A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells

Guy, Rebecca L.; Gonias, Lauren A; Stein, Murry A.

doi:10.1016/s0167-7012(00)00188-3

Cited by 4 publications

(5 citation statements)

References 12 publications

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“…The approach we determined as most likely to yield a representative set of genes mediating the phenotype was the direct examination of Salmonella transposon mutants for the loss of lgp‐tubule formation. We devised a genetic screen suitable for the efficient examination of individual Salmonella mutants for lgp‐tubule formation (Guy et al ., 2000). The key feature of the genetic screen was the use of commercially available Special Optics (SO) 96‐well plates.…”

Section: Resultsmentioning

confidence: 99%

“…As phenotypes requiring high‐magnification fluorescence microscopy are not inclined to facilitate the examination of the thousands of mutants needed to approach saturation, we devised a suitable high‐throughput methodology. The screening rationale and detailed description of the devised methodology are presented elsewhere (Guy et al ., 2000). The approach devised is likely to be applicable to a wide range of virulence‐associated phenotypes detectable by immunofluorescence.…”

Section: Discussionmentioning

confidence: 99%

“…The methodology for genetic screening to identify lgp‐tubule mutants has been described in detail elsewhere (Guy et al ., 2000). Briefly, HeLa cells were seeded into SO 96‐well plates (Corning), infected with individual Mu d J mutants, fixed with 2% formaldehyde in PBS and stained for bacterial and lgp distribution by direct immunofluorescence.…”

Section: Methodsmentioning

confidence: 99%

“…In the present study, we developed a high‐throughput immunofluorescence‐based genetic screen to identify individual Salmonella Mu d J transposon mutants negative for the lgp‐tubule formation phenotype (Guy et al ., 2000). From 11 520 individual mutants examined, 10 were deficient in lgp‐tubule formation.…”

Section: Introductionmentioning

confidence: 99%

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Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms–aroE intragenic region

Guy

Gonias

Stein³

2000

Molecular Microbiology

129

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SummarySalmonella-induced aggregation of host endosomal compartments into tubules, termed lgp-tubules, requires sifA and ompR. Lgp-tubules result from Salmonella-directed alteration of the endocytic system and typify the unique intracellular locale where Salmonella replicate. A high-throughput method devised to screen 11 520 MudJ mutants for loss of lgp-tubule formation identified one auxotrophic and nine prototrophic mutants. Molecular characterization identified four new loci required to alter epithelial endocytic structure. Salmonella pathogenicity island 2 (SPI2) is the locus central to the phenotype. A subset of SPI2 effectors is essential: SpiC and SseFG are required, but not SseE. A subset of apparatus proteins is also implicated: SsaJ, L, M, V and P are required. SPI2 was implicated further, as SifA shows similarity with known SPI2 translocation targets, and OmpR regulates SPI2. Another locus lies within the smf±aroE intragenic region. Lgp-tubule formation also involves a locus on the virulence plasmid pSLT. The pSLT-encoded SpvR negatively regulates an unknown repressor of the phenotype located on pSLT. Finally, disruption of carB leads to multiple auxotrophy that prevents lgp-tubule formation. This study demonstrates that lgp-tubule formation is a virulence mechanism that underlies the selective disruption of host endocytic trafficking and is associated with the formation of a replicationpermissive locale.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms–aroE intragenic region

Guy

Gonias

Stein³

2000

Molecular Microbiology

129

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“…The screening is carried out in multi well plates and the generated data is analysed by the software. Fluorescence spectroscopy 47 and transmitted light microscopy 48 are also routinely used to perform the phenotypic screen.…”

Section: High-throughput Screeningsmentioning

confidence: 99%

Target Identification: A Challenging Step in Forward Chemical Genetics

Das¹,

Samanta²,

Ghosh³

et al. 2011

Interdisciplinary Bio Central

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SYNOPSISInvestigation of the genetic functions in complex biological systems is a challenging step in recent year. Hence, several valuable and interesting research projects have been developed with novel ideas to find out the unknown functions of genes or proteins. To validate the applicability of their novel ideas, various approaches are built up. To date, the most promising and commonly used approach for discovering the target proteins from biological system using small molecule is well known a forward chemical genetics which is considered to be more convenient than the classical genetics. Although, the forward chemical genetics consists of the three basic components, the target identification is the most challenging step to chemical biology researchers. Hence, the diverse target identification methods have been developed and adopted to disclose the small molecule bound protein. Herein, in this review, we briefly described the first two parts chemical toolbox and screening, and then the target identifications in forward chemical genetics are thoroughly described along with the illustrative real example case study. In the tabular form, the different biological active small molecules which are the successful examples of target identifications are accounted in this research review.

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High throughput screening of protein formulation stability: Practical considerations

Capelle

Gurny

Arvinte

2007

European Journal of Pharmaceutics and Biopharmaceutics

128

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A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells

Cited by 4 publications

References 12 publications

Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms–aroE intragenic region

Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms–aroE intragenic region

Target Identification: A Challenging Step in Forward Chemical Genetics

High throughput screening of protein formulation stability: Practical considerations

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