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

Abstract

Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. 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 S. Typhi infection, the mechanisms that macrophages use to control the growth of S. 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 S. 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 S. 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 S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.

Determination of the Salmonella intracellular lifestyle by the diversified interaction of Type III secretion system effectors and host GTPases

“…In addition, shRNA‐mediated depletion of rab1 promotes the survival of S . Typhi in mouse macrophage cells (Solano‐Collado et al, 2021). However, the mechanism underlying the role of Rab1 in host restriction should be extensively studied.…”

“…The exact roles of these four Rabs in controlling the host restriction have not been determined, but SopB does not contribute to the host restriction of Salmonella (Raffatellu et al, 2005). In addition, shRNA-mediated depletion of rab1 promotes the survival of S. Typhi in mouse macrophage cells (Solano-Collado et al, 2021). However, the mechanism underlying the role of Rab1 in host restriction should be extensively studied.…”

Complete Genome Sequence of Salmonella enterica Serovar Typhi Strain ISP2825

“…There are four major S. Typhi strains extensively used for research that have helped advance our understanding of typhoid pathogenesis and evolution: Ty2 ( 4 ), CT18 ( 5 ), H58 ( 6 ), and ISP2825 ( 7 – 24 ). Complete genome sequences of these strains are available, except for ISP2825.…”