A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth
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 ma...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-04-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2021.660689/full |
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| author | Virtu Solano-Collado Rosa Angela Colamarino David A. Calderwood David A. Calderwood Massimiliano Baldassarre Stefania Spanò |
| author_facet | Virtu Solano-Collado Rosa Angela Colamarino David A. Calderwood David A. Calderwood Massimiliano Baldassarre Stefania Spanò |
| author_sort | Virtu Solano-Collado |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-19T10:17:00Z |
| format | Article |
| id | doaj.art-72a538667f73415bb587daec80cd7ab1 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-12-19T10:17:00Z |
| publishDate | 2021-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-72a538667f73415bb587daec80cd7ab12022-12-21T20:26:11ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882021-04-011110.3389/fcimb.2021.660689660689A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi GrowthVirtu Solano-Collado0Rosa Angela Colamarino1David A. Calderwood2David A. Calderwood3Massimiliano Baldassarre4Stefania Spanò5Institute of Medical Sciences, University of Aberdeen, Aberdeen, United KingdomInstitute of Medical Sciences, University of Aberdeen, Aberdeen, United KingdomDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT, United StatesDepartment of Cell Biology, Yale University School of Medicine, New Haven, CT, United StatesInstitute of Medical Sciences, University of Aberdeen, Aberdeen, United KingdomInstitute of Medical Sciences, University of Aberdeen, Aberdeen, United KingdomSalmonella 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.https://www.frontiersin.org/articles/10.3389/fcimb.2021.660689/fullhost-defenseSalmonella TyphiRab GTPasesRab1bshRNA screenmacrophages |
| spellingShingle | Virtu Solano-Collado Rosa Angela Colamarino David A. Calderwood David A. Calderwood Massimiliano Baldassarre Stefania Spanò A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth Frontiers in Cellular and Infection Microbiology host-defense Salmonella Typhi Rab GTPases Rab1b shRNA screen macrophages |
| title | A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth |
| title_full | A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth |
| title_fullStr | A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth |
| title_full_unstemmed | A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth |
| title_short | A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth |
| title_sort | small scale shrna screen in primary mouse macrophages identifies a role for the rab gtpase rab1b in controlling salmonella typhi growth |
| topic | host-defense Salmonella Typhi Rab GTPases Rab1b shRNA screen macrophages |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2021.660689/full |
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