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

P. multocida is the causative agent of several economically significant vet erinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously sc reen multiple transposon mutants of a pathogen for their inability to survi ve in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identif ied transposon insertions into genes of Pasteurella multocida that attenuat e virulence in a septicemic mouse model. A bank of 96 transposons containin g strongly-hybridizing tags was used to create 19 pools of P. multocida tra nsposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single tra nsposon insertion mutations were identified from this group. The sequence o f the disrupted ORF for each attenuated mutant was determined by either clo ning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzy mes, virulence factors, regulatory components and unknown functions. This s tudy should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibi otic targets. (C) 2000 Academic Press.