A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth

• Published in: Frontiers in cellular and infection microbiology Vol. 11; p. 660689
• Main Authors: Solano-Collado, Virtu, Colamarino, Rosa Angela, Calderwood, David A, Baldassarre, Massimiliano, Spanò, Stefania
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 07.04.2021
• Subjects: Animals; Cellular and Infection Microbiology; host-defense; macrophages; Macrophages - metabolism; Mice; rab GTP-Binding Proteins - metabolism; Rab GTPases; Rab1b; RNA, Small Interfering; Salmonella Typhi; Salmonella typhi - genetics; shRNA screen; Typhoid Fever; macrophages; shRNA screen; host-defense; Rab GTPases; Rab1b; Salmonella Typhi
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2021.660689

Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of . Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic . Typhi infection, the mechanisms that macrophages use to control the growth of . Typhi and the role of these mechanisms in the bacterium's adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of . Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled . Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict . Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls . Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in . Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of . Typhi and other intracellular pathogens in primary immune cells.