1887

Abstract

serovar Typhi (S. Typhi) is a human adapted pathogen and the causative agent of typhoid fever, a life-threatening infection that kills hundred of thousand people every year, being particularly devastating in developing countries. S. Typhi host-restriction is partly due to the Rab32-dependent antimicrobial pathway, which is crucial to prevent the growth of S. Typhi in mouse macrophages. However, the exact mechanisms used by macrophages to kill S. Typhias well as the molecular basis of these mechanisms in the adaptation to the human host are unknown.

In order to identify host genes required to kill S. Typhi, we have performed a targeted short-hairpin RNA (shRNA) screen in primary mouse macrophages, aiming to i) optimize the conditions to perform silencing screenings in primary mouse macrophages and ii) identify novel Rab GTPases involved in S. Typhi host-restriction. For this, pooled shRNAs are used to knockdown gene expression in macrophages using lentiviral-based transduction system. After infection with a fluorescently-labelled S. Typhi strain, macrophages containing different numbers of intracellular bacteria are sorted by flow cytometry and targeted genes identified by next-generation sequencing.

This small-scale screen allowed us to optimize the screening conditions to perform genome-wide screenings in primary macrophages. More importantly, we have identified other Rab GTPases required in mouse macrophages to control S. Typhi survival confirming that this approach can be used to identify genes that macrophages use to control S. Typhi infection and extending our knowledge of the immunity mechanisms controlling the growth of intracellular pathogens.

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/content/journal/acmi/10.1099/acmi.ac2020.po0493
2020-07-10
2021-08-02
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http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.ac2020.po0493
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