Adenylate Kinase as a Virulence Factor ofPseudomonas aeruginosa

Abstract

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg ⅐ ATP ؉ AMP 7 Mg ⅐ ADP ؉ ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.Pseudomonas aeruginosa is a dominant pathogen in the respiratory tract of cystic fibrosis patients (35). Unlike other bacterial infections, P. aeruginosa is more difficult to control through antibiotic therapy (26). To survive in the hostile environment of the human body, this pathogen utilizes an impressive arsenal of weapons (30). Macrophages constitute the first line of defense against infections, and the ability of P. aeruginosa to kill macrophages and other phagocytic cells such as mast cells may explain this bacterium's capability to persist and disseminate in the host. It is well known that P. aeruginosa can avoid phagocytosis by encapsulating itself with an exopolysaccharide coating, called alginate, which confers on the nonmucoid cells a mucoid phenotype (11,27,31). Earlier, it was demonstrated that a mucoid, alginate-producing strain of P. aeruginosa isolated from the lungs of a cystic fibrosis patient secretes nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase (AK), 5Ј-nucleotidase, and ATP-modifying enzymatic activities that can modulate the external ATP level of macrophages and enhance their cell death through P2Z receptor activation (40). The P2Z receptor is responsible for ATPdependent cell death of macrophages through the formation of membrane pores permeable to molecules of up to 900 daltons in size (4, 37).The role of individual enzymes secreted by the mucoid strain of P. aeruginosa (40) in the killing of macrophages is, however, unknown. In this article, we report the role of a sing...

Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents

“…Suloctidil was also detected to be active against P. aeruginosa by both primary and confirmatory AK screens, but the drug did not elicit an antimicrobial response by MIC measures. Some strains of P. aeruginosa secrete AK at high cell density, and we speculate that suloctidil may trigger a similar response (33).…”

Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype VStreptococcus agalactiae

“…The failure to detect surface expression with the remaining 84 sera may reflect masking of the proteins by the cell wall and capsule or overestimation of the surface prediction programs. One of the surface-exposed proteins is adenylate kinase (SAG0079), a ubiquitous enzyme generally intracellular, secreted by both Vibrio cholerae (33) and pathogenic strains of Pseudomonas aeruginosa, where it acts as a virulence factor through its role in macrophage cell death (34).…”

Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages

“…The released enzymes include adenylate kinase, 5′-nucleotidase (and/or ATPase/phosphatase), ecto-ATPase [15], ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) [16] and NdK. The last enzyme utilizes eATP as its principal substrate [9,10,[17][18][19][20]. eATP is known to trigger the death of immune cells, including macrophages, by its binding to purinergic receptor of the P2X family, i. e. P2X 7 [12,13,21,22].…”