Point mutation in the stop codon of MAV_RS14660 increases the growth rate of Mycobacterium avium subspecies hominissuis.

Mycobacterium avium subspecies hominissuis (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of MAV_RS14660 due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in MAV_RS14660 was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the MAV_RS14660 and MAV_RS14655 coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the MAV_RS14660- and MAV_RS14655-encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Development of Dried Emulsion/Mannitol Composite Microparticles through a Unique Spray Nozzle for Efficient Delivery of Hydrophilic Anti-tuberculosis Drug against Alveolar Macrophages.

As alveolar macrophages are attractive targets for the treatment of tuberculosis, effective methods for delivery to alveolar macrophages are under development. We investigated a pulmonary formulation for the efficient delivery of high water-soluble drugs at high concentration targeting alveolar macrophages. In this study, a surfactant-coated high water-soluble drug complex (SDC, a hydrophobic dried emulsion), which can preferably target alveolar macrophages and be expected to deliver drug at a high concentration, was prepared in the first process. OCT313, a high water-soluble sugar derivative with anti-tuberculosis activity was used. Then, a unique two-solution, mixing-type nozzle was used to prepare the SDC nanoparticles in mannitol (MAN) microparticles (SDC/MAN microparticles) because it was difficult to disperse the SDC nanoparticles in aqueous solution. The single micron size of OCT313-SDC/MAN microparticles contained OCT313-SDC nanoparticles (mean particle size of OCT313-SDC nanoparticles, 277.9 nm; drug contents, 1.31 ± 0.041 wt%). We found that the treatment of SDC/MAN microparticles exhibited significantly higher drug accumulation in macrophage cells (Raw264.7 cells, 7.5-fold, at 4 h after treatment) in vitro and in alveolar macrophages in rats (9.1-fold, at 4 h after treatment) in vivo than that of drug alone. These results suggest that the SDC/MAN microparticle formulation prepared by spray drying through a two-solution mixing-type nozzle provides efficient delivery of a water-soluble drug targeting alveolar macrophages and may be useful for tuberculosis treatment.

Identification of matrix metalloproteinase 9-interacting sequences in staphylococcal superantigen-like protein 5.

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, ß1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4ß9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved ß1-3 but not α4ß9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted ß1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored ß1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the ß1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.

5-Hydroxy-2-methylpyridine Isolated from Cigarette Smoke Condensate Aggravates Collagen-Induced Arthritis in Mice.

The risk of rheumatoid arthritis (RA) is linked to environmental and genetic factors. Cigarette smoking is an established environmental risk factor for the disease that contributes to its development and severity. Previously, we found that cigarette smoke condensate (CSC), both mainstream and sidestream, aggravates collagen type II-induced arthritis (CIA), which was observed following either intraperitoneal inoculation or nasal exposure. In the present study, we aimed to identify the compound in CSC, which aggravates CIA. By sequential fractionation and analysis, extraction with water/ether in different pH values, silica gel column chromatography, TLC, octadecyl silica (ODS) HPLC, GC/MS, and NMR, the active compound was identified as 5-hydroxy-2-methylpyridine (5H2MP). Its isomer 2-hydroxy-3-methylpyridine, but not 3-hydroxy-2-methylpyridine, was also active. 5H2MP was not mutagenic, and did not exhibit aryl hydrocarbon receptor-dependent activity. Our data help clarify the mechanism underlying the pathogenic effects of cigarette smoking on RA.

Identification of the blood coagulation factor interacting sequences in staphylococcal superantigen-like protein 10.

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10ß1-ß3 (23MEMKN ISALK HGKNN LRFKF RGIKI QVL60) bound to immobilized prothrombin, and mutants that contained SSL10ß10-ß12 (174SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK207) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.

Early-shared Mycobacterium bovis bacillus Calmette-Guérin sub-strains induce Th1 cytokine production in vivo.

