Takashi Matsuba

Expression and Characterization of the Genes Encoding Azoreductases from Bacillus subtilis and Geobacillus stearothermophilus

Azoreductases have been characterized as enzymes that can decolorize azo dyes by reducing azo groups. In this study, genes encoding proteins having homology with theazoreductase gene of Bacillus sp. OY1-2 were obtained from Bacillus subtilis ATCC6633, B. subtilis ISW1214, and Geobacillus stearotherophilus IFO13737 by polymerase chain reaction. All three genes encoded proteins with 174 amino acids. The deduced amino acid sequences of azoreductase homologs from B. subtilis ISW1214, B. subtilis ATCC6633, and G. stearotherophilus IFO13737 showed similarity of 53.3, 53.9, and 53.3% respectively to that of Bacillus sp. OY1-2. All three genes were expressed in Escherichia coli, and were characterized as having the decolorizing activity of azo dyes in a β-NADPH dependent manner. The transformation of several azo dyes into colorless compounds by recombinant enzymes was demonstrated to have distinct substrate specificity from that of azoreductase from Bacillus sp. OY1-2.

Sensitivities of ciprofloxacin-resistant Mycobacterium tuberculosis clinical isolates to fluoroquinolones: role of mutant DNA gyrase subunits in drug resistance

Minimum inhibitory concentrations of sitafloxacin, gatifloxacin, moxifloxacin, sparfloxacin, levofloxacin and ciprofloxacin against 59 ciprofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Japan were determined. The isolates were most susceptible to sitafloxacin and gatifloxacin. To understand better the basis for drug resistance, nucleotide sequences encoding the gyrA and gyrB quinolone resistance-determining region were determined. Predicted amino acid sequences revealed distinct mutational patterns likely to be responsible for fluoroquinolone resistance. Double gyrA mutations as well as mutations in both gyrA and gyrB correlated with increased resistance to all fluoroquinolones.

Simple Multiplex PCR Assay for Identification of Beijing Family Mycobacterium tuberculosis Isolates with a Lineage-Specific Mutation in Rv0679c

ABSTRACT The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains.

Induction of Apoptosis in Macrophage Cell Line, J774, by the Cell-Free Supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell‐free supernatants from P. aeruginosa (Pa) IID1117 (elastase‐positive and protease‐positive) and Pa IID1130 (elastase‐positive and protease‐negative). After 6 hr of incubation with the cell‐free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5±11%), but not Pa IID1130 (96.4±1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2±1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase‐mediated d‐UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase‐3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell‐free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.

Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis

The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains.

Difference in Antibody Responses to Mycobacterium tuberculosis Antigens in Japanese Tuberculosis Patients Infected with the Beijing/Non-Beijing Genotype

The Beijing genotype Mycobacterium tuberculosis (MTB), notorious for its virulence and predisposition to relapse, could be identified by spoligotyping based on genetic heterogeneity. The plasma samples from 20 cases of Beijing and 16 cases of non-Beijing MTB infected individuals and 24 healthy controls (HCs) were collected, and antibodies against 11 antigens (Rv0679c142Asn, Rv0679c142Lys, Ag85B, Ag85A, ARC, TDM-M, TDM-K, HBHA, MDP-1, LAM, and TBGL) were measured by ELISA. Compared to the HCs, the MTB infected subjects showed higher titers of anti-Ag85B IgG (positivity 58.2%) and anti-ACR IgG (positivity 48.2%). Of note, anti-ACR IgG showed higher titer in Beijing MTB infected tuberculosis (TB) patients than in HC (Kruskal–Wallis test, p < 0.05), while the levels of anti-Ag85B, anti-TBGL, anti-TDM-K, and anti-TDM-M IgG were higher in non-Beijing TB patients than in HC. Moreover, anti-Ag85B IgG showed higher response in non-Beijing TB patients than in Beijing TB patients (p < 0.05; sensitivity, 76.9% versus 44.4%). The sensitivity and specificity analysis showed that 78.8% Beijing infected individuals were negative in anti-TBGL-IgG or/and anti-Ag85B-IgG, while 75.0% of those were positive in anti-TBGL-IgA or/and anti-ACR-IgG tests. These results indicate the possibility of developing antibody-based test to identify Beijing MTB.

Development and evaluation of an interferon-γ release assay in Asian elephants (Elephas maximus)

We developed an interferon-γ release assay (IGRA) specific for Asian elephants (Elephas maximus). Whole blood collected from forty captive Asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (PHA), pokweed mitogen (PWM) and phorbol myristate aceteate/ionomycin (PMA/I). A sandwich ELISA that was able to recognize the recombinant elephant interferon-γ (rEIFN-γ) as well as native interferon-γ from the Asian elephants was performed using anti-elephant IFN-γ rabbit polyclonal antibodies as capture antibodies and biotinylated anti-elephant IFN-γ rabbit polyclonal antibodies as detection antibodies. PMA/I was the best mitogen to use as a positive control for an Asian elephant IGRA. The development of an Asian elephant-specific IGRA that detects native IFN-γ in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (TB) in Asian elephants.

Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection

The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection.

Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration

BackgroundTetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments.ResultsWe expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport.ConclusionsThese results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.