle control. N.A. indicates data are not available due to the high cytotoxicity Fig. 4 Dose-dependent induction of micronuclei in CHO AA8 cells infected with clb E. coli. Mean ± SD values of at least three independent experiments are shown. MOI = 0 represents the vehicle control. * indicates p < 0.05 and ** indicates p < 0.01 (versus that of MOI = 0) according to the t-test Kawanishi et al. Genes and Environment (2020) 42:12 Page 5 of 6 Fig. 5 Induction of SOS response (umuC gene) by E. coli extracts in umu test. Relative LacZ activity to the clb strain JCM1649T. Mean values of duplicated determinations are shown. 4-NQO as a positive control of DNA damaging agent (incubated for 3 h at 37 °C) 16 genes were not detected in JCM1649T, JCM1246, treated with JCM5491 and JCM20114 at MOI = 100 JCM18426 and JCM20114. The presence or absence of were 2.6% (data not shown) and 24%, respectively. the clb gene cluster in the strains was also confirmed JCM5491 and JCM20114 were hemolysin-positive with next-generation sequencing of the bacterial gen- strains (data not shown), therefore, their high cyto- omic DNA (Fig. 2). We concluded that three among toxicity might be involved in hemolysin. Since the seven strains, i.e., six strains randomly chosen from the MN test cannot be performed under such highly- Japan Collection of Microorganisms and the positive cytotoxic conditions, we tried lower-MOI treatments control strain Nissle 1917, harbored the clb gene cluster and found that at MOI = 6.25, JCM5491 induced MN (Table 1). It has been reported that 20.8% of healthy with frequency 2.5-fold greater than that at MOI = 0 people who have neither inflammatory bowel disease (Fig. 3). We concluded that clb JCM5263, JCM5491 nor colorectal cancer as well as 66.7% of colorectal can- and Nissle 1917 are MN-induction positive strains cer patients harbor clb E. coli [8]. Furthermore, this (Table 1). We also confirmed that infections with gene cluster is found not only in E. coli but also in Kleb- JCM5263 and Nissle 1917 resulted in dose- siella pneumoniae, Enterobacter aerogenes and Citrobac- dependent MN-inductions (Fig. 4). ter koseri [8, 9]. The bacterial clb gene cluster seems to Since DNA damage is known to induce MN [10], be well-distributed in nature. we examined the extracts of clb E. coli (JCM5263 and Nissle 1917) for induction of an SOS response in In vitro genotoxicity analysis the umu test. The extracts were prepared using Next, for screening of their genotoxicities, the MN- BugBuster reagent, which disrupts the cell walls and inducing activity of the E. coli strains was examined liberates the cytosol. Increased SOS responses were using the CHO AA8 cell line, since the test is a con- observed in the extracts of clb from both JCM5263 venient and reliable for evaluating genotoxicity [10]. and Nissle 1917 compared with that of clb As showninFig. 3, the degree of induction varied JCM1649T (Fig. 5). The relative SOS-induction levels among the strains. In the present study, we deter- by the extracts of both JCM5263 and Nissle 1917 mined that E. coli induces MN-frequency at least were 1.5 times higher than that of JCM1649T. The twofold compared with MOI = 0 as an MN-induction induction level by the positive control agent 4-NQO positive strain. Evidently, JCM5263 and Nissle 1917 (1.0 μg/mL) was 4.3-fold that by JCM1649T. These re- were MN-induction positive strains, that is, infection sults indicate that the clb E. coli extracts have weak of both strains at MOI = 100 induced MN with fre- potency for SOS induction. The clbSgene encodes a quency 2.5- to 7-fold greater than that at MOI = 0. resistance protein blocking the genotoxicity of coli- The level of cytotoxicity also varied. Infection of bactin and ClbS protein functions as an antidote for JCM5491 and JCM20114 led to high cytotoxic effects colibactin-autotoxicity in clb E. coli [11]. Presumably, in CHO cells. The relative growths of CHO cells the presence of ClbS protein in the extracts in the Kawanishi et al. Genes and Environment (2020) 42:12 Page 6 of 6 present study attenuated their DNA-damaging Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kyoto, Japan. potency. Received: 24 January 2020 Accepted: 17 February 2020 Conclusion Genotyping analysis revealed that two of six E. coli References strains randomly chosen from the Japan Collection of 1. Nougayrede JP, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, Gottschalk G, Buchrieser C, Hacker J, Dobrindt U, Oswald E. Escherichia coli induces DNA Microorganisms possessed a clb gene cluster. The clb double-strand breaks in eukaryotic cells. Science. 