Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14: a novel bacteriocin complemented by two extracellular components (lysin and activator)

The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL₁) ORF8 (bacL₂), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL₁, bacL₂, bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL₁ and -L₂ and bacA mutants. bacL₁ and -L₂ and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL₁-encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL₁-encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.     

Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses

Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml⁻¹ in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.     

Atg26-Mediated Pexophagy Is Required for Host Invasion by the Plant Pathogenic Fungus Colletotrichum orbiculare

The number of peroxisomes in a cell can change rapidly in response to changing environmental and physiological conditions. Pexophagy, a type of selective autophagy, is involved in peroxisome degradation, but its physiological role remains to be clarified. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare undergo peroxisome degradation as they infect host plants. We performed a random insertional mutagenesis screen to identify genes involved in cucumber pathogenesis by C. orbiculare. In this screen, we isolated a homolog of Pichia pastoris ATG26, which encodes a sterol glucosyltransferase that enhances pexophagy in this methylotrophic yeast. The C. orbiculare atg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step, implying a relationship between pexophagy and fungal phytopathogenicity. Consistent with this, its peroxisomes are degraded inside vacuoles, accompanied by the formation of autophagosomes during infection-related morphogenesis. The autophagic degradation of peroxisomes was significantly delayed in the appressoria of the atg26 mutant. Functional domain analysis of Atg26 suggested that both the phosphoinositide binding domain and the catalytic domain are required for pexophagy and pathogenicity. In contrast with the atg26 mutant, which is able to form appressoria, the atg8 mutant, which is defective in the entire autophagic pathway, cannot form normal appressoria in the earlier steps of morphogenesis. These results indicate a specific function for Atg26-enhanced pexophagy during host invasion by C. orbiculare.     

Overexpression and purification of rat peroxisomal membrane protein 22, PMP22, in Pichia pastoris

Peroxisomal membrane protein 22, PMP22, is an integral membrane protein that has four putative transmembrane-spanning regions. First reported as a major component of rat liver peroxisomal membranes and suggested to be involved in the metabolism of reactive oxygen species, its function and structure are still unknown owing to a lack of biochemical and structural experiments. Here we report the overproduction and purification of rat PMP22 (rPMP22) with the use of a methylotrophic yeast, Pichia pastoris, as a host. rPMP22 was localized not to peroxisomal membranes but to membrane compartments, such as the nuclear envelope. Highly pure rPMP22 was obtained in two steps. Several physicochemical assays indicated that the purified preparation should retain its functional structure. Furthermore, fed-batch fermentation yielded 90 mg of rPMP22 protein from 4L of culture. This is the first report to demonstrate the overproduction of a recombinant rPMP22 in the membrane compartments of P. pastoris.     

Green tea polyphenol suppresses tumor invasion and angiogenesis in N-butyl-(-4-hydroxybutyl) nitrosamine-induced bladder cancer

Background: Green tea polyphenol (GTP) suppresses malignancy in bladder cancer cell lines. However, the detail of its anti-carcinogenic effect in vivo is not fully understood. This study investigated the effect of GTP on bladder tumor size and angiogenesis in mice given N-butyl-(-4-hydroxybutyl) nitrosamine (BBN), with and without GTP. Methods: Eight-week-old female C3H/He mice were treated with and without 0.05% BBN solution for 14 or 24 weeks. In addition, they were also treated with and without 0.5% GTP solution for the same periods. Histopathological diagnosis was established using hematoxylin and eosin staining, and microvessel density (MVD) was estimated by counting CD34- and von Willebrand factor-positive vessels in the tumor area. Results: At 14 weeks, cancer cells were detected in BBN and BBN + GTP mice [5/14 (35.7%) and 3/14 (21.4%), respectively, p = 0.678]. At 24 weeks, the incidence of cancer cells was also similar between the groups (BBN + GTP: 61.9% vs. BBN: 82.6%; p = 0.179). However, the frequency of invasive tumors in BBN + GTP mice was significantly lower (23.8%; p = 0.030) than in those given BBN alone (65.2%). Tumor volume and MVD of intratumoral and stromal region in the BBN + GTP group were also significantly lower than in BBN mice. Conclusion: The results showed that GTP had no anti-carcinogenic effect, but inhibited tumor growth and invasion in mice with established bladder cancer, at least in part through the regulation of angiogenesis. Our data suggest that GTP seems to suppress tumor development in bladder cancer.     

<Emphasis Type="Italic">Legionella</Emphasis> pneumonia due to non-<Emphasis Type="Italic">Legionella pneumophila</Emphasis> serogroup 1: usefulness of the six-point scoring system

Background

Because of a limited number of reports, we aimed to investigate the clinical characteristics of patients with Legionella pneumonia due to non-Legionella pneumophila serogroup 1 and the diagnostic usefulness of the six-point scoring system for such patients compared with patients with pneumonia caused by L. pneumophila serogroup 1.

Methods

We retrospectively analysed patients diagnosed with Legionella pneumonia due to non-L. pneumophila serogroup 1 between March 2001 and June 2016. We examined the clinical characteristics, including symptoms, laboratory findings, radiologic findings, pneumonia severity, initial treatment and prognosis. We also calculated scores using the six-point scoring system in these patients. Furthermore, we compared the clinical characteristics and six-point scores between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients among hospitalized community-acquired pneumonia patients enrolled prospectively between October 2010 and July 2016.

Results

Eleven patients had pneumonia due to non-L. pneumophila serogroup 1; their median age was 66 years and 8 patients (72.7%) were male. The most common pathogen was L. pneumophila serogroup 3 (6/11), followed by L. pneumophila serogroup 9 (3/11), L. pneumophila serogroup 6 (1/11) and L. longbeachae (1/11). Non-specific symptoms, such as fever and cough, were common. Six patients (54.5%) had liver enzyme elevation, but no patient developed hyponatraemia at <130 mEq/L. Nine patients (81.8%) showed lobar pneumonia and 7 patients (63.6%) manifested with consolidation and ground-glass opacity. Patients with mild to moderate severity comprised 10 (90.9%) by CURB-65 and 5 (45.5%) by the Pneumonia Severity Index. Of all patients, 4 were admitted to the intensive care unit and 3 died despite appropriate empiric therapy. The clinical characteristics were not significantly different between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients (n?=?23). At a cut-off value of ≥?2 points, the sensitivity of the six-point scoring system was 54.5% (6/11) for non-L. pneumophila serogroup 1 patients and 95.7% (22/23) for L. pneumophila serogroup 1 patients.

Conclusions

Cases of non-L. pneumophila serogroup 1 pneumonia varied in severity from mild to severe and the clinical characteristics were often non-specific. The six-point scoring system was not useful in predicting such Legionella pneumonia cases.

     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

SH3BP2 Gain-Of-Function Mutation Exacerbates Inflammation and Bone Loss in a Murine Collagen-Induced Arthritis Model

Objective

SH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.

Methods

CIA was induced in wild-type (Sh3bp2^+/+) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2^KI/+) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-α production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.

Results

Sh3bp2^KI/+ mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-α and osteoclast marker genes were higher in the joints of Sh3bp2^KI/+ mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-γ and IL-17 production were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2^+/+ and Sh3bp2^KI/+ mice. In vitro experiments showed that TNF-α production in Sh3bp2^KI/+ BMMs is elevated compared with Sh3bp2^+/+ BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2^KI/+ BMMs associated with increased NFATc1 nuclear localization.

Conclusion

Gain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.