Interleukin-12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL-2 and IFN-γ. We have previously reported greater IL-12 production from macrophages infected with early-shared BCG sub-strains (ex. BCG-Japan, -Sweden) than from those infected with late-shared BCG (ex. BCG-Pasteur and -Connaught) . In this study, we investigated the Th1 cytokine-inducing activity of splenocytes co-cultured with BCG-infected DCs. Early-shared BCG-infected DCs produced IL-12 and TNF-α⋅ Furthermore, when they were co-cultured with purified protein derivative-stimulated DCs, the splenocytes of mice immunized with BCG-Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG-Connaught. In conclusion, early-shared BCG sub-strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.

[SENSORS IN MYCOBACTERIA FOR THE DETECTION OF REDOX STRESS].

Mycobacterium species are exposed to oxidative and nitrosylative stress from environments within and outside the host cells. After the host is infected with the bacilli, macrophages produce superoxide molecules via NADPH oxidase activity and nitric oxide (NO) via inducible NO synthase activity to kill the bacilli. The pathogenic bacilli can successfully survive in host cells via anti-oxidative and anti-nitrosylative mechanisms. In particular, Mycobacterium tuberculosis persisters pose a great problem for chemotherapy because most anti-mycobacterial drugs are ineffective against mycobacteria that are in the persistent state. In accordance with the changes in redox balance, the bacilli change their metabolic pathways from aerobic to anaerobic ones, thereby leading to a change from an actively growing state to a dormant state. Therefore, M. tuberculosis is expected to be equipped with sensors that detect redox stress in the environment such that it can switch to the dormant state and change its metabolic pathways accordingly. In this review, roles of the mycobacterial O2, NO, and CO gas sensors, DosS and DosT, consisting of the DosR regulon, and mycobacterial DNA binding proteins WhiBs, which contain iron-sulfur clusters, in latent infection are discussed.

Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Bacterial sphingophospholipids containing non-hydroxy fatty acid activate murine macrophages via Toll-like receptor 4 and stimulate bacterial clearance.

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.

Staphylococcal superantigen-like protein 8 (SSL8) binds to tenascin C and inhibits tenascin C-fibronectin interaction and cell motility of keratinocytes.

Staphylococcal superantigen-like protein (SSL), a family of exotoxins composed of 14 SSLs, exhibits no superantigenic activity despite of its structural similarity with superantigens. Several SSLs have been revealed to bind to host immune molecules such as IgA, IgG, complement and cell surface molecules expressed on immune cells, but the physiological function of SSL family has not been fully identified. In this study we attempted to isolate host target proteins of SSLs from human breast milk using SSLs-conjugated Sepharose. SSL8-conjugated Sepharose specifically recovered tenascin C (TNC), a multimodular and multifunctional extracellular matrix protein. Pull down analysis using SSL8-conjugated Sepharose and recombinant truncated fragments of TNC revealed that SSL8 interacts with fibronectin (FN) type III repeats 1-5 of TNC. The interaction of TNC with immobilized FN was attenuated, the scratch wound closure by HaCaT human keratinocytes was delayed and the inhibition of cell spreading on FN by TNC was recovered in the presence of SSL8. These findings suggest that SSL8 binds to TNC, thereby inhibits the TNC-FN interaction and motility of keratinocytes. The present study added a novel role of SSL family protein as an interrupting molecule against the function of extracellular matrix.

Reactivation of immune responses against Mycobacterium tuberculosis by boosting with the CpG oligomer in aged mice primarily vaccinated with Mycobacterium bovis BCG.

BACKGROUND: Mycobacterium bovis bacillus Calmette Guérin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation. FINDINGS: The booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-γ, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H37Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50's in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4+ CD44high CD62Lhigh, central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H37Rv. CONCLUSIONS: Th1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming.

Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments.

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.

Staphylococcal superantigen-like protein 3 binds to the Toll-like receptor 2 extracellular domain and inhibits cytokine production induced by Staphylococcus aureus, cell wall component, or lipopeptides in murine macrophages.

