牙龈卟啉单胞菌调控细胞自噬的分子机制研究进展

细胞自噬是细胞内一种保守的降解代谢途径,是宿主防御细菌感染的关键步骤。牙龈卟啉单胞菌（P. gin-givalis）作为一种牙周致病菌,能够利用其引发的细胞自噬实现其在宿主细胞内的生存和增殖,从而逃避宿主的免疫监视。本文就P. gingivalis内化并调控各种宿主细胞发生自噬的分子机制作一综述,从而深入探讨P. gingivalis的致病作用,并拓展P. gingivalis与全身疾病关系的研究思路。     

具核梭杆菌感染KB细胞影响细胞增殖和炎症因子分泌的研究

目的具核梭杆菌(Fusobacterium nucleatum,F.nucleatum),为革兰氏阴性杆菌,是牙周病的重要致病菌之一。研究发现,牙周病原体感染和口腔癌的发生发展密切相关。因此,本研究通过分析细菌感染对KB细胞增殖和细胞因子分泌的影响,为具核梭杆菌感染和口腔癌症发展之间的关系提供新的证据。方法建立具核梭杆菌感染KB细胞模型(MOI=10和100),透射电镜观察细胞和细菌形态变化,PI单染流式细胞术检测细胞周期,ELISA检测上清液中IL-6和IL-8细胞因子分泌水平。结果透射电镜观察发现,4 h具核梭杆菌侵入细胞,细菌在细胞内保持完整形态,细胞与细胞之间紧密连接破坏。感染24 h后,细胞内仍能发现细菌,菌体被膜状结构包绕,并保持完整形态。低浓度具核梭杆菌(MOI=10)感染KB细胞,细胞周期G1期进程在感染早期加速,细胞增殖率没有显著变化(P>0.05)。高浓度具核梭杆菌(MOI=100)感染KB细胞,细胞周期S期4 h至8 h细胞数增多,同时促进细胞增殖;感染24 h后抑制G1期进程,S期细胞显著减少,从而抑制细胞增殖(P<0.05)。上清液中IL-6和IL-8细胞因子分泌水平均升高,并对具核梭杆菌感染的时间和浓度存在依赖性。结论具核梭杆菌感染口腔癌细胞可能通过影响细胞增殖和细胞周期进程,促进细胞炎症反应从而加重癌细胞恶性程度。     

细菌感染影响牙周组织细胞表达环鸟苷-腺苷合成酶的实验研究

目的 研究侵入牙周组织细胞内的细菌对细胞表达环鸟苷-腺苷合成酶（cGAS）的影响。方法 将SYTO9标记的牙龈卟啉单胞菌（P. gingivalis）以感染复数（MOI）为10与人牙周膜细胞（hPDLCs）共培养24 h后,使用激光共聚焦显微镜观察P. gingivalis对hPDLCs的侵袭情况,并应用荧光标记活细胞筛选（FACS）分选出被P. gingivalis入侵的hPDLCs,利用实时定量逆转录聚合酶链反应（qRT-PCR）和Western blot对hPDLCs中cGAS的表达变化进行检测。此外还利用免疫组织化学技术对细菌感染性牙龈组织和正常牙龈组织中cGAS表达定位和表达水平变化进行分析。结果 激光共聚焦显微镜下观察,P. gingivalis和hPDLCs共培养24 h后,几乎所有的细胞内都可观察到绿色荧光;被P. gingivalis感染的hPDLCs中cGAS表达水平明显上调;在牙龈组织中cGAS主要在上皮细胞和上皮下组织中的炎性细胞中表达;且细菌感染性的牙龈组织中cGAS的表达水平明显高于正常牙龈组织。结论 侵入hPDLCs内的活体P. gingivalis,可以明显上调细胞中cGAS的表达;且在细菌感染的炎性牙龈组织中cGAS的表达也明显升高。该结果提示cGAS可能参与了牙周组织细胞中细菌来源的病原dsDNA的识别。     

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。     

外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响

目的: 观察外源性三磷酸腺苷（ATP）对牙龈卟啉单胞菌(P.gingivalis)和热灭活牙龈卟啉单胞菌(HP.gingivalis)感染的人牙龈成纤维细胞NLRP3 炎症小体（inflammasome）的活化以及下游因子IL-1β分泌的影响。方法: 组织块法体外培养人牙龈成纤维细胞（hGFs）获取原代细胞,经5 mmol/L ATP预处理,用100 MOI P.gingivalis、100 MOI HP.gingivalis体外刺激hGFs,实时定量PCR检测NLRP3、ASC、Caspase-1、IL-1β的基因表达;Western免疫印迹技术检测细胞内NLRP3、Caspase-1和IL-1β蛋白的表达;ELISA法检测白细胞介素1β（IL-1β）的分泌。采用Graphpad prism 6软件包对数据进行t检验或单因素方差分析。结果: 与对照组相比,P.gingivalis 下调NLRP3 、ASC mRNA,上调IL-1β基因表达,下调NLRP3 、IL-1β胞内蛋白水平。HP.gingivalis 诱导NLRP3 、IL-1β、ASC基因和胞内蛋白的表达,P.gingivalis或者 HP.gingivalis单独刺激对Caspase-1 mRNA水平及IL-1β分泌均无影响。ATP/P.gingivalis或ATP/HP.gingivalis 共刺激均明显上调NLRP3、ASC、Caspase-1和IL-1β基因及胞内蛋白水平,增加上清液中IL-1β的分泌水平。结论: 外源性ATP可调控牙周主要致病菌P.gingivalis感染的人牙龈成纤维细胞 NLRP3炎症小体的激活,介导炎症因子IL-1β的成熟与分泌。     

牙龈卟啉单胞菌合成环二腺苷酸的高效液相色谱-串联质谱法定性分析

目的 定性检测牙龈卟啉单胞菌（P. gingivalis）是否能产生细菌信号分子环二腺苷酸（c-di-AMP）,为探索其在P. gingivalis生命代谢以及牙周炎免疫中的作用奠定基础。方法 以P. gingivalis标准菌株ATCC33277为实验菌株,抽提细菌内核酸物质作为样品,配置c-di-AMP标准品,通过高效液相色谱-串联质谱法（HPLC-MS/MS）和高效液相色谱法（HPLC）对样品进行验证。结果 HPLC-MS/MS检出限按照信噪比（S/N）3∶1计算,c-di-AMP标准品出峰的保留时间为7.49 min,P. gingivalis提取物样品在保留时间为8.82 min时有目标峰出现（大于3 S/N）。HPLC检测结果表明,P. gingivalis核酸提取物样品及c-di-AMP标准品均在15.7 min处出现目标峰,且二者的紫外吸收光谱相同。结论 牙龈卟啉单胞菌核酸提取物中含有c-di-AMP,牙龈卟啉单胞菌可以合成产生c-di-AMP。     

病毒介导IL-24基因对口腔鳞癌耐药细胞株凋亡的影响

目的:研究杂合病毒介导的IL-24基因对口腔鳞状细胞癌耐药细胞的凋亡诱导作用,探讨其治疗耐药口腔癌的可能性。方法:通过MTT细胞活力测定鉴定口腔鳞状细胞癌细胞株KB和其耐药细胞株KBV。利用杂合病毒Ad LTR2EF1α搭载EGFP和IL-24转染KB细胞,确定最佳转染浓度,荧光倒置显微镜及RT-PCR鉴定EGFP或IL-24基因表达。以人永生化上皮细胞Ha Ca T细胞作为对照,使用Ad LTR2EF1α-IL-24转染KB、KBV及Ha Ca T细胞后,MMT法检测细胞活力,Annexin V/PI双染法检测细胞凋亡,透射电镜观察细胞凋亡形态。结果:KBV细胞对长春新碱的耐受性明显高于KB细胞。Ad LTR2EF1α-IL-24搭载的IL-24基因可以在KB及KBV细胞内过表达。IL-24基因在KB和KBV细胞中的过表达,有明显抑制细胞生长和促凋亡作用,而在Ha Ca T细胞中没有类似的作用。结论 :杂合病毒介导的IL-24对口腔鳞状细胞癌耐药细胞有较强的细胞毒性和诱导凋亡作用,并且对正常组织细胞没有影响。具有治疗耐药口腔癌的潜在价值。     

