Magnesium Links Starvation-Mediated Antibiotic Persistence to ATP

Abstract

Bacterial persisters emerge and increase in numbers over time as a bacterial culture grows from log phase to stationary phase. However, the underlying basis of the inevitable tendency is unclear. In this study, we investigated the role of nutrients in starvation-mediated persister formation of Staphylococcus aureus. By screening of nutrient components, we found that starvation-induced persister formation of log-phase cultures could be reversed by addition of magnesium (Mg 2ϩ ) but not amino acids, nucleotides, or other salts. Further, deprivation of extracellular Mg 2ϩ reduced cytoplasmic ATP, inducing persistence without affecting cytoplasmic Mg 2ϩ or membrane potential. Finally, we showed that Mg 2ϩ reduced expression of stationary cell marker genes, cap5A and arcA. These findings indicate a connection between Mg 2ϩ levels and ATP, which represents metabolic status and mediates antibiotic persistence during growth. IMPORTANCE Various genes have been identified to be involved in bacterial persister formation regardless of the presence or absence of persister genes. Despite recent discoveries of the roles of ATP and membrane potential in persister formation, the key element that triggers change of ATP or membrane potential remains elusive. Our work demonstrates that Mg 2ϩ instead of other ions or nutrient components is the key element for persistence by inducing a decrease of cytoplasmic ATP, which subsequently induces persister formation. In addition, we observed tight regulation of genes for Mg 2ϩ transport in different growth phases in S. aureus. These findings indicate that despite being a key nutrient, Mg 2ϩ also served as a key signal in persister formation during growth.

Nramp: Deprive and conquer?

“…Though Nramp1 prevents death from fulminant infection with virulent Salmonella enterica serovar Typhimurium ( S. Typhimurium) bacteria remain carried chronically in the reticuloendothelial system (RES) for as long as 1 year after infection ( Monack et al, 2004 ). Paradoxically, limiting stress resistance of ingested bacteria by depriving the phagocytic vacuole of Mn may lead to survival of a few, competent for growth related processes requiring Mg as a co-factor (DNA and RNA synthesis, energy production and intracellular signaling), a situation analogous to reversion of starvation-induced persister formation ( Xu et al, 2020 ) and reminiscent of the role of Mg in regrowth of quiescent cells ( Metaane et al, 2022 ). In turn, Mg could appear rate-limiting for intravacuolar survival, which may explain why virulent S. Typhimurium feel starved for Mg, relying on the MgtB/MgtU importer system to survive within Slc11a1 + macrophages ( Groisman and Chan, 2021 ).…”

IraM Remodels the RssB Segmented Helical Linker to Stabilize σsagainst Degradation by ClpXP

“…At the other end of the spectrum, non-limiting Mg 2+ dampens starvation-induced persistence 32 , implicating pH sensing, Mg 2+. homeostasis, and thus IraM, in the development of antibiotic resistance and expression of virulence genes.…”

“…ClpXP is composed of an unfoldase – ATP-dependent hexameric ClpX, and of a sequestered fourteen-subunit peptidase – ClpP, which collaborate to unfold, translocate and then degrade substrates of remarkably diversity 1 . Prime targets of ATP-dependent proteolysis are sigma factors such as Streptococcus mutans σ X 2 , Streptomyces albus AntA 3 , Escherichia coli σ 32 4 , and especially Escherichia coli σ s , which was the first sigma factor to be identified as a ClpXP substrate 5-9 . These proteins play important roles in bacterial physiology as they serve as alternative promoter specificity subunits of RNA polymerase that effect wholesale shifts of gene expression profiles in response to developmental or environmental cues 10 .…”

“…In fact, some of the properties characteristic of clinical isolates of Pseudomonas aeruginosa can be induced by growth under Mg 2+ limitation 30 , and the importance of Mg 2+ homeostasis extends to other bacterial species 31 . At the other end of the spectrum, non-limiting Mg 2+ dampens starvation-induced persistence 32 , implicating pH sensing, Mg 2+. homeostasis, and thus IraM, in the development of antibiotic resistance and expression of virulence genes.…”

PurN Is Involved in Antibiotic Tolerance and Virulence in Staphylococcus aureus

“…Recent studies have identified several pathways involved in persister formation in S. aureus , such as biosynthesis of amino acids (ArgJ) [ 26 ]; purine biosynthesis metabolism (PurF, PurB, and PurM) [ 27 , 28 ]; energy production (CtaB, SucA, SucB, SdhA, and SdhB) [ 29 , 30 , 31 ]; glycerol metabolism [ 32 ]; protein degradation (ClpX) [ 31 ]; and phosphate metabolism (PhoU) [ 33 ]. Numerous studies have demonstrated that persister formation in stationary phase bacteria is significantly higher than that of the bacteria in the exponential phase [ 10 , 12 , 34 , 35 , 36 , 37 ]. This indicates that there may be differences in the mechanisms of persister formation at different growth phases.…”