Colamarino, R. A. | Search bioRxiv

bioRxiv2020

A small-scale shRNA screen in primary mouse macrophages identifies a role for the Rab GTPase Rab1b in controlling Salmonella Typhi growth

Salmonella Typhi, the causative agent of typhoid fever, a life-threatening systemic infection, is a human restricted bacterial pathogen. 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 bacteriums 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 fluorescently-labeled S. Typhi, cells defective in genes important for controlling S. Typhi growth (and therefore unable to kill S. Typhi) 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.

microbiology↗

bioRxiv2020

Lipidation of a bacterial effector is critical for bacterial evasion of host-defense

The Rab32 antimicrobial pathway has been shown to restrict Salmonella Typhi, in mouse macrophages. The broad-host pathogen Salmonella Typhimurium however has evolved a strategy to evade the Rab32 antimicrobial pathway, via its effector protein GtgE. GtgE is a cysteine protease that specifically mediates the cleavage and inactivation of Rab32. Here we show that GtgE association and targeting to membranes is critical for its efficient proteolytic activity. The C-terminus of GtgE contains a CaaX motif, which can be post-translationally modified by the hosts prenylation machinery. Using a combination of confocal microscopy and subcellular fractionation we show that a cysteine in the CaaX motif is crucial for GtgE membrane targeting and, more importantly, GtgE localization to the Salmonella-containing vacuole. We also demonstrated that prenylation of CaaX is important for an effective and fast Rab32 cleavage, which in turn helps Salmonella to successfully survive in macrophages and establish an in vivo infection in mice. Our findings shed light on the importance of a host mediated post-translational modification that targets GtgE to the membranes where it can efficiently cleave and inactivate Rab32, leading to a better Salmonella survival in macrophages. Author summarySalmonella species includes a large group of bacteria that cause disease in different hosts. While some serovars are host generalists, others are restricted to humans. This is the case of Salmonella Typhi, responsible for Typhoid fever, a disease that affects millions globally. We have previously discovered an antimicrobial activity in macrophages that is controlled by Rab32. While the broad-host bacterium Salmonella Typhimurium effectively counteracts this mechanism through the delivery of two effectors, GtgE and SopD2, Salmonella Typhi does not express those effectors and cannot survive in mouse macrophages. In this article, we demonstrate how Salmonella Typhimurium exploits a host machinery to modify GtgE. We show that this host mediated modification is important for GtgE intracellular localization and effective Rab32 targeting, resulting in both a better intracellular survival and infection in vivo.

microbiology↗