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. 2006 Apr;74(4):2207-14.
doi: 10.1128/IAI.74.4.2207-2214.2006.

Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes

Marek Basler  1 , Jiri Masin, Radim Osicka, Peter Sebo
Affiliations

Pore-forming and enzymatic activities of Bordetella pertussis adenylate cyclase toxin synergize in promoting lysis of monocytes

Marek Basler et al. Infect Immun. 2006 Apr.

Abstract

Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the alphaMbeta2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC- toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC- toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.

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Figures

FIG. 1.
FIG. 1.
SDS-PAGE analysis of the purified CyaA-derived proteins. The proteins were produced in E. coli XL1-Blue cells and purified from urea extracts of cell debris by a combination of ion-exchange and hydrophobic chromatography as described previously (34). Five micrograms of the purified proteins was separated by SDS-PAGE (7.5%) and visualized by Coomassie blue staining.
FIG. 2.
FIG. 2.
J774A.1 murine monocytes are highly susceptible to CyaA toxin activity. (A) J774A.1 cells rapidly lyse upon exposure to low concentrations of CyaA. A total of 105 J774A.1 cells were incubated with CyaA at 37°C in DMEM, and the extent of cell lysis was determined at the indicated time points as the amount of LDH released into culture media, using a Cytotox 96 assay kit (Promega). (B) Low CyaA concentrations cause massive elevation of cAMP level in cells. A total of 105 J774A.1 cells were incubated at 37°C with indicated concentrations of toxin in DMEM. The reaction was stopped after 5, 10, 20, and 30 min by addition of 0.2% Tween 20 in 50 mM HCl; the samples were boiled for 15 min at 100°C, neutralized by addition of 150 mM unbuffered imidazole; and the cAMP concentration was determined by immunoassay (35). (C) cAMP accumulation is accompanied by depletion of cellular ATP. A total of 105 J774A.1 cells were incubated with 200 ng/ml of CyaA in DMEM, and the ATP level was monitored over time in cell aliquots using the ATP Bioluminescence Assay kit CLS II (Roche). (D) CyaA activity causes loss of cell viability. A total of 105 J774A.1 cells were incubated with CyaA, and cell viability was assessed spectrophotometrically at the indicated time points as the amount of the WST-1 substrate (Roche) reduced to its tetrazolium salt by mitochondrial dehydrogenases. The given results are representative of at least two independent determinations performed in triplicate.
FIG. 3.
FIG. 3.
Enzymatic depletion of ATP and not accumulation of cAMP as such accounts for rapid lysis of J774A.1 cells by CyaA. (A) Depletion of ATP in J774A.1 monocytes is due to enzymatic activity of CyaA. A total of 105 J774A.1 cells were incubated with 200 ng/ml of CyaA or CyaA-AC− in DMEM, and the ATP concentration over the time of incubation was monitored in cell aliquots using the ATP Bioluminescence Assay kit CLS II (Roche). (B) Accumulation of cAMP as such does not promote cell lysis. A total of 105 J774A.1 cells were incubated for 3 h in DMEM and with different concentrations of intact CyaA, its enzymatically inactive CyaA-AC− toxoid alone, or with CyaA-AC− plus 10 mM db-cAMP or 3 mM 8-bromo-cAMP, respectively. The extent of cell lysis was determined by LDH release assay as above. The results are representative of two independent determinations performed in triplicate.
FIG. 4.
FIG. 4.
Enzymatic activity and not the pore-forming capacity of CyaA induces depletion of ATP and vacuolization of J774A.1 cells. (A) Monocytes were grown overnight on glass coverslips in RPMI medium; the medium was changed for DMEM 2 h prior to addition of the toxins at indicated concentrations. After 90 min of exposure to toxin in DMEM at 37°C, the J774A.1 cells were viewed at a magnification of ×100 using Nomarski differential interference contrast optics with an Olympus BX60 microscope. The experiment was repeated twice, and representative images from a series of micrographs are shown. (B) Pore-forming activity of CyaA causes only moderate ATP depletion even at LC50 doses of AC− toxoids. A total of 105 J774A.1 cells were incubated with mutant CyaA-AC− toxoids, intact acylated CyaA, or the nonacylated proCyaA at protein concentrations representing the respective LC50 (see Table 1) in DMEM. The ATP level in toxin-treated cells over time was determined in cell aliquots using the ATP Bioluminescence Assay kit CLS II (Roche). The results are representative of at least three independent determinations performed in triplicate.
FIG. 5.
FIG. 5.
CyaA promotes lysis of nonmyeloid CHO cells expressing CD11b/CD18. CHO transfectants expressing the CD11b/CD18 receptor (105 cells) were incubated with 5,000 ng/ml of CyaA variants at 37°C in DMEM for 3 h, and the extent of cell lysis was determined as the amount of LDH released into culture media using the Cytotox 96 assay kit (Promega). The results are representative of at least three independent determinations performed in triplicate.
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References

    1. Bachelet, M., M. J. Richard, D. Francois, and B. S. Polla. 2002. Mitochondrial alterations precede Bordetella pertussis-induced apoptosis. FEMS Immunol. Med. Microbiol. 32:125-131. - PubMed
    1. Barry, E. M., A. A. Weiss, I. E. Ehrmann, M. C. Gray, E. L. Hewlett, and M. S. Goodwin. 1991. Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation. J. Bacteriol. 173:720-726. - PMC - PubMed
    1. Basar, T., V. Havlicek, S. Bezouskova, M. Hackett, and P. Sebo. 2001. Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase. Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC. J. Biol. Chem. 276:348-354. - PubMed
    1. Basar, T., V. Havlicek, S. Bezouskova, P. Halada, M. Hackett, and P. Sebo. 1999. The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase toxin is crucial for toxin function independently of its acylation status. J. Biol. Chem. 274:10777-10783. - PubMed
    1. Baumann, U., S. Wu, K. M. Flaherty, and D. B. McKay. 1993. Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two-domain protein with a calcium binding parallel beta roll motif. EMBO J. 12:3357-3364. - PMC - PubMed

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