title: P2Z-Independent and P2Z receptor-mediated macrophage killing by Pseudomonas aeruginosa isolated from cystic fibrosis patients.
abstract: We demonstrate that a mucoid, alginate-producing strain of Pseudomonas aeruginosa isolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5'-nucleotidase, and ATP-modifying enzymatic activities. The secretion 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 presence of 5.0 mM Ca2+ or Mg2+. Interestingly, the secretion is triggered primarily 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 intracellular Ndk and demonstrates similar enzymatic properties. The N-terminal sequence 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 of 5'-nucleotidase, adenylate kinase, and a putative ATP reductase activity. These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP 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 (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane. Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death. Thus mucoid P. aeruginosa cells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation. Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.
authors.affiliation: Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
authors.forename: O
authors.initials: O
authors.lastname: Zaborina
authors.forename: N
authors.initials: N
authors.lastname: Misra
authors.forename: J
authors.initials: J
authors.lastname: Kostal
authors.forename: S
authors.initials: S
authors.lastname: Kamath
authors.forename: V
authors.initials: V
authors.lastname: Kapatral
authors.forename: M E
authors.initials: ME
authors.lastname: El-Idrissi
authors.forename: B S
authors.initials: BS
authors.lastname: Prabhakar
authors.forename: A M
authors.initials: AM
authors.lastname: Chakrabarty
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002364:Caseins / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000382:microbiology*; D006801:Humans; D008264:Macrophages / Q000473:pathology*; D051379:Mice; D008806:Mice, Inbred AKR; D008807:Mice, Inbred BALB C; D009701:Nucleoside-Diphosphate Kinase / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000378:metabolism / Q000472:pathogenicity*; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D002364:Caseins; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D012965:Sodium Chloride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D000251:Adenosine Triphosphatases