Different sensitivity of Pseudomonas aeruginosa exotoxin A and diphtheria toxin to enzymes from polymorphonuclear leukocytes

Döring, Gerd; Müller, Evelyn

doi:10.1016/0882-4010(89)90102-2

Cited by 9 publications

(3 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These toxins are important agents of human disease and are potential agents of bioterrorism (Balint, 1974;Boulton, 2003;Bradberry et al, 2003;Clarke, 2002;Dittmann et al, 2000;Franz & Zajtchuk, 2000;Lyczak et al, 2000;Madsen, 2001;O'Brien et al, 1992;Olsnes, 2004;Pickering et al, 1994;Tarr, 1995). Consequently, there is considerable interest in understanding how these toxins interact with host cells, with the ultimate goal of developing inhibitors of their action (Doring & Muller, 1989;Laithwaite et al, 1999;Ohmi et al, 1998;Sekino et al, 2004;Valdivieso-Garcia et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis

Zhao

Haslam

2005

Journal of Medical Microbiology

View full text Add to dashboard Cite

Inhibition of protein synthesis is a common mechanism by which bacterial and plant toxins injure human cells. Examples of toxins that inhibit protein synthesis include shiga toxins of Escherichia coli, diphtheria toxin, Pseudomonas exotoxin A and the plant toxin ricin. In order to facilitate studies on toxin pathogenesis and to enable screening for inhibitors of toxin action, a quantitative and highly sensitive assay for the action of these toxins on mammalian cells was developed. The cDNA encoding destabilized luciferase was cloned into an adenoviral expression plasmid and a high-titre viral stock was prepared. Following transduction of Vero cells, luciferase expression was found to be linear with respect to viral multiplicity of infection. Luciferase expression by as few as 10 cells was readily detected. Treatment of transduced cells with either cycloheximide or shiga toxin resulted in a decrease in luciferase activity, with a half-life ranging from 1 to 2 h. Inhibition of luciferase expression was evident at toxin concentrations as low as 1 pg ml À1 . The assay was adapted for use in 24-, 96-and 384-well plates, enabling rapid processing of large numbers of samples. Using this approach, susceptibility of Vero, Hep2, Chang, A549, COS-1 and HeLa cells to three different toxins was determined. These results demonstrate that the luciferase-based assay is applicable to the study of numerous cell types, is quantitative, highly sensitive and reproducible. These features will facilitate studies on pathophysiology of toxin-mediated diseases and allow high-throughput screening for inhibitors of cytotoxicity.

show abstract

Section: Introductionmentioning

confidence: 99%

A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis

Zhao

Haslam

2005

Journal of Medical Microbiology

View full text Add to dashboard Cite

show abstract

“…A total of 1×10 5 Chinese Hamster Ovary (CHO) cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) ACC 110) or human lung carcinoma A-549 cells (DSM ACC 107) in Dulbecco's minimal essential medium (DMEM) (Gibco, Eggenstein, Germany), supplemented with 10% foetal calf serum, 1% sodium py-ruvate, 1% nonessential amino acids, and 50 µg·mL -1 gentamycin, were seeded for 48 h at 37°C and 5% CO 2 into 96 well flat bottom microtitre plates (Becton Dickinson, Lincoln Park, NJ, USA), washed and then incubated with buffer alone or in various test conditions for 5 h [20]. Cytotoxicity was assessed with 0.025% trypan blue.…”

