Identification of genes encoding glycosyltransferases involved in lipopolysaccharide synthesis in Porphyromonas gingivalis.

Porphyromonas gingivalis can synthesize both A-LPS and O-LPS lipopolysaccharides, which contain anionic O-polysaccharides and conventional O-polysaccharides, respectively. A-LPS can anchor virulence proteins to the cell surface, so elucidating the mechanism of A-LPS synthesis is important for understanding the pathogenicity of this bacterium. To identify the genes involved in LPS synthesis, we focused on uncharacterized genes encoding the glycosyltransferases, PGN_0361, PGN_1239, PGN_1240 and PGN_1668, which were tentatively named gtfC, gtfD, gtfE and gtfF, respectively, and characterized their mutants. We found that disruption of gtfC and gtfF resulted in A-LPS deficiency. In addition, a gtfD mutant had abnormal A-LPS synthesis, and a gtfE mutant exhibited a rough-type LPS that possesses a short oligosaccharide with lipid A-core. We then constructed a gtfC and gtfD double mutant, because their amino acid sequences were very similar, and this mutant similarly possessed a rough-type LPS. Cross-complementation analysis revealed that the GtfD protein is a functional homologue of the Escherichia coli WbbL protein, which is a rhamnosyltransferase. These results suggested that the GtfE protein is essential for the synthesis of both O-LPS and A-LPS, and that GtfC and GtfD proteins may work together to synthesize the two kinds of LPS. In addition, the GtfF protein was essential for A-LPS synthesis, although this may be achieved in a strain-specific manner.

Mycobacterium bovis bacillus calmette-guérin induces protective immunity against infection by Plasmodium yoelii at blood-stage depending on shifting immunity toward Th1 type and inducing protective IgG2a after the parasite infection.

Bacillus calmette-guérin (BCG)-vaccination raised dramatically the survival rates of A/J mice from infection by Plasmodium yoelii 17XL at blood-stage. The analysis of the immune response of spleen cells indicated that BCG vaccination biased the immune response toward Th1 type. Neutralization of IFN-gamma and nitric oxide abrogated the protection. The kinetics of Ab production in the course of P. yoelii 17XL infection was monitored. Surprisingly, larger amounts of parasite-specific Abs were produced in BCG-vaccinated mice than in the placebo control. The vast majority of the produced IgG against parasites in BCG-vaccinated mice was IgG2a, which was observed hardly in placebo controls. The peak of IgG2a production coincided with the clearance of infection. The naive mice transferred adoptively with IgG2a from self-cured mice survived the lethal challenge from the parasite. These data indicated that BCG vaccination protected A/J mouse from P. yoelii 17XL infection by biasing immunity toward Th1-type after parasite infection and enhancing production of IgG2a, which ultimately played a major role in protection.

The gene encoding mycobacterial DNA-binding protein I (MDPI) transformed rapidly growing bacteria to slowly growing bacteria.

Pathogenic species of Mycobacterium are slowly growing intracellular bacteria. Slow growth is important for the parasitism of these organisms and chronicity of the disease, but its precise mechanism has not been elucidated. Recently, we found that a novel DNA-binding protein (MDPI) was expressed (7-10% in total protein) in mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guérin, Mycobacterium tuberculosis, and Mycobacterium leprae. In this study, we observed that MDPI interfered with replication, transcription, and translation in the analysis in in vitro E. coli cell-free macromolecular biosynthesizing systems. Furthermore, MDPI inhibited the rapid growth of both Escherichia coli and Mycobacterium smegmatis, and NH(2)-terminal second amino acid, asparagine, was observed to be important in terms of this function. These data suggest an important role of MDPI for suppression of growth rates of mycobacteria.

Long-lasting protective immunity against rodent malaria parasite infection at the blood stage by recombinant BCG secreting merozoite surface protein-1.

Previously, we constructed a recombinant live BCG (rBCG) secreting a 15 kDa C-terminal region of MSP-1 from Plasmodium yoelii (MSP-1(15)) and succeeded in the induction of more efficient protective immunity against parasite infection than observed with artificial adjuvants (Matsumoto S, Yukitake H, Kanbara H, Yamada T. Recombinant Mycobacterium bovis bacillus Calmette-Guerin secreting merozoite surface protein 1 (MSP-1) induces protection against rodent malaria parasite infection depending on MSP-1-stimulated interferon gamma and parasite-specific antibodies. J Exp Med 1998;188:845-54 [1]). In this study, we examined the endurance of the protective effects. The protective effect generated by rBCGMSP-1(15) was observed even 9 months after final immunization, whereas the effects of immunization by MSP-1(15) together with incomplete Freund adjuvant (IFA) were found to last only 4 months.

Identification of a novel DNA-binding protein from Mycobacterium bovis bacillus Calmette-Guérin.

