Cardiotoxic and Lethal Effects of Listeria monocytogenes Hemolysin

Cardiotoxic and lethal effects of Listeria monocytogenes hemolysin were studied in CD-1 mice injected with varying doses of hemolysin. Intravenous injection of 100 complete hemolytic units (CHU) caused 100% lethality within 4 to 5 min. Doses ranging from 40 to 50 CHU caused death of approximately 50% of the animals. Adrenergic blocking agents and antihistamine failed to protect mice against lethality and thereby suggested that death was not due to release of vasoactive agents by hemolysin. Plasma levels of creatine phosphokinase increased after intravenous administration of hemolysin and suggested myopathy, possibly of the myocardium. Electrocardiograms from hemolysin-treated mice indicated serious alterations in heart rate and rhythm, suggesting damage to contractile and pacemaker cardiac tissue. In addition, there were indications of increased potassium levels influencing the heart. Presumably, death was due to functional damage to heart muscle and electrical arrest. The cardiotoxic and lethal effects could be prevented by prior incubation of hemolysin with cholesterol, heating, or failure to reactivate the preparation with cysteine.     

Biochemical and Immunological Effects of Listeria monocytogenes Hemolysin

The biochemical and immunological effects of Listeria monocytogenes hemolysin in CD-1 mice were studied. Intraperitoneal injection of 256 complete hemolytic units (CHU) caused a twofold increase in plasma β-glucuronidase levels but was not lethal. In contrast, 256 or more CHU caused 100% lethality in 4 to 5 min when administered intravenously. Intravenous administration of 50 CHU caused a 10- to 11-fold increase in plasma β-glucuronidase levels and was lethal for a variable percentage of the animals. Carbon clearance experiments showed the phagocytic index to be depressed by relatively small amounts of intravenously administered hemolysin and suggested that hemolysin may function as a leucocidal agressin during listeric infection. Increased plasma levels of ornithine carbamyltransferase after intravenous injection of hemolysin indicated hepatocellular damage. Liver carbohydrate and blood glucose determinations on fasted mice showed a reduced gluconeogenic capability in hemolysin-treated animals. Mice immunized with purified hemolysin or live vaccine were more resistant to several of the toxic parameters studied. The data indicate that hemolysin is produced during listeric infection and is antigenic, but not necessarily a protective immunogen.     

Gentamicin kills intracellular Listeria monocytogenes.

The purpose of the experiments described here was to test whether membrane-impermeant antibiotics present in the extracellular milieu could kill bacteria within macrophages. For this, mouse macrophage hybrids and elicited mouse peritoneal macrophages first were allowed to phagocytose the facultative intracellular bacterium Listeria monocytogenes. The cells were incubated with or without gentamicin, and their bactericidal activity was measured. The results show that gentamicin caused normally nonbactericidal macrophages to kill L. monocytogenes. In addition, gentamicin caused listericidal cells to kill significantly more bacteria. To determine whether gentamicin accumulated within macrophages during culture, we tested whether lysates of macrophage hybrids cultured for 72 h in gentamicin-containing medium and then washed could kill Listeria cells. When cultured with 50 to 100 micrograms of gentamicin per ml, but not when cultured with 0 to 5 micrograms of gentamicin per ml, cell lysates were extremely listericidal, demonstrating the presence of intracellular gentamicin. Because gentamicin does not penetrate cell membranes, we hypothesized that it can be internalized by the cell through pinocytosis and can enter the same intracellular compartment as does phagocytosed L. monocytogenes. To test this, macrophages which had phagocytosed L. monocytogenes were incubated with the fluorochrome lucifer yellow to trace pinocytosed medium. About half of the Listeria cells within the macrophages were surrounded by lucifer yellow, indicating delivery of pinocytosed fluid, which could contain antibiotics, to phagosomes containing bacteria. The experiments described here indicate that membrane-impermeant antibiotics can enter macrophages and kill intracellular bacteria. Thus, the use of gentamicin in macrophage bactericidal assays can interfere with the results and interpretation of experiments designed to study macrophage bactericidal activity.     

