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Updated: Sep 21, 2025
A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues
Published on: February 19, 2019
Quantification of Microbial Fluorescent Sensors During Live Intracellular Infections
1Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel. erez.mills@mail.huji.ac.il.
Abstract:
The interaction of pathogens with their eukaryotic hosts during intracellular growth is critical to many diseases. However, the relative scarcity of pathogen biomolecules versus the abundant host biomolecule concentration can make quantitative evaluation of pathogen intracellular responses difficult. Recent years have seen an explosion in utilization of fluorescent proteins to serve as transcriptional reporters and biosensors for quantification of pathogen responses. Here, we describe a method to establish a fluorescent assay quantifying pathogen behavior during intracellular infection and to quantify these results at a single cell level. The sensitivity of these fluorescent assays permits the live observation of changing pathogen responses, while the ability to measure at a single cell level uncovers subpopulations of pathogens whose existence may be missed during the population-level assays often required to accumulate sufficient pathogen biomolecules for analysis.
Insights
This study introduces a novel fluorescent assay for quantifying pathogen behavior within host cells. This method enables single-cell analysis, revealing pathogen subpopulations missed by traditional population-level assays.
Area of Science:
- Microbiology
- Cell Biology
- Infectious Diseases
Background:
- Pathogen-host interactions during intracellular growth are crucial for disease pathogenesis.
- Quantifying pathogen responses inside host cells is challenging due to low pathogen biomolecule concentrations.
- Fluorescent proteins are increasingly used as reporters and biosensors for studying pathogen behavior.
Purpose of the Study:
- To develop a sensitive fluorescent assay for quantifying pathogen intracellular behavior.
- To enable single-cell level analysis of pathogen responses during infection.
- To overcome limitations of population-level assays in studying pathogen dynamics.
Main Methods:
- Development of a novel fluorescent assay for pathogen intracellular growth.
- Utilizing fluorescent proteins as transcriptional reporters and biosensors.
- Implementing single-cell level quantification of pathogen responses.
Main Results:
- The fluorescent assay allows for sensitive, live observation of pathogen responses.
- Single-cell analysis revealed pathogen subpopulations previously undetected.
- The method overcomes challenges associated with low pathogen biomolecule concentrations.
Conclusions:
- This fluorescent assay provides a powerful tool for studying pathogen-host interactions at the single-cell level.
- The technique enhances our understanding of pathogen dynamics within host cells.
- It offers a sensitive method for quantifying pathogen behavior and uncovering hidden subpopulations.
