Biomedical subjects

E T Lally

Publications and source records attributed to E T Lally.

At least 19 recordsLinked to original sources

PubMed2002

Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a gram-negative bacterium strongly implicated in the pathogenesis of juvenile periodontitis. This periodontal pathogen synthesizes a leukotoxin that destroys human polymorphonuclear leukocytes (PMNs), and this toxin is thought to be responsible for the virulence of A. actinomycetemcomitans. It was therefore of interest to assess whether major virulence factors of periodontal pathogens were neutralized by salivary components. This study focuses on the effect of histatins, components of the nonimmune oral defense system, on leukotoxin activity. Leukotoxin was extracted with polymyxin B from freshly grown anaerobic cultures of A. actinomycetemcomitans strain Y4. PMNs isolated from blood of healthy human volunteers were incubated in a cytotoxicity assay containing PMNs (10(7) cells/ml) and leukotoxin preparation (0-500 microg/ml) in Hanks' balanced salt solution at 37 degrees C for 0-120 min with or without synthetic histatin 5 (0-500 microM). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) at different time intervals. Histatin 5 neutralized the toxic effect of the leukotoxin preparation in a concentration-dependent manner, with an IC(50) value of 150 microM. When PMNs were preincubated with histatin 5 (300 microM), washed and subsequently exposed to leukotoxin, no protective effect was observed. This observation suggests a mechanism of inhibition whereby histatin 5 either directly neutralizes the leukotoxin or interferes with the leukotoxin-PMN interaction. The inhibitory effect of histatin 5 on leukotoxic activity may suggest a new biological function of histatins in the oral cavity as a naturally occurring secondary antibiotic.

Adult↗

PubMed2002

Molecular cloning of the fur gene from Actinobacillus actinomycetemcomitans.

In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle.

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. Moreover, we have shown that CdtB impairs lymphocyte function by inducing G(2) arrest of the cell cycle. We now report that both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A. actinomycetemcomitans cdt genes (rCdtABC) induce apoptosis. Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h. No DNA fragmentation was induced in nonactivated cells. Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation. Mitochondrial function was also perturbed in cells pretreated with either CdtB or rCdtABC. An increase in the expression of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this includes a decrease in the transmembrane potential and elevated generation of reactive oxygen species. Activation of the caspase cascade, which is an important biochemical feature of the apoptotic process, was also observed in Cdt-treated lymphocytes. Overexpression of the bcl-2 gene in the human B lymphoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis. Interestingly, Bcl-2 overexpression did not block Cdt-induced G(2) arrest. The implications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis.

Subnanomolar concentrations (3 x 10(-10) M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (DeltaPsim), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat-inactivated leukotoxin (DeltaI Ltx) controls, ATP levels in Ltx-treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl-2 and bcl-xL and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/bcl-2) and 73% (JY/bcl-xL) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx-treated JY/bcl-2 and JY/bcl-xL cells, whereas its presence in the cytoplasmic fraction of JY/gen cells suggests an uncoupling of electron transport. Expression of bcl-2 and bcl-xL in cells inhibited downstream apoptotic events such as cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.

Adenosine Triphosphate↗

PubMed2001

Beyond the specific plaque hypothesis: are highly leukotoxic strains of Actinobacillus actinomycetemcomitans a paradigm for periodontal pathogenesis?

Actinobacillus actinomycetemcomitans is a facultative anaerobe implicated in a variety of periodontal diseases. Its presence is most closely associated with localized juvenile periodontitis (LIP), although the exact role of the organism in this and other periodontal diseases is not entirely clear. While A. actinomycetemcomitans produces several different putative virulence factors, the most widely studied is the leukotoxin. The leukotoxin selectively kills polymorphonuclear leukocytes and macrophages in vitro, constituting the host's first line of defense. Interestingly, even though all strains of A. actinomycetemcomitans have the genes encoding the leukotoxin, there is variability in leukotoxin expression. Differences in the structure of the promoter region of the leukotoxin gene operon were shown to correlate directly with levels of leukotoxin production. Highly leukotoxic forms appear to exhibit increased pathogenic potential, as evidenced by recent studies that have shown a significant association between the prevalence of such strains and the occurrence of LIP in several different populations. This represents the first demonstration of an association between a particular subset of a pathogenic species and a specific periodontal disease. Early identification of A. actinomycetemcomitans by microbial and genetic assays to evaluate leukotoxicity may enhance the efficacy of preventive and/or therapeutic techniques. Future investigations should continue to evaluate pathogenic variations of additional virulence factors expressed in vivo, not only of A. actinomycetemcomitans, but also of other periodontal bacteria and infectious disease pathogens.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Conformational studies of Actinobacillus actinomycetemcomitans leukotoxin: partial denaturation enhances toxicity.

