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BackgroundProving that specific genes are essential for the intracellular viability ofLeishmaniaparasites within macrophages remains a challenge for the identification of suitable targets for drug development. This is especially evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in allLeishmaniaspecies. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention.MethodsHere, we have used plasmid shuffle methodsin vivoto provide supportive genetic evidence thatN-myristoyltransferase (NMT) is essential forLeishmaniaviability throughout the parasite life-cycle. Following confirmation of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established infection.ResultsEctopically-expressedNMTwas only dispensable under negative selection in the presence of another copy. Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additionalNMTcopy, not affected by the negative selection, was expressed.ConclusionsNMTis an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.

Ly6Chiinflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chimonocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chimonocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected withLeishmania donovani, the number of bone marrow Ly6Chimonocytes increased over time. Ly6Chimonocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2-/-mice +at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+T cells. Depletion of CD4+T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+T cells. In B6.Il10-/-mice,L. donovaniinfection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+T cell production of IFNγand resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10-/-mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that duringL. donovaniinfection, Ly6Chimonocytes are primed in the bone marrow in a process driven by CD4+T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.

Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2-/- mice + at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFN-γ produced by CD4+ T cells. In B6.Il10-/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFN-γ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10-/- mice and drug (AmBisome) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFN-γ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.