Bacterial Hemolysins Research Articles

Pancreatic Expression and Mitochondrial Localization of the Progestin-AdipoQ Receptor PAQR10

Steroid hormones induce changes in gene expression by binding to intracellular receptors that then translocate to the nucleus. Steroids have also been shown to rapidly modify cell function by binding to surface membrane receptors. We identified a candidate steroid membrane receptor, the progestin and adipoQ receptor (PAQR) 10, a member of the PAQR family, in a screen for genes differentially expressed in mouse pancreatic beta-cells. PAQR10 gene expression was tissue restricted compared with other PAQRs. In the mouse embryonic pancreas, PAQR10 expression mirrored development of the endocrine lineage, with PAQR10 protein expression confined to endocrine islet-duct structures in the late embryo and neonate. In the adult mouse pancreas, PAQR10 was expressed exclusively in islet cells except for its reappearance in ducts of maternal islets during pregnancy. PAQR10 has a predicted molecular mass of 29 kDa, comprises seven transmembrane domains, and, like other PAQRs, is predicted to have an intracellular N-terminus and an extracellular C-terminus. In silico analysis indicated that three members of the PAQR family, PAQRs 9, 10, and 11, have a candidate mitochondrial localization signal (MLS) at the N-terminus. We showed that PAQR10 has a functional N-terminal MLS and that the native protein localizes to mitochondria. PAQR10 is structurally related to some bacterial hemolysins, pore-forming virulence factors that target mitochondria and regulate apoptosis. We propose that PAQR10 may act at the level of the mitochondrion to regulate pancreatic endocrine cell development/survival.

Selective killing of HIV-1-positive macrophages and T cells by the Rev-dependent lentivirus carrying anthrolysin O from Bacillus anthracis

BackgroundThe ability of Human Immunodeficiency Virus (HIV) to persist in the body has proven to be a long-standing challenge to virus eradication. Current antiretroviral therapy cannot selectively destroy infected cells; it only halts active viral replication. With therapeutic cessation or interruption, viral rebound occurs, and invariably, viral loads return to pre-treatment levels. The natural reservoirs harboring replication-competent HIV-1 include CD4 T cells and macrophages. In particular, cells from the macrophage lineage resist HIV-1-mediated killing and support sustained viral production. To develop a complementary strategy to target persistently infected cells, this proof-of-concept study explores an HIV-1 Rev-dependent lentiviral vector carrying a bacterial hemolysin, anthrolysin O (anlO) from Bacillus anthracis, to achieve selective killing of HIV-1- infected cells.ResultsWe demonstrate that in the Rev-dependent lentiviral vector, anlO expression is exclusively dependent on Rev, a unique HIV-1 protein present only in infected cells. Intracellular expression and oligomerization of AnlO result in membrane pore formation and cytolysis. We have further overcome a technical hurdle in producing a Revdependent AnlO lentivirus, through the use of β-cyclodextrin derivatives to inhibit direct killing of producer cells by AnlO. Using HIV-1-infected macrophages and T cells as a model, we demonstrate that this Rev-dependent AnlO lentivirus diminishes HIV-1- positive cells.ConclusionThe Rev-dependent lentiviral vector has demonstrated its specificity in targeting persistently infected cells. The choice of anlO as the first suicidal gene tested in this vector is based on its cytolytic activity in macrophages and T cells. We conclude that Rev-regulated expression of suicidal genes in HIV-1-positive cells is possible, although future in vivo delivery of this system needs to address numerous safety issues.

Functional Analysis of the Campylobacter jejuni cj0183 and cj0588 Genes

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with amino acid sequences predicted to be homologous to other bacterial hemolysins. The Cj0183 protein exhibits homology to Brachyspira hyodysenteriae TlyC protein, whereas the cj0588 gene product is homologous to TlyA proteins Brachyspira hyodysenteriae, Helicobacter pylori, and Mycobacterium tuberculosis, which play a crucial role in bacterial virulence. The aim of our work was to examine the hemolytic activity and determine the role of cj0183- and cj0588-encoded proteins on the adherence of chosen C. jejuni strains to the Caco-2 cell line by constructing deletion mutants in the mentioned genes. We found out there is no difference in hemolytic activity between both mutants in gene cj0183 and cj0588 and the wild strains. However, Cj0588 protein but not Cj0183 is involved in adherence to the Caco-2 cells.

