Endopeptidase activities of selected Porphyromonas spp., Prevotella spp. and Fusobacterium spp. of oral and non-oral origin

The ability of three Porphyromonas spp., seven Prevotella spp., seven Fusobacterium spp. and two related Bacteroides spp. (B. levii and B. macacae) to degrade an extensive range of synthetic endo-, amino- and diamino peptidase substrates linked to the fluorescent leaving group 7-amido-4-methylcoumarin (NHMec) was investigated. Many more species than was previously recognized exhibited peptidase activities, albeit at lower levels than those already described for Porphyromonas gingivalis. Detection of chymotrypsin-like activity was dependent on which of three NHMec-linked substrates was used, but all species exhibited degradative activity with at least one of these substrates. Elastase-like activity was detected in all species though not all species reacted with each of the elastase substrates. Glycylprolyl peptidase activity was detected in all of the species tested with the exception of F. mortiferum, F. gonidiaformans, F. naviforme and F. necrophorum. While the detection of peptidase activities does not appear to be useful for the differentiation of species within the genera Bacteroides and Prevotella, its ability to differentiate species of the genus Porphyromonas or Fosobacterium further investigation.     

Expression of the β‐adhesin part of HRgpA in Sprague Dawley rats induces a specific antibody response

The β‐adhesin part of the Porphyromonas gingivalis W50 (ATCC 53978) protease HRgpA was cloned in an eukaryotic expression vector and expressed in COS‐7 cells. The monoclonal antibody MAb (61BG1.3), specific for the hemagglutinating domain of β‐adhesin, recognized the expressed β‐adhesin in the transfected cells both by immunoblot and immunofluorescence. Sprague Dawley rats were immunized intramuscularly with β‐adhesin encoding expression plasmid and expression plasmid without β‐adhesin insert. Skeletal muscle tissue at the site of immunization in the β‐adhesin immunized animals was shown to express this protein. The immunization induced a β‐adhesin‐specific antibody response. Sera from the immunized animals were tested for hemagglutination inhibiting activity. Due to high natural inhibiting activity in all rat sera tested, no increased hemagglutination inhibition was detected in sera from the β‐adhesin immunized animals.     

Characterisation of black-pigmented anaerobes isolated from diseased and healthy periodontal sites

Prevotella intermedia has recently been re-defined and a new species, Prevotella nigrescens has been proposed. However, there is little data available on the incidence of these new species in periodontal health or disease. Black-pigmented anaerobes isolated from diseased and healthy subgingival sites were identified by serotyping, SDS-PAGE and physiological tests. In adult periodontitis subjects, 64% of active sites, 35.7% of inactive sites and 38.5% of healthy sites yielded black-pigmented anaerobes. Of these, Porphyromonas gingivalis was found in 11% of active and 5% of healthy sites in diseased patients, Prevotella intermedia in 15.5% of active and 20.5% of healthy sites, Prevotella nigrescens in 37.7% of active and 11.5% of healthy sites and Prevotella denticola in 3% of active and 1% of healthy sites. In healthy subjects, 50% of sites yielded black-pigmented anaerobes. P. gingivalis was not found in healthy subjects but P. intermedia was found in 18% and P. nigrescens in 31% of sites. SDS-PAGE proved to be a useful method for routinely differentiating P. intermedia and P. nigrescens and two sub-types of the latter species were detected on the basis of band pattern. Only one P. nigrescens sub-type was found in any given individual and one type, typified by ATCC 25261, was more commonly found in deep pockets. However, overall both P. nigrescens and P. intermedia as species were just as frequently found at healthy sites as diseased sites. Thus, these species, in contrast to P. gingivalis , appear to be common commensals but they may act as opportunistic pathogens.     

Demonstration of adherence properties of Porphyromonas gingivalis outer membrane vesicles using a new microassay

Vesicles made by Porphyromonas gingivalis possess several biological activities, including the ability to adhere to oral surfaces and to bacteria. In this study, a new and simple method was developed to measure the adherence capability of outer membrane vesicles from P. gingivalis . Vesicles were conjugated to fluorescent microspheres (0.7 μ) and added to wells of a Teflon-coated microscope slide previously covered with a variety of soluble ligands. After incubation and washes, the number of fluorescent microspheres per microscopic field were counted. Vesicle-coated microspheres attached best to gelatin (<200 per field), whereas other compounds (such as fibronectin, fibrinogen, collagen and laminin) provided moderate attachment, and no attachment was observed to bovine serum albumin. Adherence to any of the tested ligands was not observed when fluorescent micro-spheres were conjugated to bovine serum albumin or lipopolysaccharides from P. gingivalis. The adherence of vesicle-coated microspheres to ligands was not significantly affected when the pH of the reaction mixture was between 4 and 10. None of the tested carbohydrates lowered the attachment capability of vesicle-coated microspheres to substrates. When vesicle-coated microspheres were treated with trypsin and chymotrypsin or heated, this resulted in a significant loss of attachment, suggesting a possible involvement of proteinaceous molecules in the process. The present study confirms that vesicles of P. gingivalis are capable of attachment to various molecules and indicate their potential role in colonization.     

The potential role of α2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis

Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma α₂-macroglobulin (α₂M). Both 50- and 95 kDa gingipain R were efficiently inhibited by α₂M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, α₁-inhibitor-3 a-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the aminio acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of α₂M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by α₂M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.     

Proteolytic artifacts in SDS-PAGE analysis of selected periodontal pathogen:

The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution of protein bands, occur during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of cellular proteins from selected proteolytic ( Porphyromonas gingivalis, Prevotella nigrescens and Treponema denticola ) and non-proteolytic ( Fusobacterium nudeatum ) bacteria. Conditions to limit or prevent proteolysis were also investigated. Bacterial cells were incubated in solubilizing buffer (SDS +β mercaptoethanol) at room temperature for various periods of time before boiling. A control assay consisted of trichloroacetic acid-treated bacterial cells. Cellular proteins were separated by electrophoresis and stained with Coomassie blue. Proteolysis occurred very rapidly in the case of P. gingivalis (<30 s), whereas a longer incubation time (>1 h) was required to observe similar effects in P. nigrescens and T. denticola. No proteolysis was observed for F. nudeatum. In all cases, heat (100°C) and low pH (<4) treatments of bacterial cells could avoid production of proteolytic artifacts. Incorporation of specific protease inhibitors before solubilization of bacteria could also prevent proteolysis. More particularly, N -α- p -tosyl- l -lysine chloromethyl ketone (TLCK), iodoacetamide and diisopropylfluorophosphate (50 mM) were highly efficient for P. gingivalis, P. nigrescens and T. denticola , respectively. When outer membranes of P. gingivalis were prepared in the presence of TLCK, numerous additional protein bands, not seen in the absence of TLCK, were detected. The present study suggests that specific protease inhibitors, effective in preventing proteolysis. should be identified and added during cell fractionation and protein purification procedures.     

牙龈卟啉单胞菌感染对载脂蛋白e基因敲除小鼠动脉粥样硬化的影响

目的: 探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,P. gingivalis)引起的炎症和氧化应激反应对动脉粥样硬化的影响及作用机制。方法: 采用8周龄载脂蛋白e基因敲除(ApoE knockout,ApoE-/-)小鼠建立动脉粥样硬化动物模型,将小鼠随机分为两组:(1)磷酸盐缓冲液(phosphate buffered saline,PBS)健康对照组:8只ApoE-/-小鼠,普通饮食+PBS鼠尾静脉注射;(2)P. gingivalis感染组:8只ApoE-/-小鼠,普通饮食+P. gingivalis鼠尾静脉注射。1周3次,隔天1次,共10次。4周后处死,取心脏组织进行油红O染色,血清进行酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA),主动脉进行实时荧光定量PCR以及Western blot检测。结果: P. gingivalis 感染组较PBS健康对照组可以显著加重ApoE-/-小鼠动脉粥样硬化斑块的形成,增加血清中炎症介质,如白细胞介素(interleukin,IL)-1β、IL-6和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)以及氧化应激介质8-羟脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHDG)表达,增加主动脉组织中IL-1β、IL-6、TNF-α、NADPH 氧化酶(NADPH oxidase,NOX)-2和NOX-4基因的mRNA水平。P. gingivalis感染后在主动脉组织中观察到核转录因子-κB(nuclear factor-kappa B,NF-κB)表达有增高趋势。结论: P.gingivalis感染会加速ApoE-/-小鼠动脉粥样硬化进程,诱导氧化应激和炎症反应;NF-κB信号通路可能是P. gingivalis加速动脉粥样硬化形成的重要作用机制。     

定向敲除牙龈卟啉单胞菌菌毛蛋白FimA的重组质粒构建

目的 构建定向敲除牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)菌毛蛋白FimA基因的质粒pPHU281_A_Spec_B,用于后续构建菌毛蛋白FimA缺陷型Pg,为研究FimA的功能奠定分子基础.方法 厌氧培养PgATCC33277,提取基因组DNA,聚合酶链反应扩增PgFimA基因一定长度的上游A片段及下游B片段,并在扩增片段两端添加合适的酶切位点；利用定向克隆技术将A和B 基因插入到载体自杀质粒pPHU281中,并添加大观霉素抗性基因片段,重组的pPHU281 _A_Spec_B 在大肠杆菌DH-5α内扩增后酶切电泳鉴定并进行DNA测序.结果 通过对重组质粒pPHU281_A_Spec_B进行酶切鉴定以及DNA序列测定分析,证明重组质粒pPHU281_A_Spec_B构建成功.结论 成功构建了质粒pPHU281_A_Spec_B,有利于菌毛蛋白FimA缺陷型Pg的构建,为深入研究FimA的作用机制奠定了基础.     

����߲����������Ⱦ����ϸ���������������о���չ

??Porphyromonas gingivalis?? a gram-negative obligate anaerobic bacterium??has been proved to be one of the main periodontal pathogens?? participating in development of periodontitis and process of periodontal tissue destruction. Previous studies have demonstrated the close relationship between periodontal infection and systemic health and diseases. P. gingivalis infection on host cells mediated cell apoptosis by interfering with cell signal transduction and expression of related proteins. This article reviews research progress of P. gingivalis infection on mediation of apoptosis of host cells.     

Immunomodulation of dendritic cells differentiated in the presence of nicotine with lipopolysaccharide from Porphyromonas gingivalis

Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T‐cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte‐derived DCs to elucidate the mechanism of tissue destruction of smoking‐associated periodontal diseases. P. gingivalis LPS‐stimulated DCs differentiated with nicotine (NiDCs) induced lower T‐cell proliferation and human leukocyte antigen (HLA)‐DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon‐γ production but maintained interleukin (IL)‐5 and IL‐10 production in co‐cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)‐type chemokines macrophage chemotactic protein‐1 and macrophage‐derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS‐stimulated DCs.