A 114 kDa, water-soluble, cytotoxin secreted by the Gram-negative bacterium Actinobacillus actinomycetemcomitans (Aa) is similar in sequence to Escherichia coli alpha-hemolysin, but is non-hemolytic, killing leukocytes of select species, including humans. In this work, we investigated aspects of the water-soluble conformation of Aa toxin which relate to its biological, pore-forming activity. The toxin has five native tryptophans and fluorescence spectra were monitored in aqueous solutions in the presence of varying denaturants. Significant changes in the fluorescence spectra, without significant wavelength shifts, were induced by small additions of denaturants and changes in the temperature or pH. The fluorescence changes suggested that small perturbations in the aqueous environment resulted in structural changes in the toxin related not to a large unfolding but to more subtle conformational changes. Analytical ultracentrifugation showed the toxin to be a globular monomer in dilute aqueous solution. Circular dichroism spectroscopy showed about 25% alpha-helical structure which is largely maintained up to a temperature (65 degrees C) known to deactivate toxin activity. Changes in the cytotoxic properties of the toxin were monitored with flow cytometric analysis following preincubation of the toxin under mild conditions similar to those used in the fluorescence studies. These experiments showed that the pretreated toxin exhibited enhanced cell-killing potency on toxin-sensitive cells. The correlation of cytotoxicity with the changes in Trp fluorescence is consistent with the idea that partial unfolding of Aa toxin is an early, obligate step in toxin-induced cell kill.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Perturbation of mitochondrial structure and function plays a central role in Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis.

Certain pore-forming bacterial toxins, including the leukotoxin (Ltx) produced by Actinobacillus actinomycetemcomitans, induce apoptosis in susceptible target cells. Although binding to the target cell surface represents the first step in the initiation of this process, the downstream events leading to toxin-induced apoptotic cell death have not been identified. Perturbation of mitochondrial function has been shown to have a major role in regulating progression of apoptosis initiated by exposure to numerous stimuli. Using Ltx as a model, the aim of this study was to evaluate whether induction of apoptosis by pore-forming toxins follows a similar paradigm. After exposure to Ltx, Epstein-Barr virus transformed B cells (JY cell line) exhibited the classical morphological features of apoptosis including decreased cell size, plasma membrane blebbing, selective alterations in plasma membrane permeability and condensation of nuclear DNA. The morphologic changes were accompanied by swelling of the mitochondria, a decrease in mitochondrial transmembrane potential (Psi(m)), hyperproduction of reactive oxygen intermediates (ROIs) and release of cytochrome c from the intermembrane space. Subsequently, we detected activation of the c ysteine asp artate-specific prote ases (caspases)-3 and -9, cleavage of the nuclear DNA repair enzyme, poly(ADP-ribose)polymerase (PARP) and internucleosomal DNA fragmentation. These results indicate that perturbation of mitochondrial structure and function, in concert with activation of specific caspases, initiate the effector phase of Ltx-induced apoptosis.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

Animals↗

PubMed2000

Evidence for the role of highly leukotoxic Actinobacillus actinomycetemcomitans in the pathogenesis of localized juvenile and other forms of early-onset periodontitis.

BACKGROUND: Actinobacillus actinomycetemcomitans leukotoxin is thought to be an important virulence factor in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. Some highly leukotoxic A. actinomycetemcomitans strains produce 10 to 20 times more leukotoxin than other minimally leukotoxic strains. The distribution, clonality, and intrafamilial transmission of highly leukotoxic A. actinomycetemcomitans were examined in order to determine the importance of leukotoxin in the pathogenesis of periodontitis. METHODS: The polymerase chain reaction (PCR) was used to differentiate highly leukotoxic from minimally leukotoxic strains in examining 1,023 fresh A. actinomycetemcomitans isolates and strains from our culture collection. These were obtained from 146 subjects including 71 with localized juvenile periodontitis (LJP), 4 with early-onset periodontitis, 11 with post-localized juvenile periodontitis, 41 with adult periodontitis, and 19 periodontally normal subjects. The arbitrarily primed polymerase chain reaction (AP-PCR) analysis of 30 oral isolates from each of 25 subjects was used to determine the intraoral distribution of A. actinomycetemcomitans clones. AP-PCR was also used to examine the transmission of A. actinomycetemcomitans in 30 members of 6 families. The clonality of 41 highly leukotoxic A. actinomycetemcomitans strains was evaluated by both AP-PCR and ribotyping. RESULTS: Highly leukotoxic A. actinomycetemcomitans was found only in subjects with localized juvenile and early-onset periodontitis. Fifty-five percent of the LJP subjects harbored highly leukotoxic A. actinomycetemcomitans isolates. Seventy-three percent of the A. actinomycetemcomitans isolates in these subjects were highly leukotoxic. Highly leukotoxic A. actinomycetemcomitans infected younger subjects (mean age 13.95 years, range 5 to 28 years) than minimally leukotoxic (mean age 35.47 years, range 6 to 65 years). Most subjects were infected with only one A. actinomycetemcomitans genotype. However, PCR of whole dental plaques and subsequent analysis of up to 130 individual oral isolates suggested a possible shift in A. actinomycetemcomitans over time in that a few subjects harbored both highly leukotoxic and minimally leukotoxic strains. AP-PCR analysis was consistent with intrafamilial A. actinomycetemcomitans transmission. Ribotyping and AP-PCR analysis confirmed a previous report that highly leukotoxic A. actinomycetemcomitans consists of a single clonal type. CONCLUSIONS: This study suggests that localized juvenile and other forms of Actinobacillus-associated periodontitis are primarily associated with the highly leukotoxic clone of A. actinomycetemcomitans.

Actinobacillus Infections↗

PubMed1999

The interaction between RTX toxins and target cells.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type- and species-specific. These toxins offer interesting models for targeting, insertion and translocation of aqueous proteins into lipid membranes.

Animals↗

PubMed1999

Oral and systemic immunoglobulin G-subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin.

Salivary, gingival crevicular fluid and serum-specific immunoglobulin G (IgG)-subclass antibodies to Actinobacillus actinomycetemcomitans leuktoxin were quantified by enzyme-linked immunosorbent assay. Samples were taken from six patients with periodontal pockets > or = 5 mm, harboring A. actinomycetemcomitans in subgingival plaque and from six healthy, sex- and age-matched controls, who did not harbor A. actinomycetemcomitans. In individuals suffering from periodontitis, the median values of specific IgG1- and IgG2-subclass antibodies in saliva, gingival crevicular fluid and serum were, respectively IgG1 147 ng/ml, 5226 ng/ml and 7318 ng/ml and IgG2 4.8 ng/ml, 934 ng/ml and 860 ng/ml. In the patients, specific IgG3 antibodies were detected in one out of six individuals in saliva, in two individuals in gingival crevicular fluid and in five out of six patients in serum with a median value of 561 ng/ml. The median values of specific IgG4 antibodies in saliva, gingival crevicular fluid and serum were below detectable levels. The median values of the total IgG subclasses in saliva and serum were 14622 ng/ml and 10.3 g/l respectively. Individuals with periodontitis had, compared with controls, a higher ratio of specific IgG1 antibodies to total IgG1 in saliva (P < 0.05) and in serum (P < 0.05) and a higher ratio of specific IgG antibodies to total IgG in saliva (P < 0.05) and in serum (P < 0.01). The results show an elevation of both oral and systemic specific antibodies to A. actinomycetemcomitans leukotoxin.

Adult↗

PubMed1998

Characterization of Actinobacillus actinomycetemcomitans leukotoxin pore formation in HL60 cells.

The mechanism of cell death induced by Actinobacillus actinomycetemcomitans leukotoxin (LTX) has been investigated with flow cytometry and patch electrode recording using cultured HL60 cells. The kinetics of propidium iodide (PI) positive staining of HL60 cells was measured as a function of LTX concentration at 37 degreesC. Results showed a concentration-dependent decrease in the tk times. Cell kill was slow at <1 microg/ml LTX concentrations with fewer than 50% of the cells killed after 1 h; at 1 microg/ml, the tk times ranged from approximately 15 to 30 min. At higher concentrations, the tk times decreased rapidly. The rate of cell kill was appreciably slowed at 20 degreesC. HL60 whole cell currents were recorded with patch electrodes. Immediately following exposure to high concentrations of LTX, large currents were recorded suggesting that the membrane potential of these cells had collapsed due to the large conductance increases. At low toxin concentrations, rapid conductance fluctuations were seen suggestive of a limited number of toxin-mediated events. Cells exposed to low concentrations of LTX exhibited these conductance fluctuations for up to 1 h, whereas toxin-insensitive cells were unaffected by long exposures to high concentrations of toxin. Our results are consistent with LTX-induced pores in susceptible cells which overwhelm the ability of the cell to maintain osmotic homeostasis causing cell death.

