Home › Immunology › Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants
Immunology JoVE (Open Access) Citable · DOI

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

DOI: 10.3791/52851-v
What you'll learn
  • ✓ Generate fluorescent Coxiella burnetii transposon mutants via isolation and PCR validation
  • ✓ Perform high-content screening to assess bacterial internalization, replication, and cytotoxicity phenotypes
  • ✓ Analyze intracellular bacterial growth dynamics using automated microscopy image acquisition
Protocol

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Difficulty
advanced
Total time
~5–7 days (mutant generation, PCR screening, cell infection, image acquisition, analysis)
Model organism
HEK293 cells; Coxiella burnetii
Biosafety
BSL-2

Steps

1
Isolate individual transposon mutant colonies

Plate and culture Coxiella burnetii transposon mutant bacteria to obtain single-colony isolates for downstream analysis.

▶ 01:49
2
Evaluate bacterial concentration and verify mutants by PCR

Quantify bacterial cell density and confirm transposon insertion via single-primer colony PCR to validate mutant identity.

▶ 03:44
3
Challenge eukaryotic cells and monitor intracellular growth

Infect mammalian cells with Coxiella mutants and measure internalization, replication, and cytotoxic phenotypes over time.

▶ 05:34
4
Prepare infected samples for automated image acquisition

Fix and stain infected cell cultures with fluorescent markers to enable high-content microscopy screening.

▶ 07:17
5
Analyze viability and infectivity phenotype results

Review automated image data to assess survival, intracellular replication, and host cell damage across mutant library.

▶ 09:25
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