Single-cell approach dissecting agr quorum sensing dynamics in Staphylococcus aureus || Timelapse microfluidics bacteria

This repository contains example datasets for the experiments described in the associated study. It contains raw files as well as the output of the fully ran bioimage analysis pipeline for each example movie included in the supplement for mother-machine (mm) and connected chamber (cc) microfluidic devices. S. aureus agr-prototype strains were used which carry an agr-P3-YFP reporter plasmid. For mm, the four agr-type strains were exposed to multiple concentrations of homologous AIP (mm/mm_meta_processing.tsv column type == sAIP or == short) or agr-I exposed to combinations of homologous AIP and heterologous AIP (mm/mm_meta_processing.tsv column type == mAIP). For cc, agr-I was grown with a partner agr-type without exogenous AIP.
Images are in phasecontrast and YFP fluorescence channel. The raw files are in the Olympus .ets + .vsi format and contain manually selected positions of timelapse images in phasecontrast and YFP fluorescence of microfluidic chips. The code to run the pipeline is hosted on github and the training images for the two StarDist2D segmentation models as well as the DeLTA2.0 2D tracking models are hosted on zenodo (10.5281/zenodo.10694025).
The outputs of the analysis pipeline is included here as well (cc/cc_savesV1.zip for cc and mm/mm_savesV1.zip for mm) and contains registered ROIs for each raw image folder (single-page tif files) along with stardist segmented cells (label images in the subfolders named sd2 and regionprops in the stardist folders). For experiments with mm/mm_meta_processing.tsv column type == short (2.75min or lower acquisition rate), DeLTA tracking output is provided as well (deltadata subfolder).

Abstract:
Staphylococcus aureus both colonizes humans and causes severe virulent infections. Virulence is regulated by the agr quorum sensing system and its autoinducing peptide (AIP), with dynamics at the single-cell level across four agr-types – each defined by distinct AIP sequences and capable of cross-inhibition – remaining elusive. Employing microfluidics, time-lapse microscopy, and deep-learning image analysis, we uncovered significant differences in AIP sensitivity among agr-types. We observed bimodal agr activation, attributed to intergenerational phenotypic stability and influenced by AIP concentration. Upon AIP stimulation, agr III showed AIP insensitivity, while agr II exhibited increased sensitivity and prolonged generation time. Beyond expected cross-inhibition of agr I by heterologous AIP II and III, the presumably cross-activating AIP IV also inhibited agr I. Community interactions across different agr-type pairings revealed four main patterns: stable or switched dominance, and delayed or stable dual activation, influenced by community characteristics. These insights underscore the potential of personalized treatment strategies considering virulence and genetic diversity.