Genetic diversity of Mycobacterium tuberculosis isolates from foreign-born and Japan-born residents in Tokyo.

Sequences of the full genomes of 259 clinical isolates of Mycobacterium tuberculosis, obtained from foreign-born and Japan-born patients in Tokyo, Japan, were determined, and a phylogenetic tree constructed by concatenated single-nucleotide polymorphism (SNP) sequences. The 259 isolates were clustered into four clades: Lineage 2 (East Asian or "Beijing" genotype; n = 182, 70.3%), Lineage 4 (Euro-American, n = 46, 17.8%), Lineage 1 (Indo-Oceanic, n = 23, 8.9%), and Lineage 3 (East African-Indian, n = 8, 3.1%). Of the 259, 36 (13.9%) were resistant to at least one drug. There was no multi-drug-resistant isolate. Drug resistance was greater for the strains in Lineage 2 than the non-Lineage 2. The proportion of Lineage 2 isolates was significantly smaller in foreign-born (n = 43/91, 47.3%) than in Japan-born (n = 139/168, 82.7%) patients, whereas the proportion of Lineage 1 isolates was significantly larger in foreign-born (n = 19/91, 20.9%) than in Japan-born (n = 4/168, 2.4%) patients. We also found eight SNPs specific to the typical Beijing sub-genotype in Lineage 2, including 4 non-synonymous SNPs. Of the 259 isolates, 244 had strain-specific SNP(s) and small (1-30-bp) insertions and deletions (indels). The numbers of strain-specific SNPs and indels per isolate were significantly larger from foreign-born (median 89, range 0-520) than from Japan-born (median 23, range 0-415) (p 3.66E-15) patients. These results suggested that M. tuberculosis isolates from foreign-born patients had more genetic diversity than those from Japan-born patients.

Regulation of Cbl phosphorylation by the Abl tyrosine kinase and the Nck SH2/SH3 adaptor.

The Cbl proto-oncogene product is tyrosine phosphorylated in response to a wide variety of stimuli. Cbl and the Abl nonreceptor tyrosine kinase both bind to SH3 domains from the SH2/SH3 adaptor Nck, and are candidate effectors for Nck function. Numerous additional SH2- and SH3-domain-mediated interactions are also possible between Cbl, Abl, and Nck. We find that these three signaling proteins associate when overexpressed in mammalian cells and can regulate each other's activity. Co-expression of wt Cbl together with c-Abl, the activity of which is normally repressed in vivo, led to extensive Abl-dependent phosphorylation of Cbl. The major proline-rich region of Cbl was required for its phosphorylation by c-Abl, but not by a constitutively activated Abl mutant, suggesting Cbl activates c-Abl by engaging its SH3 domain. Efficient phosphorylation of Cbl and its stable association with Abl required the SH2 domain of Abl, suggesting that SH2-phosphotyrosine interactions prevent dissociation of active Abl from Cbl. We also show that overexpression of Nck could repress the phosphorylation of Cbl by Abl in vivo. Studies with Nck mutants suggested that the Nck SH2 domain is responsible for inhibiting the activity of Abl toward both Cbl and Nck itself, most likely by competing with the Abl SH2 for tyrosine-phosphorylated binding sites.

[Mechanism to cause streptococcal toxic-shock syndrome].

Group A streptococci(GAS) are responsible for a number of infectious diseases in humans. The development of a variety of antibiotic drugs decreased the number of bacterial infections dramatically. Streptococcal toxic shock-like syndrome(TSLS), which is now also called streptococcal toxic shock syndrome(STSS) has, however, become an important disease because it causes severe life-threatening clinical symptoms. Several reports have described STSS during pregnancy. Although a number of GAS virulence factors have been examined, the critical causative agents for STSS has not yet been identified. The clinical symptoms and pathological examinations of STSS suggest that STSS is caused by multiple factors. We need to study the pathogenic mechanism of STSS from both the bacterial and host side. Our study indicated that the lethal activity and anti-phagocytic activity examined in mice and SLO may be a major pathogenic factor in STSS.

Abl family kinases and Cbl cooperate with the Nck adaptor to modulate Xenopus development.

We previously showed that overexpression of the Nck Src homology (SH) 2/SH3 adaptor in Xenopus embryos induced developmental defects including anterior truncation and mesoderm ventralization. Mutagenic analysis indicated that this was due to relocalization of endogenous proteins that bind the first two SH3 domains of Nck. We therefore screened a Xenopus expression library with Nck SH3 domains to identify Nck-interacting proteins, and evaluated candidate binding proteins for a potential role in Nck-induced anterior truncation/ventralization. Of 39 binding proteins analyzed, only the Abl-related kinase Arg and the Cbl proto-oncogene product bound preferentially to the first two SH3 domains in tandem compared with the individual domains, consistent with a role in the developmental phenotype. High level overexpression of c-Abl or Arg alone induced anterior truncation, as did lower levels of an activated form of Abl; Cbl alone had no effect. In a sensitized system where subthreshold amounts of a ventralizing Nck mutant were expressed, co-expression of the combination of Abl or Arg and Cbl at modest levels strongly potentiated anterior truncation, while Arg, Abl, or Cbl alone were without effect. These results suggest a role for both Cbl and Abl family kinases in patterning the Xenopus embryo.

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.

Analysis of the superantigen-producing ability of Yersinia pseudotuberculosis strains of various serotypes isolated from patients with systemic or gastroenteric infections, wildlife animals and natural environments.

Yersinia pseudotuberculosis is a pathogen causing gastroenteritis as well as acute and systemic infections. This organism produces a superantigenic exotoxin, designated Y. pseudotuberculosis-derived mitogen (YPM). We consider this exotoxin to be the primary pathogen of the systemic type infection. In this study, we examined 101 Y. pseudotuberculosis strains isolated from various sources, patients with the systemic or the gastroenteric type of infections, wildlife animals and natural environments for the presence of the YPM gene and the production of YPM or other related superantigens. We found that all of the strains isolated from patients with systemic type infection carried the YPM gene and produced YPM. A certain proportion of the organisms isolated from patients with the gastroenteric type infection, wildlife animals or natural environments did not carry the YPM gene nor produced superantigens. These results suggest that YPM is involved in the pathogenesis of the systemic type of Y. pseudotuberculosis infection.

Post-thymic maturation of migrating human thymic single-positive T cells: thymic CD1a- CD4+ T cells are more susceptible to anergy induction by toxic shock syndrome toxin-1 than cord blood CD4+ T cells.

To determine whether human CD4+ T cells undergo post-thymic maturation, we compared the susceptibility to anergy induction in human thymic CD1a- CD4+ single-positive (CD4+), cord blood (CB) CD4+, and adult peripheral blood (APB) CD4+ T cells by stimulation with toxic shock syndrome toxin-1 (TSST-1). Most TSST-1-induced T cell blasts derived from either T cell preparation expressed TCR Vbeta2, which determines the potential reactivity to TSST-1. Most thymic CD4+ T cell blast preparations exhibited little or no production of IL-2 and IL-4 after restimulation with TSST-1 and only marginal responses after stimulation with rIL-2 or a combination of PMA and calcium ionophore, while the APB CD4+ T cell blasts showed high responses to these stimuli. The responses of CB CD4+ T cell blasts to these stimuli varied, ranging from minimal to relatively high. Studies of DNA fragmentation showed that there was no significant cell death of thymic CD4+ T cell blasts. Most thymic CD1a- CD4+ and CB CD4+ T cells were CD38 positive. APB CD4+ T cell blasts derived from the CD38+ fraction and from the CD38- fraction exhibited equally high responses to restimulation with TSST-1. These results indicate that thymic CD1a- CD4+ and CB CD4+ T cells are inherently highly susceptible to anergy induction by bacterial superantigens and that thymic CD1a- CD4+ T cells are less mature than CB CD4+ T cells, suggesting that post-thymic maturation in thymic T cells migrating to the periphery is required for acquisition of full reactivity to antigenic stimulation.

