Transposon mutagenesis of virulence regulatory genes of Bordetella pertussis

Microbiology

Ward, Mandy J

University:University of Glasgow

关键词: Microbiology;   

Others  :  http://theses.gla.ac.uk/72855/1/10647135.pdf

来源: University of Glasgow

【 摘 要 】

A suitable Tn5 delivery vector for mutagenesis of the pathogen Bordetella pertussis was identified as the ColIb-based plasmid pLG221. Insertion mutants, created using this 'suicide' vector, were screened for the loss of virulence-associated properties, or virulence- regulatory functions. No mutants were identified as having lost the heat-labile toxin alone. Additionally, no mutants were Identified as being unable to modulate under C-mode growth conditions. However, a number of mutants were identified as being unable to bind the dye Congo red (a property associated with the avlrulent phenotype). Several of these strains were subsequently shown to exhibit a pleiotropically negative phenotype for the expression of virulence-associated factors. The events causing the avirulent phenotype in the Crb mutants were initially examined by identification of the Tn5 insertion sites. Two mutants were shown to have Tn5 insertions within a region with BamHI. Sail and EcoRI restriction fragments identical to the map of the bvg locus. However, no clustering of transposon Insertions, indicative of an alternate virulence regulatory locus, were noted between the other mutants. Complementation studies, introducing the cloned bvg locus into the Crb mutants, returned virulence-associated properties to all the mutants, suggesting that the avirulent phenotype could only be created by mutations in the bvg locus, Hybridisation of radiolabeledpRMB2 DNA (which contains the cloned bvg locus) to Southern blots of the Crb mutant's DNA identified deletions of this region in two mutants. Mutants not showing obvious genetic rearrangements in the bvg locus were considered to have been caused by frameshift mutations (as previously Identified in the Tohama strain), This idea was examined by using the frameshift mutagen ICR 191 to create Crb revertants. However, only a single mutant was shown to revert to virulent characteristics after such treatment. Therefore, the events causing the loss of virulence-associated characteristics in the mutants created in this study were defined in only five of the ten Isolates. A single mutant (strain 51) showing the Crb phenotype was shown to be haemolytic, while not producing other virulence-associated factors. This mutant was shown to be avirulent in an infant mouse model. Initial studies have also indicated that a vaccine made from Tn5 mutant 51 showed some protective activity against intranasal challenge in infant mice, although not against Intracerebral challenge.