BordeteUla pertussis extract that contained adenylate cyclase toxin produced large increases in ... Substances produced by Bordetella pertussis have pro-.
INFECTION AND IMMUNITY, Apr. 1988, p. 751-755 0019-9567/88/040751-05$02.00/0
Vol. 56, No. 4
Copyright C 1988, American Society for Microbiology
Effects of Adenylate Cyclase Toxin from Bordetella pertussis on Human Neutrophil Interactions with Coccidioides immitis and Staphylococcus aureus JOHN N. GALGIANI,1* ERIK L. HEWLETT,2 AND RICHARD L. FRIEDMAN3 Department of Internal Medicine, University of Arizona College of Medicine, Tucson, Arizona 85724, and Medical and Research Services, Veterans Administration Medical Center, Tucson, Arizona 857231; Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 229082; and Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona 857243 Received 27 July 1987/Accepted 21 December 1987
BordeteUla pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophilmediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetela mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial Nacetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis.
Substances produced by Bordetella pertussis have profound physiologic effects on a variety of mammalian cell types, including human polymorphonuclear leukocytes (PMNs) (1, 3, 6, 12, 16-19, 22-26, 28, 31). One of these components, the Bordetella adenylate cyclase toxin (BACT), traverses the mammalian cell membrane, is activated by calmodulin, and catalyzes the production of extraordinarily high concentrations of intracellular cyclic AMP (cAMP) (6, 17-19, 28). Associated with these events, PMNs lose their ability to kill Staphylococcus aureus and do not produce an oxidative burst as normally occurs on contact with bacteria or fungi (6, 9, 10, 21). This association has led to the hypothesis that BACT may, under certain circumstances, be entirely responsible for the PMN defects. Furthermore, under other conditions, BACT may act in concert with additional bacterial products such as pertussis toxin (25), which is also an inhibitor of PMN activities by a different mechanism. It is presumed that the cAMP accumulation elicited by adenylate cyclase toxin is responsible for the inhibition of cell function in that increased levels of cAMP have been demonstrated previously to have such an effect on PMNs (4). Previous work has indicated that human PMNs are able to inhibit the growth of arthroconidia of Coccidioides immitis (2, 14, 15) and that such inhibition can be mimicked by incubation with 2.0 mM hydrogen peroxide (13). Additional studies demonstrated that PMNs from a patient with chronic granulomatous disease failed to inhibit arthroconidia, contrary to results with PMNs from normal donors. Furthermore, BACT-containing extracts reversed the PMN inhibition of arthroconidial incorporation of N-acetylglucosamine (GlcNAc) (15). These observations suggested that the effects of PMNs on arthroconidia may be due to oxidative reactions and metabolites produced by normal PMNs. We believed it *
important to define this possible relationship in more detail by using BACT as a probe of PMN function. In so doing, we also determined differences in sensitivities among various PMN functions to the effects of BACT. MATERIALS AND METHODS Human PMNs. Peripheral venous blood was collected in syringes from healthy consenting adult volunteers and transferred to plastic tubes containing preservative-free heparin (10 U/ml, final concentration). PMNs were isolated by the single-step Ficoll-Hypaque centrifugation procedure of Ferrante and Thong (11). After recovery, PMNs were suspended in tissue culture medium 199 (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 0.1% bovine serum albumin, and cell concentrations were adjusted on the basis of hemacytometer counts. Differential counts of Giemsastained smears of these preparations demonstrated that >95% of the leukocytes were PMNs and that >95% of the PMN excluded trypan blue. PMNs were exposed to additives or to equal volumes of phosphate-buffered saline for 20 min before use in assays. Microorganisms. Strain Silveira of C. immitis was the gift of Hillel B. Levine (University of California, Berkeley). Arthroconidia were harvested from glucose-yeast extractagar plates after 1 to 2 months of growth by the method of Sun and Huppert (29). Strict containment procedures were employed for all work with coccidioidal isolates. The strains of Candida glabrata and Staphylococcus aureus used in these studies were isolated by the clinical laboratory. BACT and pertussis toxin. A 24-h culture of B. pertussis BP338 in Stainer-Scholte synthetic medium was harvested by centrifugation, and the organisms were extracted with 4 M urea. The supernatant extract was collected and dialyzed against Dulbecco phosphate-buffered saline supplemented with 1.0 mM calcium. The protein concentration of the dialysate was 1.3 mg/ml. Another preparation of adenylate
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cyclase which was recovered from calmodulin-affinity chromatography of urea extracts from BP338 and possessed lower specific activity than the urea extract (12) was used for comparative studies. In selected studies, urea extracts of transposon TnS insertion mutants of B. pertussis BP338 (32, 33) were used to evaluate the contributions of the different components. BP347 is an avirulent mutant which produces none of the virulence factors, whereas BP348 is deficient in production of BACT and hemolysin but possesses wild-type levels of pertussis toxin and other components. Extracts were prepared from these mutants as described above; protein concentrations for extracts of BP347 and BP348 were 0.41 and 1.1 mglml, respectively. For these studies, concentrations of the mutants were selected based upon equivalent volumes of extract from comparable growths of the wild-type strain. Thus, 20% (vol/vol) BACT, BP347, and BP348 were equivalent to 260, 94, and 220 ,ug/ml, respectively. Pertussis toxin was purified from culture medium of strain 165 as described previously (7) and used at concentrations in excess of that present in BP338 extract. Measurement of cAMP. After exposure to different concentrations of BACT or extracts produced by TnS mutants, 0.7-ml mixtures of 2 x 106 PMNs were centrifuged, the supernatants were discarded, and the pellets were suspended in 1.0 ml of 0.1 N HCI. After 45 min at 37°C, mixtures were centrifuged again, and the supernatants were removed for storage at -70°C until assayed in duplicate by automated radioimmunoassay (5). PMN assays. Inhibition of GlcNAc incorporation by arthroconidia was measured as previously described (15). Briefly, fungal suspensions were mixed with PMNs in final volumes of 0.9 ml in plastic cone-tipped vials (Helena Plastics, San Raphael, Calif.). These vials were then incubated at 37°C for 45 min without agitation. Subsequently, 0.1 ,uCi of ['4C]GlcNAc (New England Nuclear Corp., Boston, Mass.) was added to each vial with sufficient unlabeled GlcNAc to result in a final concentration of 0.5 x 10-6 M. In preliminary experiments, this concentration had produced greater than half-maximal GlcNAc incorporation into arthroconidia. After 20 min of pulse-labeling, mixtures were precipitated by the addition of 0.1 ml of trichloracetic acid; and after wash procedures, precipitates were dissolved in Aquasol (New England Nuclear) for liquid scintillation counting. PMNs mixed with ethanol-killed arthroconidia or preparations in which pulse-labeling was stopped immediately after the isotope was added (background activity) had counts per minute that were
