摘要：
Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.
作者：
Su, Zehong;Lian, Gaojian;Mawatari, Kazuaki;Tang, Ping;He, Shuya;...
期刊：
Photochemistry and Photobiology,2015年91(5):1165-1172 ISSN：0031-8655
通讯作者：
Yin, Weidong
作者机构：
[He, Shuya; Lian, Gaojian; Su, Zehong; Tang, Ping; Yin, Weidong] Univ South China, Sch Pharmacol & Life Sci & Technol, Dept Biotechnol, Hengyang, Peoples R China.;[Mawatari, Kazuaki; Su, Zehong; Takahashi, Akira; Shimohata, Takaaki] Univ Tokushima, Grad Sch, Inst Hlth Biosci, Dept Prevent Environm & Nutr, Tokushima 770, Japan.;[Wu, Yimou] Univ South China, Dept Microbiol & Immunol, Hengyang, Peoples R China.
通讯机构：
[Yin, Weidong] U;Univ South China, Sch Pharmacol & Life Sci & Technol, Dept Biotechnol, Hengyang, Peoples R China.
摘要：
Photoreactivation is an error‐free mechanism of DNA repair, utilized by prokaryotes and most eukaryotes and is catalyzed by specific enzymes called DNA photolyases. Photoreactivation has been reported in Vibrio parahaemolyticus WP28; however, information on photolyases in V. parahaemolyticus (V.p) strains has not been reported. This study examined the photoreactivation in V.p RIMD2210633. The photolyase responsible for repairing cyclobutane pyrimidine dimer (CPD) in DNA was identified, and the corresponding gene was determined as VPA1471. The protein was overexpressed in Escherichia coli and was purified for functional assessment in vitro. The mRNA level and protein expression level of this gene increased after ultraviolet A (UVA) illumination following ultraviolet C (UVC) irradiation. In vitro experiments confirmed that the protein encoded by VPA1471 could reduce the quantity of CPD in DNA. We designated the corresponding gene and protein of VPA1471 phr and Phr, respectively, although the function of two other photolyase/cryptochrome family members, VPA0203 and VPA0204, remains unclear. UV (ultraviolet) irradiation experiments suggest that these two genes possess some photorepairing ability. Therefore, we hypothesize that VPA0203 and VPA0204 encode (6‐4) photolyase in V. parahaemolyticus RIMD2210633. Vibrio parahaemolyticus possesses the photoreactivation ability when it utilizes blue light as energy. However, ΔVPA1471 abolished this capacity completely, whereas complementation of VPA1417 could make this ability recover partly. Some cells of UVC radiated VPA0203 KO and VPA0204 KO strains recovered by the following UVA illumination.
通讯机构：
[Wu, Yimou] U;Univ South China, Inst Pathobiol, Dept Microbiol, Hengyang, Hunan, Peoples R China.
摘要：
Although modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity.
作者机构：
[谢小平; 易婷; 朱雅玲; 罗韬; Zeng D.; 刘双全; 刘炀] Department of Clinical Laboratory, The First Affiliated Hospital of University of South China, Hengyang, 421001, China;[吴移谋] Institute of Pathogenic Biology, University of South China, Hengyang, 421001, China
通讯机构：
[Wu, Y.] I;Institute of Pathogenic Biology, University of South China, Hengyang, China
作者机构：
[何璐] Departments of Gynecology and Obstetrics, First Affiliated Hospital, Institute of Pathogenic Biology, Medical College, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Hengyang 421001, China;[吴移谋; 朱翠明; 游晓星; 余敏君; Zeng, Yanhua; 李冉辉] Institute of Pathogenic Biology, Medical College, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Hengyang 421001, China;[李国华] Institute of Cardiovascular Disease, University of South China, Hengyang 421001, China
作者机构：
[Jiang, Chuanhao; Wu, Yimou; Liu, Liangzhuan; He, Jun; Ou, Guangli; Zhu, Cuiming; Chen, Liesong; You, Xiaoxing; Yu, Minjun; Zeng, Yanhua; Li, Ranhui; Ma, Xiaohua] Univ South China, Coll Med, Inst Pathogen Biol, Hengyang City, Peoples R China.;[Ma, Xiaohua] Changsha Cent Hosp, Dept Clin Lab, Chagnsha City, Peoples R China.;[He, Jun] Univ South China, Affiliated Nanhua Hosp, Dept Clin Lab, Hengyang City, Peoples R China.
通讯机构：
[Wu, Yimou] U;Univ South China, Coll Med, Inst Pathogen Biol, Hengyang City, Peoples R China.
关键词：
Small interfering RNAs;Transfection;Immune receptor signaling;Inflammation;Phosphorylation;Mycoplasma;Inflammatory diseases;Luciferase
摘要：
AIMS: This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes. METHODS: Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay. RESULTS: MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002. CONCLUSIONS: These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.
