Impacts of Global Transcriptional Regulators on Persister Metabolism

Abstract

Bacterial persisters are phenotypic variants with an extraordinary capacity to tolerate antibiotics, and they are hypothesized to be a main cause of chronic and relapsing infections. Recent evidence has suggested that the metabolism of persisters can be targeted to develop therapeutic countermeasures; however, knowledge of persister metabolism remains limited due to difficulties associated with isolating these rare and transient phenotypic variants. By using a technique to measure persister catabolic activity, which is based on the ability of metabolites to enable aminoglycoside (AG) killing of persisters, we investigated the role of seven global transcriptional regulators (ArcA, Cra, cyclic AMP [cAMP] receptor protein [CRP], DksA, FNR, Lrp, and RpoS) on persister metabolism. We found that removal of CRP resulted in a loss of AG potentiation in persisters for all metabolites tested. These results highlight a central role for cAMP/CRP in persister metabolism, as its perturbation can significantly diminish the metabolic capabilities of persisters and effectively eliminate the ability of AGs to eradicate these troublesome bacteria. Bacterial persisters are phenotypic variants that are highly tolerant to antibiotics (1). It is believed that they are a main culprit of the proclivity of biofilm infections to relapse, which imposes a substantial burden on the health care system (2, 3). When a bacterial population is treated with bactericidal antibiotics, biphasic killing is observed, where the death of normal cells is characterized by an initial, rapid killing rate, and the presence of persisters is illuminated by a second regime with a much lower rate of cell death (4, 5). When these survivors are recultured, the resulting population exhibits antibiotic sensitivity identical to that of the original culture, demonstrating that persisters are not antibiotic-resistant mutants but phenotypic variants (1, 5, 6).While persisters largely arise from dormant subpopulations (2,4,7,8), recent studies have demonstrated that they remain metabolically active, with the capacity to catabolize specific carbon sources and generate proton motive force through respiration (9, 10). This metabolic activity, specifically, proton motive force generation, enables aminoglycoside (AG) transport into cells that results in killing of persisters, and several enzymes needed for this process have been identified (9, 10). Knowledge of the enzymes and metabolic pathways present in persisters, as well as how they can be altered, could prove useful for the development of antipersister therapies. A fundamental question in this regard is, what are the cellular components responsible for defining the metabolic network in persisters? Due to the strong dependence of metabolism on transcriptional regulation (11, 12), the goal of this study was to determine the importance of several global transcriptional regulators to persister metabolism. To do this, we measured catabolic activity in persisters from ⌬arcA, ⌬cra, ⌬crp, ⌬dksA, ⌬fnr, ⌬lrp, and ⌬rpoS mutants and...

Comparative testing of disinfectant efficacy on planktonic bacteria and bacterial biofilms using a new assay based on kinetic analysis of metabolic activity

“…Although bacterial persisters in biofilms are not necessarily completely metabolically inactive (Mok et al . ; Silva‐Herzog et al . ), culture‐based assays neither detect inactive persisters nor persisters that are metabolically active but not growing.…”

“…However, this limitation is shared with colony-counting techniques. Although bacterial persisters in biofilms are not necessarily completely metabolically inactive (Mok et al 2015;Silva-Herzog et al 2015), culture-based assays neither detect inactive persisters nor persisters that are metabolically active but not growing. In contrast, our technique is based on the detection of metabolic activity over a prolonged observation time and therefore is able to detect metabolic activity even in cells that are not dividing, including persisters.…”

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle

“…It is also important to note that bacterial persistence with tolerance to antimicrobials may be generated by multiple and redundant mechanisms involving both regulatory and non‐regulatory genes (Lewis, ; Zhang, ; Amato and Brynildsen, ; Feng et al ., ; Mok et al ., ; Brauner et al ., ; Kaldalu et al ., ; Michiels et al ., ). In E .…”

In Vitro Emergence of High Persistence upon Periodic Aminoglycoside Challenge in the ESKAPE Pathogens

“…Recent studies have investigated the role of global regulators and the genetic basis of aminoglycoside persistence in E. coli (58,59). Analysis of the genetic basis for the high-persistence phenotype of the isolated clones is outside the scope of this report, but based on our and others' previous work (30,50,51), we expect that the high-persistence phenotype results from only one or a few discrete mutations.…”