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Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis. Cells are surrounded by a plasma membrane made mostly from oily molecules known as lipids. One of these lipids, called cholesterol, is essential for keeping this membrane stable. Cholesterol is partly produced within the cells at a specialized structure called the endoplasmic reticulum, and partly imported from the blood surrounding the cell. In the blood, cholesterol is shielded inside particles called low-density lipoprotein (or LDL for short), which is taken into the cell and then sent to another structure called the lysosome. Inside the cell, cholesterol that is freshly produced in the endoplasmic reticulum or freshly imported into the lysosome, must be moved to the plasma membrane, where most of the cholesterol is located. Cholesterol levels are regulated by a ‘control machinery’ of proteins located in the endoplasmic reticulum. To keep the cholesterol levels constant, the endoplasmic reticulum needs to be in continual communication with the plasma membrane. However, the mechanisms by which cholesterol is transported between membranes are still poorly understood. Here, Infante and Radhakrishnan report a new tool to study how cholesterol is transported in human and hamster cells. The tool, which is based on part of a bacterial protein, traps cholesterol in the plasma membrane and prevents it from moving to the endoplasmic reticulum, and thus from updating the control machinery about cholesterol levels. From this inhibition, it is inferred that a stream of cholesterol constantly travels from the plasma membrane back to endoplasmic reticulum. This way, proteins in the endoplasmic reticulum can monitor the cholesterol levels in the plasma membrane in real-time. The endoplasmic reticulum responded rapidly even to small declines in cholesterol levels by activating genes that increase cholesterol production or the amount of cholesterol imported via the LDL pathway. Further work showed that cholesterol derived from LDL travels from the lysosome directly to the plasma membrane to maintain optimal cholesterol levels. It then moves to the endoplasmic reticulum to signal that cholesterol levels in the cell have been satisfied. The findings and tools described in this study will help to further investigate the mechanisms underlying the transport of cholesterol between the different membranes and structures in a cell. A next step will be to see if the mechanisms that apply to distribution of imported cholesterol from lysosomes, also apply to the cholesterol produced in the endoplasmic reticulum.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They are secreted as water-soluble proteins that are able to bind to the host cell membrane and insert hydrophobic segments into the lipid bilayer that ultimately contribute to the formation of transmembrane pores. Ion diffusion through these pores leads then to cytotoxic and cytolytic effects on the hosts. Several reports have evidenced that the binding of several RTX toxins to the target cell membrane may take place through a high-affinity interaction with integrins of the β2 family that is highly expressed in immune cells of the myeloid lineage. However, at higher toxin doses, cytotoxicity by most RTX toxins has been observed also on β2-deficient cells in which toxin binding to the cell membrane has been proposed to occur through interaction with glycans of glycosylated lipids or proteins present in the membrane. More recently, cumulative pieces of evidence show that membrane cholesterol is essential for the mechanism of action of several RTX toxins. Here, we summarize the most important aspects of the RTX toxin interaction with the target cell membrane, including the cholesterol dependence, the recent identification in the sequences of several RTX toxins of linear motifs coined as the Cholesterol Recognition/interaction Amino acid Consensus (CRAC), and the reverse or mirror CARC motif, which is involved in the toxin–cholesterol interaction.

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead to oligomerization and end in pore-formation. Perfringolysin O (PFO), secreted by Clostridium perfringens, is the prototype for the CDCs. The molecular mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not fully understood. In particular, the location of the binding site for cholesterol has remained elusive. We have summarized here the current body of knowledge on the CDCs-cholesterol interaction, with focus on PFO. We have employed sterols in aqueous solution to identify structural elements in the cholesterol molecule that are critical for its interaction with PFO. In the absence of high-resolution structural information, site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on this interaction.