Measurement of the red blood cell lysis by bacterial hemolysin

Measurement of the red blood cell lysis by bacterial hemolysin

An in vitro cell-culture model demonstrates internalin- and hemolysin-independent translocation of Listeria monocytogenes across M cells

An ability to translocate the mucosal epithelia through M cells provides invasive pathogens with a rapid means of accessing the mucosal lymphoid tissues. In order to determine the role of M cells in Listeria monocytogenes infection, we initially assessed colonization of Peyer's patch (PP) epithelium in BALB/c mice by Vibrio cholerae Eltor, wild-type L. monocytogenes and an isogenic hemolysin mutant (LO28Δ hly). It was observed that both wild-type L. monocytogenes and Δ hly showed preferential colonization of PP epithelium in this model. Furthermore, a novel luciferase reporter system was used to show rapid site-specific localization of L. monocytogenes in intestinal Peyer's patches. To examine the role of M cells in transcytosis of L. monocytogenes we utilized an in vitro transwell model that mimics M-cell activity through differentiation of C2Bbe1 epithelial enterocytes via co-culture with murine Peyer's patch lymphocytes (PPL). It was shown that L. monocytogenes transits M cells at significantly increased rates compared to C2Bbe1 monocultures. In addition, M-cell transport occurred independently of bacterial hemolysin and internalin production. This study demonstrates rapid transcytosis of L. monocytogenes through M cells, a process that occurs independently of the action of classical virulence factors.

Dependence of the lethal effect of pore-forming haemolysins of Gram-positive bacteria on cytolytic activity

Among bacterial haemolysins, cholesterol-dependent cytolysins (CDCs) produced by various Gram-positive bacteria are known to exhibit a lethal activity in mice. In this study, recombinant CDCs of streptolysin O, pneumolysin, ivanolysin O, listeriolysin O and several listeriolysin O mutants were constructed and the relationship between cytolytic activity and the lethal activity of each recombinant protein in mice was examined. Specific activity for cytolysis was determined by a quantitative haemolytic assay. Each protein was injected intravenously into mice and the lethal activity was evaluated by measuring the time until death of the mice. The four full-length CDC proteins exhibited lethal activity and their activities were highly proportional to their cytolytic activities. Inhibition of haemolytic activity resulted in the loss of lethal activity and non-haemolytic mutants of listeriolysin O did not exhibit any lethal activity. These data clearly indicate that the lethal effect of CDC proteins is dependent on the cytolytic activity.

Possible Role of Fungal Hemolysins in Sick Building Syndrome

Publisher Summary The chapter first reviews fungal hemolysins and compares them with bacterial hemolysins. Hemolysins are molecules that have the ability to lyse red blood cells (RBCs). There are primarily two types of hemolysins: alpha (α) and beta (β). Alpha hemolysins cause a partial lysis of the RBCs, resulting in a darkening of the media around a colony on sheep's blood agar (SBA). Beta hemolysins produce a complete lysis of the RBCs, resulting in a clearing around the colony growing on SBA. The chapter discusses the biochemistry of hemolysins. Hemolysins are produced by many of the common indoor fungi. Fungal hemolysins might be useful as biomarkers of exposure to indoor fungi because they can be measured in bodily fluids and environmental samples. Fungal hemolysins are generally composed of smaller-molecular-weight monomers than most bacterial hemolysins. They usually have more acidic isoelectric points and contain more cysteine amino acids than bacterial hemolysins. Fungal hemolysins are remarkably slow acting as compared with bacterial hemolysins. However, like most bacterial hemolysins, fungal hemolysins are aggregating proteins that create pores in membranes.

The PhlA hemolysin from the entomopathogenic bacterium Photorhabdus luminescens belongs to the two-partner secretion family of hemolysins.

Photorhabdus is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae. Bacterial hemolysins found in numerous pathogenic bacteria are often virulence factors. We describe here the nucleotide sequence and the molecular characterization of the Photorhabdus luminescens phlBA operon, a locus encoding a hemolysin which shows similarities to the Serratia type of hemolysins. It belongs to the two-partner secretion (TPS) family of proteins. In low-iron conditions, a transcriptional induction of the phlBA operon was observed by using the chloramphenicol acetyltransferase reporter gene, causing an increase in PhlA hemolytic activity compared to iron-rich media. A spontaneous phase variant of P. luminescens was deregulated in phlBA transcription. The phlA mutant constructed by allelic exchange remained highly pathogenic after injection in the lepidopteran Spodoptera littoralis, indicating that PhlA hemolysin is not a major virulence determinant. Using the gene encoding green fluorescent protein as a reporter, phlBA transcription was observed in hemolymph before insect death. We therefore discuss the possible role of PhlA hemolytic activity in the bacterium-nematode-insect interactions.

