{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T22:48:18Z","timestamp":1791067698528,"version":"4.1.0"},"reference-count":24,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2009,8,1]],"date-time":"2009-08-01T00:00:00Z","timestamp":1249084800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Infect Immun"],"published-print":{"date-parts":[[2009,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            <jats:italic>Rickettsia prowazekii<\/jats:italic>\n            , the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The\n            <jats:italic>R. prowazekii pld<\/jats:italic>\n            gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a\n            <jats:italic>pld<\/jats:italic>\n            mutant of the virulent\n            <jats:italic>R. prowazekii<\/jats:italic>\n            strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted\n            <jats:italic>pld<\/jats:italic>\n            gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the\n            <jats:italic>pld<\/jats:italic>\n            mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.\n          <\/jats:p>","DOI":"10.1128\/iai.00395-09","type":"journal-article","created":{"date-parts":[[2009,6,9]],"date-time":"2009-06-09T01:28:56Z","timestamp":1244510936000},"page":"3244-3248","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":91,"title":["Directed Mutagenesis of the\n            <i>Rickettsia prowazekii pld<\/i>\n            Gene Encoding Phospholipase D"],"prefix":"10.1128","volume":"77","author":[{"given":"Lonnie O.","family":"Driskell","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, Alabama 36688"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue-jie","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lihong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609"},{"name":"Anhui Province Center for Disease Control and Prevention, Hefei, Anhui Province, People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vsevolod L.","family":"Popov","sequence":"additional","affiliation":[{"name":"Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David H.","family":"Walker","sequence":"additional","affiliation":[{"name":"Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas 77555-0609"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aimee M.","family":"Tucker","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, Alabama 36688"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David O.","family":"Wood","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, Alabama 36688"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","unstructured":"Current protocols in molecular biology 1997 vol. 1 2 and 3"},{"key":"e_1_3_2_3_2","first-page":"11","volume":"17","year":"1973","unstructured":"Balaeva, N. M., and V. N. Nikolskaya. 1973. Increased virulence of the E vaccine strain of Rickettsia prowazeki passaged in the lungs of white mice and guinea pigs. J. Hyg. Epidemiol. Microbiol. Immunol.17:11-20.","journal-title":"J. Hyg. Epidemiol. Microbiol. Immunol."},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.71.4.2095-2105.2005"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/255545a0"},{"key":"e_1_3_2_6_2","first-page":"376","volume":"5","year":"1987","unstructured":"Bullock, W. O., J. M. Fernandez, and J. M. Short. 1987. A high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. 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