Flavonoid treatment of murine macrophages does not alter ingestion or intracellular survival of Listeria monocytogenes

It was recently reported that the aryl hydrocarbon receptor (AhR) plays a role in innate immunity to Listeria monocytogenes infection in mice. In this study the authors analyzed whether incubation of murine macrophages with various flavonoids that have agonist or antagonist activity for the AhR (beta-naphthoflavone, alpha-naphthoflavone, quercetin, luteolin) affect the uptake and survival of L. monocytogenes. Using the RAW264.7 and J774 murine macrophage cell lines and bone marrow-derived macrophages, the authors found no significant difference between flavonoid-treated and control macrophages. They also found that macrophages incubated with flavonoids alone did not exhibit a significant increase in release of tumor necrosis factor (TNF)-alpha, a crucial cytokine in anti-Listeria resistance.     

Protection of mice against the intracellular bacterium Listeria monocytogenes by recombinant immune interferon

Immune interferon, available at high specific activity through recombinant DNA technology, is known to activate macrophages to intra- and extracellular cytotoxicity. We now report that murine recombinant IFN-gamma activates macrophages to cytotoxicity also when applied in vivo. Furthermore, recombinant IFN-gamma can protect mice in vivo against the intracellular bacterial pathogen Listeria monocytogenes in a local as well as in a systemic infection model. The role of T lymphocyte-produced lymphokines in acquired resistance to facultative intracellular pathogens and their possible involvement in novel immunotherapy are discussed.     

Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua.

Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow intracellularly in what appear to be phagolysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a secondary function associated with the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type state does not replicate intracellularly apparently can now allow some of the bacteria to transiently multiply inside the phagosomes of host macrophage cells.     

Mouse macrophages stimulated by recombinant gamma interferon to kill tumor cells are not bactericidal for the facultative intracellular bacterium Listeria monocytogenes.

Data presented here demonstrate that recombinant gamma interferon (rIFN-gamma) activated a single population of 10% fetal calf serum-elicited mouse peritoneal exudate cells to express tumoricidal activity but not bactericidal activity for the facultative intracellular bacterium Listeria monocytogenes. Fetal calf serum-elicited cells incubated with rIFN-gamma phagocytosed listeriae normally, suggesting that their inability to kill this bacterium is not because they cannot phagocytose it. Data also show that proteose peptone-elicited peritoneal exudate cells, which are bactericidal but not tumoricidal, acquired tumoricidal activity but lost bactericidal activity following incubation overnight with rIFN-gamma. These experiments show that under conditions sufficient for rIFN-gamma to induce macrophages to express tumoricidal activity, the same cell population does not express bactericidal activity for the facultative intracellular bacterium L. monocytogenes. This suggests that mechanisms responsible for these two biological activities may be different.     

Effects of Listeria monocytogenes Hemolysin on Phagocytic Cells and Lysosomes

The effects of Listeria monocytogenes hemolysin on lysosomes and phagocytic cells were investigated. Hemolysin caused release of β-glucuronidase and acid phosphatase from suspensions of rabbit and rat lysosomes prepared from liver homogenates. The degree of lysis was proportional to the concentration of hemolysin added. There appeared to be no significant difference between the sensitivities of rat and rabbit lysosomes to disruption. Studies on the effect of pH and temperature on lytic activity suggested that hemolysin could function under conditions which might exist within phagocytic cells. Peritoneal exudates from rabbits and mice were exposed to hemolysin and observed by phase microscopy. Hemolysin possessed leucocidal activity and caused degranulation of both rabbit and mouse cells. Optimal activity against lysosomes and peritoneal exudate cells required activation of hemolysin with a reducing agent and could be prevented if hemolysin was previously incubated with cholesterol.     

Hemolysin is required for extraintestinal dissemination of Listeria monocytogenes in intragastrically inoculated mice.

