Activation of NLRP3 inflammasome in macrophages by mycoplasmal lipoproteins and lipopeptides.

The NLRP3 inflammasome, an intracellular sensor consisting of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and procaspase-1, plays critical roles in host defense against microbial pathogens by inducing production of interleukin-1ß (IL-1ß) and IL-18. Mycoplasma salivarium and Mycoplasma pneumoniae cells activated murine bone marrow-derived macrophages (BMMs) to induce production of IL-1α, IL-1ß, and IL-18. The IL-1ß production-inducing activities of these mycoplasmas toward BMMs from Toll-like receptor 2 (TLR2)-deficient mice were significantly attenuated compared with those from C57BL/6 mice (B6BMMs). This result suggests the possibility that their lipoproteins as TLR2 agonists are involved in the activity. Lipoproteins of M. salivarium and M. pneumoniae (MsLP and MpLP), and the M. salivarium-derived lipopeptide FSL-1 induced IL-1ß production by B6BMMs, but not by BMMs from caspase-1-, NLRP3- or ASC-deficient mice. The activities of MsLP and MpLP were not downregulated by the proteinase K treatment, suggesting that the active sites are their N-terminal lipopeptide moieties. B6BMMs internalized the mycoplasmal N-terminal lipopeptide FSL-1 at least 30 min after incubation, FSL-1-containing endosomes started to fuse with the lysosomes around 2 hours, and then FSL-1 translocated into the cytosol from LAMP-1+ endosomes. The artificial delivery of FSL-1 into the cytosol of B6BMMs drastically enhanced the IL-1ß production-inducing activity. FSL-1 as well as the representative NLRP3 inflammasome activator nigericin induced the NLRP3/ASC speck, but FSL-1 located in a compartment different from the NLRP3/ASC speck.

Activation of inflammasomes in dendritic cells and macrophages by Mycoplasma salivarium.

Interleukin-1ß (IL-1ß) plays crucial roles in the pathogenesis of periodontal disease. It is produced after the processing of pro-IL-1ß by caspase-1, which is activated by the inflammasome-a multiprotein complex comprising nucleotide-binding domain leucine-rich repeat-containing receptor (NLR), the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and procaspase-1. Mycoplasma salivarium preferentially inhabits the gingival sulcus and the incidence and number of organisms in the oral cavity increase significantly with the progression of periodontal disease. To initially clarify the association of this organism with periodontal diseases, this study determined whether it induces IL-1ß production by innate immune cells such as dendritic cells or macrophages by using Mycoplasma pneumoniae as a positive control. Both live and heat-killed M. salivarium and M. pneumoniae cells induced IL-1ß production by XS106 murine dendritic cells as well as pyroptosis. The activities were significantly downregulated by silencing of caspase-1. Bone-marrow-derived macrophage (BMMs) from wild-type and NLR-containing protein 3 (NLRP3)-, ASC-, and caspase-1-deficient mice were examined for IL-1ß production in response to these mycoplasmas. Live M. salivarium and M. pneumoniae cells almost completely lost the ability to induce IL-1ß production by BMMs from ASC- and caspase-1-deficient mice. Their activities toward BMMs from NLRP3-deficient mice were significantly but not completely attenuated. These results suggest that live M. salivarium and M. pneumoniae cells can activate several types of inflammasomes including the NLRP3 inflammasome. Both M. salivarium and M. pneumoniae cells can activate THP-1 human monocytic cells to induce IL-1ß production. Hence, the present finding that M. salivarium induces IL-1ß production by dendritic cells and macrophages may suggest the association of this organism with periodontal diseases.

Age-related alteration of expression and function of TLRs and NK activity in oral candidiasis.

