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Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.  相似文献   

3.
Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys477-His444-Asp388, rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.  相似文献   

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PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant plasmids. The proteinase was recovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-d-Tyr-l-Pro-l-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis ATCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other P. gingivalis proteins and describes the properties of P. gingivalis cells defective in the production of PrtP.Periodontitis is a major cause of tooth loss in the adult population, and several recent studies suggest that it may be a significant risk factor for both cardiovascular disease and preterm labor in humans (for a review, see reference 32). The potential medical importance of these oral infections justifies an intensified effort to develop effective strategies to prevent periodontitis as well as to interfere with disease progression. Accomplishing this will require identification of the major causative agents of periodontitis, as well as an understanding of the mechanisms by which these bacteria contribute to the destruction of connective tissue and bone that characterizes periodontitis lesions. Those virulence factors which render periodontal pathogens uniquely capable of contributing to the destruction of the supporting structures of the teeth, including periodontal connective tissues, periodontal ligaments, and alveolar bone, would be logical targets for the development of novel, highly specific antimicrobial agents (20).Porphyromonas gingivalis is one of a small number of bacteria implicated in the etiology and pathogenesis of human periodontitis, and its proteinases appear to be among its most important virulence factors (16, 26, 27). These enzymes, which can be recovered from P. gingivalis cells and vesicles as well as from spent growth medium (2), are capable of degrading a variety of substrates, including gelatin, fibrinogen, fibronectin, C3, C5, and C5a receptor (14, 1719, 51). Moreover, they clearly have the potential to contribute directly, as well as indirectly (via dysregulation of host proteinase cascade systems and the inflammatory response [16]), to destruction of periodontal tissues. Results of early studies suggested that P. gingivalis elaborates a bewildering number of distinct proteolytic enzymes, including at least one which is a collagenase (4). They also suggested that some of these proteinases can function as hemagglutinins (31) as well as adhesins for several host connective tissue matrix and plasma proteins (17, 19). The proteinase that we called porphypain was isolated from P. gingivalis W12 cells as sodium dodecyl sulfate (SDS)-stable conformers (150 and 120 kDa) of a 180-kDa proteinase (6). A proteinase called Lys-gingipain (also gingipain-K) was purified from spent culture medium, following growth of P. gingivalis HG66, as a 105-kDa complex containing a 60-kDa catalytic moiety (35, 37). While these proteinases have properties in common, significant differences in some of their properties were reported. Porphypain appears to contain two types of active sites, one with Lys-X activity and one with Arg-X activity. The amidolytic activity of both types of sites is greatly stimulated by derivatives of glycine and is inhibited by EDTA (6). Lys-gingipain, on the other hand, has been reported to have amidolytic activity exclusively for Lys-X but not Arg-X; it is inhibited by derivatives of glycine and is unaffected by EDTA (37). Since these proteinases were thought to be products of the same gene (2, 35), the difference between the reported activities of the purified enzymes was difficult to explain. Moreover, in spite of the fact that results of inhibitor studies have suggested that the collagenase of P. gingivalis has Lys-X and Arg-X activity (4), it is still unclear what, if any, relationship exists between porphypain, Lys-gingipain, and the collagenase of P. gingivalis.Results of more-recent biochemical and genetic studies (2, 11, 29, 39, 42) have suggested that 85 to 95% of the total proteolytic activity of P. gingivalis is attributable to three proteinases, PrtP (also called Kgp), Rgp-1, and Rgp-2. These enzymes are the products of three related genes (prtP [kgp], rgp-1, and rgp-2, respectively), and they appear to represent a unique family of cysteine proteinases (35). The gene referred to as rgp-1 (36) has also been designated prpRI (1), prtR (45), agpA (34), and rgpA (29) and encodes a 180-kDa protein. A second gene, which we refer to as rgp-2 (GenBank accession no. U85038), is also called prR2 (42), prtRII (GenBank accession no. AF007124), agpB (28), and rgpB (29), and it encodes an 80-kDa protein that is almost identical to the catalytically active region of the product of rgp-1 (42). Finally, prtP (2), like rgp-1 (33), encodes an approximately 180-kDa proteinase. Comparisons of the deduced amino acid sequences of PrtP, Rgp-1, Rgp-2, and a fourth, related P. gingivalis protein, the hemagglutinin HagA, are shown in Fig. Fig.1.1. The N-terminal two-fifths of the PrtP and Rgp-1 molecules is thought to contain the active sites of the enzymes, and the catalytic Cys residues have been mapped to these domains (35). The C-terminal three-fifths of these two proteinases is composed of regions that are highly homologous to each other and to much of HagA (2). No reports suggesting that HagA has cysteine proteinase activity have appeared. Open in a separate windowFIG. 1Comparison of PrtP with Rgp-1, Rgp-2, and HagA. Each protein is represented in a linear fashion; HagA is shown at half-scale. Putative cleavage sites in PrtP and Rgp-1 are shown below each protein. Regions in PrtP, Rgp-1, Rgp-2, and HagA with 90% or greater identity are indicated by identical boxes; regions with 50 to 60% identity are similarly underlined. (Modified from reference 2).It is unclear exactly which regions of HagA mediate hemagglutination. It appeared that a peptide (G-907 to T-919) derived from the C-terminal three-fifths of Rgp-1 inhibited hemagglutination by spent culture medium from P. gingivalis W50 (8), and a monoclonal antibody capable of blocking hemagglutination mapped to an epitope containing the same amino acid residues (7). This same peptide is located in the C-terminal three-fifths of PrtP and can also be found in HagA (2). It is unclear whether other peptides shared by these three gene products can mediate hemagglutination, and it is also unclear what contribution each of these gene products makes to the total hemagglutinating activity of P. gingivalis. Finally, regions of these proteins that mediate binding of P. gingivalis to host connective tissue and plasma proteins have not yet been identified.The PrtP and Rgp proteinases have been challenging to deal with biochemically. The full-length forms of Rgp-1 and PrtP are the same size (molecular weight, ∼180,000; PrtP is 1,732 amino acids in length, and Rgp-1 is 1,704 amino acids in length); they share regions of amino acid identity with each other and with HagA; and they autodegrade and possibly process each other during purification procedures (2, 6). Much of the confusion regarding the number, sizes, and properties of the proteinases of P. gingivalis can be attributed to these properties. Furthermore, their proteolytic degradation products, some of which are similar in size and amino acid sequence, autoaggregate and tend to stay tightly, though noncovalently, associated in solution, even in the presence of SDS (6). For these reasons, they copurify even when many differently based methods are applied for their separation (1, 6, 11, 38). In addition, the catalytic domains of Rgp-1 and Rgp-2, which are virtually indistinguishable biochemically (42), copurify from the P. gingivalis background. The existence of Rgp-2 as a gene product distinct from Rgp-1 was revealed only after identification of a second genomic locus encoding an arginine-specific proteinase (1, 29).Absolute separation of Rgp-1, Rgp-2, PrtP, HagA, and their proteolytically processed products from wild-type P. gingivalis cultures may well prove impossible to achieve, which would seriously hamper structure-function studies of these proteins. Expression of these genes in a heterologous host could provide a means for examining the functions of each of these proteins in the clear absence of the others. Coupling the results of these studies with the results of characterizations of corresponding P. gingivalis knockout mutants could provide a means of elucidating the functions of these proteins in P. gingivalis. No reports describing the expression of catalytically active proteinase from the rgp-1, rgp-2, or prtP gene cloned in Escherichia coli or any other prokaryotic heterologous host have appeared, although we (1a) and others (1) have expressed in E. coli, from cloned rgp-1 and prtP genes, proteins immunoreactive with antibodies raised against these proteinases. To date, the expression using a Baculovirus system of only a portion of kgp (prtP), containing just the purported catalytic domain, has been reported (35). The authors stated that supernatants from infected Sf9 cells contained a low level of Lys-specific amidolytic activity, but apparently they conducted no further analysis of the recombinant enzyme. Furthermore, while P. gingivalis strains with inactivated rgp genes have been previously constructed and partially characterized (15, 29, 30, 50, 52), P. gingivalis cells lacking a functional prtP gene have not been described. The purpose of this study was to develop a system for expression of prtP in a heterologous prokaryotic host and to characterize the recombinant enzyme in terms of its specificity, behavior in the presence of stimulators and inhibitors of proteolysis, ability to cleave type I collagen, and ability to function as a hemagglutinin. In addition, we sought to determine the effect of deletion of PrtP functions on P. gingivalis cells.  相似文献   

