Enzymatic Properties of an Ecto-nucleoside Triphosphate Diphosphohydrolase from Legionella pneumophila

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, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri-and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was 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.Legionella pneumophilia is the major causative agent of Legionnaires disease, a severe systemic disease characterized by acute pneumonia. During infection of the human lung, L. pneumophila is internalized by alveolar macrophages where the bacteria replicate within an intracellular vacuole that evades fusion with the endocytic pathway. Instead the L. pneumophila containing vacuole intercepts early secretory vesicles and following maturation exhibits properties of the endoplasmic reticulum (1-3). Establishment of the unique Legionella vacuole and the ability to replicate within eukaryotic cells requires the Dot/Icm type IV secretion system, which translocates bacterial effector proteins into the cytoplasm of the host cell (4 -7).The ability of L. pneumophila to replicate within mammalian cells appears to be a consequence of its association in the natural environment with free living protozoa. The three sequenced L. pneumophila genomes encode an unusually high number of proteins with similarity to eukaryotic proteins (8 -10), which may interfere with host cell processes by functional mimicry (11). We recently showed that Lpg1905 from L. pneumophila is a secreted member of the CD39/ NTPDase1 family of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases; gene family ENTPD).5 NTPDases are extremely rare in bacteria, and Lpg1905 is the first prokaryotic...

Possible Effects of Microbial Ecto-Nucleoside Triphosphate Diphosphohydrolases on Host-Pathogen Interactions

“…Indeed, inactivation of lpg1905 results in defective replication of L. pneumophila within amoebae, epithelial cells, and macrophages (123). Substantial levels of enzyme activity are also required for full virulence of L. pneumophila in an A/J mouse model of Legionnaires' disease, although it is not clear if it is ATP/ADPase or GTP/ GDPase activity or both that contribute to L. pneumophila infection (124). The second putative secreted NTPDase, Lpg0971, is dispensable for replication within amoebae and macrophages (123) but contributes to virulence in the A/J mouse lung infection model, similar to Lpg1905 (F. M. Sansom et al, unpublished data).…”

“…Lpg1905 is secreted by the bacterium in vitro, although its site of action during infection is unclear. The enzyme has maximal activity at neutral pH (124), and given that the purpose of Legionella virulence determinants is to enhance bacterial replication in amoebae, the enzyme presumably has an intracellular function. Indeed, inactivation of lpg1905 results in defective replication of L. pneumophila within amoebae, epithelial cells, and macrophages (123).…”

“…Lpg1905 is a functional NTPDase, able to hydrolyze both ATP/ADP and GTP/GDP with similar efficiencies (123,124). Lpg1905 is secreted by the bacterium in vitro, although its site of action during infection is unclear.…”

Legionella pneumophilamultiplication is enhanced by chronic AMPK signalling in mitochondrially diseased Dictyostelium cells

“…Although AMPK activation provides a possible mechanism by which the Legionella apyrase Lpg1905 contributes to virulence, this ecto-NTPDase could also work by preventing purinergic P2X receptor activation within the host tissue (Sansom et al, 2008). Extracellular ATP promotes fusion of the phagosome and lysosome, and has been shown to stimulate killing of intracellular Mycobacterium spp.…”

“…Lpg1905 exhibited ATPase and ADPase activity and contributed to the ability of L. pneumophila to infect and replicate in macrophages, epithelial cells and amoebae. Lpg1905 may act as a virulence factor by regulating extracellular or intracellular levels of ATP, depending on whether secretion occurs before or after Legionella uptake by the host cell (Sansom et al, 2007;Sansom et al, 2008). An intracellular site of action seems probable if apyrase secretion accompanies that of other virulence factors that are known to be secreted within the host cell after pathogen uptake.…”