Enzyme-linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoalveolar fluid.

A nonradioactive method is described that detects 10 to 100 legionellae in 1 ml of bronchoalveolar lavage fluid. DNA is purified by a proteinase K-phenol protocol or with a commercial DNA preparation kit and amplified by PCR with amplimers specific for the 16S rRNA gene of Legionella pneumophila. The upstream primer is 5' biotinylated. The amplification product is immobilized on streptavidin-coated microtiter plates. Because of the high binding capacity, no removal of nonincorporated biotin from the PCR product is required. After alkaline denaturation, the single-stranded PCR product is hybridized with a 5' digoxigenin-labeled probing oligomer. The amplification product is then detected by using peroxidase-labeled anti-digoxigenin antibodies in a luminescence or colorimetric reaction. The assay detects as few as 10 legionellae in 1-ml bronchoalveolar lavage fluid specimens. It is specific for medically relevant Legionella species, including Legionella pneumophila, L. bozemanii, and L. longbeachae. Of over 250 clinical specimens examined, 8 were positive for legionellae by both culture and the PCR assay. Six further specimens were culture negative but PCR positive for legionellae; of these, five specimens were from patients receiving high-dose erythromycin therapy for suspected or previously diagnosed legionella pneumonia. None of the remaining 240 specimens that were culture negative for legionellae yielded a positive PCR test, although a total of over 30 different bacterial species were cultured from these specimens. The PCR assay therefore appears to exhibit high sensitivity and specificity and thus could prove suitable for use in the routine microbiological diagnostic laboratory.

Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus.

When applied at low concentrations (< 10 micrograms/ml), staphylococcal alpha-toxin generates a small channel in keratinocyte and lymphocyte membranes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 micrograms/ml) of alpha-toxin similarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemented with serum and at pH 7.4, the cells are able to recover from toxin attack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the presence of serum in the medium and is very sensitive towards pH. Decreases of pH in the medium to < or = 7.0 as well as increases to > or = 7.8 causes the repair mechanism to fail. The fate of cell-bound toxin molecules was investigated by using a radiolabelled tracer and by immunological detection of toxin exposed at the cell surface. The results indicated that 50-70% of the toxin was shed from cell membranes. However, there was no clear correlation between shedding and recovery, and shedding was also observed in cells that died at pH 7.8. Shedding was not decisive for repair, since cells that had recovered from toxin attack continued to carry 30-40% of initially bound toxin on their cell surface. Blockade of Na+/K(+)-ATPases with ouabain evoked similar kinetics of K(+)-depletion in control cells, compared with cells that had just recuperated from toxin attack and that still carried 30-40% alpha-toxin on their surface. We therefore tentatively concluded that repair of alpha-toxin lesions was due to closure of small pores, rather than from compensation of membrane leaks by up-regulation of Na+/K(+)-ATPase activity. We speculate that repair of small membrane lesions may extend to other agents that produce channels of similar nature in nucleated cells. Larger pores created by E. coli hemolysin or streptolysin O, both of which form larger functional transmembrane lesions, could not be repaired by fibroblasts.

[Molecular basis for the pathogenicity of S. aureus alpha-toxins]. / Molekulare Grundlage für die Pathogenität des S.aureus-alpha-Toxins.

Staphylococcal alpha-toxin is produced by most strains of S. aureus and is considered a major pathogenic factor of these bacteria. The toxin is produced as a water-soluble molecule of MW 34000. Binding to a membrane target is accompanied by the formation of ring-structured hexamers with outer and inner diameters of 10 and 2-3 nm, respectively. The toxin rings carry lipid-binding surfaces that allow for insertion into and firm embedment within the membrane. Small transmembrane channels are thus generated that can induce a variety of pathological cellular changes. Large doses of toxin will generally cause cell lysis and death. However, sub-cytolytic toxin doses can also elicit major pathophysiological reactions. When introduced into the circulation of an isolated and perfused rabbit lung, the toxin causes steep rises in the pulmonary artery pressure, and lung edema results as a consequence of increases in vascular permeability occurring in parallel. These processes are the result of the activation of the arachidonic acid cascade by alpha-toxin in the lung. Studies using cultured endothelial cells as targets subsequently led to a hypothesis that would explain how membrane channel formation by a toxin could be linked to the observed arachidonic acid cascade activation. In essence, we propose that the toxin pores serve as non-physiological calcium channels, and that calcium influx triggers the observed reactions. It is probable that many other pathophysiological processes including inflammatory tissue reactions derive from such secondary effects of toxin action.(ABSTRACT TRUNCATED AT 250 WORDS)