炎症因子通过核因子κB通路促进口腔鳞状细胞癌转移的体外研究

目的 探讨炎症因子与核因子κB(nuclear factor kappa B,NF-κB)信号转导通路在口腔鳞状细胞癌转移中的意义.方法 应用口腔鳞状细胞癌低转移细胞系Tca8113和高转移细胞系Tb为研究对象,通过蛋白质印迹法和荧光素酶报告基因法检测口腔鳞状细胞癌细胞系中NF-κB通路活性.用NF-κB抑制因子α(inhibitor of kappa B alpha,IκBa)抑制质粒第32、36位丝氨酸磷酸化位点被丙氨酸替代的质粒(pBabe-SR-IκBa)和NF-κB通路抑制剂吡咯烷二硫代氨基甲酸盐(pyrolidinedithiecar bamate,PDTC)抑制信号转导通路活性,并用侵袭小室实验(TransweH)检测口腔鳞状细胞癌高转移系Tb细胞侵袭能力的变化.此外,通过酶联免疫吸附法检测pBabe-SR-IκBα和PDTC抑制信号转导通路后,肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)、白介素(IL)-1a、IL-6、IL-8和粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)等炎症因子的分泌.结果 蛋白质印迹法检测显示:高转移Tb细胞中磷酸化IκBα和磷酸化p65的表达量明显高于低转移细胞系Tea8113,分别为Tea8113细胞的3.19倍和6.81倍.荧光素酶(luciferase)报告基因结果显示,Tb细胞NF-κB的启动子活性为Tca8113的2.12倍(P<0.01),并对TNF-α更为敏感.转染pBabe-SR-IκBα和应用PDTC抑制NF-κB通路后,对Tb细胞的体外侵袭能力抑制率分别为21.9%和69.3%.此外,酶联免疫吸附试验法显示通路抑制后,TNF-α、IL-1α、IL-6、IL-8和GM-CSF等炎症因子的分泌也明显降低.结论 TNF-α、IL-1α、IL-6、IL-8和GM-CSF等炎症因子可能通过NF-κB通路促进口腔鳞状细胞癌细胞转移.     

体外培养口腔上皮细胞感染白色念珠菌后白介素-8的表达改变

目的:观察白色念珠菌对体外培养口腔黏膜上皮细胞(KB细胞)表达白介素8(IL-8)的影响。方法:采用逆转录-聚合酶反应(RT-PCR)技术及酶联免疫吸附实验(ELISA),分别检测体外口腔上皮细胞感染菌丝型、孢子型、灭活白色念珠菌及白色念珠菌培养上清后表达、分泌IL-8的mRNA水平及上清液中的蛋白含量,对实验数据间差异进行t检验,判断分析其统计学意义。结果:菌丝型、孢子型及120℃灭活处理的白色念珠菌均能诱导KB细胞增强IL-8的表达,RT-PCR成像分析系统Mark值分别为180.23、186.36、143.41,与对照组85.57相比,具有显著性差异(P<0.05);白色念珠菌的培养上清(Mark值为80.87)却不能增加其表达。动态观察结果显示,白色念珠菌诱导KB细胞表达、分泌IL-8随时间增加而增强,72h达到高峰,随后出现下降趋势;剂量依赖性观察发现,KB细胞随着菌量增多而IL-8的分泌减少,呈现负相关性。结论:白色念珠菌能增加口腔上皮细胞分泌IL-8,IL-8可能与黏膜局部白色念珠菌感染、防御密切相关。     

白色念珠菌对口腔上皮细胞细胞周期的影响

目的:研究白色念珠菌对口腔上皮细胞增殖及细胞周期的影响。从另一种角度证明白色念珠菌感染与癌发展的关系。方法:在培养的口腔上皮细胞(KB细胞)中,加入菌丝相、孢子相的白色念珠菌,共同培养48h,采用流式细胞仪观察白色念珠菌对KB细胞增殖及细胞周期的影响。结果:与对照组相比,菌丝组G0/G1期细胞所占比例降低,S期、G2/M期所占百分比显著升高,反映细胞增殖活力的增殖指数PI增高,差异有显著性(P<0.05)。而孢子组的KB细胞PI值与对照组相比无明显变化,P>0.05。结论:白色念珠菌可引起KB细胞细胞周期的改变;白色念珠菌对KB细胞周期的改变有赖于该菌的毒性及对细胞的感染。     

Interleukin 1, interleukin 6 and transforming growth factor-β production by human gingival mononuclear cells following stimulation with Porphyromonas gingivalis and Fusobacterium nucleatum

Gingival mononuclear cell production of interleukin 1 (IL-1), interleukin 6 (IL-6) and transforming growth factor-β(TGF-β) after stimulation with the putative periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum was investigated. Using an ELISA method, gingival mononuclear cells extracted from 18 adult periodontitis subjects were found to be producing IL-1. However, IL-1 activity could only be detected in 5 out of these 18 cases when tested using a thymocyte proliferation bio-assay, suggesting the presence of IL-1 inhibitors. Depletion of monocytes from peripheral blood cultures resulted in a significant decrease in IL-1 activity following P. gingivalis stimulation while there was no effect in the level of IL-1 activity following stimulation with F. nucleatum. This suggests that P. gingivalis and F. nucleatum stimulate different cell types to produce IL-1. Like IL-1. IL-6 production by gingival mononuclear cells was significantly greater than that produced by the control peripheral blood mononuclear cells. Following P. gingivalis and F. nucleatum stimulation, higher levels of IL-6 could be detected; however, both organisms stimulated similar levels. Intracytoplasmic immunofluorescence staining demonstrated a lower percent TGF-β⁺ cells in bacterial stimulated peripheral blood mononuclear cell cultures compared with cells in medium alone. In the gingival mononuclear cell cultures, the percentage TGF-β⁺ cells peaked at day 1 in F. nucleatum -stimulated, whereas in P. gingivalis -stimulated cultures the peak TGF-β⁺ cells occurred at day 3, again suggesting stimulation of different cell subsets. These results ilustrate that different periodontopathic bacteria may stimulate different cell types to produce cytokines which may have synergistic or antagonistic effects.     

Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-8 (IL-8) plays an important role in macrophage mediated inflammatory processes including exacerbation of periodontal diseases, one of the most common complications in GM-CSF receiving cancer patients. The effect of GM-CSF supplementation on IL-8 production was investigated in a human monocyte cell line THP-1, stimulated with lipopolysaccharide extracted from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. Resting THP-1 cells were treated with lipopolysaccharide (1 microgram/ml) of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) for varying time periods. The production of IL-8 in THP-1 cells was measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). A very low level of the cytokine IL-8 was produced constitutive in THP-1 cells. Starting from 8 h of treatment and afterwards GM-CSF alone significantly increased IL-8 production in THP-1 cells. Lipopolysaccharide (1 microgram/ml) extracts from either F. nucleatum or P. gingivalis amplified IL-8 production 500-800 times in comparison to resting THP-1 cells. When lipopolysaccharide of F. nucleatum or P. gingivalis was supplemented with 50 IU/ml of GM-CSF, there was a statistically significant enhanced production of IL-8 by THP-1 cells after 1 day to 7 days of treatment as compared with lipopolysaccharide treatment alone. GM-CSF (50 IU/ml) also significantly increased IL-8 production from 2-7 days of treatment of THP-1 cells when supplemented with a positive control, phorbol-12-myristate-13 acetate (PMA), as compared to PMA treatment alone. These investigations using the in vitro THP-1 human monocyte cell model indicate that there may be an increase in the response on a cellular level to oral endotoxin following GM-CSF therapy as evidenced by enhanced production of the tissue-reactive inflammatory cytokine, IL-8.     

Porphyromonas gingivalis survives within KB cells and modulates inflammatory response

BACKGROUND/AIMS: The purpose of the study was to investigate the intracellular survival of Porphyromonas gingivalis as a possible mechanism for maintaining periodontitis. METHODS: P. gingivalis strains, the strain ATCC 33277 and seven clinical isolates, were co-cultured with KB cells. The number of intracellular bacteria was determined up to 3 days after infection. In addition, the numbers of KB cells per well, the concentrations of the cytokines interleukin-1beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha) and the arginine-specific amidolytic activity were measured. The 16S rRNA of P. gingivalis and the mRNA expression of IL-1beta, IL-6, IL-8, TNF-alpha and rgpA were also determined. RESULTS: All the P. gingivalis strains studied were able to survive within KB cells. In contrast to the reduced values of colony-forming units at day 3, equal and higher levels of 16S rRNA were seen in comparison to day 0. Arginine-specific amidolytic activity declined in all samples during infection. Expression of mRNA for rgpA was not found after infection of KB cells by P. gingivalis strains. IL-8 was detectable in all samples 2 days after infection with P. gingivalis strains. Principal components analysis underlined a correlation between the arginine-specific amidolytic activity 1 h after infection and both the released IL-8 and the mRNA expression of IL-8. Associations were found between the cultivable numbers of intracellular P. gingivalis and the mRNAs of IL-1, IL-6 and TNF-alpha at the day of infection. CONCLUSION: The results indicate survival of P. gingivalis within epithelial cells, possibly in a non-cultivable stage. Invasion into cells modulates the virulence properties of P. gingivalis as well as the inflammatory response of the cells.     

