Zinc and copper play a role in coaggregation inhibiting action of Porphyromonas gingivalis

Background/aim:  We investigated the mechanisms of adherence of salivary and serum proteins, which mimic gingival crevicular fluid (GCF), to Porphyromonas gingivalis , and the effects of these adhered proteins on coaggregation and hemagglutination properties.
Methods:  The amounts of salivary and serum proteins adhering to P. gingivalis were determined using ³H-labeled and non-labeled proteins. The coaggregation between P. gingivalis and Streptococcus oralis or Streptococcus gordonii was observed. Hemagglutination was evaluated using sheep erythrocytes. Proteins that interacted with zinc or copper in saliva and serum and on P. gingivalis were examined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Results:  The amount of salivary or serum proteins that adhered to the surface of P. gingivalis strains was increased by cations, especially zinc and copper ions. The pretreatment of bacterial cells with salivary or serum proteins before the assay inhibited coaggregation with gram-positive bacteria and hemagglutination. These phenomena were enhanced by the presence of zinc or copper ions during the pretreatment of P. gingivalis with proteins. We detected protein bands that were related to these cations in saliva and serum and on P. gingivalis.
Conclusions:  Our findings suggest that zinc and copper ions markedly enhanced the adhesion and accumulation of salivary and serum proteins on cells of P. gingivalis and inhibited the coaggregation and hemagglutination of P. gingivalis . These cations might be useful for limiting the settlement of P. gingivalis in the gingival sulcus with the goal of preventing periodontal disease.     

Kinetics of coaggregation of Porphyromonas gingivalis with Fusobacterium nucleatum using an automated microtiter plate assay

Coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum strains was previously studied using either a semi-quantitative macroscopic assay or radioactive tracer assays. A new automated microtiter plate assay is introduced, in which the plate reader (Vmax) was adapted to allow quantitative evaluation of the kinetics of coaggregation. F nucleatum PK 1594 coaggregated with P. gingivalis HG 405 with a maximal coaggregation rate of 1.05 mOD/min, which occurred at a P. gingivalis to F. nucleatum cell ratio of 1 to 2. F. nucleatum PK 1594 failed to do so with P. gingivalis strains A 7436 or ATCC 33277. Galactose inhibition of this coaggregation could be quantitatively measured over a wide range of concentrations to demonstrate its dose-dependent manner. P. gingivalis HG 405 failed to coaggregate with F. nucleatum strains ATCC 25586 and ATCC 49256. The assay used in the present study is a sensitive and efficient quantitative automated tool to study coaggregation and may replace tedious radioactive tracer assays.     

口腔粘性放线菌粘附机制的体外初探

实验比较口腔致病菌──口腔粘性放线菌Ｔ＿（１４Ｖ）的３种突变株１４７，５５１９，５９５１吸附于玻璃管壁的能力以及与变形链球菌Ｉｎｇｂｒｉｔｔ、远缘链球菌６７１５、血链菌３４、ｂｌａｃｋｂｏｖａ发生共凝集反应的结果，显示细菌表面特异性菌毛影响细菌对牙体硬组织的吸附，以及和其他细菌之间的相互凝集作用。     

Purification of arginine-sensitive hemagglutinin from Fusobacterium nucleatum and its role in coaggregation

Hemagglutinin of Fusobacterium nucleatum was extracted from Triton X-100-pronase P-treated cell envelopes, and was purified by affinity chromatography on L-arginine agarose. The hemagglutinin was inactivated by heating at 70°C for 1 min. The activity was inhibited by L-arginine but was not affected by any sugars or by EDTA. The hemagglutinin aggreggated 14 out of 17 strains of oral streptococci tested, and the bacterial aggregating activity was also inhibited by L-arginine. The results indicate the dominant role of this hemagglutinin in the adherence of this bacterium both to host cells and to other bacteria.     

Characterizing the specific coaggregation between Actinobacillus actinomycetemcomitans serotype c strains and Porphyromonas gingivalis ATCC 33277

A visual coaggregation study showed specific interspecies coaggregation between an Actinobacillus actinomycetemcomitans serotype c strain and Porphyromonas gingivalis strains ATCC 33277 and 381. We mutagenized A. actinomycetemcomitans SUNYaB 67 (serotype c) with transposon IS903phikan and isolated three transposon insertion mutants that had a reduced ability to aggregate with P. gingivalis ATCC 33277. The three transposon insertions in the mutant strains mapped to the genes at ORF12, ORF13 and ORF16 of the gene cluster responsible for producing serotype c-specific polysaccharide antigen (SPA). Western blot analysis with serotype c-specific antibody showed that these strains did not produce the high-molecular-mass smear of SPA. Furthermore, two SPA-deficient mutants and an SPA-producing mutant were constructed. The two SPA-deficient mutants were deficient for ORF12 and ORF14, which are necessary for the synthesis of serotype c-SPA, and the SPA-producing mutant was deficient for ORF17, which is not related to SPA synthesis. The ORF12- and ORF14-deficient mutants showed reduced ability to aggregate with P. gingivalis ATCC 33277, while the ORF17-deficient mutant aggregated with ATCC 33277 to the same extent as wild-type SUNYaB 67. Our findings suggest that serotype c-SPA of A. actinomycetemcomitans mediates coaggregation with P. gingivalis ATCC 33277.     

