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E T Lally

Publications and source records attributed to E T Lally.

At least 37 records · Page 2Linked to original sources
PubMed1994

Identification and immunological characterization of the domain of Actinobacillus actinomycetemcomitans leukotoxin that determines its specificity for human target cells.

Although extensive amino acid homology exists among the various Repeats in ToXin (RTX) family of bacterial cytolysins, the cellular and species specificities remain unique for individual toxins (i.e. Actinobacillus actinomycetemcomitans leukotoxin (LtxA) kills human monomyelocytes while a related toxin, Pasteurella hemeolytica leukotoxin (LktA) kills bovine lymphoid cells). To determine the Ltx domain responsible for species specificity, ltxA/lktA chimeric toxin genes were expressed in tandem with the ltxC gene under control of the P lambda promoter. The ability of lysates to kill either HL-60 (human) or BL-3 (bovine) cells was assessed by trypan blue exclusion. The critical area required for the chimeric toxins to recognize human target cells is a 253-amino acid fragment (residue 688-941) that contains the GGXGXDX(L[I[V[W[Y[F)X repeats. A panel of 12 neutralizing anti-LtxA monoclonal antibodies also recognized specificities within the 253-amino acid fragment. Epitope mapping of the monoclonal antibody panel showed that all antibodies bound to one of three sites on the LtxA molecule. One monoclonal recognized epitope A which was composed of LtxA residues 698-709 (KLDYYYTNKGFK), six antibodies recognized epitope B, a peptide composed of residues 746-757 (LIYGYDGDDRLY), whereas the remaining five monoclonals recognized epitope C, which is composed of residues 926-937 (DRARLKRQFELQ).

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Flow cytometric analysis of the cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human natural killer cells.

The goal of this investigation was to determine if human natural killer (NK) cells were susceptible to the cytolytic effects of the Actinobacillus actinomycetemcomitans leukotoxin (LTX). Following treatment with LTX (0-200 ng/ml), NK cell activation by interleukin-2 (IL-2) was evaluated. LTX inhibited the IL-2-induced expression of both CD69 and the IL-2 receptor. Furthermore, the up-regulation of CD56 was also impaired. To determine whether the observed functional deficits were the result of cell death, NK cell viability was evaluated by flow cytometry. Changes in forward and side light scatter patterns consistent with cell death were observed within 60 min. Direct analysis of cell viability by measuring propidium iodide exclusion, however, indicated little change in the viability of LTX-treated NK cells. Electron microscopic analysis of NK cells exposed to LTX revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation, and condensation of nucleoplasm. However, no change in membrane integrity was initially noted. Finally, LTX caused a rapid and sustained elevation in the intracellular levels of Ca2+. These morphological and biochemical changes are consistent with the notion of programmed cell death.

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Population structure of Actinobacillus actinomycetemcomitans: a framework for studies of disease-associated properties.

The Actinobacillus actinomycetemcomitans population consists of a large number of clones among which the ubiquitous leukotoxin gene operon appears very homogeneous. Population genetic analyses performed by multilocus enzyme electrophoresis together with DNA fingerprinting and analyses of genomic DNA restriction fragment length polymorphisms (RFLP) on 97 strains isolated over a period of 45 years revealed that each of the serotypes a, b, c, d and e comprise genetically isolated subpopulations and that successful horizontal transfer of genomic DNA between strains of different serotypes appears to be extremely rare in vivo. In contrast, recombination between strains of the same serotype in general appears to take place in nature. The results provide evidence that non-serotypeable strains are serotype antigen-deficient variants originating from strains of the known serotypes. Serotype b and c strains may contain transmittable DNA sequences not found in strains of the other serotypes.

