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RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells

Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and count...

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Main Authors: Akinsola, Rasaq Olajide, Adewoyin, Malik, Lee, Choon-Weng, Sim, Edmund Ui-Hang, Narayanan, Kumaran
Format: Article
Published: Elsevier 2021
Subjects:
Q Science (General)
QD Chemistry
Online Access:http://eprints.um.edu.my/35306/
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spelling my.um.eprints.353062022-10-14T07:04:27Z http://eprints.um.edu.my/35306/ RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells Akinsola, Rasaq Olajide Adewoyin, Malik Lee, Choon-Weng Sim, Edmund Ui-Hang Narayanan, Kumaran Q Science (General) QD Chemistry Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based highthroughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and celldependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study hostbacteria interaction. Elsevier 2021-12 Article PeerReviewed Akinsola, Rasaq Olajide and Adewoyin, Malik and Lee, Choon-Weng and Sim, Edmund Ui-Hang and Narayanan, Kumaran (2021) RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells. Analytical Biochemistry, 634. ISSN 0003-2697, DOI https://doi.org/10.1016/j.ab.2021.114432 <https://doi.org/10.1016/j.ab.2021.114432>. 10.1016/j.ab.2021.114432
institution Universiti Malaya
building UM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaya
content_source UM Research Repository
url_provider http://eprints.um.edu.my/
topic Q Science (General)
QD Chemistry
spellingShingle Q Science (General)
QD Chemistry
Akinsola, Rasaq Olajide
Adewoyin, Malik
Lee, Choon-Weng
Sim, Edmund Ui-Hang
Narayanan, Kumaran
RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
description Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based highthroughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and celldependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study hostbacteria interaction.
format Article
author Akinsola, Rasaq Olajide
Adewoyin, Malik
Lee, Choon-Weng
Sim, Edmund Ui-Hang
Narayanan, Kumaran
author_facet Akinsola, Rasaq Olajide
Adewoyin, Malik
Lee, Choon-Weng
Sim, Edmund Ui-Hang
Narayanan, Kumaran
author_sort Akinsola, Rasaq Olajide
title RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
title_short RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
title_full RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
title_fullStr RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
title_full_unstemmed RFP-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
title_sort rfp-based method for real-time tracking of invasive bacteria in a heterogeneous population of cells
publisher Elsevier
publishDate 2021
url http://eprints.um.edu.my/35306/
_version_ 1748181073886445568
score 13.211869

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