Identification of murine T cells reactive with the bacterial superantigen Yersinia pseudotuberculosis-derived mitogen (YPM) and factors involved in YPM-induced toxicity in mice.

We previously reported that Yersinia pseudotuberculosis-derived mitogen (YPM) acts as a superantigen to human T cells. In this study, we assessed the superantigenicity and toxicity of YPM using murine experimental models. YPM activated T cells to produce interleukin-2 in a major histocompatibility complex class II molecule-dependent manner. The T-cell blasts induced by YPM expressed T-cell receptor (TCR) beta-chain variable region (Vbeta)7, Vbeta8.1, Vbeta8.2 and Vbeta8.3. The injection of YPM into mice pre-sensitized with D-galactosamine induced lethal shock. This shock was blocked by the injection of monoclonal antibodies (mAbs) to CD4, TCR Vbeta7 plus Vbeta8, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not by injection to CD8 or unrelated Vbeta. These results indicate that YPM-induced shock requires the presence of CD4+ T cells bearing TCR Vbeta7 and Vbeta8, and that endogenous TNF-alpha and IFN-gamma mediate the lethal effects.

Implantation of IL-2-containing osmotic pump prolongs the survival of superantigen-reactive T cells expanded in mice injected with bacterial superantigen.

In the present study we investigated the mechanism of deletion of superantigen (sAg)-reactive T cells expanded in sAg-injected mice. In staphylococcal enterotoxin A (SEA)-injected mice, IL-2 activity in serum peaked at 1 to 3 h and the expression of IL-2R alpha-chain (IL-2R alpha) on SEA-reactive (V beta 3+, or V beta 11+) T cells peaked at 6 to 12 h after the injection. Expansion of V beta 3+ or V beta 11+ T cells peaked at 2 days after the injection when most of these T cells were IL-2R alpha negative, and IL-2 activity was not detected at all in serum, suggesting the involvement of IL-2 deprivation in the deletion of expanded T cells. Implantation of an osmotic pump containing human rIL-2 (IL-2 pump) prolonged the expanded states of V beta 3+ or V beta 11+ T cells in SEA-injected C57BL/6 mice and of V beta 8+ T cells in SEB-injected MRL +/+ and Fas Ag-defective MRL-Ipr/Ipr mice. Adult thymectomy did not change at all the effect induced by implantation of IL-2 pump. DNA fragmentation was blocked substantially in mice co-treated with SEA and IL-2 pump. In addition, CD4+ T cell blasts, obtained by in vitro stimulation with rIL-2 of splenic CD4+ T cells from mice co-treated with SEA and IL-2 pump, produced substantial amounts of IL-2 upon restimulation with SEA. These results indicate that deprivation of IL-2 is deeply involved in the deletion of expanded sAg-reactive T cells and their anergy induction in sAg-injected mice.

DNA sequencing of the gene encoding a bacterial superantigen, Yersinia pseudotuberculosis-derived mitogen (YPM), and characterization of the gene product, cloned YPM.

Previously, we found a novel bacterial superantigen from Yersinia pseudotuberculosis, designated Y. pseudotuberculosis-derived mitogen (YPM). In the present study, we analyzed the DNA sequence of the gene encoding YPM. The YPM gene was cloned into a plasmid vector pMW119 and expressed in Escherichia coli DH10B. Like the native YPM, the cloned YPM required the expression of MHC class II molecules on accessory cells in the induction of IL-2 production by human T cells. TCR-V beta repertoire of human T cells reactive with the cloned YPM was V beta 3, V beta 9, V beta 13.1, and V beta 13.2. This repertoire is the same as that of T cells reactive with the native YPM. These results indicate that the cloned YPM expressed in E. coli is identical to the native YPM. Sequencing of the YPM gene revealed that the gene contained an open reading frame of 456 base pairs encoding a precursor form of 151 amino acid residues with m.w. 16,679 that is processed into a mature form of 131 amino acid residues with m.w. 14,529. Homology analysis revealed that the homology of amino acid sequence is quite low among YPM and other well known bacterial superantigens. We designated the gene encoding YPM as ypm.