OBJECTIVE: Roles of aging or immune responses mediated by Toll-like receptors and natural killer cell in the onset or progression of human candidiasis remain unclear. This study was designed to elucidate the roles using peripheral blood mononuclear cells from healthy donors and patients with oral candidiasis. SUBJECTS AND METHODS: Subjects tested were healthy volunteers and patients who visited Dental Clinical Division of Hokkaido University Hospital. The patients with oral candidiasis included 39 individuals (25-89 years of age) with major complaints on pain in oral mucosa and/or dysgeusia. Healthy volunteers include students (25-35 years of age) and teaching staffs (50-65 years of age) of Hokkaido University Graduate School of Dental Medicine. RESULTS: Functions of Toll-like receptors 2 and 4 were downregulated significantly and the natural killer activity was slightly, but not significantly downregulated in aged healthy volunteers compared with healthy young volunteers. Functions of Toll-like receptors 2 and 4 and the natural killer activity were significantly downregulated in patients with oral candidiasis compared with healthy volunteers. CONCLUSION: Downregulation of functions of Toll-like receptors 2 and 4 as well as natural killer activity is suggested to be associated with the onset or progression of oral candidiasis in human.

Toll-like receptor 2-mediated modulation of growth and functions of regulatory T cells by oral streptococci.

This study was designed to determine whether oral streptococci modulate the growth and functions of regulatory T cells. Heat-killed cells of wild-type strains of Streptococcus gordonii and Streptococcus mutans induced the Toll-like receptor 2 (TLR2) -mediated nuclear factor-κB (NF-κB) activation, but their lipoprotein-deficient strains did not. Stimulation with these streptococci resulted in a significant increase in the frequency of CD4(+) CD25(+) Foxp3(+) regulatory T cells in splenocytes derived from both TLR2(+/+) and TLR2(-/-) mice, but the level of increase in TLR2(+/+) splenocytes was stronger than that in TLR2(-/-) splenocytes. Both strains of S. gordonii enhanced the proliferation of CD4(+) CD25(+) Foxp3(+) regulatory T cells isolated from TLR2(+/+) mice at the same level as those from TLR2(-/-) mice in an interleukin-2-independent manner. However, wild-type and lipoprotein-deficient strains of both streptococci did not enhance the suppressive activity of the isolated regulatory T cells in vitro, but rather inhibited it. TLR ligands also inhibited the suppressive activity of the regulatory T cells. Inhibition of the suppressive activity was recovered by the addition of anti-IL-6 antibody. Pretreatment of antigen-presenting cells with the NF-κB inhibitor BAY11-7082 enhanced the suppressive activity of the regulatory T cells. These results suggested that interleukin-6 produced by antigen-presenting cells inhibits the suppressive activity of the regulatory T cells. Wild-type strain, but not lipoprotein-deficient strain, of S. gordonii reduced the frequency of CD4(+)  CD25(+)  Foxp3(+) regulatory T cells in the acute infection model, whereas both strains of S. gordonii increased it in the chronic infection model mice. Hence, this study suggests that oral streptococci are capable of modulating the growth and functions of regulatory T cells in vitro and in vivo.

Impaired functional capacities of liver dendritic cells from murine hepatitis B virus (HBV) carriers: relevance to low HBV-specific immune responses.

The chronic hepatitis B virus (HBV) carrier exhibits ongoing replication of HBV and expresses abundant amounts of HBV-related antigens in the liver. However, HBV-specific immune responses are either absent or narrowly focused in these subjects. With the postulation that impaired functional abilities of liver dendritic cells (DCs) might be responsible for this, we assessed the functions of liver DCs in HBV transgenic mice (HBV-TM), an animal model of the HBV carrier state. Liver DCs were isolated from normal C57BL/6 mice and HBV-TM without the use of cytokines or growth factors. Lymphoproliferative assays were conducted to evaluate the ability of liver DCs to induce the proliferation of allogenic T lymphocytes and hepatitis B surface antigen (HBsAg)-enriched T lymphocytes. Liver DCs were stimulated with viral and bacterial products to assess their cytokine-producing capacities. In comparison to liver DCs from normal C57BL/6 mice, liver DCs from HBV-TM exhibited significantly decreased T cell proliferation-inducing capacities in allogenic mixed leucocyte reaction (P <0.05) and HBsAg-enriched T lymphocytes proliferation assays (P <0.05). Liver DCs from HBV-TM produced significantly lower levels of interleukin-12p70, tumour necrosis factor-alpha, interferon-gamma, and interleukin-6 (P <0.05) compared to liver DCs from normal C57BL/6 mice. This study provides evidence that liver DCs from HBV-TM had impaired ability to induce both innate and adaptive immune responses. This might account for a weak and almost undetectable HBV-specific immune response in chronic HBV carriers. This inspires hope that up-regulation of the functions of liver DCs in situ may have therapeutic implications in chronic HBV carriers.

