Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in ß- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

Live tularemia vaccine confers protection against lethal Legionella and Listeria infections in experimental animals.

The efficacy of a live Francisella tularensis vaccine strain to cause nonspecific immunity toward experimental legionellosis and listeriosis was studied. Immunisation with tularemia vaccine protected over 80% and 17% of experimental animals against subsequent lethal challenge with Legionella pneumophila and Listeria monocytogenes, respectively. The protection was maximal during the first month following immunisation and declined thereafter. In order to delineate the immunostimulatory moieties of the Francisella microbe, several cell wall proteins have been purified and characterized. However, isolated cell wall components failed to induce protection.

A simple colony-blot method for identification of Listeria in food samples.

A method for identification of Listeria in food samples was developed. It consisted of cultivating of suspected specimen on standard agar medium, direct absorption of grown colonies onto nitrocellulose membrane and processing of the latter with rabbit serum raised against purified cell wall protein Lm79/39 of L. monocytogenes. Analysis using anti-rabbit peroxidase conjugate and 4-chloro-1-naphthol and H2O2 solutions allowed direct detection of Listeria colonies which remained readily available for subsequent isolation.

Live tularemia vaccine but not proteins purified from Francisella tularensis can confer protection against lethal Listeria infection in mice.

Immunization of Balb/c mice with Francisella tularensis vaccine strain 15/10 conferred significant protection against subsequent listerial infection. Since immunostimulatory activities could apparently be relevant to surface components of the bacterium, a technique for purification of cell wall proteins was developed. The scheme designed consisted of Triton X-100 extraction with subsequent FPLC chromatography steps, and resulted in the isolation of homogeneous proteins with molecular masses of 54 kDa (pI = 6.7) and 82 kDa (pI = 5.3), and partially purified 50, 85 and 100 kDa components. It was shown that immunization with isolated proteins failed to protect mice against lethal Listeria monocytogenes infection. Possible reasons for failure are discussed.

Intracellular parasitism of Legionella and signaling in eukaryotic cells.

In this review the phenomenon of antibacterial activity of phagocytes and its relationship to the general system of signal transduction in eukaryotic cells is examined. Data are reviewed that support the hypothesis that interference in transduction of regulatory signals during microbial invasion could be caused by specific bacterial products and lead to an inadequate bactericidal response, and hence multiplication of the bacteria in a host. In this connection, information concerning virulence factors of the facultative intracellular parasite Legionella is discussed.

Purification and characterization of a 58-kDa cell wall-associated protein from Listeria monocytogenes.

A cell wall protein (P58) was purified from Listeria monocytogenes by detergent extraction and Superose 6 gel chromatography. It had a molecular mass of 58 kDa, was strongly hydrophobic, contained reactive thiol group(s) and was located at least partially on the surface of bacterial cells. Production of this protein varied among different Listeria, being the most prominent in NCTC 7973 of L. monocytogenes, weaker in four other strains of this species and undetectable in tested strains of L. seeligeri and L. innocua. Mice that survived experimental listerial infection produced antibodies against P58. This fact allowed us to speculate that the described protein can be used as a marker for sero-diagnosing of listeriosis.

Purification and characterization of cell wall proteins of Listeria monocytogenes.

Two proteins were purified from Listeria monocytogenes cell wall using detergent extraction, Superose 6 gel chromatography. Mono Q cation-exchange chromatography and Superose 12 gel chromatography. Proteins were shown to form complexes of ca. 300 kDa and pI 4.7. These complexes could not be dissociated in 6 M urea, however, during SDS-PAGE 79 and 39 kDa monomers were formed. Immunoblot analysis showed that the proteins under investigation were common to all listerial strains tested, but were absent in strains of other bacteria. We propose that the proteins investigated here could serve as immunoserological markers for Listeria.

Induction of the immune response to Legionella pneumophila cytolysin by monoclonal anti-idiotypic antibodies.

A panel of mAb (IgG1, IgG3, IgM) against Legionella pneumophila cytolysin (CL)-protease of 37 kDa was obtained. Subtyping of L. pneumophila strains of serogroup 1 by using mAb against CL (mAb-CL) was carried out. The results of comparative analysis of the specificity of mAb-CL and the panel of mAb kindly provided by Dr. J. M. Barbaree (Centers for Disease Control, Atlanta, GA) allowed us to recommend mAb-CL to be used as a diagnostic tool to reveal the pathogenicity of L. pneumophila strains of serogroup 1. Hybridomas were also raised in a syngenic system which produced anti-idiotypic mAb (mAb2) against anti-CL mAb B6/1. The Ab2 belonged to Ab2 gamma type: 1) Ab2 reacted with B6/1 Id only, 2) Ab2 inhibited the interaction of B6/1 Ab1 with CL, and 3) CL inhibited the reaction of Ab2 with Ab1. The use of Ab2 allowed us to show that B6/1 Id is expressed in 4 to 32% of serum antibodies during the primary and secondary immune responses of BALB/c mice to CL. Ab2 induced the production of anti-anti-idiotypic antibodies (Ab3) in BALB/c mice, and some of them reacted with CL. Thus, we have demonstrated the possibility of inducing an antibody response to CL (one of the main L. pneumophila pathogenic factors) in intact syngenic mice with anti-idiotypic antibodies.