Superantigenic properties of a novel mitogenic substance produced by Yersinia pseudotuberculosis isolated from patients manifesting acute and systemic symptoms.

A novel product from Yersinia pseudotuberculosis exhibiting mitogenic activity for human PBMC (designated Y. pseudotuberculosis-derived mitogen, YPM) was examined for its biologic activities for human lymphocytes. YPM induced a substantial proliferative response and IL-2 production at 0.1 ng/ml or more in whole PBMC but not in T-depleted PBMC. IL-2 production occurred within 12 h after YPM stimulation. T cells from PBMC produced IL-2 in the presence of L cells transfected with HLA DR genes or DP genes but not in the presence of control L cells used as accessory cells. Paraformaldehyde fixation did not abolish the AC activity of the DR+ L cells. The results suggest that YPM bind directly to HLA class II molecules. Analysis of the TCR V beta element using the polymerase chain reaction revealed that YPM selectively activated human T cells bearing V beta 3, V beta 9, V beta 13.1, and V beta 13.2 in TCR. These results indicate that YPM is a potent T cell activator and has superantigenic properties (abilities to bind directly to MHC class II molecules and selectively stimulate T cell populations bearing particular V beta elements in TCR). The role of YPM in the mechanism of pathogenesis of Y. pseudotuberculosis infections manifesting acute and systemic clinical symptoms is discussed.

Purification and partial characterization of a product from Yersinia pseudotuberculosis with the ability to activate human T cells.

We tried to purify a substance exhibiting mitogenicity for human peripheral blood lymphocytes from Yersinia pseudotuberculosis isolated from patients with Y. pseudotuberculosis infection manifesting acute and systemic clinical symptoms. The supernatant of a suspension of the bacteria disrupted by sonication was serially chromatographed on DEAE-Sepharose fast-flow, Sephacryl S-100 HR, and TSK-gel G2000SW high-pressure liquid chromatography columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified mitogenically active substance migrated as a single band corresponding to a molecular mass of 21 kDa. We designated the purified substance Y. pseudotuberculosis-derived mitogen (YPM). YPM stimulated human peripheral T cells to proliferate and produce interleukin-2 at 0.1 ng/ml or more. YPM-induced T-cell activation required the expression of HLA class II molecules on accessory cells.

Purification and properties of cytochrome bo-type ubiquinol oxidase from a marine bacterium Vibrio alginolyticus.

Ubiquinol oxidase was extracted from membranes of a marine bacterium Vibrio alginolyticus with a nonionic detergent Liponox DCH and was purified about 130-fold by DEAE-Sephacel, DEAE-5PW and Sephacryl S-300. The purified ubiquinol oxidase was composed of three subunits with apparent M(r) of 79, 36 and 13 kDa on SDS-polyacrylamide gel electrophoresis. The oxidase contained cytochrome b, cytochrome o and copper atoms. The presence of heme O was confirmed by reverse-phase HPLC analysis. Ubiquinol-1, duroquinol and tetramethylphenylene diamine, but not horse heart reduced cytochrome c, were oxidized by this enzyme. The oxidase required no salts for activity and was stimulated by several detergents and by diphosphatidyl glycerol. The activity was strongly inhibited by KCN, 2-n-heptyl-4-hydroxyquinoline N-oxide and ZnSO4. These properties were essentially similar to those of cytochrome bo-type ubiquinol oxidase from Escherichia coli, suggesting that the bo-type ubiquinol oxidase is functioning as a proton pump in the marine V. alginolyticus.