In this study we demonstrated that a hemolytic strain of Listeria monocytogenes, but not a nonhemolytic mutant derived from it, translocated in substantial numbers to the mesenteric lymph nodes, spleen, and liver after intragastric inoculation of mice. Growth at 4 degrees C prior to inoculation did not increase the virulence of the nonhemolytic mutant. These results indicate that hemolytic activity is required for the virulence of L. monocytogenes via the gastrointestinal tract, as has been shown previously for parenteral challenge.     

Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium.

The extracellular protein p60 is a major virulence factor of the intracellular bacterium Listeria monocytogenes. Its roles in pathogen survival in vivo and host cell invasion in vitro were studied. To this end, Salmonella typhimurium SL7207 was used as carrier for secreted p60-HlyA fusion protein by Escherichia coli HlyB and HlyD transport proteins. C57BL/6 mice infected intravenously with this strain suffered from increased bacterial numbers in livers and spleens compared with the p60-nonexpressing control strain, but only transiently. In vitro experiments showed that p60 promotes invasion of recombinant S. typhimurium SL7207 p60 into hepatocytes and resting macrophages independent from complement. Moreover, the uptake of wild-type L. monocytogenes EGD and L. monocytogenes BUG 8, an internalin-deficient strain, into hepatocytes was partially blocked by anti-p60 antibodies. The impaired invasion of dissociated bacterial chains of L. monocytogenes RIII, a p60 expression mutant, into hepatocytes and macrophages was partially restored by addition of p60- or p60-HlyA-enriched bacterial supernatants. These data suggest that the L. monocytogenes surface-associated proteins, p60 and internalin, act in concert to achieve optimal uptake into nonprofessional phagocytes and macrophages. Together, these experiments reveal a substantial impact of p60 on cell invasion and virulence and thus emphasize the importance of the intracellular habitat for survival of L. monocytogenes in the host.     

Expression of the inlAB operon by Listeria monocytogenes is not required for entry into hepatic cells in vivo.

Listeria monocytogenes injected intravenously into mice is taken up in the liver, where hepatocytes serve as the principal site of intracellular replication. The factors effecting entry of L. monocytogenes into hepatic cells remain to be determined. Others have shown that the protein products of the inlAB (internalin) operon are required for maximum entry of L. monocytogenes into a number of cell lines in vitro. Likewise, we report here that expression of the inlAB operon was required for maximum uptake of L. monocytogenes by primary cultures of mouse hepatocytes. Uptake of an inlAB mutant strain of L. monocytogenes was approximately 10-fold less than that of the isogenic wild-type control. In contrast, inlAB expression was not a factor in (i) clearance of L. monocytogenes injected intravenously into mice and taken up in the liver, (ii) the distribution of L. monocytogenes among hepatocytes and nonparenchymal cells in the liver, or (iii) internalization of L. monocytogenes by hepatic cells in vivo. These latter findings suggest that infection of hepatic cells by L. monocytogenes in vivo does not require the protein products of the inlAB operon.     

Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria.

The intracellular survival of the ubiquitous pathogen Listeria monocytogenes was studied in primary cultures of bone marrow-derived mouse macrophages. Bacteria were able to grow rapidly in these cells, with an apparent multiplication rate of about 40 min. Electron microscopy demonstrated that intracellular bacterial replication was the consequence of simultaneous intracellular killing and replication of bacteria in the same cells. Within the first hour following phagocytosis, most bacteria were destroyed in the phagosomal compartment to which they were confined. This was due to early transfer of hydrolytic enzymes to phagosomes, undoubtedly via phagosome-lysosome (P-L) fusion, as demonstrated by a quantitative analysis after staining for a lysosomal marker, acid phosphatase. One hour after infection, about 14% of the bacteria were free in the cytoplasm, in which they multiplied and induced actin polymerization and spreading to adjacent macrophages, as in epithelial cells. By using the 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining procedure, direct evidence is presented that all phagosomes were acidified immediately after phagocytosis, thus indicating that intraphagosomal bacteria were exposed to an acidic environment that might favor vacuolar lysis by listeriolysin O. Intracellular growth in macrophages, therefore, appears to be the result of a competition between the expression of the hydrolytic activity of these cells following P-L fusion and the capacity of L. monocytogenes to escape from the acidified phagosomal compartment before P-L fusion has occurred. The finding that concomitant intracellular killing and survival of L. monocytogenes occurs in the same macrophages might explain the high immunogenicity observed in vivo with live bacteria, as opposed to killed bacteria.     

A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin.

Listeria monocytogenes has the capacity to penetrate and multiply within professional and nonprofessional phagocytic cells, such as the Caco-2 human enterocytelike cell line. It was shown recently that shortly after listeriae have been phagocytosed, the phagosomal membrane is dissolved, probably by the action of the bacterial cytolysin listeriolysin O. The listeriae, which are then lying obviously free in the cytoplasm, become surrounded by a coat of actin filaments within a few hours. Once formed, this layer of actin filaments is reorganized in an as yet unknown way to form polar tails, which seem to be associated to the generation of listerial movement inside the cytoplasm and in intercellular spread. By using transposon Tn916 mutagenesis, a bank of L. monocytogenes mutants was generated and subsequently screened by the plaque assay system in order to select an intracellular, nonmotile mutant of L. monocytogenes. One such mutant was identified. This mutant, called L. monocytogenes M117 Imt- (for intracellular motility), like the wild type, induced actin polymerization but was not able to rearrange the actin coat to generate movement and as a result remained entrapped within the actin cloud. In a mouse virulence assay, this strain was significantly reduced in virulence. L. monocytogenes M117 is the first example to date of a Listeria mutant which is still hemolytic and invasive but reduced in virulence.     

Ultrastructural study of Listeria monocytogenes entry into cultured human colonic epithelial cells.

Evidence that Listeria monocytogenes enters Caco-2 cells through the apical surface is presented. Attachment of bacteria to host cells seems to induce modifications of microvilli which are either in direct contact with the bacterial surface or in close vicinity, resulting in the formation of lamellipodia involved in the cellular uptake of the bacteria. Such modifications are not induced by L. monocytogenes SLCC 53, which carries a deletion in the prfA gene, although attachment of this mutant to Caco-2 cells occurs. Listeria innocua does not attach well to Caco-2 cells and also fails to cause structural alterations of the microvilli. Treatment of confluent monolayers of Caco-2 cells with ethylene glycol-bis(beta-aminoethyl ether)- N,N,N1,N1-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly reduced the uptake of Listeria cells. Attachment and invasion of L. monocytogenes was not accompanied by accumulation of filamentous actin around the entering bacterial cell.     

Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.     

Growth at reduced temperatures increases the virulence of Listeria monocytogenes for intravenously but not intragastrically inoculated mice

Growth of three clinical isolates (Scott A, Murray B, and F5380) and one laboratory strain (EGD) of L. monocytogenes at 4 degrees C significantly increased their virulence for intravenously injected mice. Using the EGD strain for subsequent experiments, we determined that growth at either 4 degrees or 22 degrees C enhanced the growth of listeria in the spleen and liver. Similar numbers of listeriae were recovered from the spleens and livers of mice during the first 48 h after i.v. injection of strain EGD grown at 37 degrees C or 4 degrees C. At later timepoints (3-6 days), significantly more listeriae were recovered from the spleens and livers of mice injected i.v. with strain EGD grown at 4 degrees C. In contrast, L. monocytogenes EGD grown at 37 degrees C and 4 degrees C demonstrated similar abilities to survive in the gastrointestinal tract, to translocate to the mesenteric lymph nodes, and to disseminate to the spleen and liver in intragastrically inoculated mice. Listeria monocytogenes EGD grown at 4 degrees C released less hemolysin into the culture medium than did this strain when grown at 22 degrees C and 37 degrees C. Transfer to fresh broth and incubation at 37 degrees C for 2 h increased the release, to similar levels, of hemolysin from L. monocytogenes EGD grown at 4 degrees, 22 degrees, and 37 degrees C. Temperature-induced differences in virulence, therefore, may not reflect the amount of hemolysin released.