SUPR
Bacterial response to extracellular ATP
Dnr:

NAISS 2024/22-182

Type:

NAISS Small Compute

Principal Investigator:

Andrea Puhar

Affiliation:

Umeå universitet

Start Date:

2024-02-01

End Date:

2025-02-01

Primary Classification:

10606: Microbiology (medical to be 30109 and agricultural to be 40302)

Allocation

Abstract

We study infection and inflammation in the gut using both intestinal bacteria and host cells and tissues as experimental models. Our main focus is to understand how the small metabolite and pro-inflammatory mediator extracellular ATP (eATP) regulates the function of bacteria and host cells and ultimately the onset of inflammation. A major role for eATP in infectious diseases was not recognised until very recently. We have shown that intestinal epithelial cells infected with the Gram-negative bacterial pathogens Shigella flexneri, Salmonella Typhimurium or enteropathogenic Escherichia coli (EPEC) secrete ATP across connexin-hemichannels. We have found that epithelial ATP secretion is an early alert response to infection, acting upstream of classical pro-inflammatory mediators and provoking strong inflammation of the bowels. These data bridge the molecular mechanism regulating sterile inflammation and inflammation during infection. We focused our efforts on discovering the molecular mechanisms underlying ATP secretion triggered by infection in intestinal epithelial cells. We found that ATP secretion can also be induced by Gram-positive bacteria, provided they are invasive. We discovered that the formation of severely bent plasma membrane extension, which always accompany the uptake of invasive bacteria, are a mechanical immune signal. We found the inherently mechanosensitive plasma membrane cation channel PIEZO1 is necessary and sufficient to detect infection-induced membrane ruffles, which triggers Ca2+ influx. PIEZO1 opening then results in ATP secretion via Ca2+. Further, we have used RNA-seq and analysis on SNIC resources to show that activation of PIEZO1 reprogrmmes the transcriptome. We are currently deepening this transcriptomic analysis and are expecting data to process in early 2022 to satisfy reviewer’s comments. Together, these data indicate that PIEZO1 acts as a mechanically activated immune sensor (Tadala et al., submitted). We have used RNA-seq to study changes in the transcription landscape triggered by eATP in intestinal epithelial cells in the presence or absence of Shigella infection. In addition, we obtained the transcriptome of the infecting bacteria. Shigella forces its uptake into intestinal epithelial cells and then grows in the target cell cytosol. Hence, on SNIC resources, we performed a comparison of gene expression of intracellular Shigella vs extracellular Shigella to unravel factors that are necessary for adaptation to and survival in the target cell cytosol. To validate these results, we prepared a genome-wide barcoded transposon library. We used SNIC resources to map these barcodes on the genome. We are currently expanding this study to a second independent library, followed by sequencing-based infection experiments to assess the fitness of random mutants. To validate the hits leading to reduced bacterial fitness, we have built a medium-throughput CRISPR-based mutagenesis pipeline for Shigella and E. coli (Sharma et al., in preparation) and have established medium-throughput plate reader-based infection competition assays. Together, these data will reveal new and unexpected virulence mechanisms in Shigella and provide much needed novel targets for antibiotics. Finally, we use SNIC resources for collaborations in which we perform sequencing or genome assembly experiments.