A 4.1-kilodalton polypeptide in the cultural supernatant of Mycoplasma fermentans is one of the substances responsible for induction of interleukin-6 production by human gingival fibroblasts.

The cultural supernatant of Mycoplasma fermentans induced interleukin-6 production by human gingival fibroblasts. The active entities were divided into hydrophilic and hydrophobic substances. In this study, we purified a 4.1-kilodalton polypeptide from the hydrophilic substances. It reacted with polyclonal antibodies to M. fermentans and activated human macrophages.

Construction and characterization of a Xanthomonas oryzae pv. oryzae bacterial artificial chromosome library.

Xanthomonas oryzae pv. oryzae is an important plant pathogen which causes bacterial blight of rice. To facilitate genome studies of this bacterium, we have constructed a bacterial artificial chromosome (BAC) library of strain MAFF 311018. It consisted of 750 clones representing 16 genome equivalents, and had an insert size ranging from 20 to 220 kb with an average size of 107 kb. This library is the first to be constructed from a X. oryzae pv. oryzae strain. The usefulness of this library was demonstrated through polymerase chain reaction screening of 11 genes and the 16S--23S rDNA spacer region in a 192-clone subset, representing five genome equivalents. The results obtained showed an average of 5.9 BAC clones per screening. This result is in good agreement with the estimated size of the test library, indicating that the constructed BAC library can be used to facilitate genome analysis of X. oryzae pv. oryzae.

The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts.

The activities to induce TNF-alpha production by a monocytic cell line, THP-1, and ICAM-1 expression and IL-6 production by human gingival fibroblasts were detected in plural membrane lipoproteins of Mycoplasma salivarium. Although SDS-PAGE of the lipoproteins digested by proteinase K did not reveal any protein bands with molecular masses higher than approximately10 kDa, these activities were detected in the front of the gel. A lipoprotein with a molecular mass of 44 kDa (Lp44) was purified. Proteinase K did not affect the ICAM-1 expression-inducing activity of Lp44, but lipoprotein lipase abrogated the activity. These results suggested that the proteinase K-resistant and low molecular mass entity, possibly the N-terminal lipid moiety, played a key role in the expression of the activity. The N-terminal lipid moiety of Lp44 was purified from Lp44 digested with proteinase K by HPLC. Judging from the structure of microbial lipopeptides as well as the amino acid sequence and infrared spectrum of Lp44, the structure of the N-terminal lipid moiety of Lp44 was speculated to be S-(2, 3-bisacyloxypropyl)-cysteine-GDPKHPKSFTEWV-. Its analogue, S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF, was synthesized. The lipopeptide was similar to the N-terminal lipid moiety of Lp44 in the infrared spectrum and the ICAM-1 expression-inducing activity. Thus, this study suggested that the active entity of Lp44 was its N-terminal lipopeptide moiety, the structure of which was very similar to S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF.

Insertion of the LINE retrotransposon MGL causes a conidiophore pattern mutation in Magnaporthe grisea.

We obtained three Magnaporthe grisea morphological mutants that had the LINE transposon MGL inserted into the ACR1 locus. Sequence analysis revealed that ACR1 is homologous to medA, a developmental regulator of Aspergillus nidulans conidiation. These results demonstrated that MGL elements could transpose and cause insertional mutagenesis in M. grisea.