A novel DNA-binding protein expressed (8-10% in total protein) in Mycobacterium bovis bacillus Calmette-Guérin was observed. This protein was designated mycobacterial DNA-binding protein 1 (MDP1). MDP1 recognized bases, sugar moieties, phosphate-backbone on DNA and preferentially bound to DNA guanine and cytosine. In the gel retardation assay, MDP1 preferentially bound to closed circular plasmid DNA than open circular and linear form plasmid DNA and also bound to RNA. MDP1 formed a highly polymerized structure and localized not only in the nucleoid but also at the 50S ribosomal subunits and cell surface. MDP1 was conserved in Mycobacterium thus far examined and the expression was enhanced in stationary growth phases. These results will provide a reasonable basis for further study of the function of MDP1 in living mycobacteria.

Host immune responses to ribosome, ribosomal proteins, and RNA from Mycobacterium bovis bacille de Calmette-Gúerin.

The ribosomes from BCG strongly induced delayed type hypersensitivity (DTH) skin reactions in guinea pigs immunized with live BCG or heat killed Mycobacterium tuberculosis H37Rv, and also induced lymphocyte proliferative response in mice immunized with ribosomes. In contrast, neither ribosomal proteins nor RNA alone induced both DTH skin reactions and lymphocyte proliferative responses. Particle form consisted of ribosomal proteins and RNAs might be absolutely required for the activation of immune responses.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin secreting merozoite surface protein 1 (MSP1) induces protection against rodent malaria parasite infection depending on MSP1-stimulated interferon gamma and parasite-specific antibodies.

The merozoite surface protein 1 (MSP1) has emerged as a leading malaria vaccine candidate at the erythrocytic stage. Recombinant bacillus Calmette-Guérin (rBCG), which expressed a COOH-terminal 15-kD fragment of MSP1 of Plasmodium yoelii (MSP1-15) as a fusion protein with a secretory protein of Mycobacterium kansasii, was constructed. Immunization of mice with this rBCG induced a higher degree of protection against blood-stage parasite infection than with recombinant MSP1-15 in the RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, MT) or incomplete Freund's adjuvant systems. We studied the mechanism of protection induced by MSP1-15, and found that interferon (IFN)-gamma had a major role in protection in all adjuvant systems we examined. Mice that produced low amounts of MSP1-15 stimulated IFN-gamma and could not control parasite infection. The antibody against MSP1-15 did not play a major role in protection in this system. After parasite infection, immunoglobulin G2a antibodies, which had been produced by IFN-gamma stimulation, were induced and subsequently played an important role in eradicating parasites. Thus, both cellular and humoral immune responses were essential for protection from malaria disease. These data revealed that BCG is a powerful adjuvant to induce such a protective immune response against malaria parasites.

Shotgun cloning and characterization of the thymidylate synthase-encoding gene from Mycobacterium bovis BCG.

The shotgun cloning of a Mycobacterium bovis BCG (BCG) genome into pBluescript SK (+) successfully yielded a 0.9 kbp fragment, confirming the ability of Escherichia coli thyA mutant MH2702 to grow in a thymine-depleted medium. This DNA fragment contained a gene homologous to the thymidylate synthase (TS)-encoding genes (thyA) of other organisms. An inverted repeat sequence and open reading frame (ORF) were observed at the upstream region of the thyA. A computer analysis revealed that the protein encoded by this ORF possessed a structure unique for a DNA binding protein.

Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by immunization with ribosomal fraction and culture filtrate from Mycobacterium bovis BCG.

Immunization of mice with the ribosomal fraction from ruptured Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and the culture filtrate reduced remarkably the multiplication of Mycobacterium leprae in the foot pads of mice. This is the first reported case of the protective activity against M. leprae multiplication in mice of the BCG ribosomal fraction and culture filtrate. The inhibition was more evident with the culture filtrate than with the ribosomal fraction. When the ribosomal proteins separated from ribosomal RNA were injected into mice, only slight inhibition was observed. Ribosomal RNA alone did not inhibit at all, in contrast to the conclusion reported by Youmans and Youmans.

Long-lasting immune response induced by recombinant bacillus Calmette-Guérin (BCG) secretion system.

The recombinant bacillus Calmette-Guérin (rBCG) secretion system utilizing an extracellular alpha antigen of Mycobacterium kansasii (alpha-K) was characterized biochemically and immunologically. The human immunodeficiency virus type 1 (HIV-1) p17gag B cell epitope fused to alpha-K was secreted in extremely large amounts. At least three mice out of seven inoculated with rBCG generated high titres of antibody to the epitope. The long-lasting antibody production persisted more than 14 months.

A stable Escherichia coli-mycobacteria shuttle vector 'pSO246' in Mycobacterium bovis BCG.

The most widely used plasmid vector system in mycobacteria is based on pAL5000 from Mycobacterium fortuitum. The derivatives of the pAL5000-based shuttle vectors between Escherichia coli and mycobacteria, which we have utilized to secrete recombinant antigens, were generated. The stability of the vectors was assessed in Mycobacterium bovis BCG (BCG). The plasmid vector pSO246 was stable in BCG for at least 50 generations.

[The role of some cellular components of bacterial parasites in determining the incidence of tuberculosis: studies on mycobacterial antigens, with special reference to mycobacterial immunoreactive ribosomal and secreted proteins].

Tuberculosis remains as major disease, affecting more than 20 million people. The elimination of the disease with vaccination, rapid diagnosis, and and efficient therapy is an important objective of our study. To realize the objective, the characterization of antigens is essential. We have chosen two kinds of antigens for our study, the ribosomal antigens and and an antigenic proteins secreted by mycobacteria. The biochemical and immunological characterization of ribosomal fraction was carried out. Ribosomal proteins were purified and assessed for DTH reaction. The N-terminal amino acids sequences were determined. Total structures of S19, S7 and S12 in 30S and L7/L12 in 50S subunits were elucidated. L7/L12 had 66% homology with analogue from S. griseus which showed GTPase activity in protein synthesis. This protein was secreted in culture medium and induced strong DTH. Secreted antigenic proteins are of great interest for us. Secreted antigens may be recognized rapidly by immune system and therefore may induce rapid and high level immune response. It is also expected that it may contain protective antigens, since live BCG protect disease more efficiently than heat killed BCG. We have determined and published the total structure of four proteins (MPB64, MPB70, MPB57 and alpha antigen). We attempted to utilize this antigen for the diagnosis and the design of vaccine. The structures of alpha antigens from M. avium, M. intracellulare, M. scrofulaceum, M. kansasii and BCG were determined and its potential for application to diagnosis was presented. Using the operon of M. kansasii, alpha antigen and V3 region of HIV-1 were expressed by recombinant BCG which induced CTL in mice.

[Study on recombinant BCG].

For the purpose to establish the system to express foreign antigen from Mycobacterium bovis BCG. We have cloned, sequenced and expressed genes for secreting proteins, alpha antigen, MPB64, MPB57 and MPB70 from M. bovis BCG. The upstreams and structural genes were characterized. The gene for alpha antigen of Mycobacterium kansasii was also characterized. The gene for alpha antigen of M. kansasii (k-alpha) was chosen for the further study at first. This gene was fused with shuttle plasmid PIJ666-PAL5000 obtained from T. Kisser and transfected to M. bovis BCG (Tokyo). Transformant was obtained by a selection with kanamycin. It was able to secrete k-alpha antigen. DNA-containing a B-cell epitope (Glu-12-Leu-Asp-Arg-Trp-Glu-Lys-Ile-19) of human immunodeficiency virus type 1 P17 gag was fused to this vector at C terminal of k-alpha. Using this vector, we have succeeded to express foreign antigen in M. bovis BCG. The products were analyzed in one or two dimensional electro-phoresis. The results thus obtained will be reported elsewhere.

Establishment of a foreign antigen secretion system in mycobacteria.

In order to develop recombinant Mycobacterium bovis BCG into a useful multivaccine vehicle, we established a foreign antigen secretion system in mycobacteria in which an extracellular alpha antigen of Mycobacterium kansasii was utilized as a carrier. By using this system, a B-cell epitope (Glu-12-Leu-Asp-Arg-Trp-Glu-Lys-Ile-19) of human immunodeficiency virus type 1 p17gag, which was identified by a fusion protein-based method, has been successfully obtained from BCG along with the alpha antigen. This is the first report of expression and secretion of a foreign viral antigen from BCG. It is possible that the system can become a universal vaccination vehicle applicable to protection against various infectious diseases.

Hemagglutinating activity of Mycoplasma salivarium and its attachment to sheep red blood cells.

Mycoplasma salivarium (ATCC 23064) and 10 other strains isolated from human saliva agglutinated red blood cells of rabbits and human types A and O weakly, and those of sheep (SRBC) and human type B strongly. Glycoproteins on the surface of the organism cells and N-acetylneuraminic acid residues and some sugars on SRBC were suggested to be involved in agglutination of SRBC. Protein A-like activity was detected in the organism cells. The organism cells were also shown to attach to SRBC in PPLO broth (Difco) supplemented with 10% horse serum, and bivalent metal ions were suggested to be involved in the attachment. The organism cells attaching to SRBC activated complement through the alternative pathway and lyzed the SRBC.

Attachment of Mycoplasma salivarium and Mycoplasma orale to glass surfaces.

Mycoplasma salivarium ATCC 23064 and 24 other oral strains, and Mycoplasma orale ATCC 15539 and 22 other oral strains were examined quantitatively for attachment to glass surfaces by using ELISA method. Although all of the tested strains attached to glass surfaces, M. salivarium attached far more readily than M. orale. The results suggested that electrostatic bonds were involved in the attachment and that bivalent metal ions also played a role.