Bacterial protein toxins and lipids: role in toxin targeting and activity

All bacterial toxins, which globally are hydrophilic proteins, interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Intracellular active toxins follow various trafficking pathways, the sorting of which is greatly dependent on the nature of the receptor, notably lipidic receptor or receptor embedded into a distinct environment such as lipid microdomains. Numerous other toxins act locally on cell membrane. Indeed, phospholipase activity is a common mechanism shared by several membrane-damaging toxins. In addition, many toxins active intracellularly or on cell membrane modulate host cell phospholipid pathways. Unusually, a few bacterial toxins require a lipid post-translational modification to be active. Thereby, lipids are obligate partners of bacterial toxins.     

Pore-forming toxins and cellular non-immune defenses (CNIDs)

Pore-forming toxins (PFTs) are the most common class of bacterial protein toxin and are important for bacterial pathogenesis. Recent studies have shown that the previous model stating that epithelial cells lyse in response to these toxins and have no defenses against these pores is oversimplified. Rather, it appears that cells have sophisticated mechanisms and signal-transduction pathways with which to respond to such an attack. There is a growing body of knowledge about how cells respond to and protect themselves against PFTs; this protection against PFTs is likely to be important in host survival to attack by bacterial pathogens, but does not neatly fit into current concepts of adaptive or innate immunity. Therefore, it is proposed that the terminology cellular non-immune defenses (CNIDs) be used to describe defenses that are employed by non-immune cells to protect against bacterial attack.     

Sensing of pathogenic bacteria based on their interaction with supported bilayer membranes studied by impedance spectroscopy and surface plasmon resonance

Pathogenic bacteria secrete various virulence factors, including toxins, lipases and proteases that allow them to infect, breakdown and colonize host tissue. Among various modes of action that the pathogenic bacteria use to damage the host, pore formation (by pore forming toxins (PFTs)) and lipid hydrolysis (by phospholipases) modes are common in damaging the eukaryotic cell membrane. PFTs in their monomeric form are extracellular diffusible and able to form hydrophilic pores in cell membrane while phospholipases cleaves and hydrolyzes the ester bonds of most phospholipids in cell membrane. Both modes of action cause uncontrolled permeation of ions and molecules across cell membrane, leading to cell death by apoptosis or necrosis. In this work, the toxins secreted by two clinically important human pathogens, methicillin susceptible Staphylococcus aureus (MSSA476) and Pseudomonas aeruginosa (PAO1) were studied via their interaction with a planar tethered bilayer lipid membrane (pTBLM) using surface plasmon resonance spectroscopy (SPR) and electrochemical impedance spectroscopy (EIS). Detection and discrimination is based on lipid-loss (lipid hydrolysis by phospholipases) or non lipid-loss (pore formation by PFTs) from pTBLM upon interaction with supernatant of pathogenic bacteria. Using EIS and SPR, it is demonstrated that major toxins of S. auerus are PFTs while most of toxin associated with P. aeruginosa are more lipid damaging lipolytic enzymes. Such a format might have future utility as a simple assay for measuring the presence membrane lytic virulence factors in clinical samples.     

The MACPF/CDC family of pore-forming toxins

Pore-forming toxins (PFTs) are commonly associated with bacterial pathogenesis. In eukaryotes, however, PFTs operate in the immune system or are deployed for attacking prey (e.g. venoms). This review focuses upon two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF). CDCs are produced by Gram-positive bacteria and lyse or permeabilize host cells or intracellular organelles during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles and function in immunity, invasion and development. The structure and molecular mechanism of several CDCs are relatively well characterized. Pore formation involves oligomerization and assembly of soluble monomers into a ring-shaped pre-pore which undergoes conformational change to insert into membranes, forming a large amphipathic transmembrane β-barrel. In contrast, the structure and mechanism of MACPF proteins has remained obscure. Recent crystallographic studies now reveal that although MACPF and CDCs are extremely divergent at the sequence level, they share a common fold. Together with biochemical studies, these structural data suggest that lytic MACPF proteins use a CDC-like mechanism of membrane disruption, and will help understand the roles these proteins play in immunity and development.     

