The Rab32/BLOC-3 dependent pathway mediates host- defence against different pathogens in human macrophages

Front. Cell. Infect. Microbiol.

Colamarino

Calderwood

et al. 2021

Self Cite

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.

Section: Resultsmentioning

confidence: 99%

“…Typhi glmS::Cm::mCherry ( S . Typhi:: mCherry ) ( Baldassarre et al, 2021 ) strains have been described previously.…”

Section: Methodsmentioning

confidence: 99%

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

Front. Cell. Infect. Microbiol.

Colamarino

Calderwood

et al. 2021

Self Cite

“…HPS-1 is one of the two subunits of the BLOC-3 complex, and BLOC-3 together with Rab32, controls S . Typhi growth in macrophages (Spano and Galan., 2012, Baldassarre et al, 2020). We initially assessed S .…”

Section: Resultsmentioning

confidence: 99%

“…Typhi glmS::Cm::mCherry ( S . Typhi:: mCherry ) (Baldassarre et al, 2020) strains have been described previously.…”

Section: Methodsmentioning

confidence: 99%

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

Colamarino

Calderwood³

et al. 2020

Preprint

Self Cite

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 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 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.

“…Studies from our laboratory and others, have shown that in macrophages, the BRAM pathway controls not only infection by bacterial pathogens such as Salmonella Typhi, Staphylococcus aureus, Listeria monocytogenes, and Burkholderia pseudomallei (Seto et al, 2011;Spanò and Galań, 2012;Li et al, 2016;Hu et al, 2019) but it also has an important role in controlling Candida albicans infection in mice (Baldassarre et al, 2020). However, nothing is known about how the BRAM pathway is regulated and what other proteins are part of this important antimicrobial pathway.…”

Section: Discussionmentioning

confidence: 97%

VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing

Balci

Front. Cell. Infect. Microbiol.

Baldassarre

et al. 2020

Self Cite

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.