{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T18:29:00Z","timestamp":1791829740915,"version":"4.3.4"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T00:00:00Z","timestamp":1755475200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p><jats:italic>Legionella pneumophila<\/jats:italic>, an intracellular pathogen responsible for the pneumonia-like Legionnaires\u2019 disease in humans, inhabits aquatic environments, including man-made water systems such as water fountains, foot spas, and tap water, and exists as part of biofilms or as a protozoan parasite. As a bacterivore, <jats:italic>Tetrahymena thermophila<\/jats:italic> provides a favorable environment for <jats:italic>Legionella<\/jats:italic> to establish a replicative niche (<jats:italic>Legionella<\/jats:italic>-containing vacuole; LCV) under environmental stress. Conversely, the <jats:italic>L. pneumophila<\/jats:italic> Ofk308 strain, isolated from an Ashiyu foot spa, has been found to be cytotoxic to the ciliate <jats:italic>T. thermophila<\/jats:italic> CU427. This study aimed to identify the cytotoxicity-related genes of <jats:italic>Legionella<\/jats:italic> and elucidate their mechanisms specific to the <jats:italic>Tetrahymena<\/jats:italic> host.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>A comparative analysis using RNA-sequencing was conducted with two <jats:italic>Legionella<\/jats:italic> strains, Philadelphia-1 and Ofk308, to select several candidate genes. Deletion mutants of Ofk308 were constructed by homologous recombination. Eight out of ten candidate gene deletion mutants were successfully generated. These mutants were analyzed for cytotoxicity against <jats:italic>T. thermophila<\/jats:italic> and intracellular bacterial growth at 2 h, 24 h, and 48 h postinfection.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results and Discussions<\/jats:title><jats:p>Among the deletion mutants, <jats:italic>\u0394<\/jats:italic> vicinal oxygen chelate (VOC) and msrB\/A exhibited reduced cytotoxicity. Furthermore, LCVs formed in <jats:italic>T. thermophila<\/jats:italic> infected with DVOC and msrB\/A were smaller in size compared to those formed by the parental strain Ofk308, suggesting a role in both cytotoxicity and intracellular growth. Multiple factors contribute to the cytotoxicity exhibited by the Ofk308 strain in protozoan host cells, and gene expression analysis may reveal additional relevant factors.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2025.1643556","type":"journal-article","created":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T11:31:48Z","timestamp":1755516708000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Analysis of the genes and mechanisms responsible for the cytotoxicity of the Legionella Ofk308 strain in the Tetrahymena host"],"prefix":"10.3389","volume":"16","author":[{"given":"Istiana Nur","family":"Vidayanti","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenta","family":"Watanabe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ema","family":"Fujino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Shimizu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masahisa","family":"Watarai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,8,18]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"333","DOI":"10.1007\/s00284-013-0370-1","article-title":"The effect of growth temperature on the pathogenicity of Campylobacter","volume":"67","author":"Aroori","year":"2013","journal-title":"Curr. Microbiol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"6679","DOI":"10.1128\/JB.182.23.6679-6686.2000","article-title":"Catalase-peroxidases of Legionella pneumophila: cloning of the katA gene and Studies of KatA function","volume":"182","author":"Bandyopadhyay","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"2187","DOI":"10.1128\/AEM.01214-07","article-title":"Packaging of live Legionella pneumophila into pellets expelled by Tetrahymena spp. does not require bacterial replication and depends on a dot\/Icm-mediated survival mechanism","volume":"74","author":"Berk","year":"2008","journal-title":"Appl. Environ. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"477","DOI":"10.3389\/fcimb.2017.00477","article-title":"From many hosts, one accidental pathogen: the diverse protozoan hosts of Legionella","volume":"7","author":"Boamah","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref5","first-page":"237","article-title":"Chapter 8 - Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage","volume-title":"Methods in cell biology","author":"Cassidy-Hanley","year":"2012"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"1122","DOI":"10.1080\/21505594.2021.1903199","article-title":"Pathogenicity and virulence of Legionella: intracellular replication and host response","volume":"12","author":"Chauhan","year":"2021","journal-title":"Virulence"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"863","DOI":"10.1016\/j.bbrc.2015.12.077","article-title":"Identification of activators of methionine sulfoxide reductases a and B","volume":"469","author":"Cudic","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"3931","DOI":"10.1093\/nar\/gkt1375","article-title":"A tRNA splicing operon: Archease endows RtcB with dual GTP\/ATP cofactor specificity and accelerates RNA ligation","volume":"42","author":"Desai","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.jinf.2007.09.010","article-title":"Legionella spp. and Legionnaires disease","volume":"56","author":"Diederen","year":"2008","journal-title":"J. Infect."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"467","DOI":"10.1128\/aem.47.3.467-471.1984","article-title":"Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis","volume":"47","author":"Fields","year":"1984","journal-title":"Appl. Environ. Microbiol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"208","DOI":"10.3389\/fmicb.2011.00208","article-title":"Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host? Pathogen interactions","volume":"2","author":"Gomez-Valero","year":"2011","journal-title":"Front. Microbiol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1259","DOI":"10.1016\/j.jinorgbio.2011.06.006","article-title":"Structural and mechanistic comparisons of the metal-binding members of the vicinal oxygen chelate (VOC) superfamily","volume":"105","author":"He","year":"2011","journal-title":"J. Inorg. Biochem."