eoe sub articles

EoE Sub Articles

1. Generation of a library of green fluorescence protein, GFP-tagged&#160;Coxiella&#160;transposon mutants
Manipulate&#160;Coxiella burnetii&#160;RSA439 NMII in a biosafety containment level 2 (BSL-2) in a microbial safety cabinet (MSC) in compliance with local rules. If compatible with the bacterial model used, repeat steps from 1.4.1 to 1.4.4 to increase the probability of obtaining clonal mutants. A typical mutant library is composed (at least) of a number o...

assessment of intracellular growth of coxiella mutants within eukaryotic cells

Source: Martinez, E. et al., <a href="https://app.jove.com/v/52851">Generation and Multi-phenotypic High-content Screening of&#160;Coxiella burnetii&#160;Transposon Mutants.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates a method for assessing the intracellular growth of GFP-tagged Coxiella mutants within cultured epithelial cells. The mutant phenotype is visualized using immunostaining and epifluorescence microscopy, allowing the study of the effect of the mutation on intracellular growth.

Assessment of Intracellular Growth of Coxiella Mutants within Eukaryotic Cells

1. Generation of a library of green fluorescence protein, GFP-tagged&#160;Coxiella&#160;transposon mutants
Manipulate&#160;Coxiella burnetii&#160;RSA439 ...

Take a multi-well plate containing adhered epithelial cells and treat it with green fluorescent protein-tagged Coxiella mutants; some with mutations in the type IV secretion system gene, or T4SS gene &#8212; crucial for replication.
Replicative mutants interact with epithelial cells, triggering Coxiella engulfment, forming Coxiella-containing vacuole, or CCV, and recruiting lysosome-associated membrane glycoprotein-1, or LAMP1 to CCV.
This facilitates effector proteins&#39; secretion into the cell cytoplasm via the type IV secretion system, promoting bacterial replication.
Mutants with T4SS gene mutations remain unreplicated.
Post-incubation, stain the cells with a fluorescent dye, fix them, and permeabilize the cell membrane.
Introduce LAMP1-specific primary antibodies interacting with the LAMP1 proteins on CCV.&#160;
Overlay with red-fluorophore-labeled secondary antibodies, interacting with primary antibodies, while Hoechst dye stains the cells&#39; nuclei.
Under an epifluorescence microscope, red-fluorescence vacuoles containing fewer green Coxiella near the nucleus indicate defective replication of Coxiella mutants.
However, vacuoles containing many green Coxiella inside the cells indicate the successful intracellular growth of replicative Coxiella mutants.