Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri.

The listeriolysin gene from a weakly hemolytic but virulent strain of Listeria monocytogenes serotype 1/2a was cloned in Escherichia coli K-12. Recombinants were identified on the basis of their cross-reactivities to hyperimmune antisera raised against streptolysin O and listeriolysin. Low levels of hemolytic activity were detected in crude lysates of strains harboring the listeriolysin gene. In DNA hybridization studies with five DNA probes that encoded the listeriolysin gene and surrounding sequences, highly homologous listeriolysin genes were found to be present in the species L. monocytogenes, Listeria ivanovii, and Listeria seeligeri. Immunoblotting performed with affinity-purified antibody to listeriolysin allowed the detection of this protein in supernatants of all three species. This study demonstrates for the first time that listeriolysin is produced by L. seeligeri and documents the genetic homology between the various listeriolysins produced by Listeria spp. Sequences unique to the species L. monocytogenes were found to be located downstream of the listeriolysin gene. Furthermore, the restriction fragment length polymorphisms detected with probes flanking the hlyA gene may be useful epidemiological markers in identifying and distinguishing virulent Listeria strains from each other.     

Effect of Cell Dose and Dose of Infectious Agent on Expression of Protection Against Listeria monocytogenes and Ectromelia Virus in Cell Transfer Models

Two parameters (immune cell dose and dose of infectious agent) influencing the expression of protection by transferred immune spleen cells in Listeria monocytogenes and ectromelia virus infection in mice were investigated. First, when recipient mice were infected with a constant dose of ectromelia virus, a linear relationship between log₁₀ cells transferred and the protection obtained expressed as log₁₀ decrease in virus plaque-forming units per spleen was obtained, as has been described previously for the Listeria system. Second, the detectable protection was greatly affected by the number of viable bacteria or virus plaque-forming units relative to the number of transferred cells. An otherwise very effective number of transferred immune cells became ineffective when too great a dose of infection was used. Mouse strain differences could also have influenced the results. The impact of these and other parameters on the experimental outcome and its interpretation are discussed.     

Oral infection with signature-tagged Listeria monocytogenes reveals organ-specific growth and dissemination routes in guinea pigs

Listeria monocytogenes causes a serious food-borne disease due to its ability to spread from the intestine to other organs, a process that is poorly understood. In this study we used 20 signature-tagged wild-type clones of L. monocytogenes in guinea pigs in combination with extensive quantitative data analysis to gain insight into extraintestinal dissemination. We show that L. monocytogenes colonized the liver in all asymptomatic animals. Spread to the liver occurred as early as 4 h after ingestion via a direct pathway from the intestine to the liver. This direct pathway contributed significantly to the bacterial load in the liver and was followed by a second wave of dissemination via the mesenteric lymph nodes (indirect pathway). Furthermore, bacteria were eliminated in the liver, whereas small intestinal villi provided a niche for bacterial replication, indicating organ-specific differences in net bacterial growth. Bacteria were shed back from intestinal villi into the small intestinal lumen and reinfected the Peyer's patches. Together, these results support a novel dissemination model where L. monocytogenes replicates in intestinal villi, is shed into the lumen, and reinfects intestinal immune cells that traffic to liver and mesenteric lymph nodes, a process that occurs even during asymptomatic colonization.     

Interleukin-15 May Be Responsible for Early Activation of Intestinal Intraepithelial Lymphocytes after Oral Infection with Listeria monocytogenes in Rats

Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-γ) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection with Listeria monocytogenes in rats. In in vitro experiments, invasion of L. monocytogenes into IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation of L. monocytogenes in vivo. The numbers of T-cell receptor (TCR) γδ⁺ T cells, NKR.P1⁺ cells, and CD3⁺ CD8⁺ αα cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-γ upon stimulation with immobilized anti-TCR γδ or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-γ at an early phase of oral infection with L. monocytogenes.     

Induction of the Generalized Shwartzman Reaction in Pregnant and Nonpregnant Rats by Colchicine

The intravenous injection of colchicine (2 mg/kg body weight) into pregnant rats on the last 4 to 5 days of gestation induced disseminated intravascular coagulation, occluding glomerular capillaries with fibrin thrombi, typical of the generalized Shwartzman reaction. Thrombi did not form earlier than 9 hours after the injection of colchicine, whereas in the endotoxin-induced generalized Shwartzman reaction, thrombi were already observed 2½ hours after the injection of endotoxin. The colchicine-induced generalized Shwartzman reaction could also be produced in hysterectomized “pregnant” rats. A single injection of colchicine into nonpregnant rats did not induce disseminated intravascular coagulation. If, however, fibrinolysis was inhibited with ε-aminocaproic acid, the colchicine-induced generalized Shwartzman reaction could also be elicited in nonpregnant rats. In this regard fibrinolysis inhibition represents one mechanism by which pregnancy prepares for the generalized Shwartzman reaction.     

Growth, virulence, and immunogenicity of Listeria monocytogenes aro mutants

Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10(4)-fold for the aroA, aroB, and aroA/B mutants and >10(5)-fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 x 10(6) aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 x 10(8) aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.     

