LACK, a RACK1 ortholog, facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness

Abstract

Summary Leishmania are kinetoplastid parasites that cause the sandfly-transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK-dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK-deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK-deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function.

Sterol methyltransferase is required for optimal mitochondrial function and virulence in Leishmania major

“…S10B), suggesting that most ROS was sequestered in the mitochondria. We next examined whether the mitochondria in smtmutants could effectively carry on respiration by monitoring oxygen consumption rate using the MitoXpress oxygen probe as described previously (Cardenas et al, 2015). As indicated in Fig.…”

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

“…S4B, lso – mutants had slightly higher fluorescence signal than the WT and lso – /+LSO parasites (the difference was not statistically significant except for log phase), suggesting a modest accumulation of ROS in their mitochondria. Next, we used the MitoXpress probe to examine oxygen consumption rate (63) by incubating log phase promastigotes in a respiration buffer containing sodium pyruvate but no glucose. Under this condition, lso – showed a slightly lower oxygen consumption rate than WT and add-back parasites although the difference was not statistically significant (Fig.…”

“…Cells were washed once with PBS and analyzed by flow cytometry (18). To determine the oxygen consumption rate, log phase promastigotes were resuspended in a respiration buffer (Hank’s Balanced Salt Solution with 5.5 mM of sodium pyruvate, 5.5 mM of 2-deoxy-D-glucose) at 2.0 × 10 7 /ml and oxygen consumption was measured with 1 μM of MitoXpress as described (63). WT parasites treated with 10 μM of antimycin A were included a negative control (63).…”

The Polyamine Pathway as a Potential Target for Leishmaniases Chemotherapy

“…These kinases along with phosphatases play a major role in protein and enzyme regulation. Leishmaniaactivated kinases play an important role in parasite thermo-tolerance and virulence [88].…”