Listeria monocytogenes infections in Canada

Between 1951 and January 1972 listeriosis was diagnosed bacteriologically in 101 Canadian patients. This study adds 80 cases to the 21 reported from Metropolitan Toronto by Sepp and Roy in 1963. The Laboratory Centre for Disease Control, Ottawa, collated epidemiological and clinical data. Serotypes of Listeria monocytogenes included 4b (53), 1 (15), 1b (6), 1a (2), 2 and 3. Clinically, 54 patients had meningitis and 23 septicemia. The mortality rate was 30%.Between 1954 and January 1972 listeriosis affected 15 British Columbian patients: nine were male and six female; 12 were less than 1 or more than 45 years old. Among the patients were a pregnant mother and the son to whom she gave premature birth. A day-old infant and an elderly man died.     

Macrophage intracellular signaling induced by Listeria monocytogenes

Macrophages are critical for control of Listeria monocytogenes infections; accordingly, the interactions of L. monocytogenes with these cells have been intensively studied. It has become apparent that this facultative intracellular pathogen interacts with macrophages both prior to entry and during the intracellular phase. This review covers recent work on signaling induced in macrophages by L. monocytogenes, especially intracellular signals induced by secreted proteins including listeriolysin O and two distinct phospholipases C.     

Oxidative and phagocytic functions of macrophages during infections induced in mice by Mycobacterium intracellulare and Listeria monocytogenes

The oxidative metabolism (chemiluminescence and H2O2 release) and phagocytic activity of mouse peritoneal macrophages during chronic infections induced by Mycobacterium intracellulare and more acute infections due to Listeria monocytogenes were studied. In M. intracellulare infections, macrophage chemiluminescence in response to phorbol myristate acetate (PMA) was greatest at around 2 weeks, with a 1 week lag phase after infection, while the PMA-triggered H2O2 release was markedly enhanced even 1 d after challenge, and remained high thereafter for up to 10 weeks. The pattern of changes in the phagocytic activity of host macrophages in response to latex beads during this infection resembled the pattern seen with macrophage H2O2 release. In the L. monocytogenes infections, the PMA-triggered chemiluminescence of the host macrophages increased 4 d (in a sublethal infection) and 2 d (in a lethal infection) after bacterial challenge, whereas the PMA-triggered H2O2 release was markedly enhanced as early as 1 d after infection and the elevated level persisted until either the bacteria were eliminated or the animals died. The patterns of changes in phagocytic activity of the host macrophages during L. monocytogenes infection at sublethal and lethal doses differed. In the former, phagocytosis was most active in the early phase of infection, with a peak around day 2, followed by a rapid decrease; in the latter, the phagocytic ability increased more slowly, and remained elevated until the animals died. The results suggest that the macrophages induced by M. intracellulare are in a more activated state than are those induced by L. monocytogenes.     

Salt stress proteins induced in Listeria monocytogenes

The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).     

Protective immunity induced by a LLO‐deficient Listeria monocytogenes

Listeria monocytogenes is a food‐borne pathogen able to cause serious disease in human and animals. Listeriolysin O (LLO), a major virulence factor secreted by this bacterium, is a vacuole‐specific lysin that facilitates bacterial entrance into the host cytosol. Thus, LLO plays a key role in the translocation and intracellular spread of L. monocytogenes. To study the effect of LLO on virulence and immunopotency, a LLO‐deficient L. monocytogenes mutant was constructed using a shuttle vector followed by homologous recombination. The mutant strain had lost hemolytic activity, which resulted in an extremely reduced virulence, 5 logs lower than that of the parent strain, yzuLM4, in BALB/c mice. The number of bacteria detected in the spleens and livers of mice infected with the mutant was greatly reduced, and the bacteria were rapidly eliminated by the host. Kinetics studies in this murine model of infection showed that the invasion ability of the mutant strain was much lower than that of the parent strain. Moreover, immunization with the mutant strain conferred protective immunity against listerial infection. In particular, stimulation with Ag85B_240‐259, strong specific Th1 type cellular immunity was elicited by vaccination C57BL/6 mice with hly deficient strain delivering Mycobacterium tuberculosis fusion antigen Ag85B‐ESAT‐6 via intravenous inoculation. These results clearly show that highly attenuated LLO‐deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens.     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.     

