Biology subjects

Bruni, G. N.

Publications and source records attributed to Bruni, G. N..

3 recordsLinked to original sources

bioRxiv2021

Aminoglycosides induce a bacterial senescent state that increases antibiotic tolerance in treatment-naïve cells

1.Bacterial evolution of antibiotic resistance is facilitated by non-genetic resistance that increases drug tolerance, buying time for evolutionary innovation. Escherichia coli treated with aminoglycosides permanently lose the ability to divide within four hours, yet we discovered a majority of cells maintain membrane integrity and metabolic activity greater than two days post treatment - a bacterial senescent-like state. These cells, which we term zombies, exhibit dynamic gene expression and metabolomic profiles, even after irreversible exit from the cell cycle. Our data reveal zombies upregulate the phage shock protein pathway to maintain membrane integrity. Remarkably, though unable to form new colonies, zombies increase the antibiotic tolerance of treatment-naive cells, implying chemical communication. Chemical supplementation and genetic knockouts show that zombies communicate with treatment-naive cells by secreting indole. In summary, our study revealed a bacterial senescent-like state, induced by aminoglycosides, that decreases the antibiotic susceptibility of multiple bacterial species. Thus, E. coli zombies utilize paracrine signaling to promote non-genetic antibiotic tolerance.

microbiology↗

bioRxiv2020

Genome wide functional screen for calcium transients in E. coli identifies decreased membrane potential adaptation to persistent DNA damage

1.Calcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Unlike eukaryotic cells, far less is known about calcium signaling in bacteria, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed calcium influx is induced by voltage depolarization, which were enhanced by mechanical stimulation, suggesting a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live cell screen to monitor calcium dynamics in single cells across a genome wide knockout panel in E. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased calcium transients, and 32 genes knockouts that increased transients. Knockouts involved in energy production and regulation appeared, as expected, as well as knockouts of the voltage sink, the F1Fo-ATPase. Knockouts in exopolysaccharide and outer membrane synthesis showed reduced transients and refined our model of electrophysiology mediated mechanosensation in E. coli. Additionally, knockouts annotated in DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and suggested that only long term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long term DNA damage responses in bacteria, which has implications for mitochondrial DNA damage in eukaryotes. ImportanceAll eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in Escherichia coli, we created a single cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.

microbiology↗

bioRxiv2020

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

Aminoglycosides are broad-spectrum antibiotics whose mechanism of bactericidal activity has been under debate. It is widely accepted, however, that membrane voltage potentiates aminoglycoside activity, which is ascribed to voltage dependent drug uptake. In this paper, we measured the single cell response of Escherichia coli treated with aminoglycosides and discovered that the bactericidal action arises not from the downstream effects of voltage dependent drug uptake, but rather directly from dysregulated membrane potential. In the absence of voltage, aminoglycosides are taken into cells and exert bacteriostatic effects by inhibiting translation. However, cell killing was immediate upon re-polarization. The hyperpolarization arose from altered ATP flux, which induced a reversal of the F1Fo-ATPase to hydrolyze ATP and generated the deleterious voltage. Heterologous expression of an ATPase inhibitor from Salmonella completely eliminated bactericidal activity, while loss of the F-ATPase significantly reduced the electrophysiological response to aminoglycosides. Our data support a model of voltage induced death, which could be resolved in real-time at the single cell level, and separates the mechanisms of aminoglycoside bacteriostasis and bactericide in E. coli.

microbiology↗