Red blood cell hemolysis during processing

Red blood cell (RBC) hemolysis has been reported in units of RBC for transfusion. This has significant clinical implications for transfused patients because the free hemoglobin dissociates into dimers that have to be bound to haptoglobin to be removed by the reticuloendothelial system. Once the binding capacity of haptoglobin has been exceeded, hemoglobinemia occurs. Hemolysis is caused by the breakdown of the RBC, causing release of hemoglobin and resulting in the discoloration of the plasma. Abnormal hemolysis in an individual RBC unit may be caused by several factors including inappropriate handling during processing of blood, inappropriate storage conditions, bacterial hemolysins, antibodies that cause complement lysis, defects in the RBC membrane, or an abnormality in the blood donor. The degree of hemolysis is described as the percent of free hemoglobin in relation to the total hemoglobin with appropriate correction for the hematocrit. The acceptable level of hemolysis has not been established in North America, but the value of 1% currently is used to assess biocompatibility of blood storage materials, whereas the Council of Europe has set the standard at 0.8%. This report emphasizes the need for the adequate control of the various processes that are involved in the preparation of RBCs from whole blood to minimize the occurrence of hemolysis. Careful evaluation of manufacturing processes will minimize RBC wastage caused by hemolysis.

In vitro adherence of Enterococcus faecalis and Enterococcus faecium to urinary catheters.

The in vitro adherence of ten strains of Enterococcus faecalis and ten strains of Enterococcus faecium to siliconized latex urinary catheters and to silicone elastomer was evaluated. Bacterial suspensions (2.5x10(5) cfu/ml) in tryptic soy broth containing 0.5 cm segments from each type of catheter were incubated at 37 degrees C. At specified intervals, the segments were washed to remove nonadherent bacteria and sonicated for 1 min, and colony-forming units were quantified. Bacterial adherence occurred rapidly, reaching maximal peaks after 24 h of incubation. Enterococcus faecium adherence to both biomaterials was significantly lower than that of Enterococcus faecalis. No differences were observed between the two elastomers. Bacterial adherence was not related to bacterial surface hydrophobicity, hemolysin or gelatinase production.

Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules

Advances in our understanding of fundamental cell biological processes have facilitated an expansion of therapeutic approaches to altering cellular physiology and phenotype. As many of these methods involve macromolecular agents that act on targets within the nucleus or cytoplasm, achieving their full potential ultimately requires the efficient delivery of these agents across the cell membrane barrier into the cytosol. Various strategies have been employed to enhance cytosolic delivery. These include either directly penetrating the plasma membrane, or avoiding degradation within the hydrolytic environment of the endosomal/lysosomal pathway after endocytic uptake. Some of the more promising methods in this regard have exploited the mechanisms utilized by certain viruses and bacteria for escaping into their host cell’s cytosol. In this review, we will discuss some of these methods with an emphasis on the use of pore-forming proteins from bacteria. Particular attention will be drawn to the pH-sensitive endosomolytic bacterial hemolysins, such as listeriolysin O, and the potentiol for their use in cytosolic drug delivery systems.

Genetic Immunization of Mice Against Listeria monocytogenes Using Plasmid DNA Encoding Listeriolysin O

Abstract 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 ofListeria monocytogenes

A simple and convenient method for the purification of the hemolytic toxin listeriolysin O (LLO) fromListeria monocytogenesis described. Supernatants from bacteria cultures were purified by application to a CH2spiral 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.

Cloning and characterization of a Prevotella melaninogenica hemolysin.

Hemolysins have been proven to be important virulence factors in many medically relevant pathogenic organisms. Their production has also been implicated in the etiology of periodontal disease. Hemolytic strain 361B of Prevotella melaninogenica, a putative etiologic agent of periodontal disease, was used in this study. The cloning, sequencing, and characterization of phyA, the structural gene for a P. melaninogenica hemolysin, is described. No extensive sequence homology could be identified between phyA and any reported sequence at either the nucleotide or amino acid level. As predicted from sequence analysis, this gene produces a 39-kDa protein which has hemolytic activity as measured by zymogram analysis. Unlike many Ca2+-dependent bacterial hemolysins, both the cloned and native PhyA proteins were enhanced by the presence of EDTA in a dose-dependent fashion with 40 mM EDTA allowing maximum activity. Ca2+ and Mg2+ were found to be inhibitory. The hemolytic activity also was found to have a dose-dependent endpoint. Through recovery of hemolytic activity from a spent reaction, this endpoint was shown to be the result of end product inhibition. This is the first report describing the cloning and sequencing of a gene from P. melaninogenica.

Bacterial hemolysins and leukotoxins affect target cells by forming large exogenous pores into their plasma membrane: Escherichia coli hemolysin A as a case example.

Many bacteria include among their virulence factors exoproteins which exert leukocidal and cytolytic functions and have the ability to form pores in model membranes. We show that, at least in the case of the RTX hemolysin produced by Escherichia coli (HlyA), formation of pores in planar lipid membranes is parallelled by opening of strikingly similar channels in the plasma membrane of exposed macrophages. Formation of such lesions in leukocytes can give rise to a variety of effects leading altogether to a diminished immune response towards the invasive bacteria.

