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

Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin

Adenylate cyclase toxin (ACT, CyaA) is one of the important virulence factors secreted by the whooping cough bacterium Bordetella pertussis, and it is essential for the colonization of the human respiratory tract by this bacterium. Cytotoxicity by ACT results from the synergy between toxin's two main activities, production of supraphysiological cAMP levels by its N-terminal adenylate cyclase domain (AC domain), and cell membrane permeabilization, induced by its C-terminal pore-forming domain (hemolysin domain), which debilitate the host defenses. In a previous study we discovered that purified ACT is endowed with intrinsic phospholipase A1 (PLA) activity and that Ser in position 606 of the ACT polypeptide is a catalytic site for such hydrolytic activity, as part of G-X-S-X-G catalytic motif. Recently these findings and our conclusions have been directly questioned by other authors who claim that ACT-PLA activity does not exist. Here we provide new data on ACT phospholipase A1 characteristics. Based on our results we reaffirm our previous conclusions that ACT is endowed with PLA activity; that our purified ACT preparations are devoid of any impurity with phospholipase A activity; that ACT-S606A is a PLA-inactive mutant and thus, that Ser606 is a catalytic site for the toxin hydrolytic activity on phospholipids, and that ACT-PLA activity is involved in AC translocation.This study was supported by grants from the Basque Government (Grupos Consolidados IT849) and grant from the Spanish Ministerio de Economia y Competitividad (BFU2017-82758-P (AEI/FEDER, UE) to H.O

Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins

Permeabilization of the plasma membrane represents an important threat for any cell, since it compromises its viability by disrupting cell homeostasis. Numerous pathogenic bacteria produce pore-forming toxins that break plasma membrane integrity and cause cell death by colloid-osmotic lysis. Eukaryotic cells, in turn, have developed different ways to cope with the effects of such membrane piercing. Here, we provide a short overview of the general mechanisms currently proposed for plasma membrane repair, focusing more specifically on the cellular responses to membrane permeabilization by pore-forming toxins and presenting new data on the effects and cellular responses to the permeabilization by an RTX (repeats in toxin) toxin, the adenylate cyclase toxin-hemolysin secreted by the whooping cough bacterium Bordetella pertussis, which we have studied in the laboratory.This study was supported by grants from the Basque Government (Grupos Consolidados IT849-13) and grant from the Spanish Ministerio de Economia y Competitividad (BFU2017-82758-PAEI/FEDER, UE)

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered.This study was supported by grants from the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program KK-2015/0000089). A.E. was recipient of a fellowship from the University of the Basque Country (UPV/EH) and D.G.-B. was recipient of a fellowship from the Bizkaia Biophysics Foundation

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

Validation of LDLr Activity as a Tool to Improve Genetic Diagnosis of Familial Hypercholesterolemia: A Retrospective on Functional Characterization of LDLr Variants

Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by high blood-cholesterol levels mostly caused by mutations in the low-density lipoprotein receptor (LDLr). With a prevalence as high as 1/200 in some populations, genetic screening for pathogenic LDLr mutations is a cost-effective approach in families classified as definite' or probable' FH and can help to early diagnosis. However, with over 2000 LDLr variants identified, distinguishing pathogenic mutations from benign mutations is a long-standing challenge in the field. In 1998, the World Health Organization (WHO) highlighted the importance of improving the diagnosis and prognosis of FH patients thus, identifying LDLr pathogenic variants is a longstanding challenge to provide an accurate genetic diagnosis and personalized treatments. In recent years, accessible methodologies have been developed to assess LDLr activity in vitro, providing experimental reproducibility between laboratories all over the world that ensures rigorous analysis of all functional studies. In this review we present a broad spectrum of functionally characterized missense LDLr variants identified in patients with FH, which is mandatory for a definite diagnosis of FH.This work was supported by ELKARTEK 2016 and and the Basque Government (Grupos Consolidados IT849-13). A.B.-V. and S.J. were supported by a grant PIF (2014-2015) and (2018-2021), Gobierno Vasco respectively

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

Statin Treatment-Induced Development of Type 2 Diabetes: From Clinical Evidence to Mechanistic Insights

Statins are the gold-standard treatment for the prevention of primary and secondary cardiovascular disease, which is the leading cause of mortality worldwide. Despite the safety and relative tolerability of statins, observational studies, clinical trials and meta-analyses indicate an increased risk of developing new-onset type 2 diabetes mellitus (T2DM) after long-term statin treatment. It has been shown that statins can impair insulin sensitivity and secretion by pancreatic β-cells and increase insulin resistance in peripheral tissues. The mechanisms involved in these processes include, among others, impaired Ca2+ signaling in pancreatic β-cells, down-regulation of GLUT-4 in adipocytes and compromised insulin signaling. In addition, it has also been described that statins’ impact on epigenetics may also contribute to statin-induced T2DM via differential expression of microRNAs. This review focuses on the evidence and mechanisms by which statin therapy is associated with the development of T2DM. This review describes the multifactorial combination of effects that most likely contributes to the diabetogenic effects of statins. Clinically, these findings should encourage clinicians to consider diabetes monitoring in patients receiving statin therapy in order to ensure early diagnosis and appropriate management.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia

Pathophysiology of Type 2 Diabetes Mellitus

Type 2 Diabetes Mellitus (T2DM), one of the most common metabolic disorders, is caused by a combination of two primary factors: defective insulin secretion by pancreatic β-cells and the inability of insulin-sensitive tissues to respond appropriately to insulin. Because insulin release and activity are essential processes for glucose homeostasis, the molecular mechanisms involved in the synthesis and release of insulin, as well as in its detection are tightly regulated. Defects in any of the mechanisms involved in these processes can lead to a metabolic imbalance responsible for the development of the disease. This review analyzes the key aspects of T2DM, as well as the molecular mechanisms and pathways implicated in insulin metabolism leading to T2DM and insulin resistance. For that purpose, we summarize the data gathered up until now, focusing especially on insulin synthesis, insulin release, insulin sensing and on the downstream effects on individual insulin-sensitive organs. The review also covers the pathological conditions perpetuating T2DM such as nutritional factors, physical activity, gut dysbiosis and metabolic memory. Additionally, because T2DM is associated with accelerated atherosclerosis development, we review here some of the molecular mechanisms that link T2DM and insulin resistance (IR) as well as cardiovascular risk as one of the most important complications in T2DM.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especializaci.n de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J. and A.L-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia

Cholesterol Efflux Efficiency of Reconstituted HDL Is Affected by Nanoparticle Lipid Composition

Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo. Methods: the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal. Results: DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated. Conclusions: DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J.-B. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia