Oral Presentation Lorne Infection and Immunity 2014

Surprising roles for nucleoside triphosphate diphosphohydrolases (NTPDases) in the pathogenesis of leishmaniasis. (#5)

Fiona M Sansom 1 2 , Julie E Ralton 2 , Fleur Fleur Sernee 2 , David J Hooker 2 , Elizabeth L Hartland 3 , Naderer Thomas 2 , Malcolm J McConville 2
  1. Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, The University of Melbourne, Parkville, VIC, Australia
  2. Department of Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Parkville, VIC, Australia
  3. Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC, Australia

Nucleoside triphosphate diphosphohydrolases (NTPDases) hydrolyze a broad range of nucleoside tri- and diphosphates, and in mammals are involved in various functions including inflammation and immunity. Previously, we demonstrated that secreted NTPDases are required for full virulence of the bacteria Legionella pneumophila. A number of recent studies suggest that ecto-NTPDases are involved in the pathogenesis of parasites such as Trypanosoma and Leishmania, but definitive evidence is lacking. Leishmania parasites are responsible for a number of clinical syndromes known collectively as leishmaniasis. Leishmaniasis is an important and neglected disease, and worldwide is second only to malaria as a cause of parasite-related mortality. We characterized the two putative NTPDases encoded on the genome of Leishmania major, demonstrating that L. major possesses a secreted NTPDase  (LmNTPDase2) and, somewhat unexpectedly, a Golgi-located NTPDase (LmNTPDase1). We generated single deletion mutants of both LmNTPDase1 and LmNTPDase2, indicating that neither gene is essential for growth in rich media. Surprisingly, testing of the mutants in the mouse model of cutaneous leishmaniasis revealed that LmNTPDase2 is not required for virulence, whereas deletion of LmNTPDase1 significantly delayed lesion development following infection with the promastigote stage of the parasite. However the virulence of the amastigote parasite stage was unaffected. This phenotype mirrors that seen for L. major parasites lacking Golgi nucleoside-sugar transporters, suggesting LmNTPDase1 functions in Golgi nucleoside-sugar transport (as is the case for yeast NTPDases). Interference with Golgi nucleoside-sugar transport results in altered synthesis of important glycoconjuagates, including the promastigote virulence factor lipophosphoglycan (LPG). Accordingly, we demonstrated altered synthesis of LPG in ΔLmNTPDase1 parasites, resulting in functional changes including increased susceptibility of the ΔLmNTPDase1 parasites to complement-mediated killing. This is the first time a role in Golgi nucleoside-sugar transport has been shown for a parasite NTPDase, and challenges the previous suppositions that parasite NTPDases are ecto-enzymes directly involved in pathogenesis.