Detection of Listeria monocytogenes and the toxin listeriolysin O in food

Listeria monocytogenes is an emerging bacterial foodborne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, and septicaemia. Less commonly, infection can result in cutaneous lesions and flu-like symptoms. In pregnant women, the pathogen can cause bacteraemia, and stillbirth or premature birth of the fetus. The mortality rate for those contracting listeriosis is approximately 20%. Currently, the United States has a zero tolerance policy regarding the presence of L. monocytogenes in food, while Canada allows only 100 cfu/g of food. As such, it is essential to be able to detect the pathogen in low numbers in food samples. One of the best ways to detect and confirm the pathogen is through the detection of one of the virulence factors, listeriolysin O (LLO) produced by the microorganism. The LLO-encoding gene (hlyA) is present only in virulent strains of the species and is required for virulence. LLO is a secreted protein toxin that can be detected easily with the use of blood agar or haemolysis assays and it is well characterized and understood. This paper focuses on some of the common methods used to detect the pathogen and the LLO toxin in food products and comments on some of the potential uses and drawbacks for the food industry.     

The F pilus mediates a novel pathway of CDI toxin import

Contact‐dependent growth inhibition (CDI) is a widespread form of inter‐bacterial competition that requires direct cell‐to‐cell contact. CDI⁺ inhibitor cells express CdiA effector proteins on their surface. CdiA binds to specific receptors on susceptible target bacteria and delivers a toxin derived from its C‐terminal region (CdiA‐CT). Here, we show that purified CdiA‐CT⁵³⁶ toxin from uropathogenic Escherichia coli 536 translocates into bacteria, thereby by‐passing the requirement for cell‐to‐cell contact during toxin delivery. Genetic analyses demonstrate that the N‐terminal domain of CdiA‐CT⁵³⁶ is necessary and sufficient for toxin import. The CdiA receptor plays no role in this import pathway; nor do the Tol and Ton systems, which are exploited to internalize colicin toxins. Instead, CdiA‐CT⁵³⁶ import requires conjugative F pili. We provide evidence that the N‐terminal domain of CdiA‐CT⁵³⁶ interacts with F pilin, and that pilus retraction is critical for toxin import. This pathway is reminiscent of the strategy used by small RNA leviviruses to infect F⁺ cells. We propose that CdiA‐CT⁵³⁶ mimics the pilin‐binding maturation proteins of leviviruses, allowing the toxin to bind F pili and become internalized during pilus retraction.     

Beta2 integrin mediates entry of a bacterial toxin into T lymphocytes

Helicobacter pylori secretes a pore-forming toxin, VacA, that can cause numerous alterations in gastric epithelial cells and T lymphocytes. In this issue of Cell Host & Microbe, Sewald and colleagues report that beta2 integrin (CD18) mediates entry of VacA into human T cells. Downregulation of T cell responses by VacA may allow H. pylori to evade the adaptive immune response and establish persistent infection.     

Genetic immunization of mice against Listeria monocytogenes using plasmid DNA encoding listeriolysin O.

The development of protective immunity against many intracellular bacterial pathogens commonly requires sublethal infection with viable forms of the bacteria. Such infection results in the in vivo activation of specific cell-mediated immune responses, and both CD4+ and CD8+ T lymphocytes may function in the induction of this protective immunity. In rodent models of experimental infection with Listeria monocytogenes, the expression of protective immunity can be mediated solely by the immune CD8+ T cell subset. One major target Ag of Listeria-immune CD8+ T cells is the secreted bacterial hemolysin, listeriolysin O (LLO). In an attempt to generate a subunit vaccine in this experimental disease model, eukaryotic plasmid DNA expression vectors containing genes encoding either the wild-type or modified forms of recombinant LLO were generated and used for genetic vaccination of naive mice. Results of these studies indicate that the intramuscular immunization of mice with specifically designed plasmid DNA constructs encoding recombinant forms of LLO stimulates peptide-specific CD8+ immune T cells that exhibit in vitro cytotoxic activity. More importantly, such immunization can provide protective immunity against a subsequent challenge with viable L. monocytogenes, demonstrating that this experimental approach may have direct application in prevention of acute disease caused by intracellular bacterial pathogens.     

A method for purification of listeriolysin O from a hypersecretor strain of Listeria monocytogenes

A simple and convenient method for the purification of the hemolytic toxin listeriolysin O (LLO) from Listeria monocytogenes is described. Supernatants from bacteria cultures were purified by application to a CH2 spiral cartridge concentrator (Amicon) and ion exchange chromatography. A critical step is removal of contaminating RNA. The purified proteins had characteristics described for bacterial thiol-activated hemolysins: activation by a reducing agent (DTT) and inactivation by cholesterol. In addition, the molecular weight of 58, 000 and pH-dependent hemolytic activity of this purified protein are consistent with the previously published characteristics of LLO.     

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes.

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

The autophagic pathway: a cell survival strategy against the bacterial pore-forming toxin Vibrio cholerae cytolysin

Vibrio cholerae is the causative agent of cholera in humans. In addition to the criticalvirulence factors cholera toxin and toxin coregulated pilus, V. cholerae secretes V.cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensivevacuolation. We have shown that this vacuolation is related to the activation of autophagyin response to VCC action. Furthermore, we found that the autophagic pathway wasrequired to protect cells upon VCC intoxication. Based on additional data presented here,we propose a model aimed to explain the mechanism of cell protection. We postulatethat VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involvingfusion with lysosomes.     

SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

While severe acute respiratory syndrome coronavirus （SARS-CoV）~as initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that yirus entry may also involve endocytosis. We have found that SARS-CoV enters cells viapH- and receptor-dependent endocytosis. Treatment of cells with either SARS-COV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 （ACE2）, the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic path- way inhibitors and dominant-negative Epsl5 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.     

Expression of ActA, Ami, InlB, and listeriolysin O in Listeria monocytogenes of human and food origin

Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 x 10(-4)). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 x 10(-2)) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 x 10(-3)) and group ActA3 strains (OR = 3.91; P = 1 x 10(-3)).     

Effects of glucose, growth temperature, and pH on listeriolysin O production in Listeria monocytogenes.

Expression of listeriolysin O of Listeria monocytogenes as a function of different growth conditions was studied by performing a direct hemolysin assay, immunoblotting experiments, and an enzyme-linked immunosorbent assay. Expression of listeriolysin O was reduced at a lower growth temperatures (26 degrees C) and at higher glucose concentrations (> or = 0.3%) in the growth media. The effect of glucose appeared to be due to a change in the pH of the growth media.     

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.     

The multiple mechanisms of Ca2+ signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes

Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.     

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.     

A novel cell entry pathway for a DAF-using human enterovirus is dependent on lipid rafts

The glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein decay-accelerating factor (DAF) is used by a number of enteroviruses as a receptor during infection. DAF and other GPI-anchored proteins can be found in cholesterol-rich ordered domains within the plasma membrane that are known as "lipid rafts." We have shown, by using drugs to specifically inhibit various endocytosis routes, that infection by a DAF-using strain of echovirus 11 (EV11) is dependent upon cholesterol and an intact cytoskeleton, whereas a non-DAF-using mutant derived from it was unaffected by these drugs. Using RNA transfection and virus-binding assays, we have shown that this requirement for cholesterol, the actin cytoskeleton, and the microtubule network occurs postbinding of the virus but prior to uncoating of the RNA, indicating a role during virus entry. Confocal microscopy of virus infection supported the role of cholesterol and the cytoskeleton during entry. In addition, [(35)S]methionine-labeled DAF-using EV11, but not the non-DAF-using EV11, could be copurified with lipid raft components during infection after Triton X-100 extraction. These data indicate that DAF usage by EV11 enables the virus to associate with lipid rafts and enter cells through this novel route.     

The cytotoxic fimbrial structural subunit of Xenorhabdus nematophila is a pore-forming toxin

We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.