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins sharing structural similarity with superantigens, but no superantigenic activity. Corresponding host target proteins or receptors against a portion of SSLs in the family have been identified. In this study, we show that SSL3 specifically binds to Toll-like receptor 2 (TLR2) and inhibits the stimulation of macrophages by TLR2 ligands. An approximately 100-kDa protein was recovered by using recombinant His-tagged SSL3-conjugated Sepharose from the lysate of porcine spleen, and the protein was identified as porcine TLR2 by peptide mass fingerprinting analysis. The SSL3-conjugated Sepharose recovered human and mouse TLR2 but not TLR4 from human neutrophils and mouse macrophage RAW 264.7 cells, as well as a recombinant TLR2 extracellular domain chimera protein. The production levels of interleukin 12 (IL-12) from mouse macrophages treated with heat-killed Staphylococcus aureus and of tumor necrosis factor alpha (TNF-α) from RAW 264.7 cells induced by peptidoglycan or lipopeptide TLR2 ligands were strongly suppressed in the presence of SSL3. The mutation of consensus sialic acid-containing glycan-binding residues in SSL3 did not abrogate the binding ability to TLR2 or inhibitory activity on TLR2, indicating that the interaction of SSL3 with TLR2 was independent of the sialic acid-containing glycan-binding residues. These findings demonstrate that SSL3 is able to bind the extracellular domain of TLR2 and interfere with TLR2 function. The present study provides a novel mechanism of SSL3 in immune evasion of S. aureus via interfering with its recognition by innate immune cells.

Antitubercular activity of disulfiram, an antialcoholism drug, against multidrug- and extensively drug-resistant Mycobacterium tuberculosis isolates.

The antimycobacterial activities of disulfiram (DSF) and diethyldithiocarbamate (DDC) against multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB) clinical isolates were evaluated in vitro. Both DSF and DDC exhibited potent antitubercular activities against 42 clinical isolates of M. tuberculosis, including MDR/XDR-TB strains. Moreover, DSF showed remarkable bactericidal activity ex vivo and in vivo. Therefore, DSF might be a drug repurposed for the treatment of MDR/XDR-TB.

Staphylococcal superantigen-like protein 10 binds to phosphatidylserine and apoptotic cells.

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins that have structural similarities to staphylococcal superantigens. Although SSLs do not have superantigenic activity, some of them have been reported to bind to host immune related molecules and they have been implicated in immune evasion by S. aureus. In this study, we showed that SSL10 is capable of binding to phospholipids. SSL10 bound to phosphatidylserine (PS) containing liposome, but not to phosphatidylcholine liposome. SSL10, but not SSL7, bound to PS containing liposome, suggesting that SSL10 specifically binds to PS. Analysis of PS binding ability among recombinant truncated SSL10 fragments revealed that the ß-barrel in the N-terminal oligonucleotide/oligosaccharide-binding (OB)-fold domain contributes to PS binding capacity. Fluorescein isothiocyanate labeled OB-fold of SSL10 stained hydrogen peroxide treated Jurkat cells. Annexin V is widely utilized for detection of apoptosis. Unlike annexin V, the OB-fold domain of SSL10 also bound to apoptotic cells in the presence of EDTA, suggesting that the OB-fold of SSL10 recognizes PS and apoptotic cells in a Ca(2+) independent manner. These findings suggest SSL10 and its derived peptides may be a novel detection tool for apoptotic cells.

Etiological role of cigarette smoking in rheumatoid arthritis: Nasal exposure to cigarette smoke condensate extracts augments the development of collagen-induced arthritis in mice.

Cigarette smoking is a major environmental risk factor for rheumatoid arthritis (RA). However, the experimental bases supporting the etiological role of cigarette smoking in RA have not been fully provided. We have reported that cigarette smoke condensate (CSC), by means of subcutaneous injection into DBA/1J mice with collagen and complete Freund's adjuvant or intraperitoneal injection one day before immunization, augmented the development of arthritis in the mouse model of collagen type II-induced arthritis (CIA). However, these experimental procedures may not be appropriate for cigarette smoking. In this study, we nasally exposed mice to mainstream CSC and found that CSC augmented the induction and development of arthritis and antibody level against collagen. Histological examination confirmed the augmenting effect of CSC. These findings provide experimental bases supporting the etiological role of cigarette smoking in RA.