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"379","DOI":"10.3390\/microorganisms9020379","article-title":"Development of a fluorescent tool for studying Legionella bozemanae intracellular infection","volume":"9","author":"Head","year":"2021","journal-title":"Microorganisms"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"e02370","DOI":"10.1128\/aem.02370-21","article-title":"The Legionella Lqs-LvbR regulatory network controls temperature-dependent growth onset and bacterial cell density","volume":"88","author":"Hochstrasser","year":"2022","journal-title":"Appl. Environ. Microbiol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"5247","DOI":"10.1128\/AEM.00421-12","article-title":"Ciliates expel environmental legionella-laden pellets to stockpile food","volume":"78","author":"Hojo","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"e01424","DOI":"10.1128\/AAC.01424-17","article-title":"Inhibition of Fosfomycin resistance protein FosA by Phosphonoformate (Foscarnet) in multidrug-resistant gram-negative pathogens","volume":"61","author":"Ito","year":"2017","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"1078","DOI":"10.1099\/mic.0.001078","article-title":"Wild ciliates differ in susceptibility to Legionella pneumophila JR32","volume":"167","author":"Kawashiro","year":"2021","journal-title":"Microbiology"},{"key":"ref18","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1186\/s13071-023-05824-y","article-title":"Identifying the function of genes involved in excreted vesicle formation in Acanthamoeba castellanii containing Legionella pneumophila","volume":"16","author":"Kim","year":"2023","journal-title":"Parasit. Vectors"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"129","DOI":"10.1016\/j.micinf.2003.11.004","article-title":"Cell biology of the intracellular infection by Legionella pneumophila","volume":"6","author":"Molmeret","year":"2004","journal-title":"Microbes Infect."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"141","DOI":"10.3390\/microorganisms10010141","article-title":"Knowledge to predict pathogens: Legionella pneumophila lifecycle systematic review part II growth within and egress from a host cell","volume":"10","author":"Mraz","year":"2022","journal-title":"Microorganisms"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"800","DOI":"10.3389\/fmicb.2018.00800","article-title":"Crucial role of Legionella pneumophila TolC in the inhibition of cellular trafficking in the Protistan host Paramecium tetraurelia","volume":"9","author":"Nishida","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"30","DOI":"10.1016\/j.plasmid.2017.02.004","article-title":"Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector","volume":"90","author":"Nishida","year":"2017","journal-title":"Plasmid"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1038\/s41396-020-00774-0","article-title":"Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms","volume":"15","author":"Personnic","year":"2021","journal-title":"ISME J."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1128\/MMBR.67.1.66-85.2003","article-title":"Two families of Mechanosensitive Channel proteins","volume":"67","author":"Pivetti","year":"2003","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"307","DOI":"10.4161\/viru.24290","article-title":"Cellular microbiology and molecular ecology of Legionella \u2013amoeba interaction","volume":"4","author":"Richards","year":"2013","journal-title":"Virulence"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"fnw260","DOI":"10.1093\/femsle\/fnw260","article-title":"Contribution of methionine sulfoxide reductase B (MsrB) to Francisella tularensis infection in mice","volume":"364","author":"Saha","year":"2017","journal-title":"FEMS Microbiol. Lett."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"61","DOI":"10.3389\/fcimb.2018.00061","article-title":"Acanthamoeba and Dictyostelium as cellular models for Legionella infection","volume":"8","author":"Swart","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"514395","DOI":"10.1155\/2013\/514395","article-title":"Characterization of Legionella pneumophila isolated from environmental water and ashiyu foot spa","volume":"2013","author":"Tachibana","year":"2013","journal-title":"Biomed. Res. Int."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"417","DOI":"10.1038\/nature17991","article-title":"Structural basis for amino acid export by DMT superfamily transporter YddG","volume":"534","author":"Tsuchiya","year":"2016","journal-title":"Nature"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"24322","DOI":"10.1038\/srep24322","article-title":"Ciliate Paramecium is a natural reservoir of Legionella pneumophila","volume":"6","author":"Watanabe","year":"2016","journal-title":"Sci. Rep."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"e00474","DOI":"10.1128\/genomeA.00474-15","article-title":"Draft genome sequences of five Legionella pneumophila strains isolated from environmental water samples","volume":"3","author":"Watanabe","year":"2015","journal-title":"Genome Announc."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1093\/femsec\/fiy162","article-title":"Identification of novel Legionella genes required for endosymbiosis in Paramecium based on comparative genome analysis with Holospora spp","volume":"94","author":"Watanabe","year":"2018","journal-title":"FEMS Microbiol. Ecol."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"74","DOI":"10.3390\/microorganisms11010074","article-title":"Concept about the virulence factor of Legionella","volume":"11","author":"Yang","year":"2022","journal-title":"Microorganisms"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"1415157","DOI":"10.3389\/fpubh.2024.1415157","article-title":"A review of legionella transmission risk in built environments: sources, regulations, sampling, and detection","volume":"12","author":"Yao","year":"2024","journal-title":"Front. Public Health"},{"key":"ref35","doi-asserted-by":"publisher","first-page":"3889","DOI":"10.1128\/IAI.00165-10","article-title":"Role of methionine sulfoxide reductases A and B of Enterococcus faecalis in oxidative stress and virulence","volume":"78","author":"Zhao","year":"2010","journal-title":"Infect. Immun."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1643556\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T11:31:48Z","timestamp":1755516708000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1643556\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,18]]},"references-count":35,"alternative-id":["10.3389\/fmicb.2025.1643556"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1643556","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,18]]},"article-number":"1643556"}}