Structural analysis and comparison of the C-terminal transport signal domains of hemolysin A and leukotoxin A

NMR spectroscopy was used to study the structure of the C-terminal signal sequences of the bacterial toxins, hemolysin A(HlyA) and leukotoxin A (LktA). The two signals share little sequence homology; however, both can direct toxin transport with equal efficiency. We report here that in a membrane mimetic environment both peptides form two short non-interacting α-helices separated by a short loop. This higher order structure may be a common feature of C-terminal signals and may be required for interaction with the membrane associated transporter complex.

Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin

H-o. Tamura, M. Noto, K. Kinoshita, S. Ohkuma and H. Ikezawa. Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin. Toxicon 32, 629–633, 1994.—Sphingomyelinase of Bacillus cereus, a bacterial hemolysin, reduced nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells in a dose-dependent manner. At 200 mU/ml, sphingomyelinase repressed half the neurite outgrowth of the cells at 250 ng/ml NGF. The c-fos superinduction, one of the early responses induced by NGF, was not influenced by this treatment, suggesting that the repression by sphingomyelinase occurred via a protein kinase C-independent pathway.

Hydrophobic interaction chromatography for the purification of cytolytic bacterial toxins

The usefulness of hydrophobic interaction chromatography for the simple purification of cytolytic bacterial toxins was studied. Conditions are described for different hydrophobic interaction chromatographic media for purifying with high yields two different kinds of such haemolysins, the thiol-activated toxin listeriolysin O from Listeria monocytogenes and alpha-toxin from Staphylococcus aureus. For listeriolysin O, purification on butyl-Sepharose was followed by gel filtration chromatography. From butyl-Sepharose the recovery was 84%. The final product had a specific activity of 620 000 U/mg with an overall recovery of 22%. Alpha-toxin was obtained by a single purification step from alkyl-Superose with 80% recovery and a specific activity of 29 000 U/mg. On sodium dodecyl sulphate polyacrylamide gel electrophoresis purified listeriolysin O and alpha-toxin showed a single band. Another thiol-activated toxin, streptolysin O from group A streptococci, showed a recovery of 38% from butyl-Sepharose. The results suggest the feasibility of using hydrophobic interaction chromatography, particularly with columns of weak hydrophobicity, for the purification of bacterial haemolysins in high yield.

ATPase activity and ATP/ADP-induced conformational change in the bacterial toxin exporter hemolysin B.

ATPase activity and ATP/ADP-induced conformational change in the bacterial toxin exporter hemolysin B.

Role of bacterial hemolysin production in induction of macrophage Ia expression during infection with Listeria monocytogenes.

The production of a hemolytic exotoxin (Hly) termed listeriolysin O (LLO) is a major determinant of the virulence of the Gram-positive bacterium Listeria monocytogenes. As determined by lethal inoculum size, LLO- strains of L. monocytogenes generally are several orders of magnitude less virulent than their LLO+ counterparts. The generation of protective anti-Listeria T cell immunity also has been shown to depend on the LLO phenotype of the bacteria present during primary infection, although the cellular basis of this observation is not known. The experiments described here address the role of LLO in regulation of the expression of class II MHC (Ia) molecules by murine macrophages. Because Ia expression by macrophages and other APC is thought to be a central factor in the generation of T cells specific for bacterial Ag, we have tested the hypothesis that the failure of LLO- strains to elicit anti-Listeria T cell responses might be secondary to an inability of these strains to stimulate increases in macrophage Ia levels. Our results show that the macrophage Ia response after i.p. injection of L. monocytogenes correlates strongly with the LLO phenotype of the bacteria. The presence of LLO+ organisms, even at very small numbers (as few as 10), elicits a striking increase in Ia expression by peritoneal macrophages. In contrast, even at very high numbers (up to 10(6) per mouse), LLO- bacteria fail to stimulate a strong Ia response. We also have analyzed macrophage Ia expression after injection of lysates of Escherichia coli expressing recombinant LLO protein. Similar to the results obtained with LLO+ and LLO- L. monocytogenes, we have observed Ia induction only with LLO+ lysates. Ia induction by this crude recombinant LLO preparation can be inhibited by cholesterol or heat. Furthermore, supernatants derived from cultures of LLO+ (but not LLO-) L. monocytogenes can cause Ia induction when administered via i.p. injection. Taken together, these findings suggest that the failure of macrophages to respond to LLO- organisms with an increase in Ia expression may be a major underlying cause of the failure of these bacteria to induce Listeria-specific protective T cell immunity. Furthermore, we propose that the induction of macrophage Ia expression in response to bacterial toxins such as Hly may represent one component of a set of early, innate immune mechanisms, and that this induction may provide a critical "bridge" to later, acquired, Ag-specific immune processes.