Synthesis and evaluation of anti-tubercular activity of new dithiocarbamate sugar derivatives.

The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-ß-D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1 by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25-12.5 µg/ml. The antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Importantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bactericidal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in contrast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent compound against MTB, particularly promising to overcome drug resistance.

Dihydrotestosterone inhibits interleukin-1α or tumor necrosis factor α-induced proinflammatory cytokine production via androgen receptor-dependent inhibition of nuclear factor-κB activation in rheumatoid fibroblast-like synovial cell line.

Rheumatoid arthritis (RA) is a disease with significant gender differences in its prevalence and clinical features. Interleukin (IL)-1 and tumor necrosis factor (TNF) α produced by synoviocytes are principle inflammatory and destructive mediators of RA. We found that a potent androgen, 5α-dihydrotestosterone (DHT) inhibits IL-1α-induced production and mRNA expression of IL-8, IL-6 and IL-1ß from RA patient-derived fibroblast-like synovial cell line MH7A. Promoter analysis of the IL-8 gene revealed that nuclear factor (NF)-κB activation is critical for its transcriptional activation by IL-1α, and DHT inhibited the IL-1α-induced NF-κB activation in a manner dependent on the androgen receptor (AR). DHT also inhibited the effects of TNFα on the cells overexpressed with AR, indicating that sufficient expression level of functional AR was necessary for the inhibitory effect of DHT on TNFα. These results suggest that androgen contributes to the prevention against RA and its gender difference by inhibiting IL-1α or TNFα-induced proinflammatory cytokine production from synovial fibroblast-like cells by inhibiting NF-κB activation in a manner depending on AR.

Comparative Study of the Susceptibility to Oxidative Stress between Two Types of Mycobacterium bovis BCG Tokyo 172.

Genomic analysis revealed that the vaccine seed lot of Mycobacterium bovis bacillus Calmette-Guérin (BCG) Tokyo 172 contains two subclones (types I and II), but their phenotypic differences have not been elucidated. In this study, we compared the susceptibility of bacilli types I and II to oxidative stress in vitro and within host cells. Notably, the subclones displayed similar superoxide dismutase activity; however, foam height in the catalase test and lysate catalase/peroxidase activity were higher for type I bacilli than for type II bacilli. Additionally, type I bacilli were less susceptible to hydrogen peroxide (H2O2) than type II bacilli. After exposure to H2O2, antioxidative stress response genes katG, ahpC, sodA, and trxA were more strongly induced in type I bacilli than in type II bacilli. Further, we investigated cell survival in macrophages. Fewer type II bacilli were recovered than type I bacilli. However, in the presence of apocynin, a specific inhibitor of NADPH oxidase, type II recovery was greater than that of type I. The production of interleukin 1ß (IL-1ß), IL-12 p40, and tumor necrosis factor alpha (TNF-α) was higher in type I bacillus-infected macrophages than in type II bacillus-infected macrophages. The proportions of type I and type II bacilli in vaccine lots over 3 years (100 lots) were 97.6% ± 1.5% and 2.4% ± 1.5%, respectively. The study results illustrated that type I bacilli are more resistant to oxidative stress than type II bacilli. Overall, these findings provide important information in terms of the quality control and safety of BCG Tokyo 172 vaccine.IMPORTANCE This study revealed the difference of in vivo and in vitro antioxidative stress properties of BCG Tokyo 172 types I and II as one of the bacteriological characteristics. In particular, the bacilli exhibited differences in catalase/peroxidase activity, which could explain their different protective effects against infection. The differences correlated with survival in the host cell and the production of proinflammatory cytokines to protect against infection by Mycobacterium tuberculosis The proportion of bacilli types I and II in all commercial lots of BCG Tokyo 172 over 3 years (100 lots) was constant. The findings also highlighted the importance of analyzing their content for quality control during vaccine production.

IL-1-induced ERK1/2 activation up-regulates p21(Waf1/Cip1) protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375.

IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21(Waf1/Cip1) (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.