The protozoan parasite Leishmania donovani exists as extracellular flagellates in the sandfly vector and upon transfer to its human host, the parasite resides and replicates within macrophages as aflagellate amastigotes and inflicts the disease visceral leishmaniasis (VL), which is a potentially fatal disease. Current chemotherapy for VL is extremely restricted due to the available small repertoire of drugs, drug-resistant parasites, drug-toxicity, and the requirement for parenteral administration. Therefore, there is an urgent need to expand the choices for anti-leishmanial drugs.

For new anti-leishmanials, finding new targets in the parasite is the first choice for direct effects on the pathogen. Leishmania undergoes profound biochemical rewiring during response to the host environment [1]. In Leishmania, reversible phosphorylation- operated through protein kinases and corresponding phosphatases- regulates key cellular processes and transmits signals detected following environmental changes. In particular, mitogen-activated protein kinases (MAPKs) constitute a highly conserved and ubiquitously expressed family of kinases that phosphorylate dual serine and threonine residues of their own or those on their target substrates, resulting in activation or deactivation [2]. The MAPK signaling pathways are critical in various cellular processes, including cell growth, differentiation, apoptosis, and immune response [3], [4]. The Leishmania genome database search identifies the presence of 15 MAPKs based on the (TXYXXXRXYRXPE) motif search [2]. This motif is characterized by two features: dual phosphorylation within the TXY motif and a (P + 1)-specificity pocket that is crucial in directing substrate phosphorylation at specific kinase residues [2]. Leishmania MAPKs regulate the parasite's life cycle, flagellar length, switching between forms, drug resistance, response to environmental stress, and virulence. The MAPK1 of L. donovani (LdMAPK1) influences the antimony-sensitivity by reducing the activity of P-glycoprotein efflux pumps [5]. The LdMAPK1 phosphorylates the HSP70 and HSP90 subunits modulating parasite viability [6]. LdMAPK2 phosphorylation of L. major aquaglyceroporin (LmjAQP1) and arginine transporter (AAP3) regulates antimony resistance and arginine deprivation response (ADR), respectively [7], [8]. MAPK3 and MAPK9 regulate the flagellar length, which is essential for transformation in sandfly gut and infecting mammalian host cells [9], [10]. MAPK4, MAPK7, and MAPK10 increase their activity in response to low pH and 37○C temperature, suggesting their role in the promastigote-to-amastigote transition and regulation of parasite viability and growth [11], [12]. LmjMAPK10 was found protective against infection in susceptible BALB/c mice [13]. Therefore, Leishmania MAPKs need to be studied thoroughly to understand their role at the host-parasite interface.

Herein, by employing real-time PCR, we first studied the expression of all 15 LdMAPKs in virulent and avirulent lines of AG83, miltefosine-resistant (BHU1064), and miltefosine-sensitive (BHU1066) clinical isolates. Next, considering the importance of IFN-γ and IL-4 in the development and progression of the disease[14], we studied the expression of all 15 LdMAPKs in response to IL-4 or IFN-γ in macrophages infected with virulent or avirulent AG83 parasites. Th1 and Th2 are two counteracting T-cell subsets that require IFN-γ and IL-4 for differentiation, respectively. IFN-γ suppresses Th2 and induces iNOS-mediated killing of Leishmania. Counteractively, IL-4 suppresses Th1 and macrophage activation [14]. Further, by comparing the above MAPK expression profiles, we implicated several LdMAPKs as the molecular basis of drug resistance in L. donovani, plausible new targets, and those that may modulate macrophage response to anti-leishmanial (IFN-γ) and pro-leishmanial (IL-4) cytokines.