Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani. To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.
Polyamines are metabolites that play important roles in rapidly proliferating cells, and recent studies have highlighted their critical nature in Leishmania parasites. However, little is known about the function of polyamines in parasites. To address this question, we assessed the effect of polyamine depletion in Leishmania donovani mutants lacking ornithine decarboxylase (Δodc) or spermidine synthase (Δspdsyn). Intracellular putrescine levels depleted rapidly in Δodc mutants and accumulated in Δspdsyn mutants, while spermidine levels were maintained at low but stable levels in both cell lines. Putrescine depletion in the Δodc mutants led to cell rounding, immediate cessation of proliferation, and loss of viability, while putrescine-rich Δspdsyn mutants displayed an intermediate proliferation phenotype and were able to arrest in a quiescent-like state for 6 weeks. Supplementation of Δodc mutants with spermidine had little effect on cell proliferation and morphology but enabled parasites to persist for 14 weeks. Thus, putrescine is not only essential as precursor for spermidine formation but also critical for parasite proliferation, morphology, and viability.
Liposomal azithromycin (L-AZM) promotes macrophage polarization toward an M2-like phenotype in the context of myocardial infarction that results in improved cardiovascular outcomes in mice. To improve upon this formulation, we sought to identify optimized formulation, stability, and biological activity parameters necessary to enhance the immunomodulatory activity and efficacy of L-AZM. While our parent formulation contains a mixture of long-chain saturated phosphatidylcholine and phosphatidylglycerol lipids, we evaluated a series of formulations with different amounts of unsaturated lipids and cholesterol with the goal of improving the loading capacity and stability of the formulations. We also introduce fusogenic lipids to improve the cytosolic delivery to enhance the immune modulatory properties of the drug. To achieve these goals, we initially prepared a library of 24 formulations using thin film hydration and assessed the resultant liposomes for size and polydispersity. Five lead formulations were identified based on low polydispersity (<0.3) and stability over time. The lead formulations were then evaluated for stability in serum using dialysis and macrophage polarization activity in vitro as measured by decreased IL-12 expression. Collectively, our data indicate that the formulation components drive the balance between encapsulation efficiency and stability and that all the lead liposomal formulations improve in vitro alternative macrophage activation as compared to free AZM.
Leishmaniasis, a neglected tropical disease, affects an estimated billion people worldwide. A better understanding of parasite biology is vital for the development of much needed new therapeutic strategies. Our research addresses this critical need by focusing on the role of polyamines for growth and survival of Leishmania parasites. The main polyamine biosynthetic enzymes in Leishmania are ornithine decarboxylase (ODC) and spermidine synthase (SPDSYN), which sequentially convert ornithine to the polyamines, putrescine and spermidine. With generated gene deletion mutants, Δodc and Δspdsyn, we have demonstrated in vitro that the metabolite putrescine is essential beyond its function as the precursor for spermidine formation. Additionally, we observed that putrescine‐depleted Δodc parasites immediately ceased proliferation and died within two weeks. In contrast, putrescine‐rich Δspdsyn mutants showed an intermediate growth phenotype and entered a quiescent‐like state with cell death occurring after six weeks. Furthermore, putrescine‐depleted cells were unable to synthesize DNA and stopped replicating. We also observed two hallmarks of programmed cell death in the putrescine‐depleted cells: cell‐rounding and phosphatidylserine exposure. To reduce the variability associated with working with two cell lines and to further understand the functions polyamines play, we are in the process of generating a double gene deletion mutant, ΔodcΔspdsyn. Support or Funding Information Pacific University School of Pharmacy Research Incentive Grant
A better understanding of parasite biology and host‐parasite interactions is critical for the development of much needed new therapeutic strategies against the neglected tropical disease leishmaniasis, which affects an estimated 12 million people annually. Polyamines are metabolites that play central roles in the biology of all eukaryotes, and recent studies have highlighted their critical nature in Leishmania parasites. In Leishmania, the polyamine biosynthetic pathway consists of four enzymes: arginase (ARG), ornithine decarboxylase (ODC), spermidine synthase (SPD), and S‐adenosylmethionine decarboxylase (ADOMETDC). These enzymes sequentially generate ornithine, putrescine and spermidine. We have generated a complete set of gene deletion mutants in Leishmania donovani (Δarg, Δodc, LdΔspd, and Δadometdc), in order to evaluate the polyamine biosynthetic enzymes as therapeutic targets and to study the functions of polyamines. In vivo studies revealed surprising differences in infectivity phenotypes, with the Δodc deletion exhibiting the most profound reduction in infectivity. In vitro studies showed that the gene deletion mutants depend on the provision of downstream metabolites to survive. An evaluation of cell growth between the different cell lines uncovered that the only vital role of ornithine is as precursor for polyamine synthesis. However, putrescine, which has previously been postulated to be merely a substrate for spermidine formation, has additional essential functions. Our studies revealed that putrescine is especially important for replication and proliferation. Both putrescine and spermidine were essential for cell survival, but the provision of either polyamine alone allowed parasites to enter a quiescent‐like state for several weeks. Future studies utilizing the gene deletion mutants will continue to elucidate the functions of polyamines for cellular proliferation, survival, and persistence.Support or Funding InformationNational Institute of Allergy and Infectious Disease Grant AI041622This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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