The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution.

Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063. The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence. IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp. The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence. IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family. Southern blot analysis of DNAs from B. glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B. glumae and some other related species such as B. plantarii. The polymorphisms exhibited in B. glumae isolates suggest that those elements are useful for molecular epidemiological studies of B. glumae infections.

Partial purification and characterization of the active entity responsible for inducing interleukin-6 production by human gingival fibroblasts from Mycoplasma salivarium cells.

The active entity responsible for inducing interleukin-6 production by human gingival fibroblasts was partially purified by ion-exchange chromatography from the water-soluble fraction of Mycoplasma salivarium cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation revealed one densely stained band with a molecular weight of 20.6 kilodaltons and two faint bands with molecular weights of 40.5 and 82.5 kilodaltons. The specific activity of the final preparation was 34-fold higher than that of the starting water-soluble fraction. The interleukin-6-inducing activity was destroyed by proteinase K and reduced 70% by lipoprotein lipase and heat treatment, but was not affected by deoxyribonuclease I or endoglucosidase D. The final preparation induced small amounts of tumor necrosis factor-alpha and interleukin-lbeta in a myelomonocytic cell line, THP-1 cells, but did not induce interleukin-6. The ability of Escherichia coli lipopolysaccharide to stimulate human gingival fibroblasts to release interleukin-6 was dependent upon the presence of serum in the assay medium, but that of the final preparation from M. salivarium was not. Thus, we partially purified the protein(s) from M. salivarium which were capable of stimulating human gingival fibroblasts to release interleukin-6 by a mechanism different from that of E. coli lipopolysaccharide.

Prevention of aerobic spoilage of maize silage by a genetically modified killer yeast, Kluyveromyces lactis, defective in the ability to grow on lactic acid.

In this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructed Kluyveromyces lactis killer strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth of Pichia anomala inoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.

Detection of Mycoplasma fermentans in saliva sampled from infants, preschool and school children, adolescents and adults by a polymerase chain reaction-based assay.

Attempts were made to detect Mycoplasma fermentans in saliva sampled from 201 subjects (108 males and 93 females) aged from 4 months to 59 years by a polymerase chain reaction-based assay. M. fermentans was detected in saliva from 110 (54.7%) of 201 subjects, and 10 (28.6%) of 35 subjects aged from 4 months to 3 years. Of ten positive subjects, three were aged from 16 to 23 months and five were from 26 to 31 months. The incidence tended to increase with age up to the teens. The incidence was significantly greater in teenagers than in subjects aged from 7 to 12 years, but there was no significant difference in the incidence between the group of teenagers and each of the groups of subjects older than the teenagers. Thus, it was suggested that M. fermentans colonized the mouth at the age of about 16 months up to the age of 19 years.

Transcriptional activation of mRNA of intercellular adhesion molecule 1 and induction of its cell surface expression in normal human gingival fibroblasts by Mycoplasma salivarium and Mycoplasma fermentans.

Lipoproteins in the cell membranes of both Mycoplasma salivarium and Mycoplasma fermentans were demonstrated to trigger the transcription of intercellular adhesion molecule-1 mRNA in normal fibroblasts isolated from human gingival tissue and to induce its cell surface expression by a mechanism distinct from that of Escherichia coli lipopolysaccharide. The lipid moiety of the lipoproteins was suggested to play a key role in the expression of the activity.

Detection of Mycoplasma salivarium and Mycoplasma fermentans in synovial fluids of temporomandibular joints of patients with disorders in the joints.

Thirty-six synovial fluid samples of temporomandibular joints were obtained from 33 patients with pain and anterior disk displacement (closed lock) in the joints. DNAs were prepared from the samples and amplified by a PCR-based assay specific for Mycoplasma salivarium or Mycoplasma fermentans. Of the 36 samples, five (14%), three (8%), and 19 (53%) were positive for M. salivarium, M. fermentans and both, respectively.

