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.     

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.     

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.     

Actinobacillus actinomycetemcomitans possesses an antigen binding to anti-human IL-10 antibody

It is well known that the cell components of periodontopathic bacteria are able to induce several cytokines and possibly to affect the cytokine network. In order to determine the presence of the periodontopathic Actinobacillus actinomycetemcomitans components recognized by antibodies against cytokine molecules, ELISA reactivities of sonic extracts from the bacterial cells were determined by use of ELISA kits specific for human interleukin (IL)-1beta, IL-4, IL-5, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma. The ELISA analysis demonstrated that the sonic extracts from eight strains of A. actinomycetemcomitans bound with anti-human IL-10 monoclonal antibody. Western blotting analysis revealed that the molecular mass of the antigen was approximately 65 kDa. IL-10 is produced by type 2 helper T cells and mainly down-regulates the type 1 helper T cell response. The present study suggests that the 65-kDa antigen of A. actinomycetemcomitans may affect the host defense function through binding to IL-10 receptor as an agonist or an antagonist for IL-10.     

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.     

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.     

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 .     

Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation

Actinobacillus actinomycetemcomitans is an important pathogen in periodontitis. In the present study we localized the GroEL- and DnaK-like heat shock proteins (Hsp) in subcellular fractions of 12 A. actinomycetemcomitans strains of various clinical origin and compared their effects on periodontal epithelial cell proliferation and viability. In all strains, GroEL-like protein was found in the membrane, cytoplasm, and periplasm, whereas DnaK-like protein was present in the cytoplasm and periplasm. No correlation was observed between the Hsp expression and the serotype or origin of A. actinomycetemcomitans strains. The bacterial membrane fractions that expressed the GroEL-like protein moderately or strongly induced epithelial cell proliferation more strongly than strains that expressed the protein weakly. The results suggest that GroEL-like Hsp may play a role in the virulence of A. actinomycetemcomitans by increasing epithelial proliferation.     

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h⁻¹ and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl-d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.     

Humoral immune response in lactating dairy cows after repeated exposure to human chorionic gonadotropin

Our objective was to determine if repeated exposure of lactating dairy cows to human chorionic gonadotropin (hCG) would induce an antibody (Ab) response against hCG. Cows either received an hCG injection (hCG; n = 24, each given 2000 IU im) or no treatment (CON; n = 22) 18 days after a timed AI (TAI) and 7 days before initiation of Ovsynch for resynchronization of ovulation and TAI. A subgroup of cows continued in the experiment to receive a second hCG injection (n = 17) 35 days after the first exposure to hCG, whereas another subgroup served as controls (n = 9). Another subgroup of cows continued in the experiment to receive a third hCG injection (n = 11) 35 days after the second exposure to hCG, whereas cows not receiving hCG served as controls (n = 8). A binding radioimmunoassay was used to detect hCG antibodies in serum samples collected 0, 7, 14, 21, and 28 days after treatment. A positive Ab response (>6.2% bound) was defined as three standard deviations above CON binding. No cows had hCG antibodies at Day 0 before the first exposure to hCG. After the first hCG treatment, there was no difference (P = 0.52) between Ab positive cows in CON (0%) and hCG (4%) treatments. At the second hCG treatment, on Day 0 there was no difference (P = 0.65) between CON (0%) and hCG (6%) cows, whereas, more (P = 0.02) hCG cows (47%) were positive than CON cows (0%) within 28 days of the hCG injection. At the third hCG injection, hCG cows tended (P = 0.09) to have a greater percentage of Ab positive (36%) than CON cows (0%), whereas after the injection, a greater (P < 0.01) percentage of hCG cows were positive (hCG = 73% vs. CON = 0%). After the second and third exposure to hCG, 8 of 17 and 8 of 11 cows within the hCG group had greater percent Ab bound at 7, 14, 21, and 28 days after hCG than cows in CON and those with no Ab response. The greatest percent Ab binding occurred at 14 days after the second and third hCG exposure. We concluded that some but not all lactating dairy cows developed an Ab response after repeated exposure to hCG and that maximum response occurred within 14 days after hCG exposure.     

Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii, which is widely distributed in nature, and presents a saprophytic mycelial form on plant debris and soil. The immunological mechanisms involved in the prevention and control of sporotrichosis are not yet fully understood. In this study, mice were studied after infection with Sporothrix schenckii. In the first week after infection, fungal loading increased and thence decreased drastically 14 days after infection. Analysis by immunoblotting showed that the sera of all mice tested had antibodies reacting only with a 70 kDa antigen, with predominance of IgG1 and IgG3. Taken together, our results show that antigens from S. schenckii induced a specific humoral response in infected mice.     

Involvement of caspase activation through release of cytochrome c from mitochondria in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans

We previously reported that infection with the periodontopathic bacterium Actinobacillus actinomycetemcomitans induced apoptosis in a mouse macrophage cell line J774.1. In the present study, we examined the involvement of cytochrome c and caspases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells. Following infection, the expression levels of cytochrome c, and cleaved forms of caspase-3 and caspase-9 in the cells were examined using immunoblot analysis. Cytochrome c was released from mitochondria into the cytoplasm after A. actinomycetemcomitans-infected J774.1 cells were cultured for 6 h, and caspase-3 and caspase-9 were found to be cleaved forms in the cells. Further, caspase-9 activity was markedly increased, and phosphorylated p53 was detected in the cells 30 h following infection. These results suggest that apoptosis in A. actinomycetemcomitans-infected J774.1 cells is regulated by the release of cytochrome c from mitochondria into cytoplasm and the subsequent activation of caspases through phosphorylation of p53.     

Trypanosoma cruzi: role of the immune response in the natural resistance of inbred strains of mice.

Nine inbred strains of mice were challenged with 10⁴ or 10⁵ trypomastigotes of the Brazil strain of Trypanosoma cruzi. A spectrum of resistance was evident ranging from highly susceptible strains, e.g., C3H, which developed high parasitemias and died within 3 to 4 weeks, to resistant strains, e.g., C57BL/10, which developed low parasitemias and survived. Impairment of the immune system in resistant C57BL/10 mice by X-irradiation, splenectomy, or treatment with silica led to high, often fatal parasitemias. Athymic nude mice, in particular, attained exceptionally high parasitemias before dying. The immune response appears to be necessary for survival and to play a role in the natural resistance of some mouse strains by effectively eliminating parasites and minimizing parasitemia. Using congenic strains of mice, it was shown that the principal genetic determinant of resistance is not associated with their H-2 haplotype.     

结核分枝杆菌海藻糖磷酸磷酸酶诱导小鼠体液和细胞免疫应答

目的 研究结核分枝杆菌(M．tuberculosis)海藻糖磷酸磷酸酶(TPP)诱导小鼠体液和细胞免疫。方法 差速离心分离结核分枝杆菌H37Rv和卡介苗(BCG)的各细胞组分,通过Western杂交检测抗原TPP在结核分枝杆菌H37Rv和BCG中的亚细胞定位情况。分别用5×10~6CFU的BCG和50μg的TPP蛋白免疫C57BL/6小鼠,检测小鼠血清中抗TPP的IgG1和IgG2a抗体效价。取免疫小鼠的脾细胞,体外抗原刺激,用酶联免疫斑点试验(ELISPOT)检测γ干扰素(IFN-γ)分泌细胞。结果 TPP亚细胞定位于结核分枝杆菌H37Rv和BCG的胞壁和细胞膜组分。TPP蛋白免疫后小鼠产生的TPP特异性IgG1和IgG2a抗体效价明显高于BCG免疫小鼠,并且IgG2a的抗体效价高于IgG1。体外抗原刺激TPP蛋白和BCG免疫小鼠的脾细胞,都能诱导较高的IFN-γ分泌。结论 结核分枝杆菌细胞壁蛋白TPP能诱导小鼠Ⅰ型辅助性T细胞介导的免疫反应,可作为抗结核疫苗的候选抗原。     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

Heterogeneity of the immune response to adenovirus-mediated factor VIII gene therapy in different inbred hemophilic mouse strains

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies (inhibitors) is a critical concern when considering gene therapy as a potential treatment modality for hemophilia A. We used a hemophilia A mouse model bred on different genetic backgrounds to explore genetically controlled differences in the immune response to FVIII gene therapy. METHODS: C57BL/6 FVIII knockout (C57-FVIIIKO) mice were bred with normal BALB/c (BAL) mice, to generate a recombinant congenic BAL-FVIIIKO model of hemophilia A. Early generation adenoviral (Ad) vectors containing the canine FVIII B-domain-deleted transgene under the control of either the CMV promoter or a tissue-restricted (TR) promoter were administered to C57-FVIIIKO, C57xBAL(F1)-FVIIIKO crosses, and BAL-FVIIIKO mice. FVIII expression, inhibitor development, inflammation, and vector-mediated toxicity were assessed. RESULTS: In response to administration of Ad-CMV-cFVIII, C57-FVIIIKO mice attain 3-fold higher levels of FVIII expression than BAL-FVIIIKO. All strains injected with Ad-CMV-FVIII displayed FVIII expression lasting only 2 weeks, with associated inhibitor development. C57-FVIII-KO mice that received Ad-TR-FVIII expressed FVIII for 12 months post-injection, whereas FVIII expression was limited to 1 week in C57xBAL(F1)-FVIIIKO and BAL-FVIIIKO mice. This loss of expression was associated with anti-FVIII inhibitor development. BAL-FVIIIKO mice showed increased hepatotoxicity with alanine aminotransferase levels reaching 4-fold higher levels than C57-FVIIIKO mice. However, C57-FVIIIKO mice initiate a more rapid and effective cell-mediated clearance of virally transduced cells than BAL-FVIIIKO, as evidenced by real-time PCR analysis of transduced tissues. Overall, strain-dependent differences in the immune response to FVIII gene delivery were only noted in the adaptive response, and not in the innate response. CONCLUSIONS: Our results indicate that the genetic background of the murine model of hemophilia A influences FVIII expression levels, the development of anti-FVIII inhibitors, clearance of transduced cells, and the severity of vector-mediated hepatotoxicity.     

Prevalence and distribution of bacteriophage φAa DNA in strains of Actinobacillus actinomycetemcomitans

Abstract φAa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans . Since the discovery of phage φAa , additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of φAa or φAa -related temperate phages in this species, a φAa -specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans . Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage φAa probe. A bacteriophage designated φAa ₃₃₃₈₄ was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the φAa probe. The φAa probe hybridized with the DNA extracted from bacteriophage φAa ₃₃₃₈₄. The distribution of the phage φAa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage φAa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.     

Cloning, sequence analysis and heterologous expression of the DNA adenine-(N(6)) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans

We cloned and sequenced the DNA adenine-N(6) methyltransferase gene of the human pathogen Actinobacillus actinomycetemcomitans (M.AacDAM). Restriction digestion shows that the enzyme methylates adenine in the sequence GATC. Expression of the enzyme in a DAM(-) background shows in vivo activity. A PSI-BLAST search revealed that M.AacDAM is most related to M.HindIV, M.EcoDAM, M.StyDAM, and M.SmaII. The ClustalW alignment shows highly conserved regions in the enzyme characteristic for type a MTases. Phylogenetic tree analysis shows a cluster of enzymes recognizing the sequence GATC, within a branch of orphan MTases harboring M.AacDAM. The cloning and sequencing of this first methyltransferase gene described for A. actinomycetemcomitans open the path for studies on the potential regulatory impact of DNA methylation on gene regulation and virulence in this organism.     

以Candin为佐剂的人乳头瘤病毒多肽治疗疫苗诱导小鼠的体液免疫应答

目的 研究以Candin为佐剂的人乳头瘤病毒（Human papillomavirus,HPV）多肽治疗疫苗诱导小鼠产生的体液免疫应答。方法 分别采用PBS（对照）、佐剂Candin（150 μL/只）、HPV16 E7多肽[3个E7多肽片段,50 μg/(段·只)]和包含佐剂Candin与HPV16 E7多肽的疫苗对C57BL/6小鼠进行免疫接种实验,在第21天和第42天各加强1次免疫,剂量同第1次免疫。第3次免疫后2周,颈脱位处死小鼠,取血清。以ELISA法检测血清总IgG、IgG1和IgG2a抗体浓度。结果 经多肽（P=0.001,P<0.001）和疫苗（P=0.001,P=0.008）免疫的小鼠总IgG抗体、IgG1抗体水平均较对照组高,差异有统计学意义。与对照组相比,疫苗（P=0.008）可明显提高小鼠血清中IgG2a抗体水平。结论 包含佐剂Candin及HPV16 E7多肽的疫苗能诱导小鼠产生明显的体液免疫应答。