P2Z-independent and P2Z receptor-mediated macrophage killing by Pseudomonas aeruginosa isolated from cystic fibrosis patients

We demonstrate that a mucoid, alginate-producing strain of Pseudomonas aeru ginosa isolated from the lungs of a cystic fibrosis (CF) patient secretes m ultiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5'-nucleotidase, and ATE-modifying enzymatic activities. The secre tion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presen ce of 5.0 mM Ca2+ or Mg2+. Interestingly, the secretion is triggered primar ily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1, The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracell ular Ndk and demonstrates similar enzymatic properties, The N-terminal sequ ence of the purified ATPase isolated from an ndk knockout mutant shows its identity with that of the heat shock chaperonin Hsp60, During fractionation , the flowthrough fraction from a Mono Q column demonstrates the presence o f 5'-nucleotidase, adenylate kinase, and a putative ATP reductase activity, These fractions demonstrate high cytotoxic activities for murine peritonea l primary macrophages which can be further stimulated in the presence of AT P or inhibited by pretreatment of macrophages with oxidized ATP (oATP), The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X(7)) receptors, whi ch are one of the purinergic receptors responsible for pore formation on ma crophage membrane, Blocking of these receptors by pretreatment with oATP bl ocks ATP-induced macrophage cell death. Thus mucoid P, aeruginosa cells ela borate enzymes that modulate the external ATP levels of macrophages, thereb y modulating macrophage cell death through P2Z receptor activation, Evidenc e for the presence of secreted cytotoxic agents that act independently of P 2Z receptor activation is also presented.