Aggregatibacter actinomycetemcomitans↗

PubMed1998

Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death.

Pasteurella haemolytica leukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a beta2 integrin since pre-incubating cells with anti-beta2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.

Animals↗

PubMed1998

Actinobacillus actinomycetemcomitans leukotoxin induces apoptosis in HL-60 cells.

Actinobacillus actinomycetemcomitans leukotoxin (Ltx) is a member of the repeats-in-toxin (RTX) family of pore-forming toxins and kills human immune cells. Currently, it remains unclear whether toxin-mediated killing of target cells involves the induction of necrosis or apoptosis. Therefore, the goal of this investigation was to determine whether Ltx is capable of causing apoptotic cell death in toxin-sensitive promyelocytic HL-60 cells. Multiparameter flow cytometric analysis of toxin-treated cells stained with Hoechst 33258 (or 33342) and 7-aminoactinomycin D allowed us to identify four populations: viable cells, early apoptotic cells, late apoptotic and/or secondarily necrotic cells, and a final population that was composed of cellular debris. Compared with control cells, HL-60 cells treated with Ltx exhibited a gradual decrease in forward light scatter with a coincident increase in side light scatter, indicative of a decrease in cell size and organelle condensation, respectively. Additional experiments demonstrated that Ltx-treated cells showed evidence of internucleosomal DNA fragmentation and phosphatidylserine translocation. The results of our studies clearly demonstrate that Ltx can kill HL-60 cells by inducing apoptosis. We hypothesize that elimination of acute inflammatory cells via this mechanism plays a critical role in the pathogenesis of diseases caused by A. actinomycetemeomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed1997

RTX toxins recognize a beta2 integrin on the surface of human target cells.

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coli alpha-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the beta2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins.

Amino Acid Sequence↗

PubMed1997

Antibody reactive with Actinobacillus actinomycetemcomitans leukotoxin in early-onset periodontitis patients.

The objective of this study was to determine whether a relationship exists between antibody reactive with the Actinobacillus actinomycetemcomitans leukotoxin and the severity of periodontal disease. Serum concentrations of antibody reactive with the leukotoxin were determined for 119 early-onset periodontitis patients and 59 non-periodontitis subjects using limiting dilution analysis on Western blots. Immunoglobulin G (IgG) antibody reactive with the A. actinomycetemcomitans leukotoxin ranged from undetectable to 29 micrograms/ml (mean = 3.13 +/- 0.97 micrograms/ml for the generalized early-onset periodontitis and 2.17 +/- 0.86 micrograms/ml for the localized juvenile periodontitis patients vs 0.32 +/- 0.24 ng/ml for 59 non-periodontitis controls), and the dominant subclass was IgG1. Analysis of the relationship between antibody reactive with A. actinomycetemcomitans sonicate, A. actinomycetemcomitans leukotoxin and attachment loss patterns indicates that seropositive generalized early-onset periodontitis patients had decreased attachment loss compared with patients lacking this antibody. The statistical relationship appeared to be stronger for the sonicate than the purified leukotoxin. These data suggest that antibody reactive with A. actinomycetemcomitans leukotoxin may be protective in early-onset periodontitis, but given that the sonicate appeared better than the leukotoxin alone, it is not likely that leukotoxin is the only antigen of importance to host defense.

Adolescent↗

PubMed1996

Construction of pYGK, an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector.

A shuttle vector that is capable of replicating in Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was constructed by modifying the Actinobacillus pleuropneumoniae (Ap) plasmid pYG53. A DNA fragment containing the KmR gene was inserted into pYG53 to generate pYGK, which confers resistance to kanamycin in both Aa and Ec. By electroporation, Ec DH5alpha and 17 strains of Aa were transformed with pYGK with efficiencies ranging from 0.5 to 3 X 10(6) colonies per microgram of DNA. Plasmid pYGK exists at approx. 3-4 copies per cell in Ec. This plasmid will facilitate the genetic manipulation of Aa strains and the molecular analysis of virulence factors expressed by this organism.

Actinobacillus↗

PubMed1995

Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gated ion channels when incorporated into planar lipid bilayers.

Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species- and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118, 262 and 406 pS in solutions containing 0.140 M NaCl. The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented.

Aggregatibacter actinomycetemcomitans↗