Actinobacillus Infections↗
PubMed1994

Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated as a virulence determinant in various human infections and is encoded by a multigene operon consisting of four known genes, designated ltxC, ltxA, ltxB, and ltxD. The ltx operon appears to be present in all A. actinomycetemcomitans strains, but levels of toxin expression vary greatly among strains. Thus, to gain a better understanding of the expression and regulation of the ltx operon, we have analyzed the ltx promoters of a highly toxic (JP2) and a minimally toxic (652) strain of A. actinomycetemcomitans. The nucleotide sequence of the JP2 ltx promoter contains -10 and -35 elements situated 350 bases upstream of ltxC, and primer extension of JP2 RNA confirmed that they are functional in vivo. However, a second primer extension product of 40 bases was present, and analysis of a series of truncated JP2 promoters fused to lacZ suggested that the region immediately upstream of ltxC also promotes transcription in Escherichia coli. These results suggest that two promoters may direct ltx expression in JP2. In addition, a small open reading frame capable of encoding a peptide of 78 amino acids was identified upstream of ltxC. Northern blots showed that this open reading frame is transcribed as part of a 4.2-kb mRNA, a transcript not previously identified as being derived from the ltx operon. In contrast, strain 652 expresses low steady-state levels of ltx mRNA, and its intact ltx promoter was inefficient in transcribing lacZ in E. coli. The nucleotide sequence of the 652 promoter is similar to that of the JP2 promoter but contains a region of 530 bp that is not present in JP2. Of 15 additional strains of A. actinomycetemcomitans that were analyzed, 13 contained promoters resembling the 652 sequence and 2 possessed JP2-like promoters. Both strains possessing the JP2-like promoter expressed 10- to 20-fold-higher levels of leukotoxin than did the strains possessing promoters resembling the 652 promoter. These results suggest that high levels of leukotoxin expression may correlate with the presence of the JP2-like promoter.

Actinobacillus Infections↗
PubMed1992

Lytic effects of Actinobacillus actinomycetemcomitans leukotoxin on human neutrophil cytoplasts.

Actinobacillus actinomycetemcomitans leukotoxin lysed human neutrophil cytoplasts. The reaction was associated with a rapid influx of extracellular calcium, a collapse in membrane potential, release of lactate dehydrogenase, and overt disintegration of the plasma membrane. These functional and structural alterations in the plasmalemma of neutrophil cytoplasts reinforce the hypothesis that A. actinomycetemcomitans leukotoxin acts as a pore-forming, membranolytic agent and indicate that neutrophil cytoplasts are useful tools in studying the biology of membrane-active toxins.

Aggregatibacter actinomycetemcomitans↗
PubMed1992

Electron immunocytochemical localization of Actinobacillus actinomycetemcomitans leukotoxin.

The Actinobacillus actinomycetemcomitans leukotoxin was localized in A. actinomycetemcomitans bacteria using an electron immunocytochemical thin-section labeling method. An immuno-dot blot procedure was initially used to ascertain the optimal specimen fixation. This consisted of a periodate-lysine-paraformaldehyde (2%) fixative in a phosphate buffer followed by embedding in LR White. Affinity-purified toxin was used to produce a monospecific polyclonal antibody. The reaction sites were visualized with a colloidal gold-tagged reporter antibody. The leukotoxin was found to be localized either in the cell envelope and/or in membranous vesicles on the outer surface of the bacterial cell. These results support previous observations indicating the same location.

Aggregatibacter actinomycetemcomitans↗
PubMed1991

Early changes in cytosolic calcium and membrane potential induced by Actinobacillus actinomycetemcomitans leukotoxin in susceptible and resistant target cells.

