Life Sciences & Biotechnology
Title : | Deciphering the role of innate immune genes in non-immune cells during intracellular pathogen mediated infection: Using Leishmania as a model |
Area of research : | Life Sciences & Biotechnology |
Principal Investigator : | Dr. Debanjan Mukhopadhyay, Presidency University, West Bengal |
Timeline Start Year : | 2022 |
Timeline End Year : | 2024 |
Contact info : | debmicro543@gmail.com |
Details
Executive Summary : | Intracellular protozoan Leishmania preferentially replicates within parasitophorous vacuoles (PV) inside the macrophages despite the hostile intra-macrophage environment. Although Leishmania infection occurs through the skin where the epidermal keratinocytes and dermal fibroblasts do get infection, but the parasite cannot grow efficiently in these cell types as compared to macrophages. The molecular basis for this cellular tropism of Leishmania is not known. Furthermore, different species of Leishmania causes different clinical diseases where some are limited to skin (Cutaneous leishmaniasis, CL) with pro-inflammatory immune response while others cause systemic infection with immune suppression (Visceral leishmaniasis, VL). Information regarding the ability of different Leishmania species to infect and multiply into these non-immune cells are also limited. This study will work on the hypothesis that non-immune cells like keratinocytes and fibroblasts have imprinted cell-intrinsic immunity that interferes with the growth of the Leishmania and hence does not support its growth. Our preliminary RNA-Seq data analysis from public databases suggested that guanylate binding proteins (GBPs) are expressed more in these non-immune cells than human monocytes. GBPs are GTPases that are known to be induced by interferons (IFNs) and play important roles in many bacterial and protozoal infections. Therefore, this study aims to investigate that whether the GBPs play a key role on cellular tropism of Leishmania species. Human dermal fibroblasts and keratinocytes will be infected along with differentiated THP1 human macrophages with different strains of Leishmania causing different spectrum of diseases to compare the differential infectivity and parasite growth. RNA-Seq analysis will be performed from Leishmania infected cells to find out the key differences in the immune gene expression in these cell types. The expression of different members of GBPs (GBP 1-7) upon different strains of Leishmania infection in these cell types will be evaluated by quantitative rt-pcr (qPCR) and western blotting. Localization of the GBPs will be checked by immunofluorescence. We will investigate whether GBP induced autophagy limits the parasite growth in these cells. Role of the GBPs on Leishmania growth will be confirmed by making targeted GBP knock out cells using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 based approach. This study will address the persisting knowledge gap for the basis of the selective macrophage-tropism of the Leishmania parasites. The insights gained from this study will be helpful to the community of intracellular pathogen researchers as other intracellular pathogens of bacterial or protozoal origin also show selective cellular tropism. Furthermore, this study will also shed the light on importance of cell-intrinsic immunity of non-immune cells which could be studied for determination of genetic susceptibility of skin infection. |
Total Budget (INR): | 28,16,000 |
Organizations involved