Jakub Novak

The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5′-untranslated region (5′-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis. These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways. IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis. Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the α_Mβ₂ integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin–hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (α_Mβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3′,5′-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

A major outcome of the canonical Wnt/β-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with β-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/β-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.