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of from phagosomes and enables the bacteria to grow within the host. LLO is a versatile tool allowing to trigger several cellular responses. In this study, rapid phosphorylation of ERK1/2 on Caco-2 cells caused by infection was demonstrated to be highly dependent on LLO activity. The effect could be strongly induced by adding purified recombinant LLO alone and could be inhibited by exogenous cholesterol. Lack of the PEST sequence, known to tightly control cytotoxicity of LLO, did not affect ERK1/2 activation. However, the recombinant non-cytolytic LLO , with mutations in the cholesterol-binding motif, was unable to trigger this response. Recombinant LLO , which lacks most of the cytolytic activity, also failed to activate ERK1/2 phosphorylation, and this effect could be rescued when the protein concentration reached a cytolytic level. Infection with an LLO-deficient mutant (Δ ) or the mutant complementing LLO abrogated the capacity of the bacteria to activate ERK1/2. However, infection with the Δ mutant complementing LLO , which retained partial pore-forming ability and could grow intracellularly, was capable of triggering ERK1/2 phosphorylation. Collectively, these data suggest that ERK1/2 activation by depends on the permeabilization activity of LLO and more importantly correlates with the cholesterol-binding motif of LLO.

Pore-forming toxins are widely distributed proteins which form lesions in biological membranes. In this review, bacterial pore-forming toxins are treated as a paradigm and discussed in terms of the structural principles on which they work. Then, a large family of bacterial toxins, the cholesterol-binding toxins, are analyzed in depth to provide an overview of the processes involved in pore formation. The ways in which the cholesterol-binding toxins (cholesterol-dependent cytolysins) interact with membranes and form pores, the structure of the monomeric soluble and oligomeric pore-forming states, and the effects of the toxin on membrane structure are discussed. By surveying the range of work which has been done on cholesterol-binding toxins, a working model is elaborated which reconciles two current, apparently diametrically opposed, models for their mechanism.

Background Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O. Methods To detect cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO–positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified cholesterol in NPC cells. Results GST-PFO recognized cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of cholesterol with methyl-β-cyclodextrin. Conclusions Our data indicate that a recombinant protein GST-PFO can be used to detect cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing cholesterol deposits in cells and can be useful in diagnostics of NPC disease.

The Streptococcus pneumoniae toxin pneumolysin belongs to the group of cholesterol-dependent cytolysins. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. In cell membranes, it forms prepores and pores. Here, we show that sublytic concentrations of pneumolysin produce rapid activation of Rho and Rac GTPases and formation of actin stress fibers, filopodia, and lamellipodia. That Rac1-specific and Rho-associated kinase (ROCK)-specific inhibitors reverted the formation of lamellipodia and stress fibers, respectively, identifies RhoA and Rac1 as key toxin effectors. Live imaging excluded macropore formation (as judged by membrane impermeability toward calcein) but indicated very early membrane depolarization [as judged by bis-(1,3-dibutylbarbituric acid)trimethine oxanol staining], indicative of formation of micropores with ion channel properties. That Rac1-dependent lamellipodia formation was reverted by the voltage-gated calcium channel inhibitor SKF96365 and by toxin exposure in calcium-free medium suggests a role for calcium influx via endogenous calcium channels in the Rac1 activation. Cellular cholesterol depletion by methyl-β-cyclodextrin or incubation of the toxin with cholesterol before cell treatment eliminated its membrane binding and the subsequent GTPase activation. Thus, that our experiments show small GTPase activation by a cholesterol-dependent cytolysin suggests a membrane cholesterol-dependent activation mechanism.

We have constructed recombinant listeriolysin O (rLLO) and seeligeriolysin O (rLSO) from Listeria monocytogenes and Listeria seeligeri, respectively. In hemolysis and cholesterol-binding assays, the specific activity of recombinant toxin was lower for LSO as compared to LLO. To understand the molecular basis of this difference, in particular with respect to the conserved Trp-rich undecapeptide, a naturally occurring Ala to Phe substitution in LSO was introduced into rLLO. The rLLO:A488F hemolysin exhibited a reduced activity in both hemolysis and cholesterol-binding. The reverse mutation, inserted into rLSO, also increased the hemolytic activity of this mutant LSO. These results suggested that the natural replacement of Ala to Phe is involved in the weak cytolytic activity of LSO.