sirolimus and Leishmaniasis--Cutaneous

Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 μg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica.

Topics: Amphotericin B; Animals; Antiprotozoal Agents; Cell Line, Tumor; Cytokines; Female; Humans; Inhibitory Concentration 50; Leishmania tropica; Leishmaniasis, Cutaneous; Lymph Nodes; Meglumine Antimoniate; Mice; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Parasite Load; Random Allocation; Sirolimus; TOR Serine-Threonine Kinases

Leishmania mexicana has a large family of cyclin-dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2-related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co-expression of a CRK3 transgene during rapamycin-induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3(T178E) mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.

Topics: Amino Acid Sequence; Animals; CDC2 Protein Kinase; Cell Cycle; Cell Cycle Checkpoints; Cyclin-Dependent Kinases; Cyclins; G2 Phase Cell Cycle Checkpoints; Gene Deletion; Integrases; Leishmania mexicana; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Proto-Oncogene Proteins c-crk; Reverse Genetics; Sirolimus

Autophagy is the primary mechanism of degradation of cellular proteins and at least two functions can be attributed to this biological phenomenon: increased nutrient supply via recycling of the products of autophagy under nutrient starvation; and antimicrobial response involved in the innate immune system. Many microorganisms induce host cell autophagy and it has been proposed as a pathway by which parasites compete with the host cell for limited resources. In this report we provide evidence that the intracellular parasite Leishmania amazonensis induces autophagy in macrophages. Using western blotting, the LC3II protein, a marker of autophagosomes, was detected in cell cultures with a high infection index. Macrophages infected with L. amazonensis were examined by transmission electronic microscopy, which revealed enlarged myelin-like structures typical late autophagosome and autolysosome. Other evidence indicating autophagy was Lysotracker red dye uptake by the macrophages. Autophagy also occurs in the leishmaniasis skin lesions of BALB/c mice, detected by immunohistochemistry with anti-LC3II antibody. In this study, autophagy inhibitor 3-methyladenine (3MA) reduced the infection index, while autophagy inductors, such as rapamycin or starvation, did not alter the infection index in cultivated macrophages, suggesting that one aspect of the role of autophagy could be the provision of nutritive support to the parasite.

Topics: Adenine; Amines; Animals; Autophagy; Blotting, Western; Bone Marrow Cells; Cells, Cultured; Female; Leishmania mexicana; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microtubule-Associated Proteins; Sirolimus; Staining and Labeling

The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; Cell Cycle Proteins; Cell Line; Eukaryotic Initiation Factors; Host-Parasite Interactions; Leishmania major; Leishmaniasis, Cutaneous; Macrophages; Mechanistic Target of Rapamycin Complex 1; Metalloendopeptidases; Mice; Multiprotein Complexes; Phosphoproteins; Polymerase Chain Reaction; Protein Biosynthesis; Proteins; Sequence Deletion; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases