Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: Substrate specificity and requirement for virulence

Fiona Margaret Sansom, Patrice Riedmaier, Hayley Newton, Michelle Anne Dunstone, Christa E Muller, Holger Stephan, Emma Byres, Travis Clarke Beddoe, Jamie Rossjohn, Peter J Cowan, Anthony J F d'Apice, Simon C Robson, Elizabeth Louise Hartland

Research output: Contribution to journalArticleResearchpeer-review

49 Citations (Scopus)

Abstract

Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes that are characterized by the presence of five apyrase-conserved regions (ACR) and the ability to hydrolyse nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalysed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP and UDP. Based on amino acid residues conserved in the ACR regions of eukaryotic NTPDases, we generated five site directed mutants, Lpg1905E159A, R122A, N168A, Q193A and W384A. While the mutations, E159A, R122A, Q193A and W384A abrogated activity completely, N168A resulted in decreased activity due to reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A (and not the mutants E159A, R122A, Q193A and W384A) partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905E159A, R122A, Q193A, W384A or N168A mutants were able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.
Original languageEnglish
Pages (from-to)12909 - 12918
Number of pages10
JournalJournal of Biological Chemistry
Volume283
Issue number19
Publication statusPublished - 2008

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