Pore-forming protein toxins: from structure to function

Pore-forming protein toxins (PFTs) are one of Nature's most potent biological weapons. An essential feature of their toxicity is the remarkable property that PFTs can exist either in a stable water-soluble state or as an integral membrane pore. In order to convert from the water-soluble to the membrane state, the toxin must undergo large conformational changes. There are now more than a dozen PFTs for which crystal structures have been determined and the nature of the conformational changes they must undergo is beginning to be understood. Although they differ markedly in their primary, secondary, tertiary and quaternary structures, nearly all can be classified into one of two families based on the types of pores they are thought to form: alpha-PFTs or beta-PFTs. Recent work suggests a number of common features in the mechanism of membrane insertion may exist for each class.     

细菌成孔毒素研究进展

成孔毒素是多种致病性细菌分泌的一种重要毒力因子,通过在真核细胞膜上形成孔道结构,引起细胞裂解。基于毒素产生孔径的大小和与细胞作用方式的不同,可将其分为大成孔毒素、小成孔毒素和RTX毒素。成孔毒素主要通过改变细胞膜的通透性发挥毒性效应,导致细胞死亡,然而失去细胞通透性屏障的早期后果通常是细胞因子的释放,细胞内蛋白激酶的激活,有时会诱导细胞凋亡。     

Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo

Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.     

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.     

Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin

Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.     

CDC/MACPF家族成孔毒素研究进展

成孔毒素(Pore-forming toxins,PFTs)通常与细菌等原核生物的致病机理相关,在真核生物中,这类蛋白在免疫、胚胎发育、神经细胞迁移等方面发挥着重要作用。其中球状蛋白成孔毒素中的两大家族,胆固醇结合细胞溶素(Cholesterol-dependent cytolysins,CDCs)和攻膜复合体/穿孔素超家族(Membrane attack complex/perforin superfamily,MACPF)以其独特的分子构型和功能受到研究者的关注。CDCs由革兰氏阳性细菌产生,在细菌的侵染过程中发挥裂解靶细胞膜的作用;在真核生物中,MACPF蛋白同时具有裂解和非裂解两种形式。目前,对于以产气荚膜梭菌溶血素(PFO)为代表的CDCs蛋白的结构及分子机制了解得较为透彻,近年来晶体学及生物化学研究揭示,尽管CDCs和MACPF这两大家族成孔毒素在氨基酸序列水平上存在较大差异,但它们共同的折叠模式预示:MACPF蛋白以一种类似于CDCs的成孔机制来行使其裂解靶膜的功能。文章对这两大家族成孔毒素的结构、成孔机制以及目前的研究热点问题予以综述,为该领域今后的研究提供有益参考。     

Pore-forming toxins induce multiple cellular responses promoting survival

Pore-forming toxins (PFTs) are secreted proteins that contribute to the virulence of a great variety of bacterial pathogens. They inflict one of the more disastrous damages a target cell can be exposed to: disruption of plasma membrane integrity. Since this is an ancient form of attack, which bears similarities to mechanical membrane damage, cells have evolved response pathways to these perturbations. Here, it is reported that PFTs trigger very diverse yet specific response pathways. Many are triggered by the decrease in cytoplasmic potassium, which thus emerges as a central regulator. Upon plasma membrane damage, cells activate signalling pathways aimed at restoring plasma membrane integrity and ion homeostasis. Interestingly these pathways do not require protein synthesis. Cells also trigger signalling cascades that allow them to enter a quiescent-like state, where minimal energy is consumed while waiting for plasma membrane damage to be repaired. More specifically, protein synthesis is arrested, cytosolic constituents are recycled by autophagy and energy is stored in lipid droplets.     

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

SUMMARY

Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins and are required for virulence in a large number of important pathogens, including Streptococcus pneumoniae, group A and B streptococci, Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis. PFTs generally disrupt host cell membranes, but they can have additional effects independent of pore formation. Substantial effort has been devoted to understanding the molecular mechanisms underlying the functions of certain model PFTs. Likewise, specific host pathways mediating survival and immune responses in the face of toxin-mediated cellular damage have been delineated. However, less is known about the overall functions of PFTs during infection in vivo. This review focuses on common themes in the area of PFT biology, with an emphasis on studies addressing the roles of PFTs in in vivo and ex vivo models of colonization or infection. Common functions of PFTs include disruption of epithelial barrier function and evasion of host immune responses, which contribute to bacterial growth and spreading. The widespread nature of PFTs make this group of toxins an attractive target for the development of new virulence-targeted therapies that may have broad activity against human pathogens.     

