A bacterial pore-forming toxin forms aggregates in cells that resemble those associated with neurodegenerative diseases

Listeria monocytogenes is a bacterial, facultative intracellular pathogen, which secretes a pore-forming toxin called listeriolysin O (LLO). LLO mediates the dissolution of the phagosomal membrane allowing L. monocytogenes to reach and grow in the host cytosolic compartment. In this study we report the localization of LLO secreted in infected cells. We described that LLO (i) forms small perinuclear aggregates, (ii) accumulates in large autophagosome-like structures and (iii) sequesters to large protein aggregates. The formation of protein aggregates required full LLO activity. Further characterization of protein aggregates indicated that they not only contained the active form of LLO but also polyubiquitinated proteins and p62, which are both common components of protein aggregates found in neurological diseases. Hence, a protein of bacterial origin could potentially follow the same fate as a toxic protein associated with neurodegenerative disease.     

IFN-beta increases listeriolysin O-induced membrane permeabilization and death of macrophages

Type I IFN (IFN-I) signaling is detrimental to cells and mice infected with Listeria monocytogenes. In this study, we investigate the impact of IFN-I on the activity of listeriolysin O (LLO), a pore-forming toxin and virulence protein released by L. monocytogenes. Treatment of macrophages with IFN-beta increased the ability of sublytic LLO concentrations to cause transient permeability of the plasma membrane. At higher LLO concentrations, IFN-beta enhanced the complete breakdown of membrane integrity and cell death. This activity of IFN-beta required Stat1. Perturbation of the plasma membrane by LLO resulted in activation of the p38MAPK pathway. IFN-beta pretreatment enhanced LLO-mediated signaling through this pathway, consistent with its ability to increase membrane damage. p38MAPK activation in response to LLO was independent of TLR4, a putative LLO receptor, and inhibition of p38MAPK neither enhanced nor prevented LLO-induced death. IFN-beta caused cells to express increased amounts of caspase 1 and to produce a detectable caspase 1 cleavage product after LLO treatment. Contrasting recent reports with another pore-forming toxin, this pathway did not aid cell survival as caspase 1-deficient cells were equally sensitive to lysis by LLO. Key lipogenesis enzymes were suppressed in IFN-beta-treated cells, which may exacerbate the membrane damage caused by LLO.     

Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O

Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.     

Listeriolysin O: a phagosome-specific lysin

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin family and a primary virulence factor of the gram-positive, facultative intracellular pathogen Listeria monocytogenes. During the intracellular life cycle of L. monocytogenes, LLO is largely responsible for mediating rupture of the phagosomal membrane, thereby allowing the bacterium access to the host cytosol, its replicative niche. In the host cytosol, LLO activity is controlled at numerous levels to prevent perforation of the plasma membrane and loss of the intracellular environment. In this review, we focus primarily on the role of LLO in phagosomal escape and the multiple regulatory mechanisms that control LLO activity in the host cytosol.     

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h. We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The second LLO construct contained an N-terminus His tag; it gave a yield of 3.5–8 mg l⁻¹. The third contained a C-terminus His tag; it gave a yield of 2.5 mg l⁻¹ LLO. The purified LLO from the latter two constructs retained its activity at 4°C for over a year as determined by bovine red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant LLO, and is the first to provide evidence of long-term stability of the toxin for further applications.     

Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications

The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol. This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria. A protocol for large-scale expression and purification of recombinant LLO was previously optimized. By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l(-1) of cell culture) in a hemolytically active form (1.2 x 10(6) HU mg(-1) of protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media. Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis.     

The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization

The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.     

Effect of pH on stability of anthrax lethal factor: correlation between denaturation and activity

Anthrax is caused by Gram positive bacterium Bacillus anthracis. Pathogenesis is result of production of three protein components, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to animals, while PA in combination with EF (edema toxin), causes edema. PA, LF, and EF are very thermolabile. Differential scanning calorimetry (DSC) was used to unravel the energetics of LF denaturation as a function of pH ranging from 7.8 to 5.5. Transition temperature (T(m)) of LF was found to be approximately equal to 42 degrees C and onset of denaturation occurs at approximately equal to 30 degrees C. The ratio of calorimetric to van't Hoff's enthalpy was nearly equal to unity at pH 7.0, indicative of presence of single structural domain in LF at pH 7.0, unlike PA which has been structurally observed to consist of 4 domains. It was found by cytotoxicity studies using J774A.1 macrophage like cells that LF was most stable at pH approximately 6.5. This paper reports for the first time the denaturation of LF at different pH values at 37 degrees C and tries to establish a correlation between denaturation and loss of LF activity at different pH values.     

