The reduced IFN production seen with

The reduced IFNγ production seen with BPAL10-primed splenocytes could potentially be attributed to the impaired intracellular survival of B. pertussis, thereby leading to a weakened induction of cell-mediated immunity. Earlier studies have suggested an antimicrobial role of IFNγ in mediating the elimination of intracellular B. pertussis bacteria within monocytes/macrophages [51,52]. In addition, IFNγ-producing Th1 cells have been shown to play a key role in protection against pertussis [53,54]. However, our data showed that the reduced level of IFNγ produced by BPAL10-primed splenocytes could not be attributed to the CD4+ T cell population. This finding is consistent with an earlier study where most of the IFNγ was found to be produced by CD4-/CD8- T cells [55]. In this study, the authors showed that B. pertussis infection induces high frequencies of IFNγ-producing NKT cells and that these cells play an important role in protection, especially against disseminated B. pertussis infection [55]. Therefore, it is tempting to speculate that the reduced protective efficacy observed with the CyaA-deficient BPZE1 strain may be attributed to sub-optimal priming of NKT cells.
Acknowledgments This work was supported by the Singapore National Medical Research Council (Exploratory Developmental Grant # EDG/0073/2009 allocated to SA).
Introduction The CyaA secreted by Bordetella pertussis influences bacterial pathogenesis in respiratory infections. CyaA is a 188kDa protein composed of the C-terminal domain, which is necessary for interaction with host cell receptors, and the N-terminal domain, that is an adenylate cyclase toxin (CyaA-ACD) that is inserted through the membrane into the host cell cytosol [1]. Upon interaction with CaM, CyaA-ACD is activated and intracellular cyclic adenosine monophosphate concentration increases to pathophysiological levels, which is known to impair the host immune response [1], [2], [3], [4]. CaM has four metal kinetin synthesis and is known to engage numerous effector proteins in response to calcium (Ca2+) stimulation. Isolated N-CaM or C-CaM triggers CyaA-ACD activity, but association with intact CaM promotes a 400 fold increase in binding affinity and activation by unknown mechanisms emphasizing the importance of intermolecular associations between N-CaM and CyaA-ACD [5]. Structural and functional comparisons between CyaA-ACD and another CaM-dependent adenylate cyclase toxin from Bacillus anthracis (EF), reveal that distinct molecular mechanisms have evolved for the activation of these two enzymes [6], [7], [8], [9], [10]. In direct contrast to CyaA-ACD, EF requires both high levels of Ca2+ and interaction with intact CaM for full activation [11]. EF forms an extended complex such that the N-CaM makes extensive contacts with the helical domain of EF that stabilize the catalytic loop. CyaA-ACD lacks the helical domain, but association between the C-tail of CyaA-ACD and C-CaM stabilizes its catalytic loop. While crystal structures of C-CaM bound to CyaA-ACD reveal the mode by which it likely controls CyaA-ACD function, no structural information is available detailing the role of N-CaM in toxin activation. CaM mutants defective in N-terminal opening stimulate CyaA-ACD, suggesting domain opening is not necessary for maximal activation [9]. Biochemical and molecular modeling studies showed that interaction between N-CaM and the β-hairpin (residues 259–273) of CyaA-ACD likely promotes full activation via conformational modulation of the catalytic pocket [9], but the mechanism remains to be determined. Here, we investigate the molecular interactions occurring between intact CaM and CyaA-ACD using NMR and DLS experiments. Our findings are interpreted within the context of the available structural data and the role of N-CaM interaction in CyaA-ACD activation is discussed.
Materials and methods
Results and discussion
Acknowledgments
Introduction MLTC-1 cells (mouse Leydig Tumor Cells) have retained sensitivity to the gonadotropins with LH activity, i.e. pituitary Luteinizing Hormones (LHs) and chorionic gonadotropins (CGs) (Rebois, 1982). These hormones bind to the common mouse LH receptor (LHR) that binds both LHs and CGs from various species (Klett et al., 2016), and stimulate intracellular cyclic AMP accumulation as well as progesterone secretion.