Burkholderia cepacia produces a hemolysin that is capable of inducing apoptosis and degranulation of mammalian phagocytes

Abstract

Burkholderia cepacia is an opportunistic pathogen that has become a major threat to individuals with cystic fibrosis (CF). In approximately 20% of patients, pulmonary colonization with B. cepacia leads to cepacia syndrome, a fatal fulminating pneumonia sometimes associated with septicemia. It has been reported that culture filtrates of clinically derived strains of B. cepacia are hemolytic. In this study, we have characterized a factor which contributes to this hemolytic activity and is secreted from B. cepacia J2315, a representative of the virulent and highly transmissible strain belonging to the recently described genomovar III grouping. Biochemical data from the described purification method for this hemolysin allows us to hypothesize that the toxin is a lipopeptide. As demonstrated for other lipopeptide toxins, the hemolysin from B. cepacia was surface active and lowered the surface tension of high-pressure liquid chromatography-grade water from 72.96 to 29.8 mN m(-1). Similar to reports for other pore-forming cytotoxins, low concentrations of the hemolysin were able to induce nucleosomal degradation consistent with apoptosis in human neutrophils and the mouse-derived macrophage-type cell line J774.2. Exposure of human neutrophils to higher concentrations of toxin resulted in increased activities of the neutrophil degranulation markers cathepsin G and elastase. Based on the results obtained in this study, we suggest a role that allows B. cepacia to thwart the immune response and a model of the events that may contribute to the severe inflammatory response in the lungs of CF patients.

FIG. 1

(a) Reverse-phase HPLC trace showing peak separation of hemolysin-containing culture filtrates of B. cepacia J2315. Peaks with hemolytic activity are labelled A and B. Partial purification prior to HPLC was as described in Materials and Methods. (b) Peak B was used for all subsequent experiments and reanalysed by HPLC to test purity. The concentration of solvent B (acetonitrile in 0.1% [vol/vol] trifluoroacetic acid) is shown by the broken line and the right-hand y axis. Detection was carried out at 210 nm with a flow rate of 1.3 ml min⁻¹.

FIG. 2

Quantification of the surface-active properties of the hemolysin. The surface tension of solutions of HPLC-purified toxin was determined by the drop-weight method originally described by Harkins and Brown (12), as described in Materials and Methods. All points are the result of at least 10 separate replicates. Error bars are ± the standard deviation of at least 10 replicates.

FIG. 3

Effect of B. cepacia hemolysin on fresh human erythrocytes. (A) Control cells washed in PBS. (B) Cells exposed to 5 μg of hemolysin. Both samples were incubated for 1 h at 37°C. The horizontal white bars in the lower half of the plates are 10⁻⁶ m each.

FIG. 4

Effect of HPLC-purified B. cepacia hemolysin on erythrocytes. Suspensions of cells were mixed with aliquots of toxin, and the rates of lysis were calculated by measuring the time-dependent decrease in OD₆₀₀. Each data point was the result of at least six individual replicate determinations performed as two separate trials.

FIG. 5

Hemolysin-induced nucleosomal degradation. Freshly isolated neutrophils (5 × 10⁶) were exposed to 1 μg of HPLC-pure toxin (lane B); control neutrophils were not exposed to the toxin (lane C). Molecular size standards (lane A) were generated by digestion of λ phage by HindIII and are 23.13, 9.41, 6.56, 4.36, 2.32, 2.03, and 0.56 kbp. DNA was resolved with a 1.2% (wt/vol) agarose gel.

FIG. 6

Effect of B. cepacia hemolysin on the activity of neutrophil degranulation marker enzymes. (A) Activity of granulocyte elastase. (B) Activity of cathepsin G. Each data set is obtained with samples from individual donors; each data point is the mean of at least three replicate determinations. Error bars are ±SE.