Barcoded Consortium Infections: A Scalable, Internally Controlled Method to Study Host Cell Binding and Invasion by...

Area of Science:

• Microbiology
• Infectious Diseases
• Cell Biology

Background:

• Traditional gentamicin protection assays for bacterial host cell invasion are labor-intensive and prone to significant experimental variability.
• Studying bacterial virulence factors, such as those involved in host cell invasion, is crucial for understanding pathogenesis and developing countermeasures.

Purpose of the Study:

• To describe a novel, internally controlled, medium- to high-throughput method for quantifying bacterial host cell binding and invasion.
• To provide a flexible and broadly applicable tool for the genetic analysis of bacterial virulence in host-pathogen interactions.

Main Methods:

• The method combines consortia of genetically tagged isogenic bacterial strains with a modified gentamicin protection assay.
• This approach allows for the simultaneous assessment of multiple bacterial mutant strains in a single experiment.
• The protocol is adaptable for both in vitro cultured host cells and in vivo animal models.

Main Results:

• The developed method enables stringent quantification of host cell binding and invasive properties of different bacterial mutants.
• It significantly reduces experimental noise compared to conventional methods.
• The comparable genetic readout across different experimental models facilitates cross-model comparisons.

Conclusions:

• This internally controlled, high-throughput method offers a robust and versatile tool for studying bacterial host cell interactions.
• It is widely applicable to various pathogens beyond Salmonella, aiding in the comparative analysis of virulence factors.
• The method streamlines research on bacterial pathogenesis and the development of novel therapeutic strategies.