Nucleoside diphosphate kinase (NDK), responsible for the maintenance of NTP pools, is an ATP-utilizing enzyme secreted by different pathogens. We found that NDK from Salmonella enterica serovar Typhimurium (S. Typhimurium) is also secretory in nature. Secretory NDK is known to play a crucial role in the survival of pathogenic microbes within host cells through their interaction with extracellular ATP. To elucidate this aspect, we assessed the contribution of secretory products containing NDK from intracellular (Mycobacterium tuberculosis and S. Typhimurium) and extracellular (Vibrio cholerae) pathogens to the process of ATP-induced J774 mouse macrophage cell lysis by monitoring lactate dehydrogenase (LDH) release in the culture medium. Compared with an untreated control, our results demonstrate that S. Typhimurium secretory products caused a greater than twofold decrease in LDH release from J774 macrophage cells treated with ATP. Furthermore, the secretory products from an ndk-deleted strain of S. Typhimurium did not display such behaviour. Contrary to this observation, the secretory products containing NDK of V. cholerae were found to be cytotoxic to J774 cells. At the amino acid level, the sequences of both the NDKs (S. Typhimurium and V. cholerae) exhibited 65 % identity, and their biochemical characteristics (autophosphorylation and phosphotransfer activities) were indistinguishable. However, to our surprise, the secretory product of an ndk-deleted strain of S. Typhimurium, when complemented with V. cholerae ndk, was able to prevent ATP-induced cytolysis. Taken together, our results unambiguously imply that the intrinsic properties of secretory NDKs are identical in intra- and extracellular pathogens, irrespective of their mode of manifestation.
Berrêdo-PinhoM.,
Peres-SampaioC. E.,
ChrispimP. P.,
Belmont-FirpoR.,
LemosA. P.,
MartinyA.,
Vannier-SantosM. A.,
Meyer-FernandesJ. R.(2001). A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Arch Biochem Biophys 391:16–24 [View Article][PubMed]
BiggsJ. P. S.,
HerspergerE.,
SteegP. S.,
LiottaL. A.,
ShearnA.(1990). A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase. Cell 63:933–940 [View Article][PubMed]
BlanchardD. K.,
WeiS.,
DuanC.,
PericleF.,
DiazJ. I.,
DjeuJ. Y.(1995). Role of extracellular adenosine triphosphate in the cytotoxic T-lymphocyte-mediated lysis of antigen presenting cells. Blood 85:3173–3182[PubMed]
BradfordM. M.(1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
ChiozziP.,
MurgiaM.,
FalzoniS.,
FerrariD.,
Di VirgilioF.(1996). Role of the purinergic P2Z receptor in spontaneous cell death in J774 macrophage cultures. Biochem Biophys Res Commun 218:176–181 [View Article][PubMed]
Coutinho-SilvaR.,
PerfettiniJ. L.,
PersechiniP. M.,
Dautry-VarsatA.,
OjciusD. M.(2001). Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci. Am J Physiol Cell Physiol 280:C81–C89[PubMed]
Di VirgilioF.(1995). The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death. Immunol Today 16:524–528 [View Article][PubMed]
DubyakG. R.,
el-MoatassimC.(1993). Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 265:C577–C606[PubMed]
FerrariD.,
ChiozziP.,
FalzoniS.,
Dal SusinoM.,
MelchiorriL.,
BaricordiO. R.,
Di VirgilioF.(1997). Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages. J Immunol 159:1451–1458[PubMed]
HeidelbergJ. F.,
EisenJ. A.,
NelsonW. C.,
ClaytonR. A.,
GwinnM. L.,
DodsonR. J.,
HaftD. H.,
HickeyE. K.,
PetersonJ. D. et al.& other authors (2000). DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477–483 [View Article][PubMed]
LammasD. A.,
StoberC.,
HarveyC. J.,
KendrickN.,
PanchalingamS.,
KumararatneD. S.(1997). ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors. Immunity 7:433–444 [View Article][PubMed]
MillerV. L.,
MekalanosJ. J.(1988). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575–2583[PubMed]
MolloyA.,
LaochumroonvorapongP.,
KaplanG.(1994). Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guérin. J Exp Med 180:1499–1509 [View Article][PubMed]
MukherjeeK.,
SiddiqiS. A.,
HashimS.,
RajeM.,
BasuS. K.,
MukhopadhyayA.(2000). Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome. J Cell Biol 148:741–754 [View Article][PubMed]
ProstL. R.,
SanowarS.,
MillerS. I.(2007).Salmonella sensing of anti-microbial mechanisms to promote survival within macrophages. Immunol Rev 219:55–65 [View Article][PubMed]
RaychaudhuriS.,
JainV.,
DongreM.(2006). Identification of a constitutively active variant of LuxO that affects production of HA/protease and biofilm development in a non-O1, non-O139 Vibrio cholerae O110. Gene 369:126–133 [View Article][PubMed]
SteegP. S.,
PalmieriD.,
OuatasT.,
SalernoM.(2003). Histidine kinases and histidine phosphorylated proteins in mammalian cell biology, signal transduction and cancer. Cancer Lett 190:1–12 [View Article][PubMed]
ThompsonJ. D.,
GibsonT. J.,
PlewniakF.,
JeanmouginF.,
HigginsD. G.(1997). The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 [View Article][PubMed]
ZambonA.,
BronteV.,
Di VirgilioF.,
HanauS.,
SteinbergT. H.,
CollavoD.,
ZanovelloP.(1994). Role of extracellular ATP in cell-mediated cytotoxicity: a study with ATP-sensitive and ATP-resistant macrophages. Cell Immunol 156:458–467 [View Article][PubMed]