Extending the host range of Listeria monocytogenes by rational protein design

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. I. Activation of Listeria antigen-responsive T cells

Soon after rats are infected with Listeria monocytogenes (LM), Listeria antigen- (LMA) responsive lymphocytes are delivered to the animal's thoracic duct. These LM-responsive lymphocytes can be restimulated in vitro by LMA to generate cells that have a potent cytolytic capability. The activation of LMA responsive lymphocytes is immunologically specific and dependent upon the activity of histocompatible accessory cells. Neither cell-free LMA nor LMA-pulsed allogeneic accessory cells can promote a significant cytotoxic response by negatively selected responder lymphocytes. LM-dependent cytolytic lymphocytes differ from natural killer (NK) cells inasmuch as their activation is not facilitated by interferon (IF). Likewise, supernatants from cultures containing specifically sensitized thymus-derived (T) lymphocytes and histocompatible LMA-pulsed accessory cells fail to augment (day 2 and day 3 supernatants actually inhibit) the activation process. The results imply that the successful activation of LM dependent cytolytic lymphocytes requires the cooperative interplay of responder T cells and specific-antigen-pulsed accessory cells.     

Species specificity of the Listeria monocytogenes InlB protein

InlA and InlB mediate L. monocytogenes entry into eukaryotic cells. InlA is required for the crossing of the intestinal and placental barriers. InlA uses E-cadherin as receptor in a species-specific manner. The human E-cadherin but not the mouse E-cadherin is a receptor for InlA. In human cells, InlB uses Met and gC1qR as receptors. By studying the role of InlB in vivo, we found that activation of Met by InlB is species-specific. In mice, InlB is important for liver and spleen colonization, but not for the crossing of the intestinal epithelium. Strikingly, the virulence of a DeltainlB deletion mutant is not attenuated in guinea pigs and rabbits. Guinea pig and rabbit cell lines do not respond to InlB, although expressing Met and gC1qR, but support InlB-mediated responses upon human Met gene transfection, indicating that InlB does not recognize or stimulate guinea pig and rabbit Met. In guinea pig cells, the effect of human Met gene transfection on InlB-dependent entry is increased upon cotransfection with human gc1qr gene, showing the additive roles of gC1qR and Met. These results unravel a second L. monocytogenes species specificity critical for understanding human listeriosis and emphasize the need for developing new animal models for studying InlA and InlB functions in the same animal model.     

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro

Background

Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.

Methodology/Principal Findings

The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp^LAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp^LAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp^LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp^LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp^LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.

Conclusions/Significance

Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp^LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.     

Transport of glycine-betaine by Listeria monocytogenes

Uptake of [¹⁴C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4–0.5 M. The glycine-betaine transport system had a K _m of 22 M and a V _max of 11.7 nmol^-1 min^-1 mg^-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the V _max increased to 27.0 nmol^-1 min^-1 mg^-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K⁺ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.     

Mechanism of polarization of Listeria monocytogenes surface protein ActA

The polar distribution of the ActA protein on the surface of the Gram-positive intracellular bacterial pathogen, Listeria monocytogenes, is required for bacterial actin-based motility and successful infection. ActA spans both the bacterial membrane and the peptidoglycan cell wall. We have directly examined the de novo ActA polarization process in vitro by using an ActA-RFP (red fluorescent protein) fusion. After induction of expression, ActA initially appeared at distinct sites along the sides of bacteria and was then redistributed over the entire cylindrical cell body through helical cell wall growth. The accumulation of ActA at the bacterial poles displayed slower kinetics, occurring over several bacterial generations. ActA accumulated more efficiently at younger, less inert poles, and proper polarization required an optimal balance between protein secretion and bacterial growth rates. Within infected host cells, younger generations of L. monocytogenes initiated motility more quickly than older ones, consistent with our in vitro observations of de novo ActA polarization. We propose a model in which the polarization of ActA, and possibly other Gram-positive cell wall-associated proteins, may be a direct consequence of the differential cell wall growth rates along the bacterium and dependent on the relative rates of protein secretion, protein degradation and bacterial growth.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

Utilization of transferrin-bound iron by Listeria monocytogenes

Abstract It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes . This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.