Actinobacillus actinomycetemcomitans produces a cytolytic peptide leukotoxin which kills susceptible target cells, including human neutrophils, monocytes, lymphocytes, and HL-60 promyelocytic leukemia cells. Cell death occurs as a consequence of colloid osmotic lysis. In the present investigation early leukotoxin-induced changes in membrane permeability were studied by flow cytometry and quantitative spectrofluorimetry in leukotoxin-susceptible and resistant targets. Within 5 s toxin-susceptible cells exhibited concentration-dependent, sustained increases in systolic free Ca2+, and this was rapidly followed by a progressive fall in membrane potential. These early manifestations of membrane injury occurred approximately 10-15 min before cell death, as reflected by flow cytometric analysis of propidium iodide stained cells. The rise in cytosolic Ca2+ was almost entirely due to an influx of extracellular Ca2+. The results of Hill plots for the action of leukotoxin on Ca2+ permeability in human neutrophils or HL-60 cells suggested that two or more toxin molecules participate in the assembly of an ion conducting pore in the plasma membrane. Changes in membrane permeability or cell viability were not observed in response to heat-inactivated toxin. Under appropriate conditions toxin-induced membrane abnormalities were inhibited by leukotoxin-neutralizing mAb or relatively high concentrations (greater than or equal to 2.5 mM) of extracellular Ca2+. Leukotoxin-resistant target cells showed no evidence of membrane injury even when exposed to high concentrations of leukotoxin for prolonged periods of time. These included resistant human K562 erythroleukemia cells and murine SP2 myeloma cells which have previously been shown to adsorb the toxin, suggesting that they possess a protective mechanism(s) which impedes toxin insertion or assembly in the lipid bilayer. These data support the concept that A. actinomycetemcomitans leukotoxin acts as a cell-specific, pore-forming protein which permeabilizes the plasma membrane of susceptible target cells.

Aggregatibacter actinomycetemcomitans↗
PubMed1991

Structure and function of the B and D genes of the Actinobacillus actinomycetemcomitans leukotoxin complex.

The Actinobacillus actinomycetemcomitans leukotoxin gene complex, consisting of four genes, has been cloned and the sequence of the AaLtC and AaLtA genes reported. The present paper details the sequences of the AaLtB and AaLtD genes which, like AaLtC and AaLTA, are also homologues of genes found in other cytolytic toxin complexes of several other Gram-negative bacterial pathogens. When tested in a recombinant expression system, the AaLtB and/or AaLtD genes are required for the translocation and insertion of the A. actinomycetemcomitans leukotoxin (AaLtA) into the cell membrane of Escherichia coli.

Actinobacillus↗
PubMed1991

Lethal effects of Actinobacillus actinomycetemcomitans leukotoxin on human T lymphocytes.

The majority of strains of Actinobacillus actinomycetemcomitans isolated from patients with periodontal diseases secrete a leukotoxin that destroys human myeloid cells within minutes but has no effect on viability of peripheral blood lymphocytes in culture for 1.5 h. However, since this organism persists in the gingival crevice and thus may continuously release toxin over extended periods of time, we assessed the viability of T cells cultured with leukotoxin (0 to 250 ng/ml) for up to 2 days. Although the total numbers of cells recovered from cultures with or without leukotoxin were equivalent, leukotoxin killed up to 70% of the T cells in a time- and concentration-dependent manner. Cell death was associated with uptake of propidium iodide, release of 51Cr from the cytoplasm, and morphological evidence of damage to the plasma membrane and apoptosis. Leukotoxin also induced increased cleavage of chromosomal DNA into nucleosome-sized fragments, suggesting activation of an endogenous nuclease in the T cells. These data suggest that leukotoxin kills T cells by pathways resembling necrosis and programmed cell death. Leukotoxin-induced lymphotoxicity may represent a critical mechanism by which A. actinomycetemcomitans suppresses the host local immune response and contributes to the pathogenesis of diseases involving this microorganisms.

Actinobacillus↗
PubMed1990

Comparison of Streptococcus mutans and Streptococcus sanguis receptors for human salivary agglutinin.

Oral streptococci vary in their susceptibility to salivary agglutinin-mediated aggregation. To understand the molecular basis of this specificity, the structure and function of receptors for agglutinin from Streptococcus mutans KPSK2 (MSL-1) and Streptococcus sanguis M5 (SSP-5) were compared. Immunological screening of an S. mutans KPSK2 genomic DNA library yielded two identical clones expressing a streptococcal protein that co-migrated with a 220 kDa peptide in SDS extracts from this organism. This protein inhibited agglutinin-mediated aggregation of S. mutans KPSK2 in a dose-dependent manner. The MSL-1 gene is homologous to the S. mutans SpaP and pac genes although single base substitutions alter several amino acids. MSL-1 is also similar to the agglutinin receptor (SSP-5) cloned from S. sanguis M5. All three proteins, MSL-1, P1, and SSP-5 share at least one epitope since monoclonal and polyclonal anti-SSP-5 antibodies react with both MSL-1 and P1. However, other monoclonal antibodies are specific for SSP-5 and appear to react with a peptide domain exhibiting little homology to MSL-1 or P1. Sugar inhibition studies showed that agglutinin-mediated aggregation of S. mutans KPSK2 was most potently inhibited by fucose and lactose. Sialic acid, a potent inhibitor of S. sanguis aggregation, had no effect on the interaction of agglutinin with S. mutans KPSK2. These results suggest that while the MSL-1 and SSP-5 proteins are genetically and immunologically related, their specificity for binding sites on agglutinin differs.

