Virulence factors of Actinobacillus actinomycetemcomitans relevant to the pathogenesis of inflammatory periodontal diseases

Abstract: There is strong evidence implicating Actinobacillus actinomycetemcomitans as the causative agent of localised juvenile periodontitis (LJP), a disease characterised by rapid destruction of the tooth-supporting tissues. This organism possesses a large number of virulence factors with a wide range of activities which enable it to colonise the oral cavity, invade periodontal tissues, evade host defences, initiate connective tissue destruction and interfere with tissue repair. Adhesion to epithelial and tooth surfaces is dependent on the presence of surface proteins and structures such as a microvesicles and fimbriae. Invasion has been demonstrated in vivo and in vitro although the mechanisms involved are poorly understood. The organism has a number of means of evading host defences which include: (i) inhibiting polymorphonuclear leukocyte (PMN) chemotaxis; (ii) killing PMNs and monocytes; (iii) producing immunosuppressive factors; (iv) secreting proteases capable of cleaving IgG; and (v) producing Fc-binding proteins. Surface components of A. actinomycetemcomitans are potent stimulators of bone resorption and can induce the release of a range of cytokines which can initiate tissue destruction. A number of surface components can also inhibit the proliferation of fibroblasts and their production of components of the extracellular matrix. Little is known, however, regarding the way in which these factors operate in vivo to produce the pathological features of the disease.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

放线共生放线杆菌粗糙型与光滑型菌落的主要外膜蛋白

目的：观察放线共生放线杆菌粗糙型与光滑型菌株菌体蛋白表达上的差异。方法：聚丙稀酶胺凝胶电泳观察两型细菌全细胞蛋白及超高速离心提取的细菌主要外膜蛋白差异。结果：全细胞蛋白电泳两型细菌蛋白带无明显差异;提取的主要外膜蛋白电泳粗糙型存在18、29、45kDa蛋白带,实验室参考菌株不存在,临床光滑型菌株存在少量,光滑型实验室参考菌株存在的蛋白带在所有实验菌株中均存在。结论：18、29、45kDa蛋白带可能与放线共生放线杆菌粗糙型菌株相关。     

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2

The gene coding for recA in the oral pathogen Actinobacillus actinomycetemcomitans SUNY 465 was cloned and sequenced. The DNA sequence coded for a 352-amino acid protein that was homologous to RecA of a variety of bacterial species. A derivative of a non-replicating mobilizable plasmid was constructed for directed mutagenesis in A. actinomycetemcomitans. A recA-deficient strain of A. actinomycetemcomitans was developed by homologous recombination of an internal recA fragment contained on the mobilizable suicide vector. The recA mutant strain was more sensitive to UV radiation and showed a reduced recombinatorial proficiency than the isogenic parent strain. These data suggest that recA of A. actinomycetemcomitans SUNY 465 is involved in the repair of DNA damage caused by UV irradiation and homologous recombination as determined for other bacteria.     

Identification of in vivo essential genes from Pseudomonas aeruginosa by PCR-based signature-tagged mutagenesis

We adapted PCR-based signature-tagged mutagenesis (STM) to Pseudomonas aeruginosa. A collection of 1056 mutants was screened in a chronic lung infection rat model. Thirteen mutants were confirmed to be attenuated. Analysis revealed that these STM mutants represented transposon insertions into eight genes previously described in databases, three genes encoding proteins sharing identity with hypothetical proteins and two genes that shared no significant identity with sequences in databases. Five strains mutated in genes involved in protein degradation, stress tolerance, cation transport, ABC transporter, and an unknown protein were shown to be highly attenuated when tested individually in the rat chronic lung infection model.     

Isolation and characterization of hemolytic genes from Actinobacillus actinomycetemcomitans

Abstract Periodontopathic Actinobacillus actinomycetemcomitans produces hemolysin and other leukotoxins. In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A. actinomycetemcomitans ATCC 43718 on blood agar plates. DNA hybridization analysis indicates that there were two distinct hemolytic genes present. Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit. Rabbit antiserum to A. actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.     

Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae

Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS). Some strains can also phenotypically switch from a smooth to a 'rugose' phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions. High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology. VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS). However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood. We report that rugose strains of both classical and El Tor biotypes of epidemic V. cholerae are defective in the in vitro production of extracellular collagenase activity. In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity. Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis. Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis. Additionally, VpsR might regulate genes with roles in virulence. Rugose strains appear to be a subpopulation of cells that might act as a 'helper' phenotype promoting the pathogenesis of certain strains. Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V. cholerae.     

