Unaltered expression of the major protease genes in a non‐virulent recA‐defective mutant of Porphyromonas gingivalis W83

Porphyromonas gingivalis FLL32, a recA mutant, was isolated during construction of a recA defective mutant of P. gingivalis W83 by allelic exchange mutagenesis. In contrast to W83 and FLL33, the typical recA^? mutant previously reported, FLL32 was non‐pigmented, lacked β‐hemolytic activity on blood agar and produced significantly less proteolytic activity. The proteolytic activity in FLL32 was mostly soluble. Expression of the rgpA, rgpB and kgp protease genes was unaltered in FLL32 when compared to FLL33 and the wild‐type strain. FLL32 exhibited reduced virulence in a murine model and partially protected the animals immunized with that strain against a subsequent lethal challenge by the wild‐type strain. These results indicate that the reduced level of proteolytic activity in FLL32 may be due to a defect in the processing of the proteases. Further, immunization with a non‐virulent recA defective mutant of P. gingivalis can partially protect against a lethal wild‐type challenge. The results from this study suggest that the recA locus may be involved in expression and regulation of proteolytic activity in P. gingivalis.     

Alkyl hydroperoxide peroxidase subunit C (ahpC) protects against organic peroxides but does not affect the virulence of Porphyromonas gingivalis W83

The cloned Porphyromonas gingivalis alkyl hydroperoxide reductase (ahpC) gene complemented an ahpC defect in Escherichia coli. To study the role of ahpC in protecting against oxidative stress in P. gingivalis a 1.8 kb fragment containing the ahpC gene was amplified from the chromosome of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic resistance cassette and used to create a ahpC-deficient mutant by allelic exchange. One mutant strain, designated FLL141, demonstrated no change in the growth rate, black pigmentation, beta-hemolysis or level of proteolytic activity compared to the parent strain. Although P. gingivalis FLL141 was more sensitive to hydrogen peroxide than the parent strain, there was no change in its virulence potential in the mouse model compared to the wild-type strain. These findings suggest that the ahpC gene plays a role in peroxide resistance in P. gingivalis but does not contribute significantly to virulence.     

The bcp gene in the bcp-recA-vimA-vimE-vimF operon is important in oxidative stress resistance in Porphyromonas gingivalis W83

The ability of Porphyromonas gingivalis to overcome oxidative stress in the inflammatory environment of the periodontal pocket is critical for its survival. We have previously demonstrated that the recA locus, which carries the bacterioferritin co-migratory protein (bcp) gene and has a unique genetic architecture, plays a role in virulence regulation and oxidative stress resistance in P. gingivalis. To further characterize the bcp gene, which was confirmed to be part of the bcp-recA-vimA-vimE-vimF operon, we created a P. gingivalis bcp-defective isogenic mutant (FLL302) by allelic exchange. Compared with the wild-type, FLL302 had a similar growth rate, black pigmentation, β-hemolysis and UV sensitivity. Although there was no change in the distribution of gingipain activity, there was a 30% reduction in both Arg-X and Lys-X activities in the mutant strain compared with the wild-type. When exposed to 0.25 mm hydrogen peroxide, P. gingivalis FLL302 was more sensitive than the wild-type. In addition, the cloned P. gingivalis bcp gene increased resistance to 0.25 mm hydrogen peroxide in a bcp-defective Escherichia coli mutant. The mutant also demonstrated decreased aerotolerance when compared with the wild-type. Porphyromonas gingivalis FLL302 and the wild-type strain had similar virulence profiles in a mouse model of virulence. These observations suggest that the bcp gene may play a role in oxidative stress resistance but has a decreased functional significance in the pathogenic potential of P. gingivalis.     

牙龈卟啉单胞菌唾液酸酶基因突变株黏附与侵入KB细胞能力研究

目的    研究PG0352基因缺失对牙龈卟啉单胞菌（P. gingivalis）黏附和侵入人口腔上皮癌KB细胞能力的影响。方法    本研究于2014年7—12月在中国医科大学口腔医学院中心实验室完成。实验组：将P. gingivalis W83菌株（W83菌株组）和PG0352基因突变株（PG0352突变株组）分别以100∶1的比例与KB细胞共培养2 h,制备P. gingivalis与KB细胞的黏附和侵入模型;对照组：单纯KB细胞培养。透射电镜观察KB细胞表面及细胞内是否有P. gingivalis存在;用PBS清除未黏附于KB细胞的细菌,裂解细胞后涂板检测P. gingivalis黏附和侵入KB细胞情况,抗生素保护法检测P. gingivalis侵入KB细胞情况。结果    透射电镜结果发现,W83菌株组和PG0352突变株组KB细胞内均有细菌侵入;通过涂板后菌落计数发现P. gingivalis W83菌株和PG0352基因突变株对KB细胞的黏附率分别为（15.559 ± 2.020）%和（9.309 ± 1.750）%,侵入率分别为（0.651 ± 0.287）%和（1.517 ± 0.233）%,两者差异均有统计学意义（均P < 0.05）。结论    P. gingivalis W83菌株和PG0352基因突变株均能够黏附和侵入KB细胞;与W83菌株相比,PG0352基因突变株黏附上皮细胞的能力较弱而侵入能力较强。     

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??Objective    To study the adhesive and invasive potential of PG0352 mutant strain interacting with KB cell. Methods    This study was carried out at Center Laboratory of School and Hospital of Stomatology??China Medical University from July to December 2014. Porphyromonas gingivalis??P. gingivalis??W83 or PG0352 mutant strain and KB cells were cocultured by MOI 100??1 for 2 h. Transmission electron microscopy was used to see whether P. gingivalis W83 and PG0352 mutant strain existed in KB cell. P. gingivalis in the coculture medium were removed??and P. gingivalis??which adhered to and invaded KB cell were released after lysis KB cells and then P. gingivalis were coated to the BHI plate. The invasive ability of P. gingivalis W83 and PG0352 mutant strain was measured by means of an antibiotic protection assay. Results    Both P. gingivalis W83 and PG0352 mutant strain adhered to and entered KB cells. ??15.559 ± 2.020??% P. gingivalis W83 and ??9.309 ± 1.750??% PG0352 mutant strain adhered to KB cell??and ??0.651 ± 0.287??% P. gingivalis W83 and ??1.517 ± 0.233??% PG0352 mutant strain  invaded into KB cell??both with statistical difference. Conclusion    Both P. gingivalis W83 and PG0352 mutant strain can adhere to and enter KB cell. Compared to P. gingivalis W83??less PG0352 mutant strain can adhere to KB cell and more PG0352 mutant strain can invade into KB cell.     

Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

The ability for DNA mismatch repair, after oxidative stress‐induced DNA damage, is critical for the persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket. Our previous report demonstrated that, in contrast to other organisms, the repair of oxidative stress‐induced DNA damage involving 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) may occur by a yet‐to‐be described mechanism in P. gingivalis. 8‐oxoG does not block DNA replication; rather, it mispairs with adenine, which can be repaired by the MutY glycosylase. To determine the function of the P. gingivalis MutY homologue in DNA repair, it was insertionally inactivated using the ermF‐ermAM antibiotic cassette and used to create a mutY‐deficient mutant (FLL147) by allelic exchange mutagenesis. FLL147 had an increased rate of spontaneous mutation and was more sensitive to hydrogen peroxide compared with the wild‐type W83 strain. DNA oligomers containing a site‐specific 8‐oxoG:A mispair was repaired similarly in both the P. gingivalis mutY‐defective mutant and wild‐type strains. The P. gingivalis mutY homologue was shown to complement the mutY mutation in Escherichia coli. In a gel mobility shift assay, the purified recombinant MutY is able to bind an oligo containing an 8‐oxoG:A mispair. Taken together, MutY may play the expected role in oxidative stress resistance in P. gingivalis. However, there may exist other redundant mechanism(s) for the removal of 8‐oxoG:A mismatch in this organism.     

Failure of Bacteroides gingivalis W83 to accumulate bound C3 following opsonization with serum

Our previous studies have demonstrated that strains of Bacteroides gingivalis are capable of proteolytic degradation and inactivation of complement proteins including the third component of complement C3. Since a crucial step in the ability of complement to control bacterial infections is the binding of C3 fragments to the bacterial surface with subsequent enhancement of phagocytosis, further examination of the importance of the proteolytic capacity of Bacteroides in interactions with complement proteins was carried out by quantitating the amount of C3 bound to two proteolytic Bacteroides gingivalis strains. Pooled normal human serum (NHS) containing 125I-C3 was incubated with strains of B. gingivalis (W83 and ATCC 33277) and the non-proteolytic pathogen A. actinomycetemcomitans strain Y4, and samples of the reaction mixtures were removed at various time intervals for determination of bound C3. B. gingivalis 33277 bound only half the number of C3 molecules as did A. actinomycetemcomitans, while B. gingivalis W83 bound very little C3. A large increase in the number of C3 molecules bound to B. gingivalis W83 was noted in assays carried out in the presence of the protease inhibitor TLCK, indicating that bacterial proteases may be responsible for the lack of binding of C3 to strain W83. TLCK treatment modestly increased the accumulation of C3 on strain 33277, but had no effect on A. actinomycetemcomitans. Analysis of 125I-C3 in supernatants from reaction mixtures of strain 33277, W83, or a proteolytic strain of B. intermedius demonstrated no qualitative differences in the C3 fragments amongst the tested strains or in the presence or absence of TLCK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation in vivo of the C3 protein of guinea-pig complement by a pathogenic strain of Bacteroides gingivalis

Abstract – The pathogenicity of five black-pigmented strains of Bacteroides was tested in subcutaneously implanted Teflon cages in guinea pigs. The tissue reaction around the cages was registered and the contents of the fluid of the cages were analyzed. Two strains of B. intermedius produced a localized abscess around the cages, while one strain (381) of B. gingivalis and an asaccharolytic strain (BN11a-f) different from B. gingivalis did not induce any signs of abscess formation. One strain (W83) of B. gingivalis caused extensive purulent breakdown of the tissues. When the inoculum of strain VV83 contained more than 109 cells, the animals were killed. Strain W83 was the only strain that increased in number in the cage. The fluid of cages inoculated with strain W83 was also remarkably different from the fluid of cages inoculated with the other strains. The fluid had a high proteolytic activity. No C3 protein of complement and only traces of immunoglobulins could be detected in the fluid. Both strain W83 and strain 381 had a high proteolytic activity against whole guinea-pig serum and when bacteria of these two strains were incubated with guinea-pig serum for 24 h, almost all serum proteins, including the C3 protein, were degraded. These two strains might thus have similar capacity in perturbing the host defence when inoculated into the tissue cages. The actual difference in pathogenicity between the strains might be explained by a recent finding that the pathogenic strain W83, but not strain 381, requires complement in activating polymorphonuclear leukocytes. The degradation of the C3 protein by the pathogenic strain W83 of B. gingivalis thus may be the crucial event in its perturbation of the host defence. A degradation of the C3 protein by strain 381 would be of no help in eluding the host defence, since this strain activates polymorphonuclear leukocytes in the absence of complement.     

牙龈卟啉单胞菌唾液酸酶基因缺失影响其感染上皮细胞后炎症相关microRNA的表达

目的:检测牙龈卟啉单胞菌(Porphyromonas gingivalis,P. gingivalis)唾液酸酶基因(PG0352)缺失是否影响其感染上皮细胞后对上皮细胞炎症相关小RNA的调控。方法:厌氧培养P. gingivalis W83及其唾液酸酶基因突变株(△PG0352),以MOI为100∶1的比例分别与永生化牙龈上皮细胞epi4进行共培养,PCR array的方法检测88个与炎症相关的小RNA的表达。结果:△PG0352组epi4的hsa-miR-9、hsa-miR-23a、hsa-miR-23b和hsa-miR-410的表达分别是P. gingivalis W83组的4.3387、3.3978、4.7930和4.3137倍。结论:与P. gingivalis W83野生株相比,唾液酸酶基因缺陷株对牙龈上皮细胞炎症相关小RNA的表达有不同的调控作用。     

Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization technique

Background/aims:  Porphyromonas gingivalis , a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P .  gingivalis .
Methods:  The subtractive hybridization technique was employed to identify the genes specific to P .  gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P .  gingivalis ATCC 33277 was the driver strain. The prevalence of W83-specific genes was determined by Southern blot analysis of several P. gingivalis strains.
Results:  We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50.
Conclusions:  The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P .  gingivalis strains.     

Hemoglobin hydrolysis and heme acquisition by Porphyromonas gingivalis

Porphyromonas gingivalis has been implicated in the progression of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. This bacterium is a gram-negative, black-pigmented, asaccharolytic anaerobe that relies on the fermentation of amino acids for the production of metabolic energy. The Arg- and Lys-specific extracellular cysteine proteinases of P. gingivalis, RgpA, RgpB and Kgp have been implicated as major virulence factors. In this study we investigated the hydrolysis of human hemoglobin by whole cells of P. gingivalis W50 and the mutants W501 (RgpA-), W50AB (RgpA-RgpB-) and W50ABK (RgpA-RgpB-Kgp-) under strictly anaerobic conditions in a physiological buffer (pH 7.5) using mass spectrometric analysis. Incubation of P. gingivalis W50 with hemoglobin over a period of 30 min resulted in the detection of 20 hemoglobin peptides, all with C-terminal Arg or Lys residues. The majority of the hemoglobin alpha- and beta-chain sequences were recovered as peptides except for two similar regions of the C-terminal half of each chain, alpha(92-127) and beta(83-120). The residues of the unrecovered sequences form part of the interface between the alpha- and beta-chains and an exposed surface area of the hemoglobin tetramer that may be involved in binding to P. gingivalis. P. gingivalis W501 (RgpA-) produced similar peptides to those seen in the wild-type. All identified peptides from the hydrolysis of hemoglobin by the P. gingivalis W50AB (RgpA-RgpB-) mutant were the result of cleavage at Lys. The triple mutant W50ABK was unable to hydrolyze hemoglobin under the assay conditions used, suggesting that on whole cells the major cell surface activity responsible for hydrolysis of hemoglobin is from the RgpA/B and Kgp proteinases. However, the triple proteinase mutant W50ABK grew as well as the wild-type in a medium containing hemoglobin as the only iron source, indicating that the RgpA/B and Kgp proteinases are not essential for iron assimilation from hemoglobin by P. gingivalis.     

Complement factor -like activity of Porphyromonas gingivalis W83

Porphyromonas gingivalis is a proteolytic gram-negative anaerobic bacterium that is frequently isolated from lesions of human periodontal disease. Previous studies have shown that P. gingivalis strain W83 inactivates C3 in pooled normal human serum (NHS) by a mechanism that is inhibitable by EDTA, yet it degrades purified complement proteins by a mechanism that is not EDTA-inhibitable. Furthermore, during complement activation, only a small number of C3 molecules accumulate on the surface of this organism unless the bacteria are treated with the protease inhibitor TLCK prior to complement activation. The hypothesis was tested that P. gingivalis W83 contains protease activity mimicking that of complement factor D, thus enabling it to activate C3 in serum without significant C3 accumulation on the cell surface. It was first noted that incubation of P. gingivalis W83 in absorbed human serum that was depleted of factor D resulted in C3 consumption that was reversed in the presence of the protease inhibitor TLCK. To directly demonstrate that factor B-dependent C3 consumption occurs in the absence of factor D, P. gingivalis W83 was incubated with purified C3 or a mixture of C3 and B. Although some proteolysis of C3 was noted, increased C3 consumption was noted in mixtures containing both C3 and B. This increment in C3 consumption was inhibited by both EDTA and TLCK. Furthermore, the addition of purified factor H to this mixture inhibited the increment in C3 consumption, indicating that a C3 convertase was probably formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteome analysis of Porphyromonas gingivalis cells placed in a subcutaneous chamber of mice

Introduction:  Porphyromonas gingivalis , an oral anaerobic bacterium, is considered a major pathogen for chronic periodontitis. Pathogenic bacteria usually upregulate or downregulate gene expression to combat the protective responses of their hosts.
Methods:  To determine what protein is regulated when P. gingivalis cells invade host tissues, we analyzed the proteome of P. gingivalis cells that were placed in a mouse subcutaneous chamber by two-dimensional gel electrophoresis and mass spectrometry.
Results:  Fourteen proteins were upregulated, while four proteins were downregulated. We focused on three upregulated proteins, PG1089 (DNA-binding response regulator RprY), PG1385 (TPR domain protein), and PG2102 (immunoreactive 61-kDa antigen), and constructed mutant strains that were defective in these proteins. Mouse abscess model experiments revealed that the mutant strain defective in PG1385 was clearly less virulent than the wild-type parent strain.
Conclusion:  These results indicate that the PG1385 protein is involved in P. gingivalis virulence and that the method used here is useful when investigating the P. gingivalis proteins responsible for virulence.     

