Effect of methyl-cyclodextrin on adenylate cyclase activity of Bordetella pertussis

Abstract

The activity of Bordetella pertussis extracytoplasmic adenylate cyclase (AC) decreased during decelerating growth phase in a Stainer-Scholte medium. Neither proteolytic activity nor virulence variation (phase variation; antigenic modulation) appears to be responsible for the observed activity fall. The addition of methyl-β-cyclo-dextrin enhances AC activity and prevents the inhibition of AC activity by fatty acids. Cyclodextrin could entrap inhibitors increasing in this way the AC activity. These results show that the inclusion of cyclodextrin in the culture medium increases the AC activity.

Release of Outer Membrane Vesicles from Bordetella pertussis

“…To examine the presence of vesicles in the supernatant, the Stainer Scholte medium supplemented with cyclodextrin was used. This medium, in which the levels of extracellular proteins [7,13,16] and LPS [14] were enhanced, would increase the possibility of finding OMVs. Culture samples from the decelerating growth phase were centrifuged at 10,000 g for 20 min at 4°C.…”

“…Therefore, research directed to gain a better understanding of the mechanisms of AC-Hly secretion into the medium might be useful to devise strategies to improve AC-Hly yields. We have previously reported that the addition of methyl-␤-cyclodextrin [7,15,16,32] to the culture medium increases the extracellular level of the AC-Hly [13]. Since the latter compound also enhances the release of lipopolysaccharide (LPS), one of the major outer membrane components [14,26], it seemed probable that the increased shedding of outer membrane blebs or vesicles (OMVs) might explain the increased levels of both cell-free AC-Hly and LPS in the presence of methyl-␤-cyclodextrin.…”

“…B. pertussis strains were grown in Stainer-Scholte liquid medium (SS) [36] as indicated previously [13]. In some experiments, 3 mg of methyl-␤-cyclodextrin per ml were added to the SS medium (SS ϩ Me␤CD) [13].…”

Use of cyclodextrin as an agent to induce excretion of Bordetella pertussis antigens

“…In a previous paper [7] we demonstrated that the addition of cyclodextrin (1 g 1-1 ) to SS medium produced 1.9-fold increase in the extracellular AC volumetric activity (pmol cAMP/min ml of supernate) and 1.7-fold increase in AC specific activity (pmol cAMP/min mg of protein). With SS and SS + Me/3CD media, the activity was maximal during the decelerating growth phase but, with the latter medium, the activity reduction at 48 h (stationary growth phase) was lower.…”

“…However, vaccine production based on this protein, using cultures of the virulent organism as the source of material, has been rather difficult because of the low yields of this protein secreted into culture fluids. The discovery that addition of 2,6-O-dimethyl-/3-cyclodextrin (Me/~CD) to the Stainer-Scholte (SS) [6] medium led to an enhanced level of AC, proved to be helpful [7]. Other authors have reported that cyclodextrin also increased the level of the other important soluble immunogens of B. pertussis such as pertussis toxin (PT) and filamentous hemagglutinin (FHA) [8][9][10][11].…”