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo‐induced antigen technology (IVIAT). Methods and Results: The convalescent‐phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo‐induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion‐mutated, and the growth and 50% lethal dose (LD₅₀) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo‐expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Microaerophilic property of Actinobacillus actinomycetemcomitans in fructose-limited chemostat cultures

Abstract The effect of oxygen on the growth, metabolism, and leukotoxin production of Actinobacillus actinomycetemcomitans 301-b was examined using a chemostat equipped with a redox potential control system. Steady states were obtained with fructose-limited cultures grown at a dilution rate of 0.1 h⁻¹ under strictly anaerobic ( E _h=−460 mV) and microaerobic conditions ( E _h≤ 150 mV) but not under highly aerated conditions ( E _h≥ 100 mV). The optimum growth was recorded at E _h=−300 to − 200 mV and the recorded Y _fructose value was about 1.3 times the Y _fructose of anaerobic cultures. Although the organism contains a respiratory chain, the increased Y _fructose under the microaerobic conditions might result from the increased substrate-level phosphorylation at the site of acetate kinase but not from electron transport phosphorylation. After passing threshold aeration ( E _h=−100 mV), the culture yielded a variant with transparent colony morphology. Under anaerobic conditions, the Y _fructose of the variant was about 1.6 times that of the original opaque colony-forming cells. The optimum growth of the variant was also recorded at E _h=− 300 to − 200 mV. In both types of cells, the production of leukotoxin reached a maximum at E _h=−350 to − 200 mV. These findings suggested the microaerophilic nature of A. actinomycetemcomitans .     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Heat shock response in Actinobacillus actinomycetemcomitans

Abstract The heat shock response in Actinobacillus actinomycetemcomitans , a capnophilic Gram-negative bacterial species that is implicated in the development of certain forms of periodontitis, was characterized. Different strains of A. actinomycetemcomitans were grown at 37, 42 and 48°C in the presence of ³⁵S-methionine. The bacterial cells were lysed, run on SDS-PAGE and subsequently blotted on nitrocellulose paper. After autoradiography of the blots, several protein bands from the cultures at 42°C showed an increased intensity; major bands were observed at 90, 70, and 60 kDa, but increased protein synthesis was also detected at 54, 28 and 17 kDa. Nitrocellulose blots were also incubated with a panel of monoclonal and polyclonal antibodies directed to epitopes on different heat shock proteins. Strong reactivity was found with several antibodies at the position corresponding to a molecular mass of 60 kDa. The protein is probably the GroEL homologue in A. actinomycetemcomitans , a member of the ‘common bacterial antigen’ family.     

一株益生性粪肠球菌的安全性评价

目的 研究1株微生态活菌制品生产用粪肠球菌的安全性。 方法 采用目前肠球菌安全性评价主要方法,测定粪肠球菌GMCC 0460.3株的全基因组序列并分析毒力和耐药性相关基因;以生物化学方法测定其耐药性和有毒代谢产物的产生情况;经口灌胃小鼠测定其动物体内毒力。 结果 该株肠球菌对卡那霉素和磺胺类药物耐药,耐药谱窄;基因组存在多种肠球菌毒力基因;生化试验表明其溶血性阴性、氨基脱羧酶活性阴性、硝基还原酶活性较低、细胞表面疏水性较低、生物膜形成能力较弱;动物实验结果表明该株菌在小鼠体内无明显毒性作用。 结论 粪肠球菌GMCC0 460.3株实验评价的结果显示了良好的安全性。     

Altered antigenicity is seen in the lipopolysaccharide profile of non-serotypeable Actinobacillus actinomycetemcomitans strains

Non-serotypeable Actinobacillus actinomycetemcomitans strains may be derived from the serotypeable ones. In the present study, we compared the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of serotypeable and non-serotypeable A. actinomycetemcomitans strains (n=24) of the same genotype in the same subject (n=6) to find out if alterations on the cell-surface contribute to the non-serotypeability. Serotypeable and non-serotypeable A. actinomycetemcomitans strains showed great similarity in the OMP patterns both within and between subjects. Using serotype-specific antisera, clear immunoblotting LPS profiles in the O-antigenic region were seen in serotype b and c strains but not in non-serotypeable strains from the same subjects. The results suggest that changes in LPS lead to the altered antigenicity of non-serotypeable A. actinomycetemcomitans strains.