牙龈卟啉单胞菌不同毒力株基因差异的比较研究

目的比较牙龈卟啉单胞菌（Porphyromonas gingivalis，Pgingivalis）高毒力株W83与低毒力株标准参考菌ATCC 33277之间的差异基因。方法采用抑制消减杂交技术（SHH）对比牙龈卟啉单胞菌高毒力株W83与低毒力株标准参考菌ATCC 33277的基因差异。以高毒力株W83为被检菌，低毒力株ATCC 33277为参考菌，将提取的基因组DNA用内切酶Rsa Ⅰ酶切，连接特殊设计的接头进行两次消减杂交和PCR扩增，得到消减混合物，与TA克隆载体连接，转化到JM109中，建立消减文库，经PCR筛选鉴定阳性克隆，进而对部分片段进行测序和同源分析。结果经SSH筛选鉴定得到36个片段大小为88～372bp的阳性克隆基因片段。结论从全基因角度研究牙龈卟啉单胞菌高毒力株W83与标准株ATCC 33277之间的分子遗传差异，为牙龈卟啉单胞菌致病基因的筛选及今后牙周病预防、诊治的靶标提供依据。     

细菌array-CGH技术用于比较牙龈卟啉单胞菌不同菌株基因组差异的研究

目的:构建牙龈卟啉单胞菌(简称P.gingivalis)全基因组规模的微阵列比较基因组杂交(简称array-CGH)芯片平台,分析不同菌株间基因组的差异,为后续检测临床分离株的毒力基因,进一步阐述牙周炎发病机制提供依据。方法:综合已经全基因组测序的12株P. gingivalis菌的序列信息,设计探针序列,定制芯片。提取P. gingivalis高毒力株W83和低毒力株ATCC 33277的基因组DNA,利用array-CGH检测其全基因组的差异DNA片段,并运用PCR验证结果的准确性。结果:Array-CGH结果显示两组菌株间存在几个连续片段的拷贝数不同。P.gingivalis W83的部分特异片段参与编码毒力致病因子。在P.gingivalis ATCC 33277的特有片段中,部分编码蛋白可增强细菌表明粘附力,使其具有高粘附力。从中挑选12个基因进行PCR差异性验证,证实与芯片结果一致。结论:用array-CGH芯片的方法来分析全基因组拷贝数的变化具有分辨率高,能精确定位异常片段的特性。本文成功构建了array-CGH技术检测P.gingivalis不同毒力株基因差异的平台,为后续比较临床分离株的差异DNA片段,筛查毒力基因,深入阐述牙周炎的发病机制奠定基础。     

Clonal analysis of Porphyromonas gingivalis by the arbitrarily primed polymerase chain reaction

Genetic analysis of Porphyromonas gingivalis strains may distinguish between virulent and nonvirulent strains and also may be used to trace individual strains in epidemiological studies. The present study examined the utility of the arbitrarily primed polymerase chain reaction for genotypic fingerprinting of P. gingivalis. DNA was extracted according to conventional methods. Ten-base oligonucleotide primers with arbitrary sequences were used with the polymerase chain reaction to amplify P. gingivalis genomic DNA. The amplification products were analyzed by agarose gel electrophoresis. The primer GACCGCTTGT grouped 73 P. gingivalis strains into 23 genotypes, including 16 genotypes containing a single strain each. The primer AGGGGTCTTG identified 45 different genotypes, 33 of which contained a single strain. P. gingivalis strains ATCC 332771^T and 381 belonged to the same genotype. Likewise, strains W50 and W83 were of the same genetic clone. The present study indicates that the arbitarily primed polymerase chain reaction represents a valuable and easy method for clonal analysis of P. gingivalis.     

Studies on the virulence properties and metabolism of pleiotropic mutants of Porphyromonas gingivalis (Bacteroides gingivalis) W50

Porphyromonas gingivalis (Bacteorides gingivalis) strain W50 and variants isolated from continuous culture designated W50/BP1 (black pigmented), W50/BR1 (brown pigmented) and W50/BE1 (beige or non-pigmented) were previously shown to lose virulence with the loss of pigmentation (8). Major properties which may affect the virulence and metabolism of P. gingivalis were compared amongst the 4 strains. The non-pigmented strain lost the ability to hemagglutinate sheep erythrocyte, had a reduced hydrophobicity and possessed lower levels of proteolytic activity. Defects in the electron transport system occurred at the level of cytochrome b but not menaquinone synthesis and resulted in an altered metabolic end product profile of the non-pigmented strain.