Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins.This research was funded by the Spanish Ministerio de Economía y Competitividad [grant number BFU2017–82758-P] and by the Basque Government [grant number IT1264-19]. J.A. was recipient of a fellowship from the University of Basque Country (UPV/EHU). RA holds a contract funded by the Fundación Biofisika Bizkaia

Cholesterol stimulates the lytic activity of Adenylate Cyclase Toxin on lipid membranes by promoting toxin oligomerization and formation of pores with a greater effective size

Several toxins acting on animal cells present different, but specific, interactions with cholesterol. Bordetella pertussis infects the human respiratory tract and causes whooping cough, a highly contagious and resurgent disease. Its virulence factor adenylate cyclase toxin (ACT) plays an important role in the course of infection. ACT is a pore-forming cytolysin belonging to the Repeats in ToXin (RTX) family of leukotoxins/hemolysins and is capable of permeabilizing several cell types and lipid vesicles. Previously, we observed that in the presence of cholesterol ACT induces greater liposome permeabilization. Similarly, recent reports also implicate cholesterol in the cytotoxicity of an increasing number of pore-forming RTX toxins. However, the mechanistic details by which this sterol promotes the lytic activity of ACT or of these other RTX toxins remain largely unexplored and poorly understood. Here, we have applied a combination of biophysical techniques to dissect the role of cholesterol in pore formation by ACT. Our results indicate that cholesterol enhances the lytic potency of ACT by promoting toxin oligomerization, a step which is indispensable for ACT to accomplish membrane permeabilization and cell lysis. Since our experimental design eliminates the possibility that this cholesterol effect derives from toxin accumulation due to lateral lipid phase segregation, we hypothesize that cholesterol facilitates lytic pore formation, by favoring a toxin conformation more prone to protein-protein interactions and oligomerization. Our data shed light on the complex relationship between lipid membranes and protein toxins acting on these membranes. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins.Rocío Alonso is gratefully acknowledged for excellent technical assistance. This study was supported by grants from the Spanish Ministerio de Economia y Competitividad BFU2017-82758-P (H.O.) and of Basque Government (Grupos Consolidados IT1264-19). D.G.B was recipients of a fellowship from the Bizkaia Biophysics Foundation, and JA was recipient of a fellowship from the Basque Government

Kukutxeztula: "gainditutako" gaitz baten itzulera

Arnasbideetako infekzioek heriotza tasa altuak eragiten dituzte munduan, COVID-19 birusa izanik azken urteetako adibiderik argiena. Hala ere, ez da libre dabilen patogeno bakarra eta berrikuspen honetan Bordetella pertussis bakterio patogenoak eragindako kukutxeztulaz arituko gara. Kukutxeztula arnasbideen infekzio larri eta kutsakorra da, bertako jariakinekin edota listu tantekin kontaktua izatean transmititzen dena. Helduetan arrisku berezirik ez duten sintomak eragiten dituen arren (etengabeko eztul gertakariak, arnasteko zailtasuna edota sukarra, besteak beste), heriotza eragin dezake urte batetik beherako haurretan batez ere. Kukutxeztula fase goiztiar batean gainditu ez duten gaixoek bigarren mailako pneumonia bezalako konplikazioak garatu ditzakete, horixe izaten delarik kukutxeztulaz hiltzen diren gehienen arrazoi nagusia. Lehen txertoa garatu zenetik kontrolpean zegoela uste izan den arren, kalte handiak eragin ditu garapen bidean dauden herrialdeetan batez ere. Herrialde garatuetan ere badu bere inpaktua, eta 2011tik gaur arte Ipar Amerikan eta mendebaldeko Europan epidemia egoera zabaldu da. Berrikuspen honetan kukutxeztulari buruzko informazioaren eguneratze bat aurkezten da, B. pertussisek erabiltzen dituen hainbat mekanismo infekziosotan arreta jarriz. Mekanismo hauen oinarri molekularra ulertzeak lagundu dezake zeluletan gertatzen diren beste hainbat prozesuren ulerkeran, gaixotasunaren aurkako sendagai berrien garapenean edota biologia molekularreko tresna berrien garapenean.; Respiratory infections cause high mortality rates throughout the world, being the COVID-19 virus the clearest example in recent years. However, there are other several pathogens not fully controlled and in this review we will focus on whooping cough, the sickness caused by the pathogenic bacterium Bordetella pertussis.Whooping cough is a severe and contagious respiratory infection that is transmitted by coming into contact with local secretions or with saliva drops. Despite the symptoms are not of special risks for adults (continuous episodes of cough, difficulty breathing or fever), it can cause death especially in children younger than one year of age. Patients who have not overcome cough at an early stage may develop complications such as secondary pneumonia, which is the main cause of most who die with whooping cough. Although since the development of the first vaccine it has been considered to be under control, whooping cough has caused significant damage especially in developing countries. Surprisingly, it has also an impact on developed countries and since 2011 the epidemic has spread in North America and Western Europe. This review presents an update on whooping cough, focusing on the different infective mechanisms used by B.pertussis. Understanding of the molecular basis of these mechanisms may contribute to the understanding of other cellular processes, the development of new anti-disease drugs or the development of new molecular biology instruments

Membrane Permeabilization by Pore-Forming RTX Toxins: What Kind of Lesions Do These Toxins Form?

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. The pore-forming cytolysins of the RTX (repeats in toxin) family belong to a steadily increasing family of proteins characterized by having in their primary sequences a number of glycine- and aspartate-rich nonapeptide repeats. They are secreted by a variety of Gram-negative bacteria and form ion-permeable pores in several cell types, such as immune cells, epithelial cells, or erythrocytes. Pore-formation by RTX-toxins leads to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. The pores formed in lipid bilayers by the RTX-toxins share some common properties such as cation selectivity and voltage-dependence. Hemolytic and cytolytic RTX-toxins are important virulence factors in the pathogenesis of the producing bacteria. And hence, understanding the function of these proteins at the molecular level is critical to elucidating their role in disease processes. In this review we summarize the current state of knowledge on pore-formation by RTX toxins, and include recent results from our own laboratory regarding the pore-forming activity of adenylate cyclase toxin (ACT or CyaA), a large protein toxin secreted by Bordetella pertussis, the bacterium causative of whooping cough.This study was supported by grant from the Spanish Ministerio de Economia y Competitividad BFU2017-82758-P. D.G.-B. and A.B.G.A. were recipients of a fellowship from the Bizkaia Biophysics Foundation