Systemic mitochondrial disruption is a key event in the toxicity of bacterial pore-forming toxins to Caenorhabditis elegans

Pore-forming toxins (PFTs) are important weapons of multiple bacterial pathogens to establish their infections. PFTs generally form pores in the plasma membrane of target cells; however, the intracellular pathogenic processes triggered after pore-formation remain poorly understood. Using Caenorhabditis elegans as a model and Bacillus thuringiensis nematicidal Cry PFTs, we show here that the localized PFT attack causes a systemic mitochondrial damage, important for the PFT toxicity. We find that PFTs punch pores only in gut cells of nematodes, but unexpectedly mitochondrial disruption is able to occur in distal unperforated regions, such as the head and muscle tissues. We demonstrate that PFTs affect the activity of the mitochondrial respiratory chain (MRC) complex I resulting in the loss of mitochondrial membrane potential (ΔΨ_m), which causes further mitochondrial fragmentation and the reduction of total mitochondrial content. Worms with decreased ΔΨ_m or inhibited MRC activity show higher sensitivity to PFTs. The inhibition of mitochondrial fission or the increase of mitochondrial content markedly improves the survival of animals treated with PFTs. These findings suggest that mitochondrial changes underpin PFT-mediated toxicity against nematodes and that systemic mitochondrial disruption caused by localized pore-formation represents a conserved key intracellular event in the mode of action of PFTs.     

Beta-barrel membrane protein folding and structure viewed through the lens of alpha-hemolysin

The beta-barrel is a transmembrane structural motif commonly encountered in bacterial outer membrane proteins and pore-forming toxins (PFTs). Alpha-hemolysin (alphaHL) is a cytotoxin secreted by Staphylococcus aureus that assembles from a water-soluble monomer to form a membrane-bound heptameric beta-barrel on the surface of susceptible cells, perforating the cell membranes, leading to cell death and lysis. The mechanism of heptamer assembly, which has been studied extensively, occurs in a stepwise manner, and the structures of the initial, monomeric form and final, membrane-embedded pore are known. The toxin's ability to assemble from an aqueous, hydrophilic species to a membrane-inserted oligomer is of interest in understanding the assembly of PFTs in particular and the folding and structure of beta-barrel membrane proteins in general. Here we review the structures of the monomeric and heptamer states of LukF and alphaHL, respectively, the mechanism of toxin assembly, and the relationships between alphaHL and nontoxin beta-barrel membrane proteins.     

Pore formation: an ancient yet complex form of attack

Bacteria, as well as higher organisms such as sea anemones or earthworms, have developed sophisticated virulence factors such as the pore-forming toxins (PFTs) to mount their attack against the host. One of the most fascinating aspects of PFTs is that they can adopt a water-soluble form at the beginning of their lifetime and become an integral transmembrane protein in the membrane of the target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation. These points will be addressed in this review.     

Bacterial protein listeriolysin O induces nonmonotonic dynamics because of lipid ejection and crowding

Membrane-bound protein complexes involving pore forming toxins (PFTs) released by virulent bacteria are known to form transmembrane pores leading to host cell lysis. Developing alternative strategies against PFT mediated bacterial virulence factors requires an understanding of the cellular membrane response. However, membrane disruption and related lipid reorganization events during attack by PFTs remain largely unexplored. We report counterintuitive and nonmonotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane-bound oligomers of a prototypical cholesterol-dependent cytolysin, listeriolysin O (LLO). The observed dynamical crossover is correlated with concentration dependent transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area-based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states. At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodeling, and mechanical property variations, providing unique insights into the pore forming mechanisms of cholesterol-dependent cytolysins.     

Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization

Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.     

Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa

Type III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation.     

β-Barrel membrane protein folding and structure viewed through the lens of α-hemolysin

The β-barrel is a transmembrane structural motif commonly encountered in bacterial outer membrane proteins and pore-forming toxins (PFTs). α-Hemolysin (αHL) is a cytotoxin secreted by Staphylococcus aureus that assembles from a water-soluble monomer to form a membrane-bound heptameric β-barrel on the surface of susceptible cells, perforating the cell membranes, leading to cell death and lysis. The mechanism of heptamer assembly, which has been studied extensively, occurs in a stepwise manner, and the structures of the initial, monomeric form and final, membrane-embedded pore are known. The toxin's ability to assemble from an aqueous, hydrophilic species to a membrane-inserted oligomer is of interest in understanding the assembly of PFTs in particular and the folding and structure of β-barrel membrane proteins in general. Here we review the structures of the monomeric and heptamer states of LukF and αHL, respectively, the mechanism of toxin assembly, and the relationships between αHL and nontoxin β-barrel membrane proteins.