Identification of the minimum requirements for the survival of Leishmania in host cells: RNA-seq of enucleated L929 fibroblasts infected with Leishmania amazonensis

• Grant number: 14/25296-2
• Support Opportunities: Regular Research Grants
• Start date: May 01, 2015
• End date: April 30, 2017
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Renato Arruda Mortara
• Grantee: Renato Arruda Mortara
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Clara Lúcia Barbiéri Mestriner ; Cristina Mary Orikaza Toqueiro ; Fernando Roberto Oliveira Real ; José Franco da Silveira Filho ; Marjorie Mendes Marini e Souza ; Michel Pinkus Rabinovitch

Abstract

Enucleated cells or cytoplasts (cells which nucleus is removed in vitro) are na unprecedented and unexplored biological model for the study of intracellular pathogens. Cell enucleation, performed by centrifugation in the presence of cytochalasin B before infection assays, completely interrupts the transcription of host chromossomical genes in response to intracellular pathogens. As result, important nucleus-dependent signaling pathways, such as NF-ºB response, become inoperative. Host cell enucleation does not exclude, however, the possibility that an intracellular pathogen could interfere in the fate of the available messenger RNAs or in their translation in host cytoplasts.This project aims to identify which transcripts remain after cell enucleation and are preserved during Leishmania amazonensis intracellular infection, sustaining parasite survival and multiplication. We will establish a minimal network of host cells factors sufficient for intracellular parasitism, based on mRNA preserved in infected cytoplasts. This objective will be approached by i) cytoplast infection for 48 hours, which allows for two cycles of parasite multiplication in the absence of de novo transcription; ii) sequencing of cytoplasts and parasites transcripts by RNA-seq, in order to identify which transcripts are conserved during 48 hours of intracellular infection in enucleated cells; and iii) compare the transcriptional profile of cytoplasts or nucleated cells (L929 fibroblasts), infected or not.The results will establish which host cells factors are sufficient for L. amazonensis intracellular parasitism and will possibly elucidate the participation of the parasites in the preservation and modulated half-life of host cell transcripts. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

REAL, FERNANDO. Isolation of intact Leishmania amazonensis large parasitophorous vacuoles from infected macrophages by density gradient fractionation. Experimental Parasitology, v. 218, NOV 2020. (14/25296-2)

ORIKAZA, CRISTINA M.; PESSOA, CARINA C.; PALADINO, V, FERNANDA; FLORENTINO, V, PILAR T.; BARBIERI, CLARA L.; GOTO, HIRO; RAMOS-SANCHEZ, EDUARDO MILTON; SILVEIRA, JOSE FRANCO; RABINOVITCH, MICHEL; MORTARA, RENATO A.; et al. Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life o Host Cell Transcripts by the Parasite. Infection and Immunity, v. 88, n. 11, NOV 2020. (14/25296-2, 16/15000-4)