Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis

• Published in: Microbial pathogenesis Vol. 29; no. 1; pp. 25 - 38
• Main Authors: Fuller, Troy E, Kennedy, Michael J, Lowery, David E
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.07.2000; Elsevier
• Subjects: Animals; Bacterial diseases; Bacteriology; Biological and medical sciences; Blotting, Southern; DNA Transposable Elements; Experimental bacterial diseases and models; Female; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Genetics; Infectious diseases; Lethal Dose 50; Medical sciences; Mice; Mice, Inbred Strains; Microbiology; Mutagenesis, Insertional; Pasteurella multocida; Pasteurella multocida - genetics; Pasteurella multocida - pathogenicity; Pasteurella multocida, signature-tagged mutagenesis (STM), virulence, attenuated; Plasmids; Polymerase Chain Reaction; Sepsis - microbiology; Virulence - genetics; Pasteurella multocida, signature-tagged mutagenesis (STM), virulence, attenuated; Animal model; Rodentia; Site directed mutagenesis; Identification; Vaccine; Transposable element; Pasteurellosis; Pasteurella multocida; Infection; Pasteurellaceae; Pathogenicity; Screening; Vertebrata; Mammalia; Gene; Mouse; Insertion mutation; Bacteriosis; Bacteria; Attenuated strain; Signature tagged mutagenesis
• ISSN: 0882-4010; 1096-1208
• DOI: 10.1006/mpat.2000.0365

P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70–90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in thePasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets.