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 (Mg²⁺) but not amino acids, nucleotides, or other salts. Further, deprivation of extracellular Mg²⁺ reduced cytoplasmic ATP, inducing persistence without affecting cytoplasmic Mg²⁺ or membrane potential. Finally, we showed that Mg²⁺ reduced expression of stationary cell marker genes, cap5A and arcA These findings indicate a connection between Mg²⁺ 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 Mg²⁺ 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 Mg²⁺ transport in different growth phases in S. aureus These findings indicate that despite being a key nutrient, Mg²⁺ also served as a key signal in persister formation during growth.

Keywords: ATP; Staphylococcus aureus; antibiotic persistence; magnesium.

FIG 1

Magnesium dampens starvation-induced persistence. (a to d) Persister levels of S. aureus Newman log-phase cultures treated with saline or supernatant of stationary-phase cultures. Bacterial samples were treated with different antibiotics as described in the text for 8 h, and CFU counting was performed every 2 h. (e) Persister levels of Newman log-phase cultures treated with saline and different groups of HHWm. (f) Persister levels of Newman log-phase cultures treated with saline and different components from the major salt group of HHWm. (g) Levels of persisters against levofloxacin of Newman exponential cultures treated with saline and different concentration of Mg²⁺. (h) Levels of persisters against levofloxacin from Newman exponential cultures treated with EDTA and Mg²⁺ or Ca²⁺. (i) Levels of persisters against levofloxacin from exponential cultures of the yhdP mutant strain or Newman with induced asRNA against mgtE. Results are expressed as CFU count with comparison to untreated cultures. Data are the average results from at least two independent experiments, each with three biological replicates. Significant differences are indicated with asterisks: *, P < 0.05; **, P < 0.01 (two-way ANOVA). Standard deviations are represented with error bars.

FIG 2

ATP but not membrane potential mediates Mg²⁺-associated persistence. (a) Quantitation of cytoplasmic and extracellular concentrations of Mg²⁺ as well as cytoplasmic ATP in growth. (b) Detection of cytoplasmic Mg²⁺ and ATP levels of Newman strain treated with saline or EDTA. (c) Antisense RNA of mgtE decreases cytoplasmic Mg²⁺ and ATP. (d to f) Impacts of growth phase, saline, or EDTA on membrane potential of the Newman strain. The red fluorescence that indicates membrane potential was analyzed by fluorescence-activated cell sorting. sta, stationary phase; exp, exponential phase; CCCP, carbonyl cyanide m-chlorophenylhydrazone. (g) Mg represses expression of arcA and cap5A in Newman exponential cultures treated with saline or EDTA, detected by qRT-PCR. (h) Expression levels of mgtE and yhdP in different growth phases. Data represent the results from three independent experiments with standard deviations represented with error bars. Significant differences are indicated with asterisks: *, P < 0.05; **, P < 0.01 (two-way ANOVA); ND, no data.