Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes.

Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.     

Expression of listeriolysin O by intracellular Listeria monocytogenes following infection of lipopolysaccharide-treated or untreated J774.1 macrophage-like cells

Abstract Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes , which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 4 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased between 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-treated host cells was also induced at 2 h post-infection, suggesting that listeriolysin O was newly synthesized in the macrophage-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes .     

Listeriolysin O secretion by Listeria monocytogenes in the presence of cysteine and sorbate

The influence of cysteine or cysteine-HCl and their combination with potassium sorbate on growth of Listeria monocytogenes and listeriolysin O (LLO) secretion, and on activation of LLO in the haemolysin assay system was studied. Both cysteine and cysteine-HCl (10 and 20 mmol 1^-1) enhanced LLO secretion in tryptic soy broth supplemented with yeast extract during 24 h incubation at 35°C. While sorbate did not affect growth, it suppressed both LLO secretion and LLO activation by cysteine in the haemolysin activity assay. These findings provide further evidence that sulphydryl-containing enzymes are implicated in the mechanism of microbial inhibition of sorbate. Addition of sorbate to foods has the potential of inactivating listeriolysin and other thiol-dependent toxins.     

Interactions between the environmental pathogen Listeria monocytogenes and a free-living protozoan (Acanthamoeba castellanii)

Listeria monocytogenes can cause severe disease in animal hosts, but it has no recognized animal host reservoir. We tested the hypothesis that L. monocytogenes retains virulence traits to survive predation by amoebae and that listeriolysin O plays a crucial role in this process. Co-culturing of L. monocytogenes and Acanthamoeba castellanii demonstrated that L. monocytogenes does not actively kill amoebae, but in the presence of amoebae, high bacterial population densities can be maintained over a period of at least 96 h. A gentamicin protection assay demonstrated that there is no significant difference in the ability to survive predation between serovars (4b versus 1/2a and 1/2c; P  = 0.08) and between five species of Listeria ( P  = 0.14). Three of these species do not harbour the hly gene responsible for listeriolysin O production. A hly knockout strain had poorer survival compared with the parental strain ( P  = 0.04 at 24 h; P  = 0.04 at 48 h; P  = 0.02 at 72 h) and electron microscopy was consistent with a wild-type strain being able to escape the phagosome whereas the hly knockout strain did not appear to have this ability. Thus, while there is weak evidence that listeriolysin O can contribute to improved survival after ingestion by amoebae, listeriolysin O does not appear to provide a significant selective advantage under the conditions of this study.     

T cell recognition of listeriolysin O is induced during infection with Listeria monocytogenes

During bacterial multiplication, Listeria monocytogenes (strain EGD) secretes sulfhydryl-dependent cytotoxin, termed listeriolysin O, a virulence factor presumable promoting intracellular growth of this ubiquitous pathogen. The role of this exotoxin in the process of T cell activation was studied in vivo during the course of an experimental infection in the mouse. By using highly purified listeriolysin O, it was found that infection with viable, replicative bacteria induced in vivo the emergence of T cells specifically reacting against this exotoxin, as demonstrated by eliciting the expression of delayed-type hypersensitivity to listeriolysin O in Listeria-immune mice. The kinetics of this inflammatory reaction followed the same pattern as that observed with crude Listeria antigenic preparation classically used for the detection of delayed-type hypersensitivity, with a peak of expression by day 6 and a slow decline over the next 3 wk to a residual level, indicating the presence of memory T cells reacting with the exotoxin. This result, therefore, allowed us to identify for the first time that a pure immunogenic molecule secreted by L. monocytogenes is specifically recognized by sensitized T cells induced during the course of infection by L. monocytogenes. The expression of T cell-mediated immunity to listeriolysin O was generated by very low amounts of replicative bacteria, indicating that the exotoxin released in host tissues during the process of intracellular growth is highly immunogenic. Our data favor the view that the binding of listeriolysin O to the membrane cholesterol might be a critical event potentiating the in vivo expression of delayed sensitivity against this exotoxin. Indeed, the insertion of listeriolysin O into the cell membrane induced resistance to enzymatic proteolysis and membrane-bound listeriolysin O was significantly more effective in inducing delayed inflammatory reaction in Listeria-immune mice.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Radiometric assays for glycerol, glucose, and glycogen

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.     

Toxicity of nitric oxide and peroxynitrite to Photobacterium damselae subsp. piscicida

Virulent strains of Photobacterium damselae subsp. piscicida (Pdp) were grown in media with or without glucose supplementation (to enhance polysaccharide capsule formation) and the bactericidal action of nitric oxide (NO) and peroxynitrites was evaluated in a cell-free assay. Treatment with the NO-donor S-nitroso-acetyl-penicillamine (SNAP) induced a dose-and time-dependent decrease in Pdp survival. This effect was greater for strains grown without glucose supplementation (C forms) than for their counterparts grown with glucose supplementation (C(+) forms). Addition of superoxide anion (O2(-)) generating systems (Xanthine/Xanthine oxidase, glucose/glucose oxidase) to the culture media further enhanced the bactericidal effect of NO. A similar bactericidal effect, with the same pattern of sensitivity, was observed when C+ and C forms of the bacteria were treated with 3-morpholino-sydonimide hydrochloride (SIN-1), a compound which simultaneously generates NO and O2(-). Addition of superoxide dismutase (SOD) or SOD plus catalase (CAT) did not fully reverse the toxic action of SIN-1 and the bactericidal effect was similar for both C and C(+) forms suggesting that while NO alone is sufficient to cause damage in all strains of the pathogen tested, growth in glucose supplemented medium enhanced protection to reactive oxygen intermediates rather than NO.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to a _w 0.93 were more rapid than in unsupplemented media ( a _w 0.99). Although growth in unsupplemented medium was lower at 35°C, incubation at 21°C or 35°C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 μg potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35°C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35°C than in cultures incubated at 21° in media both with and without 300 μg potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at - 18°C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 μg potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21°C. Sensitivity to freezing increased when cultures were incubated at 21°C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Growth medium-independent genetic competence mutants of Bacillus subtilis.

The development of competence in Bacillus subtilis is normally dependent on the growth medium. Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media. Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose. Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables. Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated. Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable. One of the mecB mutations (mecB31) conferred oligosporogenicity. The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14. The mecA42 mutant was linked by transduction to argC4.     

Dense Crithidia Growth and Heme Sparing: Relation to Fe,Cu, Mo Chelation*

SYNOPSIS. Heme, intrinsically required by Trypanosomatidae, is unstable, especially in conventional alkaline (pH 7.2–8.0) media. Low solubility of heme in a pH 6.5 basal medium (developed to assay biopterin with Crithidia fasciculata) posed a problem: in media acidified during growth because of glycolysis, heme precipitated, perhaps contributed to acid-limited growth and interfered with densitometric estimation of growth. The remedy was to: replace glucose with less rapidly metabolized mannitol; distribute media in thin layers to promote oxidation of acetate, fumarate, and malate (presumably leaving an alkaline residue); and buffer heavily with histidine + Good zwitterionic buffers, and superimpcse physiological buffering by arginine + asparagine whose catabolism appeared to yield an excess of NH⁺₄ over acid. Thereupon, Fe and Cu deficiencies sharply limited growth in the medium whose main chelators were: (a) 2,3–dihydroxybenzoic + 5-sulfosalicylic acids (which preferentially bind transitional elements at their higher valences; (b) malic and gluconic acids; and (c) histidine. With unconventionally heightened concentrations of Fe, Cu, and Mo (the latter serving as Cu buffer as well as nutrient per se), the hemin concentration could be lowered, widening the margin of safety for heme solubility. Growth then reached 1.4 × 10⁸ cell/ml. This medium may serve to screen for ligands promoting uptake or release of Fe and Cu. The increased growth is a step towards improving the assay medium for biopterin and practical use of Crithidia to assay several B vitamins and essential amino acids for metazoa.     

Induction of macrophage interleukin-1 production by Listeria monocytogenes hemolysin.

