Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model. In the present study, we characterized the activity of a second eukaryotic-type NTPDase, Lpg0971, from L. pneumophila. We observed that recombinant Lpg0971 hydrolysed only ATP and exhibited divalent cation preference for manganese (II) ions. Similar to lpg1905, an lpg0971 mutant carrying the plasmid pMIP was attenuated in a mouse lung infection model and impaired for replication in human macrophages and amoebae. Increased trafficking of the LCV (Legionella-containing vacuole) to a LAMP-1 (lysosome-associated membrane protein-1)-positive compartment was observed for both the lpg1905 and lpg0971 mutants carrying pMIP. Complementation with either lpg1905 or lpg0971 restored intracellular replication, suggesting that a minimum level of ATPase activity was required for this function. A double lpg1905/0971 mutant was not more impaired for intracellular replication than the single mutants and complementation of the double mutant with lpg0971, but not lpg1905, restored intracellular replication. This suggested that although the NTPDases have overlapping activities they have distinct functions. Unlike many eukaryotic-type proteins from L. pneumophila, neither Lpg1905 nor Lpg0971 were translocated into the host cell by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IV secretion system. Overall our data suggest that the ability of L. pneumophila to replicate in eukaryotic cells relies in part on the ability of the pathogen to hydrolyse ATP within an intracellular compartment.
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Research Article|
August 07 2014
Multiple ecto-nucleoside triphosphate diphosphohydrolases facilitate intracellular replication of Legionella pneumophila
Patrice Riedmaier;
Patrice Riedmaier
*Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia
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Fiona M. Sansom;
Fiona M. Sansom
*Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia
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Trifina Sofian;
Trifina Sofian
†The Protein Crystallography Unit, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia
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Travis Beddoe;
Travis Beddoe
†The Protein Crystallography Unit, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia
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Ralf Schuelein;
Ralf Schuelein
*Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia
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Hayley J. Newton;
Hayley J. Newton
*Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia
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Elizabeth L. Hartland
Elizabeth L. Hartland
1
*Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia
1To whom correspondence should be addressed (email hartland@unimelb.edu.au).
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Publisher: Portland Press Ltd
Received:
July 15 2013
Revision Received:
June 09 2014
Accepted:
June 24 2014
Accepted Manuscript online:
June 24 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 462 (2): 279–289.
Article history
Received:
July 15 2013
Revision Received:
June 09 2014
Accepted:
June 24 2014
Accepted Manuscript online:
June 24 2014
Citation
Patrice Riedmaier, Fiona M. Sansom, Trifina Sofian, Travis Beddoe, Ralf Schuelein, Hayley J. Newton, Elizabeth L. Hartland; Multiple ecto-nucleoside triphosphate diphosphohydrolases facilitate intracellular replication of Legionella pneumophila. Biochem J 1 September 2014; 462 (2): 279–289. doi: https://doi.org/10.1042/BJ20130923
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