0000000000022229
AUTHOR
Daniel Jonas
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 prese…
Comparative evaluation of three different genotyping methods for investigation of nosocomial outbreaks of Legionnaires' disease in hospitals.
ABSTRACT The increased incidence of nosocomial Legionnaires' disease in two hospitals prompted investigation of possible environmental sources. In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of three methods— Sfi I macrorestriction analysis (MRA), amplified fragment length polymorphism (AFLP), and arbitrarily primed PCR (AP-PCR)—were compared. Twenty-nine outbreak-associated and eight nonassociated strains of Legionella pneumophila with 13 MRA types and subtypes were investigated. These strains comprised isolates from bronchoalveolar lavages, from environmental, patient-related sources, and type strains. All three typing…
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 …
Pathogenesis of Sepsis Syndrome: Possible Relevance of Pore-Forming Bacterial Toxins
This review focuses on a group of bacterial products whose very existence is known to only a minority of clinicians, and whose potential significance as inducers of the sepsis syndrome has eluded the attention of most microbiologists. This is unfortunate because pore-forming bacterial toxins possess all the properties for contributing to the pathogenesis of local and systemic inflammatory reactions. Because pore formers generally are highly immunogenic proteins, the prospects for immune intervention are described that may eventually be of benefit to patients. The subject is therefore of interest not only from a theoretical but also from a practical point of view.
A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes
Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above. S. aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required. This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied. If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small. In this case, high concentrations of alpha-toxin should be tried. If this still does not work, we recommend the use of HlyA. When very large pores are to be created, e.g. for introduction of antibodies into the cell…
Interferon alfa-2c in chronic myelogenous leukemia (CML): hematologic, cytogenetic and molecular-genetic response of patients with chronic phase CML previously resistant to therapy with interferon gamma.
Alpha- and gamma-interferons have been shown to actively suppress hematopoiesis in patients in the chronic phase of chronic myelogenous leukemia in vitro and in vivo. Since both interferons act through different receptors on their hematopoietic target cells, they are expected to be capable of independently inhibiting abnormal blood cell development in patients with chronic myelogenous leukemia. We have utilized recombinant human interferon alfa-2c to treate 11 patients with Philadelphia chromosome positive chronic myelogenous leukemia in chronic phase, who were resistant to previous interferon gamma therapy. Ten of the patients were evaluable for hematologic, cytogenetic and molecular-genet…
Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions
Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb+ is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca2+ or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in…
Cytocidal effects of Escherichia coli hemolysin on human T lymphocytes.
Escherichia coli hemolysin is the prototype of a large family of pore-forming toxins produced by gram-negative organisms. Besides its known cytotoxic activities against granulocytes, monocytes, endothelial cells, and renal epithelial cells, we now demonstrate that the toxin potently kills human T lymphocytes. Evidence based on different and independent approaches indicates that lymphocidal activity is due to formation of transmembrane pores. Additionally, cells prestimulated with phytohemagglutinin respond to low doses of E. coli hemolysin with DNA fragmentation similar to that observed in cells undergoing programmed cell death. Kinetic considerations lead us to conclude that DNA degradatio…
Infection of the upper extremity by Mycobacterium marinum in a 3-year-old boy--diagnosis by 16S-rDNA analysis.
A 3-year-old boy developed several subcutaneous nodular lesions on his right arm. Based on the histological examination of one of these nodules furunculosis was suspected and cefuroxime was tentatively given. However, acid-fast bacilli were then detected in the tissue specimen and a few colonies of acid fast, gram-positive rods grew on blood agar. Definitive species diagnosis (Mycobacterium marinum) was rapidly achieved by automated sequencing of amplified 16S-rDNA and antimicrobial therapy was adjusted according to the available literature. After 3 weeks of treatment with clarithromycin, rifampicin and protionamid regression of the nodular lesions was evident.
Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation.
Peripheral-blood human T lymphocytes were treated with Staphylococcus aureus alpha-toxin. Membrane permeabilization was assessed by measuring efflux of K+ and Rb+ and influx of Na+, Ca2+, and propidium iodide. Cellular ATP and [3H]thymidine incorporation following lectin stimulation were measured as parameters for cell viability. Internucleosomal cleavage characteristic of programmed cell death was assessed by agarose gel electrophoresis and by quantifying low-molecular-weight, [3H]thymidine-labeled DNA fragments. Nanomolar concentrations of alpha-toxin evoked protracted, irreversible ATP depletion in both activated and resting T lymphocytes. Toxin-damaged cells also lost their ability to i…