T-cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection

Listeria monocytogenes infection of mice leads to a rapid expansion of activated T cells, followed by a decline in specific cells once the bacteria are eliminated. In order to define the relationship between T-cell proliferation and activation, and to investigate the role of apoptosis in limiting the expansion, the expression of activation markers, uptake of 5-bromo-2'-deoxyuridine (BrdU) in vivo and the incidence of apoptosis was investigated. Increased numbers of T cells expressing the activated phenotype CD25+, CD44hi and CD62Llo were detected 4 days after infection. Expression of CD25 (IL-2Ralpha chain) on CD4+ and CD8+ T cells peaked at this time and returned to normal by day 7. In contrast, CD44hi and CD62Llo persisted, with the maximum proportion occurring at 7 days after infection. This was accompanied by a burst of in vivo proliferation of CD4+ and CD8+ T cells occurring between day 5 and 7. Apoptosis, which is presumably needed to control this expansion of T cells, also peaked at 7 days after infection. Apoptosis occurred preferentially amongst T cells which had proliferated. Most but not all proliferating T cells had down-regulated their CD62L marker. While most apoptotic T cells were CD62Llo, again not all had down-regulated this marker. Hence, CD25 expression peaked early, but expression of other activation markers, in vivo proliferation and apoptosis coincided after Listeria infection. T cells that had proliferated were over-represented in the apoptotic population.     

Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes

Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC betaI and betaII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2, 3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.     

Bacteriological and histopathological evaluation of guinea pigs after infection with Listeria monocytogenes.

Randomly bred guinea pigs were infected with Listeria monocytogenes using the intracardial, intravenous and intraperitoneal routes of infection. Doses of Listeria ranged from 5 to 1,000 x the 50% lethal dose based on the 50% lethal dose for intracardially injected Listeria. A complete necropsy was performed on all animals that died after infection. Gross and microscopic examination of tissues revealed major pathological features which include myocarditis, edema and congestion with interstitial pneumonitis present in the lungs, and fatty hepatic changes with focal necrosis. For all or a majority of the animals, large numbers of Listeria were likewise recovered from these organs and from lymph nodes, spleen, kidneys, and adrenal gland tissue. Of the three routes of infection used, guinea pigs were most susceptible to Listeria injected via the intracardial route. The relatively high lethal dose of listeric for the quinea pig, however, suggests that the organism is a low-grade pathogen for this species.     

Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.     

iactA of Listeria ivanovii, although distantly related to Listeria monocytogenes actA, restores actin tail formation in an L. monocytogenes actA mutant.

A gene homologous to the actA gene of Listeria monocytogenes was cloned from Listeria ivanovii (strain CLIP257) by chromosome walking starting from the ilo gene that encodes the pore-forming toxin ivanolysin. The nucleotide sequence revealed that this gene, named iactA, encodes a protein of 1,044 amino acids (IactA) comprising a central region with seven highly conserved tandem proline-rich repeats of 47 amino acids. Although IactA and ActA share an overall similar structure, these two proteins are only distantly related. Like ActA, IactA migrates aberrantly on sodium dodecyl sulfate gels. When expressed in an L. monocytogenes actA deletion mutant strain, iactA restored actin polymerization.     

Interaction of Listeria monocytogenes with Human Brain Microvascular Endothelial Cells: InlB-Dependent Invasion, Long-Term Intracellular Growth, and Spread from Macrophages to Endothelial Cells

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D₁ macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.     

Disruption of the Cellular Inflammatory Response to Listeria monocytogenes Infection in Mice with Disruptions in Targeted Genes

The results of this study to dissect the nature of the acquired immune response to infection with Listeria monocytogenes in mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of αβ T cells and the capacity to elaborate gamma interferon. In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days. The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; αβ-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in γδ-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower. In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the γδ-T-cell-knockout animals. These data further support the hypothesis that γδ T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.     

The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.     

Oral inoculation of A/J mice for detection of invasiveness differences between Listeria monocytogenes epidemic and environmental strains

Four-week-old Harlan A/J mice were orally infected with six epidemic and six environmental strains of Listeria monocytogenes. Epidemic strains were significantly more invasive as a group than were environmental strains (P < 0.05), and the intestines of some mice infected with epidemic strains had extensive hemorrhage. Mice inoculated with epidemic strains were significantly more likely to become systemically infected than mice inoculated with environmental strains (P < 0.01).     

LipA, a tyrosine and lipid phosphatase involved in the virulence of Listeria monocytogenes

Intracellular bacterial pathogens manipulate host cell functions by producing enzymes that stimulate or antagonize signal transduction. The Listeria monocytogenes genome contains a gene, lmo1800, encoding a protein with a conserved motif of conventional tyrosine phosphatases. Here, we report that the lmo1800-encoded protein LipA is secreted by Listeria and displays tyrosine as well as lipid phosphatase activity in vitro. Bacteria lacking LipA are severely attenuated in virulence in vivo, thus revealing a so-far-undescribed enzymatic activity involved in Listeria infection.     

Impaired Bactericidal Activity and Host Resistance to Listeria monocytogenes and Borrelia burgdorferi in Rats Administered an Acute Oral Regimen of Ethanol

A rat model was used to examine how ethanol ingestion may interfere with antimicrobial immunity both in vitro and in vivo. Nonimmune Long-Evans rats were given a short-course treatment orally with excessive amounts of ethanol. Their spleens were removed at the time of sacrifice, and separate spleen cell suspensions were prepared and tested in vitro for their ability to kill two bacterial pathogens, Listeria monocytogenes and Borrelia burgdorferi. After the bacteria were mixed separately with various concentrations of spleen cells, it was found that spleen cells from the ethanol-treated rats killed fewer bacteria than matching pair-fed controls, based on counts of the number of cultured CFU (for Listeria) or based on microscopic examination (for Borrelia). For the in vivo studies, ethanol-treated and control rats were infected intraperitoneally with Listeria, and then, 1 to 3 days later, they were assessed for systemic infection based on the numbers of organisms present in their livers and spleens. Numbers of bacterial CFU for both organs were significantly higher in the group fed ethanol for the first 2 days after listerial challenge. These results support the concept that acute exposure to high levels of ethanol can impair host defense mechanisms, especially those expressed at the cellular level, which could lead to increased susceptibility to certain types of infections.