Adenylate cyclase activity during modulation of Bordetella pertussis

Brownlie, Robert M (1983) Adenylate cyclase activity during modulation of Bordetella pertussis. PhD thesis, University of Glasgow.

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During the phenotypic and genotypic changes which occur during the processes respectively known as C modulation and phase degradation, several virulence-associated properties of Bordetella pertussis and adenylate cyclase (AC) are lost (Parton and Durham, 197B; Wardlaw and Parton, 1979). The main aim of the present investigation was to assess whether AC plays a causal role in these two distinct processes as this enzyme is known to play a key regulatory role in the Enterobacteriaceas. Growth of B. pertussis in Stainer and Scholte medium (SS-X) containing high levels of MgSO4, Na2 504, butyrate, Na caprylate, Na succinate or nicotinic acid resulted in C modulation as shown by marked reductions in AC activity, histamine-sensitizing activity (HSA) and the 28k and 3Dk cell-envelope polypeptides (X polypeptides). Although there was some variation between the susceptibility of strains to pro-C-mode salts, there was no instance of components being lost independently of each other. The level of cAMP in the supernate of C-mode cultures was less than 5x of that of X-inode cultures. Magnesium chloride, present at four times the molar concentration of MgSO4 required to induce C modulation, was ineffective at inducing loss of AC activity HSA, or the X polypeptides. During MgSO4-induced modulation, AC activity in three compartments (viz. culture supernate, cell-associated but extracytoplasmic, and cytoplasmic) was reduced to the same extent. Time-course studies on the rate of loss of AC activity, HSA, and the X-polypeptides during ngSO4-induced modulation indicated that these properties were lost simultaneously. Furthermore, losses could be accounted for by complete repression of the synthesis of the components when cells were introduced into C-medium. The low levels of cAMP in the supernate of C-mode cultures could not be accounted for by the ability of pro-C-mode salts to inhibit vitro AC activity, or by inhibition of cAMP excretion by C medium. Loss of AC activity during C modulation required growth and was not due to prolonged exposure to C medium, as chloramphenicol added to C medium prevented loss of AC activity. Loss of HSA and the X polypeptides also required growth or protein synthesis. C-mode cell lysate did not inhibit AC activity of X-mode cell lysate suggesting that loss of AC activity during C modulation is not due to production of an inactivating or inhibitory factor. Similarly, C-mode cell lysate did not destroy HSA present in X-mode cell lysate. Sodium fluoride caused marked inhibition of vitro AC activity, but, at the same concentration, had little effect on the synthesis of cAMP during growth. Growth in X medium containing B. pertussis AC activator (calmodulin) resulted in a four fold-increase in culture supernate cAMP levels. The critical concentration of MgSO4 required to induce loss of the X polypeptides was 10 - 11 mH. In one culture, containing 10 mH MgSD4 and AC activator, partial loss of the X polypeptides occurred yet cAMP levels in the supernate were twice that which normally occurred in X-mode cultures without activator. Respiration rates of amino acids by B. pertussis variants was investigated to determine whether cARP played a role in amino acid catabolism in the organism. The ability of washed suspensions of X- and C-mode and phase IV B. pertussis to respire L-glutamate, L-aspartate, L-proline, L-alanine and L-serine was demonstrated. While differences were found between the respiration rates of different amino acids, there were no significant differences between the ability of variants of B. pertussis to respire any particular amino acid. Phosphonomycin resistance has been used as a convenient method to isolate AC mutants of Escherichia coli. However, eight independently isolated phosphonomycin resistant mutants of B. pertussis possessed the same AC activity as the original strain. Exogenous cAMP and dibutyryl cAnP, in X and C media, had no effect on the production of AC, the X polypeptides or haeraagglutinin. Attempts were made to determine if a cyclic AnP-receptor protein (CRP) analogous to that in E. coli exists in B, pertussis. [3h] cAMP binding activity was demonstrated in several strains of B. pertussis and was about half that obtained for E. coli. Anti-E. coli CRP gave two precipitin lines with E. coli cell extract but none with B. pertussis cell extract. In conclusion, the results of this study suggest that AC does not play a causal role in modulation, and that the mechanism responsible for repressing synthesis of X-mode specific components (such as pertussigen) during modulation, also represses synthesis of AC.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Advisers: R Perton; J Coote
Keywords: Biochemistry
Date of Award: 1983
Depositing User: Enlighten Team
Unique ID: glathesis:1983-72659
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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