Construction of a BAC library of the rice blast fungus Magnaporthe grisea and finding specific genome regions in which its transposons tend to cluster.

We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones. The insert size ranged from 35 to 175 kbp, with an average of 120 kbp. The library is about 18 genomes equivalent, therefore covering more than 99.999% of the genome. This library is the first to be constructed using a rice pathogenic wild type isolate. Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency. Total library clones were arranged onto two nylon membranes for efficient screening. Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions. As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed. Of all the BAC clones, 10%, 13%, 18%, 12%, and 23% contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively. The percentage of clones possessing more than five kinds of transposons was 1.4%, 215 times greater than the expected number. The results show that these transposons were distributed in clusters in the M. grisea genome.

Isolation and characterization of IS1416 from Pseudomonas glumae, a new member of the IS3 family.

Isolation and characterization of four different insertion sequence (IS) elements from Pseudomonas glumae MAFF 302744 through transposition into the entrapment vector pSHI1063 are described. One of the elements, IS1416, was further characterized. IS1416 is 1322 bp long and carries 29-bp terminal inverted repeats flanked by a 3-bp direct duplication. IS1416 contains three open reading frames (ORFs), which are designated ORFA1, ORFA2, and ORFB, on one strand. Both DNA sequence of IS1416 and the deduced amino acid sequences of its ORFs strongly suggest that IS1416 is a member of the IS3 family, and is closely related to IS401 from Pseudomonas cepacia and IS51 from Pseudomonas syringae. To our knowledge, IS1416 is the first IS element isolated from P. glumae. The gene organization and possible regulation of transposition functions of IS1416 are also discussed.

Mycoplasma salivarium induces interleukin-6 and interleukin-8 in human gingival fibroblasts.

Analysis by an enzyme-linked immunosorbent assay for cytokines indicated that whole cells, intracellular materials and cell membranes of Mycoplasma salivarium induced interleukin-6 and interleukin-8 in a human gingival fibroblast cell line, Gin-1 cells. This was confirmed by reverse transcription-polymerase chain reaction analysis of mRNAs of these cytokines. Studies with inhibitors of second-messenger pathway indicated that a protein kinase C-dependent pathway was involved in the expression of the activity of the cell membranes. In addition, whole cells of other mycoplasmas (M. hominis, M. arthritidis, M. arginini, M. fermentans, M. penetrans, M. pirum and M. pneumoniae) tested for comparative purposes were also shown to possess the activity. Thus, this study demonstrated that mycoplasmas possess the activity to induce interleukin-6 and interleukin-8 in human fibroblasts.

[Study on the effects of influenza virus vaccines--relation between the rate of two-fractional vaccination and the overall rate of absenteeism].

In 1987 and 1988, in 9 elementary schools, the percentage of children who received two sessions of vaccination and the overall rate of absenteeism resulting from influenza were determined for each class, and their relationship was investigated. The following results were obtained. 1) The mean vaccination rate was 58.6% among 157 classes in 1987, whereas it was 29.9% among 151 classes in 1988, the rate being significantly higher in 1987. 2) The mean overall rate of absenteeism was 1.524% in 1987, which was significantly lower than the corresponding rate, 2.802%, in 1988. 3) There was a significant negative correlation between the vaccination rate and the overall rate of absenteeism in 7 of the 9 schools in 1987; the overall rate of absenteeism became significantly low with an increase in the vaccination rate. 4) No such trend, however, was noted in any of the schools in 1988. 5) The difference between the results in 1987 and those in 1988 seems to be attributable to the facts that variability of the prevailing strains of influenza was low (V0, 82%) in 1987, in addition to the high vaccination rate in that year, and that influenza virus type B having a high variability (V3, or more, 78%) prevailed in 1988, when the vaccination rate was low.