Listeriolysin O: cholesterol inhibits cytolysis but not binding to cellular membranes

Listeriolysin O (LLO) binds to cholesterol-containing membranes in which it oligomerizes to form pores. Preincubation of the toxin with cholesterol is known to inhibit haemolysis, whereas the oxidized form of cholesterol has no inhibitory effect. Using immunoblot analyses and flow cytometry we demonstrate that preincubation with cholesterol does not influence binding of the listeriolysin–cholesterol complex to red blood cells, eukaryotic cells or artificial membranes. Lytic activity of membrane-bound LLO inactivated by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. To determine the step at which cholesterol inhibits lytic activity, we looked for pore formation using electron microscopy. Pores formed by purified listeriolysin could be directly visualized using erythrocyte ghosts. This property was lost upon incubation of the toxin with cholesterol. Quantitative analysis strongly suggest that inhibition of lysis by cholesterol is not due to decreased binding of listeriolysin to target membranes, but rather to an interference with a subsequent step leading to polymerization of the toxin.     

The pH threshold in the dissolution of beta-lactoglobulin gels and aggregates in alkali

The existence of a practical minimum pH for the dissolution of heat-induced whey gels in alkaline solutions has been studied using beta-lactoglobulin (betaLg) as a model protein. A sharp transition in solubility was observed between pH 11 and 12; this transition shifts to higher pHs for gels formed at higher temperatures and for longer gelling times. The breakdown reactions of heat-induced aggregates in alkali were monitored with size exclusion chromatography. The destruction of large aggregates was faster at higher pH and also showed a transition between pH 11 and 12. Using tryptophan fluorescence and near- and far-UV circular dichroism, this transition was assigned to the base-induced denaturation observed in solutions of aggregates (pK 11.53). It is suggested that the high protein repulsion caused by the large number of charges at pH > 11.5 drives the unfolding of the protein and the disruption of the intermolecular noncovalent bonds. Concentrated urea and GuHCl were found to be less effective than a pH 12 solution in destroying large aggregates. Aggregates formed for a long time (80 degrees C for 24 h) contained a larger number of intermolecular disulfide bonds that hinder the dissolution process. Gels formed at low temperatures (65 degrees C for 60 min), with fewer intermolecular noncovalent bonds, showed a similar solubility-pH profile to that observed for the base-induced denaturation of unheated beta-lactoglobulin (betaLg) (pK 10.63).     

Salt-induced oligomerization of partially folded intermediates of equinatoxin II

Equinatoxin II (EqTxII) is a cytolytic, water-soluble protein which binds to and forms cation-selective pores in lipid membranes. To characterize the native and denatured states of EqTxII and to elucidate the biological role of its oligomers, we have studied salt-dependent heat-induced conformational transitions of EqTxII. To this end, we have employed a variety of experimental techniques, including differential scanning calorimetry, circular dichroism and light absorption spectroscopy, ultrasonic velocimetry, electron microscopy, PAGE, and a hemolytic activity assay. This experimental combination has enabled us to monitor and structurally and thermodynamically characterize temperature-induced conformational transitions and oligomerization of EqTxII at different concentrations of NaCl. At pH 3.0 and 25 degrees C, EqTxII retains its native conformation and remains hemolytically active over a broad range of NaCl concentrations. However, an increase in the salt concentration results in a diminution of the thermal stability of EqTxII. Specifically, the calorimetrically determined denaturation temperature, T(d), and enthalpy, DeltaH(cal), of the toxin decrease with an increase in the salt concentration. Our CD data suggest that the heat-induced denatured state of EqTxII lacks rigid tertiary structure while exhibiting well-defined secondary structure. The amount of the induced, non-native secondary structure of EqTxII depends on the solution ionic strength, temperature, time of incubation at an elevated temperature, and protein concentration. Our combined results suggest that, in the presence of salt, an increase in temperature results in formation of the partially unfolded state of the toxin that oligomerizes and forms biologically inactive, water-soluble aggregates.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

Characterization of a transferrin-diphtheria toxin conjugate

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.     

Listeriolysin O as cytotoxic component of an immunotoxin

Monoclonal antibodies (mAbs) have been developed over the past years as promising anticancer therapeutics. The conjugation of tumor specific mAbs with cytotoxic molecules has been shown to improve their efficacy dramatically. These bifunctional immunotoxins, consisting of covalently linked antibodies and protein toxins, possess considerable potential in cancer therapy. Many of them are under investigation in clinical trials. As a result of general interest in new toxic components, we describe here the suitability of the bacterial protein Listeriolysin O (LLO) as cytotoxic component of an immunotoxin. Unique characteristics of LLO, such as its acidic pH optimum and the possibility to regulate the cytolytic activity by cysteine‐oxidation, make LLO an interesting toxophore. Oxidized LLO shows a substantially decreased cytolytic activity when compared with the reduced protein as analyzed by hemolysis. Both oxidized and reduced LLO exhibit a cell‐type‐unspecific toxicity in cell culture with a significantly higher toxicity of reduced LLO. For cell‐type‐specific targeting of LLO to tumor cells, LLO was coupled to the dsFv fragment of the monoclonal antibody B3, which recognizes the tumor‐antigen Lewis Y. The coupling of LLO to dsFv‐B3 was performed via cysteine‐containing polyionic fusion peptides that act as a specific heterodimerization motif. The novel immunotoxin B3‐LLO could be shown to specifically eliminate antigen positive MCF7 cells with an EC₅₀ value of 2.3 nM, whereas antigen negative cell lines were 80‐ to 250‐fold less sensitive towards B3‐LLO.     

Delivery of protein to the cytosol of macrophages using Escherichia coli K-12

Listeriolysin O (LLO) is an essential determinant of pathogenicity whose natural biological role is to mediate lysis of Listeria monocytogenes containing phagosomes. In this study, we report that Escherichia coli expressing cytoplasmic recombinant LLO can efficiently deliver co-expressed proteins to the cytosol of macrophages. We propose a model in which subsequent or concomitant to phagocytosis the E. coli are killed and degraded within phagosomes causing the release of LLO and target proteins from the bacteria. LLO acts by forming large pores in the phagosomal membrane, thus releasing the target protein into the cytosol. Delivery was shown to be rapid, within minutes after phagocytosis. Using this method, a large enzymatically active protein was delivered to the cytosol. Furthermore, we demonstrated that the E. coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the major histocompatibility (MHC) class I pathway for antigen processing and presentation, greater than 4 logs compared with E. coli expressing OVA alone. Moreover, the time required for processing and presentation of an OVA-derived peptide was similar to that previously reported when purified OVA was introduced directly into the cytosol by other methods. Using this system, potentially large amounts of any protein that can be expressed in E. coli can be delivered to the cytosol without protein purification. The potential use of this system for the delivery of antigenic protein in vivo and the delivery of DNA are discussed.     

Structural stability of E. coli transketolase to temperature and pH denaturation

We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism.     

High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein

Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures, which is a difficult task, mainly owing to the low levels of toxin released in the culture media. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.     

Identification of a PEST-like motif in listeriolysin O required for phagosomal escape and for virulence in Listeria monocytogenes

The hly-encoded listeriolysin O (LLO) is a major virulence factor secreted by the intracellular pathogen Listeria monocytogenes, which plays a crucial role in the escape of bacteria from the phagosomal compartment. Here, we identify a putative PEST sequence close to the N-terminus of LLO and focus on the role of this motif in the biological activities of LLO. Two LLO variants were constructed: a deletion mutant protein, lacking the 19 residues comprising this sequence (residues 32-50), and a recombinant protein of wild-type size, in which all the P, E, S or T residues within this motif have been substituted. The two mutant proteins were fully haemolytic and were secreted in culture supernatants of L. monocytogenes in quantities comparable with that of the wild-type protein. Strikingly, both mutants failed to restore virulence to a hly-negative strain in vivo. In vitro assays showed that L. monocytogenes expressing the LLO deletion mutant was strongly impaired in its ability to escape from the phagosomal vacuole and, subsequently, to divide in the cytosol of infected cells. This work reveals for the first time that the N-terminal portion of LLO plays an important role in the development of the infectious process of L. monocytogenes.     

Listeriolysin O Affects the Permeability of Caco-2 Monolayer in a Pore-Dependent and Ca2+-Independent Manner

Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.     

Phosphorylation, ubiquitination and degradation of listeriolysin O in mammalian cells: role of the PEST-like sequence

Listeria monocytogenes is a bacterial pathogen that grows within the cytosol of infected host cells. Entry into the cytosol is largely mediated by a secreted bacterial cytolysin, listeriolysin O (LLO). In order to prevent host cell damage, the pore-forming activity of LLO is restricted to the phagosome. Compartmentalization of LLO requires a PEST-like sequence; PEST sequences can direct eukaryotic proteins for proteasomal degradation. Here we test the hypothesis that LLO's PEST-like sequence compartmentalizes pore-forming activity by targeting this bacterial protein for degradation in the host cytosol. We show that intracellular LLO was degraded in a proteasome-dependent manner, and that, prior to degradation, LLO was ubiquitinated and was phosphorylated within the PEST-like sequence. However, wild-type LLO and PEST region mutants had similarly short intracellular half-lives and both the wild-type and mutant proteins were stabilized by inhibitors of host proteasomes. Additionally, blocking host proteasomes did not cause toxicity in a wild-type infection, but enhanced the cytotoxicity of PEST region mutants. Together with the observation that PEST region mutants exhibit higher intracellular LLO levels than wild-type bacteria, these data suggest that LLO's PEST-like region does not mediate proteasomal degradation by the host, but controls LLO production in the cytosol.