Section: Cytotoxicity Assaysmentioning

confidence: 99%

Catalase, myeloperoxidase and hydrogen peroxide in cystic fibrosis

Worlitzsch

Herberth²,

Ulrich³

et al. 1998

Eur Respir J

View full text Add to dashboard Cite

aaIn cystic fibrosis (CF), the most common autosomal recessive disorder of white populations, abnormal exocrine gland secretions lead to persisting endobronchial bacterial infections [1]. Consequently, large numbers of polymorphonuclear leukocytes (PMN) are recruited to the infected airways where PMN activation and lysosomal enzyme release occur [2]. In particular PMN-elastase (PMN-Ela) has been implicated in chronic lung destruction [2], the major cause of morbidity and mortality in CF, and, consequently, trials of aerosol therapy with a 1 -proteinase inhibitor (α 1 -PI, synonymous: α 1 -antitrypsin) have been initiated [3].It is not yet certain whether toxic oxygen metabolites produced by stimulated PMN also contribute to lung injury in CF, as has been suggested in other airway diseases [4][5][6][7]. However, indirect evidence may support this hypothesis; high sputum concentrations of extracellular myeloperoxidase (MPO), a PMN-derived enzyme which transforms hydrogen peroxide (H 2 O 2 ) into highly reactive oxygen metabolites, have been detected in CF patients [8][9][10][11], and lung function has been inversely correlated with MPO levels [9][10][11]. In addition, increased lipid peroxidation [12], reduced free-radical-trapping capacity [13] and altered plasma antioxidant status [12][13][14] have been reported to occur in CF patients.In this study, we have attempted to test this hypothesis by measuring H 2 O 2 concentrations in breath condensates of CF patients, to determine in CF sputum samples activities and concentrations of MPO and catalase (CAT), an enzyme that detoxifies H 2 O 2 to oxygen and water, and, also, to investigate a possible cytotoxic effect of CF sputum on cells and on α 1 -PI in vitro. Materials and methods PatientsA total of 63 CF patients from six German CF clinics were studied. Patients (mean±SD (range) age 24±8 (8-37) yrs, males n=30; females n=33) had significantly increased sweat electrolyte levels [15], and were chronically infected by Pseudomonas aeruginosa (n=30) or other pathogens (n=33). All patients were receiving pancreatic enzyme replacement therapy, but no anti-inflammatory medication. All hospitalized patients (n=23) were being treated with parenteral antibiotics against P. aeruginosa. Of the hospitalized patients 10 suffered from acute exacerbations. Mean±SD maximal vital capacity (VCmax) (75±19%) and forced expiratory volume in one second (FEV1) (69±25%) in the CF patients ranked below normal age-predicted values. A total of 54% of the patients had moderately reduced lung function and 46% had severely reduced lung function. Controls included 51 healthy, nonsmoking subjects (27±8 (12-61) yrs) and 23 patients with bronchial asthma (36±16 (12-61) In conclusion, catalase, sulphated glycoconjugates and deoxyribonucleic acid may prevent myeolperoxidase-mediated oxygen radical generation in cystic fibrosis sputum.

show abstract

“…In comparison to diphtheria toxin which reveals an identical enzymatic mechanism. exotoxin A is significantly more sensitive to lysosomal-derived PMN-enzymes ( 13). The different susceptibility to proteolytic cleavage may explain the diiTerent courses of clinical diphtheria where diphtheria toxin is responsible for many clinical features.…”

Section: P Aeruginosa Virulence In Chronic Infectionmentioning

confidence: 99%

Host Response to Pseudomonas aeruginosa

Döring

1989

Acta Paediatrica

Self Cite

View full text Add to dashboard Cite

Patients with cystic fibrosis (CF) do not reveal a primary immune defect and respond with high numbers of functional polymorphonuclear leukocytes (PMN) and specific antibodies to lung infection with Pseudomonas aeruginosa. The mucoid character of P. aeruginosa, an altered epithelial cell surface, and high concentrations of PMN‐derived lysosomal enzymes contribute to impaired bacterial lung clearance and result in chronic infection. Released PMN‐elastase inactivates exotoxin A, the major toxin of P. aeruginosa, thus reducing its virulence. The imbalance between PMN‐proteinases and their inhibitors leads to lung tissue damage, impaired opsonophagocytosis, and T‐cell and B‐cell imbalance. New therapeutical concepts in CF therefore combine anti‐inflammatory drugs with effective antibiotics.

show abstract

Different sensitivity of Pseudomonas aeruginosa exotoxin A and diphtheria toxin to enzymes from polymorphonuclear leukocytes

Cited by 9 publications

References 23 publications

A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis

A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis

Catalase, myeloperoxidase and hydrogen peroxide in cystic fibrosis

Host Response to Pseudomonas aeruginosa

Contact Info

Product

Resources

About