作者机构：
[Yanhua Zeng; Xiaoxing You; Cuiming Zhu; Yimou Wu] Institute of Pathogenic Biology, University of South China, Hengyang 421001, China;[Jun He] Department of Clinical Laboratory, the Affiliated Nanhua Hospital of University of South China, Hengyang 421001, China
会议名称：
The 6th Meeting of the Asian Organization for Mycoplasmology (AOM)The 7th Meeting of the Chinese Society for Mycoplasmology (CSM)(第七届全国支原体学术会议暨第六届亚洲支原体学术会议)
会议时间：
2014-8-22
会议地点：
张家界
会议主办单位：
中华医学会微生物与免疫学分会;亚洲支原体学组织
会议论文集名称：
The 6th Meeting of the Asian Organization for Mycoplasmology (AOM)The 7th Meeting of the Chinese Society for Mycoplasmology (CSM)(第七届全国支原体学术会议暨第六届亚洲支原体学术会议)论文集
摘要：
Objective To analyze the the humoral and cellular immune response levels of multiple antigen peptides (MAPs) containing the mimic epitope of the adhesion protein of Mycoplasma genitalium Pa (MgPa) in order to provide the theoretical basis for the development of multi-epitope-baseded marker vaccines.
作者机构：
[谢婧] Departments of Obstetrics, Center Hospital of Hengyang, Hengyang 421001, China;[吴移谋; 陈列松; 朱翠明; 游晓星; 余敏君; Zeng, Yanhua; 李冉辉] Institution of Pathogenic Biology, Medical College, Hengyang 421001, China;[何军] Institution of Pathogenic Biology, Medical College, Department of Clinical Laboratory, Affiliated Nanhua Hospital, University of South China, Hengyang 421001, China
作者机构：
[Guozhi Dai; Yimou Wu] Institute of Pathogenic Biology, University of South China, Hengyang 421001, China;[Guozhi Dai; Hongliang Chen] Department of Inspection, The First People's Hospital of Chenzhou, Chenzhou 423000, China
会议名称：
The 6th Meeting of the Asian Organization for Mycoplasmology (AOM)The 7th Meeting of the Chinese Society for Mycoplasmology (CSM)(第七届全国支原体学术会议暨第六届亚洲支原体学术会议)
会议时间：
2014-8-22
会议地点：
张家界
会议主办单位：
中华医学会微生物与免疫学分会;亚洲支原体学组织
会议论文集名称：
The 6th Meeting of the Asian Organization for Mycoplasmology (AOM)The 7th Meeting of the Chinese Society for Mycoplasmology (CSM)(第七届全国支原体学术会议暨第六届亚洲支原体学术会议)论文集
摘要：
Objective As obligate parasites, Ureaplasma urealyticum are continuously exposed to oxidative damage due to host generated peroxides and reactive oxygen species (ROS).In addition, the ROS could be induced by U.urealyticum which indicated the U.urealyticum should have antioxidant system that can protect the bacteria from the oxidative damage to maintain the infection.
作者机构：
[余益本] Department of Gerontology, Dongfeng Hospital, Hubei University of Medicine, Shiyan 442000;[余益本] Laboratory of Pathogenic Biology, National Center of Clinical Medicine Research, University of South China, Hengyang 421001, China;[吴移谋] Laboratory of Pathogenic Biology, University of South China, Hengyang 421001, China;[文格波] National Center of Clinical Medicine Research, University of South China, Hengyang 421001, China;[杨文琼] Department of Neurology, Dongfeng Hospital, Hubei University of Medicine, Shiyan 442000, China
作者：
Bo He;Xilin Xiao;Yanhua Zeng;Lifu Liao;Yimou Wu;...
作者机构：
[Bo He; Xilin Xiao; Lifu Liao; Shuqin Gao] College of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China;[Yanhua Zeng; Yimou Wu] Institute of Pathogenic Bio1ogy, Medical College, University of South China, Hengyang 421001, China
会议名称：
The 6th Meeting of the Asian Organization for Mycoplasmology (AOM)The 7th Meeting of the Chinese Society for Mycoplasmology (CSM)(第七届全国支原体学术会议暨第六届亚洲支原体学术会议)
会议时间：
2014-8-22
会议地点：
张家界
会议主办单位：
中华医学会微生物与免疫学分会;亚洲支原体学组织
会议论文集名称：
The 6th Meeting of the Asian Organization for Mycoplasmology (AOM)The 7th Meeting of the Chinese Society for Mycoplasmology (CSM)(第七届全国支原体学术会议暨第六届亚洲支原体学术会议)论文集
关键词：
Mycoplasma genitalium;Wireless;Quantum dot
摘要：
A wireless immunosensor for the detection of Mycoplasma genitalium was fabricated by immobilizing polyclonal antibody onto the surface of a magnetostrictive strip.In response to a time-varying magnetic field, the immunosensor longitudinally vibrates at a resonance frequency, emitting magnetic flux that can be remotely detected by a pickup coil.