2006;313:848–51. JCM5263, JCM5491 and Nissle 1917 (as clb control 2. Cuevas-Ramos G, Petit CR, Marcq I, Boury M, Oswald E, Nougayrede JP. strain) exhibited MN induction in CHO cells. The cell Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. Proc Natl Acad Sci U S A. 2010;107:11537–42. extracts of JCM5263 and Nissle 1917 also had DNA- 3. Vizcaino MI, Crawford JM. The colibactin warhead crosslinks DNA. Nat damaging potency in a bacterial umu test. These results Chem. 2015;7:411–7. support the observations that clb gene clusters are 4. Hirayama Y, Tsunematsu Y, Yoshikawa Y, Tamafune R, Matsuzaki N, Iwashita Y, Ohnishi I, Tanioka F, Sato M, Miyoshi N, Mutoh M, Ishikawa H, Sugimura widely distributed in nature and that clb E. coli, which H, Wakabayashi K, Watanabe K. Activity-based probe for screening of high- has genotoxic potency, is not rare among colibactin producers from clinical samples. Org Lett. 2019;21:4490–4. microorganisms. 5. Kawanishi M, Hisatomi Y, Oda Y, Shimohara C, Tsunematsu Y, Sato M, Hirayama Y, Miyoshi N, Iwashita Y, Yoshikawa Y, Yagi T, Sugimura H, Abbreviations Wakabayashi K, Watanabe K. In vitro genotoxicity analyses of E. coli 4-NQO: 4-nitroquinoline 1-oxide; CHO: Chinese hamster ovary; producing colibactin isolated from a Japanese colorectal cancer patient. J clb : colibactin-producing; MN: micronucleus; MOI: multiplicity of infection Toxicol Sci. 2019;44(12):871–6. 6. Oda Y. Development and progress for three decades in umu test systems. Acknowledgements Genes Environ. 2016;38:24. Not applicable. 7. Oda Y, Nakamura S, Oki I, Kato T, Shinagawa H. Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens. Mutat Res. 1985;147:219–29. Authors’ contribution 8. Arthur JC, Perez-Chanona E, Muhlbauer M, Tomkovich S, Uronis JM, Fan TJ, MK, K. Wakabayashi and K. Watanabe designed the study. MK, Y Hisatomi Campbell BJ, Abujamel T, Dogan B, Rogers AB, Rhodes JM, Stintzi A, and CS performed micronucleus assays with mammalian cells. YO carried out Simpson KW, Hansen JJ, Keku TO, Fodor AA, Jobin C. Intestinal inflammation umu tests. YT, MS, Y Hirayama, NM and YY conducted genotyping. HS, targets cancer-inducing activity of the microbiota. Science. 2012;338:120–3. YI, MM, HI and TY critically discussed the study. MK wrote the manuscript. All 9. Putze J, Hennequin C, Nougayrede JP, Zhang W, Homburg S, Karch H, authors read and approved the final manuscript. Bringer MA, Fayolle C, Carniel E, Rabsch W, Oelschlaeger TA, Oswald E, Forestier C, Hacker J, Dobrindt U. Genetic structure and distribution of the Authors’ information colibactin genomic island among members of the family Enterobacteriaceae. Not applicable. Infect Immun. 2009;77:4696–703. 10. Hayashi M. The micronucleus test -most widely used in vivo genotoxicity Funding test. Genes Environ. 2016;38:18. This study was supported by Grants-in-Aid for Scientific Research (Grant 11. Bossuet-Greif N, Dubois D, Petit C, Tronnet S, Martin P, Bonnet R, Oswald E, Number: 17 K08841) from the Japan Society for the Promotion of Science Nougayrede JP. Escherichia coli ClbS is a colibactin resistance protein. Mol (JSPS) to Y.Y. and the Development of Innovative Research on Cancer Thera- Microbiol. 2016;99:897–908. peutics from Japan Agency for Medical Research and Development (AMED) (K. Watanabe, 16ck0106243h0001), Innovative Areas from MEXT, Japan (K. Watanabe, 16H06449), the Takeda Science Foundation (K. Watanabe), the In- Publisher’sNote stitution of Fermentation at Osaka (K. Watanabe), the Princess Takamatsu Springer Nature remains neutral with regard to jurisdictional claims in Cancer Research Fund (K. Watanabe, 16–24825), and the Yakult Bio-Science published maps and institutional affiliations. Foundation (K. Watanabe). Availability of data and materials Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details Graduate School of Science and Radiation Research Center, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai-shi, Osaka 599-8570, Japan. Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan. Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan. Department of Tumor Pathology, Hamamatsu University School of Medicine, Shizuoka, Japan. School of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Tokyo, Japan. Division of Prevention, Center for Public Health Sciences, National Cancer Center, Tokyo, Japan.

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Published: Mar 11, 2020

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