This study was undertaken to determine the frequency of Legionella infection in a dental clinic setting. Serum samples from 270 dental clinic personnel were evaluated using an enzyme-linked immunosorbent assay to detect Legionella-specific IgM and IgG antibodies. The pooled-species whole-cell-antigen preparation used in these assays was derived from six Legionella pneumophila strains and one strain each from Legionella bozemanii and Legionella micdadei. Significant levels of IgG and IgM antibodies were found in 20% and 16%, respectively, of the samples. This compares with 8% and 10%, respectively, for a randomly selected non-clinical group from the region (P less than 0.005). Samples from clinic personnel with significant IgG titers (greater than 1:128) were also evaluated for activity to each of the eight single-species antigens, with the following results: L. pneumophila, 45% (combined six strains); L. micdadei, 37%; and L. bozemanii, 18%. Comparing individuals' "years spent in the clinic environment" with the incidence of significant antibody levels strongly suggests that the risk of Legionella infection increases proportionately with increased clinic exposure time (P less than 0.05). Analysis of these data implies that Legionella may be present in the dental clinic environment, thus creating an increased risk for clinical personnel or patients.

The primary structure of Escherichia coli hemolysin (HIlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity. Hemolysin also requires an unknown modification by an accessory protein, HlyC, for hemolytic activity. The role of calcium in the interaction of HlyA with erythrocytes was investigated by using recombinant strains which produced inactive hemolysins unmodified by EllyC or deleted of the repeat sequences. 45Ca2' autoradiography of the recombinant hemolysins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium. Inactive hemolysin, unmodified by ElyC, contained the repeated sequences and bound calcium as efficiently as the active, full-length toxin. The binding of the inactive toxins to erythrocytes was investigated by immunoblotting saline-washed, toxin-treated cells with monoclonal antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. The binding of full-length, active hemolysin to erythrocytes was calcium dependent. Inactive hemolysin deleted of the repeat units did not bind to cells. The inactive hemolysin, unmodified by HlyC, bound calcium but did not bind to erythrocytes. These results highlight the importance of calcium in the binding of hemolysin to erythrocytes and suggest that the binding of hemolysin to cells requires an interaction between the calcium-binding repeat domain and the modification produced by the HlyC protein.

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.

To gain further evidence that Escherichia coli alpha-hemolysin has a role in pathogenesis, its effect on human peripheral leukocyte viability was studied in vitro. Viability of leukocytes exposed to low doses of alpha-hemolysin decreased nearly 10-fold within 15 min of exposure. This response was dose and time dependent and was neutralized by antiserum, heat, proteases, and lipase. To gain further evidence that alpha-hemolysin was the molecule responsible for leukotoxicity, preparations of alpha-hemolysin were passed through a hydrophobic interaction chromatographic column. Alpha-hemolysin prepared in this way retained its leukotoxic activity. Both hemolytic and leukotoxic activities copurified at the same ratio and were inactivated to the same degree by heating at 56 degrees C. Lysis of leukocytes, if it occurs in vivo, would enhance the chances of survival for an invading hemolytic E. coli.

The ability of Escherichia coli which possess or lack mannose-sensitive adherence factors (adhesins) to associate with human peripheral leukocytes in vitro in the absence of serum was studied. E. coli 19+, which have mannose-sensitive adhesins, were derived from E. coli strain 19 by culturing in static Trypticase soy broth at 37 degrees C. E. coli 19-, which lack mannose-sensitive adhesins, were derived from E. coli 19 by culturing in agitated Trypticase soy broth at 30 degrees C. E. coli 19+ attached to leukocytes and stimulated the release of lysozyme but not beta-glucuronidase or lactate dehydrogenase. In contrast, E. coli 19- showed poor attachment to the leukocytes and failed to stimulate lysosomal enzyme release. During a 60-min incubation with the leukocytes, the number of viable 19+ organisms decreased, whereas the number of viable 19- remained constant. Purified type 1 pili from E. coli 19+ agglutinated the leukocytes but did not stimulate lysosomal enzyme release. Pretreatment of leukocytes with type 1 pili failed to prevent the adherence of E. coli 19+. The association of 19+ with leukocytes and subsequent release of lysozyme could be blocked by alpha-methyl-D-mannoside but not by equivalent concentrations of dextrose and sucrose. These results show that mannose-sensitive adhesins on E. coli mediate association of the organisms with leukocytes in the absence of serum components. The identity of the adhesins involved in leukocyte association has yet to be determined.

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SUMMARY. Alpha haemolysin, produced by Escherichia coli, grown in a chemically defined medium, was purified 19-fold and the endotoxin content reduced 2 176-fold by ultrafiltration and glycerol-gradient ultracentrifugation. Immunodiffusion of purified a haemolysin (PH) against antiserum to crude haemolysin (CH) revealed only one precipitation line. PH was cytotoxic in nanogram amounts for mouse-fibroblast 3T3 cells, and the cytotoxicity exhibited proportional dose-response and time-course kinetics. The cytotoxic and haemolytic activities of PH were neutralised by immunoglobulins to CH. A mutant, produced by treating the haemolytic wild type with mitomycin C, possessed all of the biochemical characteristics of the wild type with the exception that the extracellular products of the mutant were non-haemolytic and noncyto toxic.

The calcium requirement for hemolytic activity of Escherichia coli hemolysin was investigated by using hemolytic assays and immunoblotting of toxin-treated erythrocytes. The hemolytic activity of cell culture supernatants obtained during growth of E. coi in Luria-Bertani (LB) broth or calcium-free LB broth was calcium dependent. The hemolytic activity of culture supernatants obtained during growth in LB broth supplemented with calcium was calcium independent. Osmotic protection experiments using Dextran 4 to prevent cell lysis indicated that calcium was required for the binding of hemolysin to erythrocytes at both 4 and 37°C. The binding efficiency at 4°C was 50% of that occurring at 37C. The calcium-dependent binding was confirmed by immunoblotting saline-washed, toxin-treated erythrocytes with a monoclonal antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. Bound hemolysin increased the calcium permeability of the cell membranes as evidenced by calcium-induced membrane protein alterations. The alterations in membrane proteins did not directly cause lysis of the cells. The results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells.