Deletion of Vitamin C biosynthesis enzyme, Arabino-1, 4-lactone oxidase in Leishmania donovani results in increased pro-inflammatory responses from host immune cells.
Recently, we reported molecular characterization, localization and functional importance of Arabino-1, 4-lactone oxidase (ALO) enzyme from Leishmania donovani that catalyses the last step in ascorbate biosynthesis pathway. Vitamin C (l-ascorbic acid) is implicated in several crucial physiological processes. To elucidate the biological role of d-arabinono-γ-lactone oxidase in Leishmania, we made L. donovani ALO null mutant (ΔALO) by double targeted gene replacement. This mutant lacked ALO activity, showed transient growth defect and reduced ascorbate levels. ΔALO grown in ascorbate depleted media further enhanced growth defect with no detectable levels of ascorbate, implying that parasites have the ability to scavenge ascorbate. ΔALO mutants showed reduced survival in mouse macrophages and are impaired in their infectivity in vivo. Furthermore, the ΔALO mutant induced production of pro-inflammatory cytokines gamma interferon (IFN-gamma), interleukin-12 (IL-12) and tumour necrosis factor-α (TNF-α) by infected mouse macrophages. These mutants were susceptible to oxidative stresses in vitro as revealed by the decreased survival inside macrophages by increased production of reactive oxygen or nitrogen species. Complementation of the ΔALO mutants restored the phenotypic effects in these parasites. Our description of ALO null mutant parasite that triggers pro-inflammatory host responses provides a novel platform for targeting ALO in anti-parasitic strategies.
School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.
Reetika
R
Manhas
Sneha
S
Anand
Pankaj
P
Tripathi
Rentala
R
Madhubala
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D016207:Cytokines / Q000378:metabolism*; D005816:Genetic Complementation Test; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D008264:Macrophages / Q000469:parasitology*; D051379:Mice; D017384:Sequence Deletion; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D013401:Sugar Alcohol Dehydrogenases; C090492:D-arabinonolactone oxidase; D001205:Ascorbic Acid