**3. Intracellular life of** *L. donovani***: the role of Wnt5A signaling therein**

Intracellular parasitism is a strategy by which parasites build a niche to sustain within the host. Parasites such as *L. donovani* have developed sophisticated strategies to counteract host defense machinery. One such strategy to adapt to a parasitic mode of life is the dimorphic life cycle in *L. donovani*. *L. donovani* resides in the gut of its vector (Phlebotomine sand flies) in a flagellated infective form (promastigote). During a blood meal of the sand fly on its mammalian host these promastigotes are transferred to the blood, where they are phagocytosed by neutrophils and macrophages. While residing in macrophages, the parasites lose their flagella and transform to amastigotes. Amastigotes divide and thrive within the host, causing disease. The parasite life cycle is repeated with blood meal of sandflies from parasite-infected patients. The mechanism of entry of *L. donovani* into macrophages has been debated for long. It has been shown that host cell receptors (for example Complement receptors and Fcγ) influence *L. donovani* internalization and this interaction is partially dependent on the presence of promastigote flagella [49]. It is also documented that host cell membrane microdomains influence internalization of the parasite [5, 7, 50] . In order to hijack the cellular defense machinery *L. donovani* interacts with components of endoplasmic reticulum and the trans-Golgi network (TGN) [7, 51]. *L. donovani* containing vacuoles take up necessary nutrients like glucose, amino acid and essential ions like Fe2+ from the trans-Golgi network (TGN). These parasite harboring vacuoles/parasitophorous vacuoles (PV) while acquiring nutrients also disrupt the transport of different proteins to their designated vacuoles (endosomes/ lysosomes) from the TGN and endoplasmic reticulum (ER), thus compromising their function [51]. The internalized parasite also delays the fusion of PV with the lysosomes through the action of lipophosphoglycan (LPG), a parasite derived molecule. Parasitophorous vacuoles accordingly become encapsulated with host F-actin, myosin and F-actin nucleating factors, thus producing a halo of F-actin surrounding the vacuole and inhibiting its lysosomal fusion [6]. The parasitophorous vacuole also expresses the early endosomal marker EEA1, and the small GTPases Rab5 and Rab 7 [52] preventing lysosomal degradation. The altered acidification of parasitophorous vacuoles is instrumental in promastigote to amastigote transformation and sustenance of infection [53]. Such remodeling of PV may lead to alteration in host lipid microdomains and alter assembly of the NADPH oxidase complex, which holds a key to microbial elimination through generation of microbiocidal Reactive Oxygen Species (ROS). The influence of Wnt5A signaling on actin cytoskeletal dynamics, organization of lipid raft microdomains and organelle polarity and assembly [30, 40, 41] suggests that host macrophages can potentially counteract the establishment and progression of *L. donovani* infection through Wnt5A signaling.

*L. donovani* infection is accompanied by increase in anti-inflammatory cytokine expression, which may help the intracellular amastigotes to build a safe niche within the macrophage. Increase in anti-inflammatory cytokines is often associated with decrease in production of ROS or Nitric oxide, which is unfavorable for amastigote growth [54–56]. Host macrophage Wnt5A signaling may be instrumental in attenuating the effect of anti-inflammatory cytokines by maintaining a proinflammatory cytokine signature [37, 40, 41].