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构建抗牙龈卟啉单胞菌的牙周炎基因疫苗p VAX1-HA2、pVAX1-HA2/IL-15,体外检测其在293T细胞的表达。以HA2基因(牙龈卟啉单胞菌牙龈素—血凝素基因编码区的核心功能区)为目的基因与IL-15基因为免疫佐剂构建真核表达质粒,用Lip2000介导瞬时转染293T细胞,RT-PCR检测目的基因mRNA水平及酶联免疫吸附试验检测IL-15蛋白表达水平。重组质粒p VAX1-HA2、pVAX1-HA2/IL-15经酶切及DNA测序鉴定构建正确,转染的293T细胞能够检测到目的基因的表达,也可以检测到IL-15蛋白的表达。说明我们成功构建了真核共表达质粒pVAX1-HA2和p VAX1-HA2/IL-15,为下一步研制抗牙龈卟啉单胞菌DNA疫苗奠定了基础。  相似文献   

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人小肠三叶因子(hITF)基因在生菜中的整合与表达   总被引:10,自引:0,他引:10  
用根癌土壤杆菌(Agrobacterium tumefaciens(Smith et Townsend)conn)介导的叶盘法,将人小肠三叶因子(hITF)导入生菜(Lactuca sativa L.)中,在含有除草上培养基上筛选,获得抗性植株,通过PCR和Southern印迹分析证明,hITF cDNA已整合到生菜基因组中,Western印迹分析证明hITF在生菜中的表达。ELISA检测表明,hITF在生菜新鲜叶片中的表达量为200-300ng/g,最高达700ng/g,约占总可溶性蛋白的0.1%。  相似文献   

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Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site.  相似文献   

10.
细菌毒力基因体内表达检测技术研究进展   总被引:1,自引:0,他引:1  
病原菌入侵宿主是一个及其复杂的过程。为了深入了解病原菌的致病机理,人们需要鉴定那些在感染过程中特异表达的细菌毒力基因。为此,多种体内实验模型被建立起来分析细菌在宿主体内的基因表达,它们包括了体内表达技术、信号标签突变技术、差异荧光诱导、体外转座进行基因组分析和作图技术以及体内诱导抗原技术等。文章对目前运用的这些研究方法进展进行综述,并讨论了它们的优点与不足。  相似文献   

11.
将bdnf基因克隆入逆转录病毒载体pLNCX,构建得到pLNC/BDNF,经PA317细胞包装后,感染大鼠成肌细胞L6TG,G418筛选2周后,得到稳定表达bdnf基因的细胞克隆L6TG/BDNF。DNA印迹结果证实bdnf基因已经整合入L6TG染色体中,RNA印迹和斑点印迹结果分别从mRNA水平和蛋白水平证明了bdnf基因的表达,且L6TG/BDNF培养上清中BDNF的含量约为25ng(106细胞数每ml每24h)。  相似文献   

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Porphyromonas gingivalis, a Gram-negative anaerobic rod, has been closely associated with the initiation and progression of periodontal disease. This organism has been shown to produce a large number of proteolytic enzymes which can degrade a variety of tissue proteins, and these are considered to be major virulence factors. One of the proteinases produced by this organism, referred to as gingipain-1, has been purified to homogeneity from P. gingivalis culture medium by a combination of gel filtration and ion-exchange chromatography. The enzyme was found to have a molecular mass near 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for cysteine for activation and Ca2+ for stabilization. Amino-terminal sequence analysis indicated that gingipain belongs to a new, so far unknown, subfamily of cysteine proteinases. Three unusual features of this proteinase are: (a) the stimulation of amidolytic activity by glycine-containing dipeptides; (b) a narrow specificity which is limited to peptide bonds containing arginine residues; and (c) resistance to inhibition by proteinase inhibitors in human plasma.  相似文献   

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为了发展一种新型的融合蛋白(RGD)3/tTF用于肿瘤血管的选择性栓塞治疗,利用PCR技术重组(RGD)3/tTF融合基因,克隆于pET22b( )载体,表达于E.coliBL21(DE3)。用镍柱纯化融合蛋白。凝血实验与FⅩ活化实验检测融合蛋白tTF组分的活性。间接ELISA分析(RGD)3/tTF与αvβ3的特异结合能力。pET22b( )/(RGD)3/tTF重组质粒成功获得并表达于E.coliBL21(DE3)。纯化蛋白(RGD)3/tTF能有效诱发血液凝固,活化FⅩ。(RGD)3/tTF与αvβ3的特异结合能力比RGD/tTF提高了32%。新型融合蛋白(RGD)3/tTF已在E.coli系统成功表达,表达蛋白保持tTF的活性并显示比RGD/tTF更高的与αvβ3的结合能力。  相似文献   

16.
The ability of Porphyromonas gingivalis to biosynthesize tetrapyrroles de novo has been investigated. Extracts of the bacterium do not possess activity for 5- aminolevulinic-acid dehydratase or porphobilinogen deaminase, two key enzymes involved in the synthesis of uroporphyrinogen III. Similarly, it was not possible to detect any genetic evidence for these early enzymes with the use of degenerate polymerase chain reaction. However, the bacterium does appear to harbor some of the enzymes for cobalamin biosynthesis since cobyric acid, a pathway intermediate, was converted into cobinamide. Furthermore, degenerate polymerase chain reaction with primers to cbiP, which encodes cobyric-acid synthase, produced a fragment with a high degree of identity to Salmonella typhimurium cbiP. Indeed, the recently released genome sequence data confirmed the presence of cbiP together with 14 other genes of the cobalamin pathway. A number of these genes were cloned and functionally characterized. Although P. gingivalis harbors all the genes necessary to convert precorrin-2 into cobalamin, it is missing the genes for the synthesis of precorrin-2. Either the organism has a novel pathway for the synthesis of precorrin-2, or more likely, it has lost this early part of the pathway. The remainder of the pathway may be being maintained to act as a salvage route for corrin synthesis.  相似文献   

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Porphyromonas gingivalis, and organism implicated in the etiology and pathogenesis of human periodontal diseases, produces a variety of potent proteolytic enzymes, and it has been suggested that these enzymes play a direct role in the destruction of periodontal tissues. We now report that two cell-associated cysteine proteinases of P. gingivalis W12, with molecular masses of approximately 150 kDa (porphypain-1) and 120 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable conformational variants of a 180-kDa enzyme, and they are the largest cysteine proteinases yet purified from P. gingivalis. The purified proteinases hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tosyl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agents, is inhibited by EDTA, and is stimulated in the presence of derivatives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the Lys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl ketone and insensitive to leupeptin. These data suggest that porphypains contain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal tissue destruction.  相似文献   

19.
A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.  相似文献   

20.
Probiotics and Antimicrobial Proteins - Thaumatin-like proteins (TLPs) are the products of a large, highly complex gene family involved in host defense. TLPs also belong to the pathogenesis-related...  相似文献   

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