Inhibition of Pertussis Toxin by Human α-Defensins-1 and -5: Differential Mechanisms of Action.

Whooping cough is a severe childhood disease, caused by the bacterium Bordetella pertussis, which releases pertussis toxin (PT) as a major virulence factor. Previously, we identified the human antimicrobial peptides α-defensin-1 and -5 as inhibitors of PT and demonstrated their capacity to inhibit the activity of the PT enzyme subunit PTS1. Here, the underlying mechanism of toxin inhibition was investigated in more detail, which is essential for developing the therapeutic potential of these peptides. Flow cytometry and immunocytochemistry revealed that α-defensin-5 strongly reduced PT binding to, and uptake into cells, whereas α-defensin-1 caused only a mild reduction. Conversely, α-defensin-1, but not α-defensin-5 was taken up into different cell lines and interacted with PTS1 inside cells, based on proximity ligation assay. In-silico modeling revealed specific interaction interfaces for α-defensin-1 with PTS1 and vice versa, unlike α-defensin-5. Dot blot experiments showed that α-defensin-1 binds to PTS1 and even stronger to its substrate protein Gαi in vitro. NADase activity of PTS1 in vitro was not inhibited by α-defensin-1 in the absence of Gαi. Taken together, these results suggest that α-defensin-1 inhibits PT mainly by inhibiting enzyme activity of PTS1, whereas α-defensin-5 mainly inhibits cellular uptake of PT. These findings will pave the way for optimization of α-defensins as novel therapeutics against whooping cough.

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin.

Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not ß-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo.

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.

Combined Pharmacological Inhibition of Cyclophilins, FK506-Binding Proteins, Hsp90, and Hsp70 Protects Cells From Clostridium botulinum C2 Toxin.

The Clostridium botulinum C2 toxin is an exotoxin causing severe enterotoxic symptoms. The C2 toxin consists of the binding/translocation component C2II, and the enzymatic active component C2I. After proteolytic activation, C2IIa forms heptamers that bind C2I. The C2IIa/C2I complex is taken up into mammalian target cells via receptor-mediated endocytosis. Acidification of endosomes leads to conformational changes in both components. C2IIa heptamers form a pore into the endosomal membrane, and C2I becomes unfolded and translocates through the narrow C2IIa pores into the cytosol of the cell. Here, C2I covalently transfers an ADP-ribose moiety from its co-substrate NAD+ onto G-actin, which leads to depolymerization of F-actin resulting in rounding up of adherent cells. Translocation of C2I into the cytosol depends on the activity of the chaperones Hsp90 and Hsp70 and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506-binding protein (FKBP) families. Here, we demonstrated that C2I is detected in close proximity with Hsp90, Cyp40, and FKBP51 in cells, indicating their interaction. This interaction was dependent on the concentration of C2 toxin and detected in mammalian Vero and human HeLa cells. Moreover, the present study reveals that combination of radicicol, VER-155008, cyclosporine A, and FK506, which are specific pharmacological inhibitors of Hsp90, Hsp70, Cyps, and FKBPs, respectively, resulted in a stronger inhibition of intoxication of cells with C2 toxin compared to application of the single inhibitors. Thus, the combination of inhibitors showed enhanced protection of cells against the cytotoxic effects of C2 toxin. Cell viability was not significantly impaired by application of the inhibitor combination. Moreover, we confirmed that the combination of radicicol, VER-155008, CsA, and FK506 in particular inhibit the membrane translocation step of C2I into the cytosol whereas receptor binding and enzyme activity of the toxin were not affected. Our findings further characterize the mode of action of Hsp90, Hsp70, Cyps, and FKBPs during membrane translocation of bacterial toxins and furthermore supply starting points for developing of novel therapeutic strategies against diseases caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs.

P2X4 receptor re-sensitization depends on a protonation/deprotonation cycle mediated by receptor internalization and recycling.

KEY POINTS: Re-sensitization of P2X4 receptors depends on a protonation/de-protonation cycle Protonation and de-protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartments Protonation and de-protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptors Re-sensitization is blocked in the presence of the receptor agonist ATP ABSTRACT: P2X4 receptors are members of the P2X receptor family of cation-permeable, ligand-gated ion channels that open in response to the binding of extracellular ATP. P2X4 receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re-sensitization is required to return the receptor into its resting state. Desensitization and re-sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re-sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X4 receptor re-sensitization. Re-sensitization depends on a protonation/de-protonation cycle of critical histidine residues within the extracellular loop of P2X4 receptors that is mediated by receptor internalization and recycling. Interestingly, re-sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X4 receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X4 receptors regulates the cellular responsiveness in the sustained presence of ATP.

Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c.

During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons.