Unraveling a synthetic rescue process involved in persisters formation

A common strategy that bacteria utilize to increase their survival under stressful conditions in their natural environments, including antibiotic treatment, is the entry into quiescence, a state of reversible cell growth arrest that offers protection against many environmental insults. Understanding quiescence is an important fundamental question, with relevance in the medical and environmental fields. Little is known about the molecular and physiological determinants that orchestrate survival during this temporary arrest of proliferation, or those that allow a rapid transition back to the proliferating state when conditions again become favorable. In the wide host-range pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and other Gram-negative bacteria, this temporary arrest of proliferation induces the expression of the alternative sigma subunit of RNA polymerase, sigma S/RpoS, which remodels global gene expression to reshape the cell physiology and ensure survival under starvation and various stress conditions (i.e. the general stress response).

In S. Typhimurium, sigma S is required for stress resistance, biofilm formation and virulence. The incidence of human infections by non-typhoidal Salmonella such as S. Typhimurium increases. These serovars can infect farm animals, thus contaminating animal products, and can be transferred from animal carriers to the environment through fecal matter where they contaminate vegetables, fruits, nuts, and roots. Foodborne diseases caused by Salmonella represent a severe problem to the food supply as well as the public health. S. Typhimurium actively cycles through host and nonhost environments, where it is exposed to a wide variety of stresses, and where sigma S likely plays a crucial role in its persistence.

One important aspect of persistence is the phenotypic differentiation of quiescent populations into sub-population(s) of "persisters" that survive in the presence of lethal concentrations of antibiotics. This phenomenon is worsening the worldwide antibiotic crisis, by causing therapy failure and chronic infections and potentially favoring the development of antibiotic resistance. Understanding mechanisms governing bacterial persisters is thus an important topic and a key issue for drug developments. However, despites many studies, the physiological and molecular mechanisms controlling the formation of persisters are poorly understood and controversial. In the present study we explore the recently discovered and unexpected functional interaction between sigma S and succinate dehydrogenase (Sdh), in the formation of persisters. 

Persisters are phenotypic variants within a population that survive in the presence of lethal concentrations of antibiotics.  When a bacterial population is diluted into fresh medium containing bactericidal antibiotics, a biphasic killing is observed. The bulk of the population, consisting of sensitive cells, dies rapidly and the surviving persisters are killed much more slowly, or do not die during the time course of the experiment. Their regrowth after antibiotic removal yields a new population that has the same sensibility to the antibiotics, as did the parental one. Persisters formation is critically dependent on the growth phase. Despite the identification of a number of genes and pathways involved in persisters formation (toxin-antitoxin modules, SOS and stringent responses, efflux systems, metabolic functions and global regulators), the underlying molecular mechanisms are still poorly understood and controversial. In particular, persisters formation in Escherichia coli K-12 has been reported to be increased, decreased or not affected by a rpoS deletion. A better understanding of physiological parameters favoring persisters formation is critical to develop antipersisters strategies.

Succinate dehydrogenase (Sdh), a membrane bound complex that connects the TCA cycle and respiratory chain, is one major target down regulated by sigma S. Negative regulation by sigma S likely targets housekeeping genes that might be deleterious when fully expressed in quiescent cells. Understanding why full expression of those genes has a fitness cost might provide insights into survival mechanisms and weaknesses of quiescent cells, with potential application for antibacterial strategies. To tackle this issue, we used Sdh as a model system. Our study led us to unravel a synthetic rescue process of a sdh mutation in the formation of persisters. 

Stationary-phase Salmonella form persisters with a higher frequency than actively growing bacteria, after transfer to fresh medium in the presence of lethal concentrations of ampicillin and ciprofloxacin, but not significant effect of the rpoS deletion on this phenomenon was observed. Surprisingly however, the rpoS deletion suppressed the defect in persister formation of a sdh deletion mutant. Similar results were obtained with independent sdh and sdhrpoS constructs and the mutations did not significantly affect the minimum inhibitory concentration (MIC) of Salmonella for ampicillin and ciprofloxacin. It is very likely that the rpoS deletion compensates for a metabolic perturbation provoked by the sdh deletion, and key for persister formation. Since a sdh mutation also decreases persister formation by E. coli  and Staphylococcus aureus, the underlying physiological effect might be common to Gram-negative and Gram-positive bacteria. Understanding the molecular and physiological bases of this phenomenon should provide insights into key features driving persisters formation and revival. 

For references , see the STUDY pdf file.  

The persister assay is described in the PROTOCOL pdf file.

Strains used are described in the STRAINS AND PRIMERS .xlsx file.

Results are summarized in the STUDY pdf file 

For detailed data, see the M1 to M101 persisters .xlsx files. 

This work was supported by the French National Research Agency (ANR-19-CE44-0005-01, PERIOMET project).

See also:

NOREL Francoise, MONTEIL Veronique, DOUCHE Thibaut, & MATONDO Mariette. (2023). Global effects of deletion of the sdh genes, encoding succinate dehydrogenase, and of cobalt on protein abundance in stationary phase Salmonella enterica serovar Typhimurium. [Data set]. Zenodo. https://doi.org/10.5281/zenodo.8279681. 

Phégnon, L., Uttenweiler-Joseph, S., & Létisse, F. (2024). Key physiological and metabolic characteristics for the differentiation of quiescent Salmonella's cells into persisters [Data set]. Zenodo. https://doi.org/10.5281/zenodo.10885905