Thiazolidinedione (pioglitazone) blocks P. gingivalis- and F. nucleatum, but not E. coli, lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production in adipocytes

An elevated level of C-reactive protein (CRP) predicts the future development of coronary heart disease. Periodontitis appears to up-regulate CRP. CRP is produced by hepatocytes in response to interleukin-6 (IL-6). A major source of IL-6 in obese subjects is adipocytes. We hypothesized that lipopolysaccharide (LPS) from periodontal pathogens stimulated adipocytes to produce IL-6, and that the production was suppressed by the drugs targeted against insulin resistance, thiazolidinedione (pioglitazone), since this agent potentially showed an anti-inflammatory effect. Mouse 3T3-L1 adipocytes were stimulated with E. coli, P. gingivalis, and F. nucleatum LPS. The IL-6 concentration in culture supernatants was measured. All LPS stimulated adipocytes to produce IL-6. Although pioglitazone changed adipocyte appearance from large to small, and completely suppressed P. gingivalis and F. nucleatum LPS-induced IL-6 production, E. coli LPS-induced IL-6 production was not efficiently blocked. Thus, pioglitazone completely blocked periodontal-bacteria-derived LPS-induced IL-6 production in adipocytes, a major inducer of CRP.     

Different responses in B cells induced by Porphyromonas gingivalis and Fusobacterium nucleatum.

A phenotypic study had shown that gingival B cells respond differently to two periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum. Further investigation now shows a reduction in the percentage of Ki-67 + T cells in cultures of gingival and peripheral blood mononuclear cells stimulated with P. gingivalis for 3 and 6 days, respectively, but no suppression of Ki-67 expression in B cells in response to either P. gingivalis or F. nucleatum. Depletion studies of cultures of peripheral blood mononuclear cells showed that in the absence of CD4 cells, the percentage of CD19+ and CD20+ B cells stimulated with P. gingivalis increased after 6 days whereas depletion of CD8 cells resulted in a rise in the percentage of F. nucleatum- and P. gingivalis-stimulated B cells, although this was not significant in the case of P. gingivalis. Specific antibody to P. gingivalis and F. nucleatum was found in culture supernatants of gingival but not of peripheral blood mononuclear cells, indicating a possible higher frequency of antigen-specific B cells in periodontal lesions. IgG was the predominant isotype in both gingival and control peripheral blood cultures, followed closely by IgA in gingival cultures. F. nucleatum stimulated higher levels of Ig in cultures of peripheral blood mononuclear cells than P. gingivalis or cells cultured in medium only, whereas in gingival cell cultures, stimulation by P. gingivalis appeared to result in higher levels of IgG. Also Ig was present at day 3 in gingival cultures, whereas in the blood cell cultures, Ig was only detected at day 6, further suggesting a degree of activation of of gingival B cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Epithelial cell pro-inflammatory cytokine response differs across dental plaque bacterial species

Aim: The dental plaque is comprised of numerous bacterial species, which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines, which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of interleukin (IL)-1 β , IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential.
Materials and Methods: HGECs were challenged with the four bacterial species, live or heat killed, at various multiplicity of infections and the elicited IL-1 β , IL-6, IL-8 and IL-10 responses were assayed by enzyme-linked immunosorbent assay.
Results: Primary HGECs challenged with live P. gingivalis produced high levels of IL-1 β , while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory.
Conclusion: We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary HGECs that may reflect their individual virulence or commensal status.     

Induction of systemic murine B-cell responses by Fusobacterium nucleatum and Porphyromonas gingivalis

The purpose of this study was to examine the antigenic abilities of Fusobacterium nucleatum strain ATCC 25586 and Porphyromonas gingivalis strain W50 black inbred BALB/cABom mice immunized subcutaneously. Furthermore, we aimed to analyze whether the outer membranes (OM) and whole cells (WC) of F. nucleatum or P. gingivalis had an effect on the levels of antibody response and whether a combination of both could either enhance or suppress the B-cell response. A single-cell assay, solid-phase enzyme-linked immunospot (ELISPOT), was used to analyze the splenic B-cell response (immunoglobulin A (IgA), IgG and IgM). Enzyme-linked immunosorbent assay (ELISA) and immunoblotting were used to verify the specific antibody response in the sera. A statistically significant lower level of spontaneous antibody production was observed in the group immunized with P. gingivalis OM compared with groups immunized with F. nucleatum and saline. The specific antibody titers measured by ELISA indicated that the bacterial preparations were able to induce IgG and IgM response. The preparations containing P. gingivalis OM induced higher humoral response than the preparations containing P. gingivalis WC, but for F. nucleatum such a difference was not observed. The prominent proteins revealed had apparent molecular masses of 40 kDa for F. nucleatum and 115, 55–56 and 43 kDa for P. gingivalis ; whereas the immunoreactive proteins were 70, 65 and 40 kDa for mice immunized with F. nucleatum and 115, 55–56, 43 and 33–34 kDa for mice immunized with P. gingivalis . Quantitative analysis of B-cell response at the single cell level with ELISPOT revealed that some component(s) of P. gingivalis OM may have a suppressive ability on splenocytes incubated for a short time.