Listeriolysin O produced by a hemolytic strain of Listeria monocytogenes was purified from the ammonium sulfate precipitate of a culture supernatant through the steps of ion-exchange chromatography and gel filtration. The purified hemolysin finally gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 58,000. When peritoneal exudate macrophages were stimulated with purified hemolysin, we found a high level of IL-1 activity as determined by thymocyte costimulator assay in the culture supernatant. Cell-associated and intracellular IL-1 activity was also detected. The activity in the supernatant or membrane was blocked by polyclonal antibody to murine IL-1 alpha. Moreover, IL-1-specific mRNA expression could be detected in the macrophages stimulated with listeriolysin O by Northern blot analysis. Possible contamination by LPS of the listeriolysin O preparation did not seem to contribute to the induction of macrophage IL-1 production.     

Paradoxical effects of 17β-estradiol on glucose transport in primary cell cultures of a rat mammary tumor

Primary cell cultures of rat mammary carcinoma R3230AC exhibited a rapid, reversible and dose-related inhibition of carrier-mediated 3-0-methyl-D-glucose transport when estrone, 17β-estradiol, estriol, diethylstilbestrol or phloretin was present during the transport assay. With 17β-estradiol, maximal transport inhibition (66%) was observed at 40μM, a concentration also effective in preventing cell growth when present in the media. Cultures preincubated in growth media containing 5mM glucose plus 40μM 17β-estradiol for one day displayed enhanced rates of carrier mediated 3-0-methyl-D-glucose transport. This increase was prevented by 50mM glucose and can be explained as an adaptive response to a condition simulating glucose starvation.     

Manganese and Defenses against Oxygen Toxicity in Lactobacillus plantarum

Lactobacillus plantarum is aerotolerant during log-phase growth on glucose, but is an obligate aerobe on polyols. Respiration was cyanide resistant and under certain conditions was associated with the accumulation of millimolar concentrations of H(2)O(2). On glucose, optimal growth was observed in the absence of O(2). Extracts of L. plantarum did not catalyze the reduction of paraquat by reduced nicotinamide adenine dinucleotide, but plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was readily reduced. Such extracts produced O(2) (-) in the presence of NADH plus plumbagin. Plumbagin caused a 10-fold increase in the rate of respiration of intact cells in the presence of glucose and also imposed a loss of viability which was dependent upon both glucose and O(2). Although extracts of L. plantarum were devoid of true superoxide dismutase activity, this organism was comparable to superoxide dismutase-containing species in its resistance toward hyperbaric O(2) and toward the oxygen-dependent lethality of plumbagin. L. plantarum required Mn-rich media and actively accumulated Mn(II). Soluble extracts were found to contain approximately 9 mug of Mn per mg of protein and 75 to 90% of this Mn was dialyzable. Such extracts exhibited a dialyzable and ethylenediaminetetraacetic acid-inhibitable ability to scavenge O(2) (-). This O(2) (-)-scavenging activity was due to the dialyzable Mn(II) present in these extracts and could be mimicked by MnCl(2). Cells grown in Mn-rich media were enriched in dialyzable Mn and were more resistant toward oxygen toxicity and toward the oxygen-dependent plumbagin toxicity than were cells grown in Mn-deficient media. L. plantarum exhibited no nutritional requirement for iron and little or no iron was present in these cells, even when they were grown in iron-rich media. L. plantarum thus appears to use millimolar levels of Mn(II) to scavenge O(2) (-), much as most other organisms use micromolar levels of superoxide dismutases.     

Plasminogen Activator Inhibitor-1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose

Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 m M glucose compared with BBEC cultured in media with 5.5 m M glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.     

Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin

Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymatic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells. Being a hydrophilic macromolecule, however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degradation within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g. ricin). These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent. Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells. Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol. In in vitro experiments, co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of approximately 0.1 nM with an extreme efficiency requiring an incubation time of only 1 h. By contrast, cells treated with equivalent concentrations of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity. Moreover, treatment by direct intratumor injection into subcutaneous solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes containing both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.