Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium

Porwollik, Steffen; Santiviago, Carlos A.; Cheng, Pui; Long, Fred; Desai, Prerak; Fredlund, Jennifer; Srikumar, Shabarinath; Silva-Valenzuela, Cecilia A.; Wu, Chu; Chen, Xin; Canals, Rocı́o; Reynolds, Mark; Bogomolnaya, Lydia M.; Shields, Christine; Cui, Ping; Guo, Jingyi; Zheng, Yi; Endicott-Yazdani, Tiana; Yang, Hee-Jeong; Maple, Aimee; Ragoza, Yury; Blondel, Carlos J.; Valenzuela, Camila; Andrews‐Polymenis, Helene; McClelland, Michael

doi:10.1371/journal.pone.0099820

Cited by 158 publications

(152 citation statements)

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“…novicida, and B. pseudomallei (42)(43)(44), indicating that energy and nutrient sources are key to the rapid doubling time of bacteria in the mammalian cytosol. Surprisingly, we did not identify such genes in our Salmonella MGD screen, although if these are required for S. Typhimurium growth in broth, they would not have been targeted in the MGD library collection (21). However, we identified CorA, the primary Mg influx channel in S. Typhimurium, as being required for the efficient colonization of the epithelial cell cytosol, implicating magnesium uptake as a requirement for Salmonella growth within the host cell cytosol.…”

Section: Discussionmentioning

confidence: 78%

“…Salmonella enterica serovar Typhimurium SL1344 (19) and 14028s (20) were used as wild-type strains. The collection of targeted MGD mutants and SGD mutants of 14028s was described previously (21). Bacteria were grown in Luria-Bertani-Miller (LB-Miller) broth or on LBMiller agar supplemented with carbenicillin (100 g/ml), chloramphenicol (30 g/ml), kanamycin (50 g/ml), or streptomycin (100 g/ml), where appropriate.…”

Section: Methodsmentioning

confidence: 99%

“…This library contains deletions of 2,543 nonessential genes of S. Typhimurium 14028s with 3 or more contiguous genes replaced by a kanamycin resistance cassette in each mutant (21). We screened the MGD mutants in Caco-2 C2BBe1 cells, a colonic epithelial cell line, using the CHQ resistance assay at 7 h p.i.…”

Section: Screening Of Mgd Mutants For Cytosolic Proliferationmentioning

confidence: 99%

“…To pinpoint the individual gene(s) within each of these regions responsible for the observed phenotype, we screened the corresponding 14028s SGD mutants (21) in HeLa cells using the CHQ resistance assay. The ⌬STM1450 -STM1464 MGD mutant has 15 genes deleted and exhibits a lower proportion of cytosolic bacteria than the wild type in HeLa epithelial cells (29% Ϯ 7.9% versus 59% Ϯ 15%) (Fig.…”

Section: Screening Of Mgd Mutants For Cytosolic Proliferationmentioning

confidence: 99%

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Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells

et al. 2016

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Intestinal epithelial cells provide an important colonization niche for Salmonella enterica serovar Typhimurium during gastrointestinal infections. In infected epithelial cells, a subpopulation of S. Typhimurium bacteria damage their internalization vacuole, leading to escape from the Salmonella-containing vacuole (SCV) and extensive proliferation in the cytosol. Little is known about the bacterial determinants of nascent SCV lysis and subsequent survival and replication of Salmonella in the cytosol. To pinpoint S. Typhimurium virulence factors responsible for these steps in the intracellular infectious cycle, we screened a S. Typhimurium multigene deletion library in Caco-2 C2Bbe1 and HeLa epithelial cells for mutants that had an altered proportion of cytosolic bacteria compared to the wild type. We used a gentamicin protection assay in combination with a chloroquine resistance assay to quantify total and cytosolic bacteria, respectively, for each strain. Mutants of three S. Typhimurium genes, STM1461 (ydgT), STM2829 (recA), and STM3952 (corA), had reduced cytosolic proliferation compared to wild-type bacteria, and one gene, STM2120 (asmA), displayed increased cytosolic replication. None of the mutants were affected for lysis of the nascent SCV or vacuolar replication in epithelial cells, indicating that these genes are specifically required for survival and proliferation of S. Typhimurium in the epithelial cell cytosol. These are the first genes identified to contribute to this step of the S. Typhimurium infectious cycle.

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Section: Discussionmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Section: Screening Of Mgd Mutants For Cytosolic Proliferationmentioning

confidence: 99%

Section: Screening Of Mgd Mutants For Cytosolic Proliferationmentioning

confidence: 99%

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Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells

et al. 2016

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“…Seeking a method for screening a larger fraction of the genes in the Salmonella genome, we constructed 182 mutants with targeted deletions in regions of multiple contiguous genes (multigene deletion [MGD] mutants) in Salmonella Typhimurium ATCC 14028s using lambda red recombination (28)(29)(30). Collectively, these MGD mutants are deleted for ϳ2,069 genes, and they can be pooled to identify those mutants under selection during infection.…”

mentioning

confidence: 99%

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

Yang¹,

Bogomolnaya

Elfenbein³

et al. 2016

Infect Immun

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dContaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

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Murine Models of Salmonella Infection

et al. 2023

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The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium–infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella‐infected intestinal tissues

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Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium

Cited by 158 publications

References 53 publications

Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells

Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

Murine Models of Salmonella Infection

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