Coaggregation to prevent dental caries

This application claims that selected species of Lactobacillus, capable of co- aggregating with the cariogen Streptococcus mutans, can be administered therapeutically to prevent colonisation with S. mutans or to significantly diminish the presence of S. mutans within dental plaque. This strategy has the potential for success as S. mutans do not need to be totally eliminated to significantly diminish the probability of caries development. Mechanistically, it is predicted that the Lactobacillus will bind to S. mutans and interfere with adhesins on the S. mutans cell surface that are specific for receptors on the tooth pellicle. Aggregates of the two species will be eliminated from the oral cavity by swallowing. The most effective delivery of this technology may be during the critical ‘window of infectivity’ in young children where preventing S. mutans colonisation promotes the establishment of a healthy, non-cariogenic plaque biofilm. The utility of this technology as a therapeutic for individuals with ongoing caries is less certain. Although either appliance is theoretically feasible, the application does not venture much beyond demonstrating in vitro coaggregation of the two species. Consequently, there are still several issues that must be experimentally addressed before the effectiveness of the technology can be established.     

Interactions between Streptococcus oralis,Actinomyces oris,and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual‐species biofilms, or three‐species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non‐fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.     

COAGGREGATION AND COAGGREGATION INHIBITION BETWEEN PERIO-PATHOGENIC AND CARIOGENIC BACTERIA

Objective To screen the coaggregating pairs between perio-pathogenic and cariogenic bacteria and to investigate the susceptibility of these coaggregations to inhibitors. Methods 4 strains of perio-pathogenic bacteria, Fusobacterium nuleatum （Fn） ATCC 10953, Actinobacilllus actinomycetem comitans （Aa） Y4, Porphyromonas gingivalis （Pg） ATCC 33277,Prevotella intermedia （Pi） ATCC 25261 and 4 strains of cariogenic bacteria, Streptococcus mutans （Sm） lngbritt, Streptococcus sanguis （Ss） 34, Actinomyces viscosus （ Av） 19246 and Lactobacillus acidophilus （La） ATCC 4356 were used to determine the coaggregating degrees of various combinations of the above bacteria by a visual assay and a turbidimetric assay. Then more than ＋ 2 （ or 20% ） coaggregation degrees＇ pairs were used to investigate the inhibitory effect of lactose and arginine and to identify the minimum of their coaggregation-inhibitory concentration. Results The coaggregation degrees of Fn-Av, Pg-Av, Fn-Sm, Fn-Ss, Fn-La and Pg-Ss pairs were higher than ＋ 2 （ 20% ）. 3.0 - 6.0mmol/L of arginine were considerably effective to the above pairs except Fn-Av pair and the disaggregation degrees were 49% - 92%. The maximum of their disaggregation degree to Fn-Av pair was just 18%. 120 - 300mmol/L of lactose were significantly effective to Pg-Ss pair, the disaggregation degrees were 57% - 91%. They partially inhibited Pg-Av pair and were almost ineffective to FnG^＋pairs. Conclusion The coaggregations between perio-pathogenic and cariogenic bacteria are highly specific. Most of them are relatively sensitive to arginine.     

Evidence for the existence of two classes of corncob (coaggregation) receptor in Fusobacterium nucleatum

Oral isolates of Fusobacterium nucleatum have the ability to form distinct coaggregation units, termed corncobs, when mixed with Streptococcus sanguis. Saturation binding kinetics determined for 12 strains of F. nucleatum revealed that these strains could be divided into two groups. One group, represented by F. nucleatum ATCC 10953 reproducibly bound twice as many streptococci as the other group, exemplified by F. nucleatum FDC 364. Other Fusobacterium species tested, including F. mortiferum, Fusobacterium periodonticum, Fusobacterium simiae and Fusobacterium necrophporum , failed to form corncobs with S. sanguis. In inhibition experiments lipoteichoic acid (LTA)-enriched cell extracts from S. sanguis blocked corncob formation between this bacterium and strain FDC 364 with material containing as little as 50 ng phosphate exhibiting greater than 50% inhibition. Alternatively, 40 times as much material failed to inhibit corncob formation between S. sanguis and F. nucleatum ATCC 10953 or Bacterionema matruchotti. Deacylation of the LTA destroyed its ability to inhibit the fusobacterial coaggregation but produced an active inhibitor in the case of B. matruchotii corncobs. Concerning the fusobacterial coaggregation partner, membrane fractions obtained from strain ATCC 10953 blocked corncob formation while the inhibitory activity of strain FDC 364 was confined to the soluble protein fraction. Corncob-deficient mutants of F. nucleatum FDC 364 were produced by treating the cells with acridine orange. However, this agent had no effect on the corncob-forming ability of strain ATCC 10953. The results of these experiments provide convincing evidence that at least 2 independent strain-specific receptors on F. nucleatum are involved in corncob formation between this bacterium and S. sanguis.     

牙周病和龋病致病菌的共聚及共聚抑制

目的 :研究牙周病和龋病致病菌的共聚和共聚抑制关系及特征 ,筛选有效共聚抑制剂。方法 :以 4株牙周致病菌 (具核梭杆菌、伴放线放线杆菌、牙龈卟啉单胞菌和中间普氏菌 )和 4株致龋菌 (变形链球菌、血链球菌、粘性放线菌和嗜酸乳杆菌 )的国际标准菌株为研究对象 ,通过目测法和比浊法确定各组合细菌的共聚度并研究乳糖和精氨酸对共聚度 > 2 (或 >2 0 % )共聚对的共聚抑制作用。结果 :Fn Av、Pg Av、Fn Sm、Fn Ss、Fn La和Pg Ss共聚度 >2 0 % ;4.5～ 6.0mM精氨酸对除Fn Av外 5对有明显抑制作用 ,解聚度为 49%～ 92 % ;12 0～ 3 0 0mM乳糖仅对上述 6对共聚对中的Pg Ss有明显抑制作用 ,解聚度为 5 7%～ 91% ,对Pg Av有部分抑制作用 ,最大解聚度约为 3 5 % ,但对Fn G 菌几乎无效。结论 :牙周致病菌和致龋菌之间存在高度特异的共聚反应 ,精氨酸为有效共聚抑制剂。