Adhesins, Bacterial↗
PubMed1990

Effects of cations and osmotic protectants on cytolytic activity of Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans leukotoxin permeabilized the plasma membrane of HL-60 promyelocytic leukemia cells, resulting in colloid osmotic lysis. These events were associated with efflux of 51chromium (from prelabeled cells), influx of propidium iodide, and ultrastructural evidence of cellular damage. Target cell lysis was inhibited by procedures which may interfere with the initial interaction of the toxin with the plasma membrane. For example, washing cultures (to dilute and remove toxin) or the addition of monoclonal antibodies (to neutralize toxin) or trypsin (to inactivate toxin) limited lysis when undertaken within the first 5 min of the reaction. The extent of injury was also diminished when radiolabeled HL-60 cells were exposed to toxin in the presence of unlabeled, toxin-sensitive cells (e.g., HL-60 cells or human neutrophils) or certain toxin-resistant target cells (e.g., human K562 erythroleukemia cells). This suggests that the association of the toxin with the cell membrane may not be sufficient to cause lysis without activation of additional effector mechanisms. The addition of specific trivalent (e.g., La3+) or divalent (e.g., Ca2+ and Zn2+) cations to toxin-treated cells appeared to enhance their capacity to repair or minimize the extent of toxin-mediated membrane damage. Depending on size, certain saccharides served as osmotic protectants: maltose almost completely inhibited radiolabel release, while smaller molecules provided correspondingly less protection. The results imply that the leukotoxin has membranolytic activity, producing pores in target cells with a functional diameter approximately the size of maltose (0.96 nm).

Actinobacillus↗
PubMed1989

Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins.

Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.

Amino Acid Sequence↗
PubMed1989

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene.

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.

Actinobacillus↗
PubMed1988

The occurrence of a fibro-osseous lesion and multiple dental anomalies in a patient with the Greig cephalopolysyndactyly syndrome.

The Greig cephalopolysyndactyly syndrome is an uncommon dysmorphic syndrome characterized by preaxial and postaxial polysyndactyly and minor craniofacial anomalies. It has an autosomal dominant inheritance. A review of the literature has failed to show documentation of any dental or oral abnormalities. Therefore, we report on a patient with Greig syndrome who had a maxillary fibro-osseous lesion and multiple dental and oral abnormalities.

Abnormalities, Multiple↗
PubMed1988

An ELISA for PGE2 utilizing monoclonal antibody.

An ELISA for PGE2 has been developed which is sensitive to concentrations of 0.5 to 20.0 ng PGE2/ml. Mouse monoclonal anti-PGE2 ascites is utilized in a binding competition between the test sample and an adsorbed conjugate of PGE2-BSA. The antibody which remains bound to the solid phase is quantitated colorimetrically by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG followed by incubation with p-nitro-phenylphosphate. PGE1, PGA1, PGA2, PGB2, 6-keto-PGF1 alpha, PGF2 alpha, 13,14-dihydro-15-keto-PGE2, thromboxane B2 and arachidonic acid showed minimal cross-reactivity with the anti-PGE2. The PGE2 ELISA permits the quantitative analysis of large numbers of samples at a fraction of the cost and time required to process a commercial RIA kit. When linked to the appropriate computer software, data collection and analysis can be performed in less than 10 minutes per 96-well plate. Furthermore, the use of an ELISA system eliminates the radioactive and toxic chemical waste generated by RIA methods.

Antibodies, Monoclonal↗