• Journal of Microbiology and Biotechnology
• /
• v.21 no.11
• /
• pp.1193-1198
• /
• 2011

Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.1
• /
• pp.122-135
• /
• 2018

Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of $CD8^+$ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.

• Korean Journal of Food Science and Technology
• /
• v.31 no.6
• /
• pp.1628-1634
• /
• 1999

The polymerase chain reaction (PCR) for the sensitive and specific detection of Listeria monocytogenes was employed by using LM 1 and LM 2 primers which were based on the listeriolysin O gene. The direct use of cell suspension as DNA template, without DNA extraction or lysis step, was suitable and specific enough to detect L. monocytogenes at the level of $10^2$ CFU or less per PCR for the pure culture and milk sample, however, the detection sensitivity became blunt for other food samples such as kimchi and chicken. The nested PCR, in which L-1 and L-2 (both designed from listeriolysin O gene) were employed as inner primers, was specific for detecting L. monocytogenes and enhanced the detection limit by 10 times. The PCR using LM 1 and LM 2 primers was very effective to detect L. monocytogenes from foods in terms of the specificity and time consumed, i. e. within $4{\sim}8\;hrs$ (nested PCR).

• IMMUNE NETWORK
• /
• v.5 no.3
• /
• pp.172-178
• /
• 2005

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and trans activator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. Methods: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-${\gamma}$ producing T lymphocytes were measured. Results: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-${\gamma}$ secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+T cell to $CD4^+$ cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. Conclusion: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.

• Korean Journal of Veterinary Research
• /
• v.43 no.2
• /
• pp.231-237
• /
• 2003

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.4
• /
• pp.398-403
• /
• 1999

In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase(AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua ($prifA^+hly^+$) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua ($prifA^{+}hly{^}+$) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).