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Title: Sequence variation and conservation in virulence-related genes and expression of adenylate cyclase toxin of Bordetella pertussis
Author: Packard, Erica Ruth
ISNI:       0000 0001 3462 6907
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2004
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The aetiological agent of pertussis, Bordetella pertussis, is still a major cause of disease today and occurs even in countries that have had vaccination programmes implemented since about the 1950s. Several countries have reported increasing levels of pertussis in recent years and one possible reason for this is that an antigenic shift has occurred in the B. pertussis population since the introduction of pertussis whole-cell vaccines. Changes have been described in sequence types of two virulence-related genes, encoding pertactin (prnA) and pertussis toxin SI subunit (ptxA), amongst B. pertussis from several countries and the United Kingdom (UK). B. pertussis population was expanded in this study by investigating other virulence-related genes. An already established multilocus sequence typing (MLST) scheme (based on a combination of alleles of ptxA, pertussis toxin S3 subunit (ptxC) and tracheal colonisation factor (tcfA) genes) was applied to the UK B. pertussis population. The same predominating tcfA allele was found in all time periods studied, however, there has been a shift in predominating sequence types in the UK population relating to ptxC and prnA types. The predominating alleles found in recent isolates were not seen in isolates from the pre vaccination era or from the era just after the introduction of whole-cell vaccine. New, multilocus virulence gene sequence typing (MVGST) schemes are proposed in this study. These include the prnA gene and another polymorphic gene encoding fimbrial antigen 2 and these schemes are compared to the MLST scheme. Increasing the number of gene targets increased the discriminatory power of the typing scheme. All schemes identified sequence types in recent B. pertussis isolates that were not found in the prevaccination era or the era just after the introduction of vaccination. However, no increased incidence of pertussis has been described for the UK. It is accepted that particular sequence types found within the UK B. pertussis population and other B. pertussis populations may not be identical, and that there may be unknown genes that are affecting pertussis levels within different countries. Other genes were investigated, namely those encoding adenylate cyclase toxin, virulence-activated gene 8, bordetella resistance to killing protein A, bordetella autotransporter protein C, outer membrane protein Q, but they showed insufficient variation to be useful for inclusion in the MVGST schemes. A novel conductimetry tool was used to develop a rapid procedure for detecting adenylate cyclase (AC) enzymic activity from adenylate cyclase toxin (CyaA) xvii preparations and the optimisation of this assay is described. The conductimetry assay was found to be useful for determining the phenotypic phase of Bordetella strains as CyaA is expressed only in the virulent (Bvg+) phase. However, the conductimetry assay was not sufficiently sensitive for use as a diagnostic tool. Two recent (1999) isolates of B. pertussis were found to be different from other isolates due to the presence of a 6 bp insertion between cyaA and its accessory gene cyaC. These isolates were screened for differences in AC activity by the conductimetry assay and for differences in cytotoxicity. No clear differences were found between these two isolates and a laboratory strain of B. pertussis laboratory strain, BP338 strain, which was considered to be a typical strain and representative of the B. pertussis population. Further studies should be carried out to determine if this 6 bp insertion has an effect on the phenotype of the strains and their virulence. Novel plasmid constructs were made containing the cyaA promoter region inserted upstream of promoterless reporter genes, namely the lux operon and gfp, and these were maintained in Escherichia coli. A plate mating method was used to transfer the lux construct from the donor E. coli strain to the recipient B. pertussis BPS 3 8 strain but the plasmid was not maintained in B. pertussis possibly due to the HindIII restriction/modification system known to be present in this species.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available