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iMAD, a genetic screening strategy for dissecting complex interactions between a pathogen and its host.

Tamara J O'Connor¹, Ralph R Isberg²

¹Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

Nature Protocols

|July 18, 2014

Summary

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Insertional mutagenesis and depletion (iMAD) is a genetic screening strategy to uncover interactions between organisms. This method identifies genetic interactions between pathogen and host gene disruptions, revealing functional relationships.

Area of Science:

* Genetics
* Molecular Biology
* Microbiology

Background:

* Complex interactions between organisms are difficult to study using traditional genetic methods.
* Understanding pathogen-host interactions is crucial for developing new treatments.
* Existing genetic screening strategies lack the capacity for simultaneous manipulation of both interacting organisms.

Purpose of the Study:

* To present a detailed protocol for the insertional mutagenesis and depletion (iMAD) genetic screening strategy.
* To demonstrate the application of iMAD for dissecting interactions between a pathogen, Legionella pneumophila, and its host, Drosophila melanogaster cells.
* To provide a framework for adapting iMAD to various experimental systems.

Main Methods:

* Simultaneous genetic manipulation of both pathogen and host organisms.

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* Employing bacterial mutagenesis and host RNA interference (RNAi) for gene disruption.

* Utilizing hierarchical clustering and genetic interaction networks for data analysis.

Main Results:

* The iMAD strategy enables the identification of both aggravating and alleviating genetic interactions.
* Subsets of genes with common behavioral patterns can be identified, elucidating functional relationships.
* The protocol covers key stages from screen design to data analysis.

Conclusions:

* iMAD is a powerful genetic screening strategy for dissecting complex inter-organismal interactions.
* The presented protocol facilitates the study of pathogen-host relationships.
* Adaptation to specific systems requires 6-18 months, with screens completed in 4-8 weeks post-establishment.