Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K(d) values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold.

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αM β2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3',5'-cyclic monophosphate (cAMP). We show that the signalling of CyaA-produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP-1 and activates the pro-apoptotic BimEL-Bax cascade. Mitochondrial hyperpolarization occurred in human THP-1 macrophages within 10 min of exposure to low CyaA concentrations (e.g. 20 ng ml(-1) ) and was accompanied by accumulation of BimEL and association of the pro-apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP-1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP-1 but not of the SHP-2 phosphatase. Moreover, signalling of CyaA-produced cAMP inhibited the AKT/protein kinase B pro-survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP-1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro-apoptotic programme of macrophages and subverting the innate immunity of the host.

Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells.Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producingB. pertussisbacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression onin vitrodifferentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiatedin vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCEMacrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agentBordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion ofB. pertussisby suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

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Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis adenylate cyclase toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

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Purpose of review RTX toxin action often defines the outcome of bacterial infections. Here, we discuss the progress in understanding the impacts of RTX toxin activities on host immunity. Recent findings Bordetella pertussis CyaA activity paralyzes sentinel phagocytic cells by elevating cellular cAMP levels and blocks differentiation of infiltrating monocytes into bactericidal macrophages, promoting also de-differentiation of resident alveolar macrophages into monocyte-like cells. Vibrio cholerae multifunctional autoprocessing repeats-in-toxins (MARTX), through Rho inactivating and α/β-hydrolase (ABH) domain action blocks mitogen-activated protein kinase signaling in epithelial cells and dampens the inflammatory responses of intestinal epithelia by blocking immune cell recruitment. The action of actin crosslinking effector domain and Ras/Rap1-specific endopeptidase (RRSP) domains of MARTX compromises the phagocytic ability of macrophages. Aggregatibacter actinomycetemcomitans LtxA action triggers neutrophil elastase release into periodontal tissue, compromising the epithelial barrier and promoting bacterial spreads into deeper tissue. Summary Action of RTX toxins enables bacterial pathogens to cope with the fierce host immune defenses. RTX toxins often block phagocytosis and bactericidal reactive oxygen species and NO production. Some RTX toxins can reprogram the macrophages to less bactericidal cell types. Autophagy is hijacked for example by the activity of the V. cholerae ABH effector domain of the MARTX protein. Subversion of immune functions by RTX toxins thus promotes bacterial survival and proliferation in the host.

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