Maintenance and generation of proton motive force are both essential for expression of phenotypic antibiotic tolerance in bacteria.

Bacterial antibiotic tolerance, a phenomenon first observed in 1944, is known to be responsible for both onset and exacerbation of recurrent and chronic bacterial infections. The development of antibiotic tolerance was previously thought to be due to a switch to physiological dormancy when bacteria encounter adverse growth conditions. Our recent laboratory findings, however, showed that a set of genes related to the maintenance of proton motive force (PMF) are up-regulated under starvation, indicating that the tolerant sub-population, which are commonly known as persisters, can actively maintain their tolerance phenotypes. In this study, we investigated the relative functional roles of proteins involved in the maintenance and active generation of PMF in mediating tolerance formation in bacteria and found that the PspA and RcsB proteins play a key role in PMF maintenance in persisters, as deletion of genes encoding these two proteins resulted in significantly lower tolerance levels. Consistently, expression of the OsmC and Bdm proteins, which is under regulation by RcsB, is required to maintain PMF and the antibiotic tolerance phenotypes. On the other hand, the NuoL, Ndh, AppC, CyoB, and NuoF proteins, which are electron transport chain (ETC) components, were also found to be actively expressed in persisters in order to generate PMF to support functioning of various tolerance mechanisms such as efflux activities. Our data show that active generation of PMF is even more important than the PMF maintenance functions of PspA and RcsB in the expression of antibiotic tolerance phenotypes in persisters. Assessment of double- and triple-gene knockout strains, in which the PMF maintenance genes and those encoding ETC components were simultaneously deleted, confirms that these two groups of genes are both required for the expression of antibiotic tolerance phenotypes and that a lack of these functions would result in complete PMF dissipation and accumulation of antibiotics in the intracellular compartment of persisters and eventually cell death. Products of these genes are, therefore, ideal targets for future development of anti-tolerance agents. IMPORTANCE In this work, bacteria were found to undergo active generation and maintenance of proton motive force (PMF) under adverse conditions, such as starvation so as to support a range of physiological functions in order to survive under such conditions for a prolonged period. The ability to maintain a substantial level of PMF was found to be directly linked to that exhibiting phenotypic antibiotic tolerance under nutrient starvation or other adverse conditions. These findings infer that bacteria do not simply become physiologically dormant when they become antibiotic tolerant, instead they need to produce a wide range of proteins including those which help prevent PMF dissipation, such as PspA and RcsB, and the electron transport chain components, such as NuoL and Ndh, that actively generate PMF even during long-term starvation. As antibiotic tolerant sub-population is known to play a role in eliciting recurrent and chronic infections, especially among patients with a weakened immune system, the PMF maintenance mechanisms identified in this work are potential targets for the development of new strategies to control recurrent and chronic infections.

Membrane Transporters of the Major Facilitator Superfamily Are Essential for Long-Term Maintenance of Phenotypic Tolerance to Multiple Antibiotics in E. coli.

Antibiotic tolerance is not only the key underlying the cause of recurrent and chronic bacterial infections but it is also a factor linked to exacerbation of diseases, such as tuberculosis, cystic fibrosis-associated lung infection, and candidiasis. This phenomenon was previously attributed to a switch to physiological dormancy in a bacterial subpopulation triggered by environmental signals. However, we recently showed that expression of phenotypic antibiotic tolerance during nutrient starvation is highly dependent on robust production of proteins that actively maintain the bacterial transmembrane proton motive force (PMF), even under a nutrient-deficient environment. To investigate why PMF needs to be maintained for expression of phenotypic antibiotic tolerance, we tested the relative functional role of known transporters and efflux pumps in tolerance development by assessing the effect of deletion of specific efflux pump and transporter-encoding genes on long-term maintenance of antibiotic tolerance in an Escherichia coli population under starvation. We identified eight specific efflux pumps and transporters and two known efflux pump components, namely, ChaA, EmrK, EmrY, SsuA, NhaA, GadC, YdjK, YphD, TolC, and ChaB, that play a key role in tolerance development and maintenance. In particular, deletion of each of the nhaA and chaB genes is sufficient to totally abolish the tolerance phenotypes during prolonged antimicrobial treatment. These findings therefore depict active, efflux-mediated bacterial tolerance mechanisms and facilitate design of intervention strategies to prevent and treat chronic and recurrent infections due to persistence of antibiotic-tolerant subpopulations in the human body. IMPORTANCE We recently showed that the antibiotic-tolerant subpopulation of bacteria or persisters actively maintain the transmembrane proton motive force (PMF) to survive starvation stress for a prolonged period. This work further shows that the reason why antibiotic persisters need to maintain PMF is that PMF is required to support a range of efflux or transportation functions. Intriguingly, we found that tolerance-maintaining efflux activities were mainly encoded by 10 efflux or transporter genes. Because our study showed that deletion of even one of these genes could cause a significant reduction in tolerance level, we conclude that the products of these genes play an essential role in enhancing the survival fitness of bacteria during starvation or under other adverse environmental conditions. These gene products are therefore excellent targets for future design of antimicrobial agents that eradicate antibiotic tolerant persisters and prevent occurrence of chronic and recurrent human infections.

Active maintenance of proton motive force mediates starvation-induced bacterial antibiotic tolerance in Escherichia coli.

Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. In an attempt to investigate the range of starvation-induced physiological responses underlying tolerance development, we found that active maintenance of the transmembrane proton motive force (PMF) is essential for prolonged expression of antibiotic tolerance in bacteria. Eradication of tolerant sub-population could be achieved by disruption of PMF using the ionophore CCCP, or through suppression of PMF maintenance mechanisms by simultaneous inhibition of the phage shock protein (Psp) response and electron transport chain (ETC) complex activities. We consider disruption of bacterial PMF a feasible strategy for treatment of chronic and recurrent bacterial infections.