The finding that antibiotics are pumped out of drug-tolerant bacterial cells by the TolC protein complex provides insight into how some cells, known as persisters, survive in the face of antibiotic treatments.
In bacterial persistence, a small fraction of an antibiotic-sensitive cell population has switched to a slow-growing or dormant state, and is drug tolerant1,2. This differs from antibiotic resistance in that regrowth of a persistent population results in the same percentage of drug-sensitive cells as before. Persistence has been interpreted as a bet-hedging strategy that increases the survival rates of bacterial populations3,4, and is medically relevant because it might sustain recurrent and chronic infections. Writing in Molecular Cell, Pu et al.5 challenge the widespread view that persistence is a passive state. The authors demonstrate that persister cells use an energy-dependent efflux pump protein called TolC to actively reduce the intracellular accumulation of antibiotic — a finding that might have both fundamental and therapeutic relevance.
Pu and colleagues isolated persisters and labelled them with a fluorescent antibiotic called BOCILLIN, which is derived from penicillin. They observed the cells using microscopy and found that the antibiotic could penetrate persister cells. However, the average antibiotic concentration in the persisters was about 20% of that in the drug-sensitive population.
TolC is the outer-membrane component of a family of efflux pumps that can move small molecules out of the cell from both the cytoplasm and the periplasmic space between the inner and outer bacterial membranes. Using sophisticated microfluidics combined with fluorescence microscopy, Pu et al. showed that TolC is responsible for the rapid export of BOCILLIN from persisters (Fig. 1). An alternative explanation could be that less of the antibiotic is taken up into cells in the first place, but the authors found that lower membrane permeability owing to depletion of porin proteins only slightly decreased BOCILLIN uptake. These observations raised the possibility that increased TolC levels contribute to drug tolerance in persisters.
Next, an analysis of cells in which TolC was labelled with a fluorescent dye called FlAsH revealed that persisters do have higher TolC levels than the drug-sensitive subpopulation. Moreover, when the authors isolated the subpopulation of cells with relatively high TolC levels, they found that this fraction contained nearly 20 times more persisters than the rest of the population. Thus, there is a clear correlation between a high level of TolC and persistence. The researchers then tracked cells using the FlAsH-labelled TolC: these experiments suggested that most persister cells emerged from a subpopulation that had increased levels of TolC even before treatment with the antibiotic. This important result warrants further study, and raises the question of whether the molecular mechanism that underlies the drug-independent variation of TolC is separate from, or an integral component of, other pathways that are already known to regulate stochastically induced persistence.
The current study leaves little doubt that TolC is involved in persistence. Most convincingly, perhaps, Pu and colleagues showed that deletion of the tolC gene or inhibition of TolC with a chemical compound drastically reduced the level of persisters. Because TolC is an outer-membrane protein, such inhibitors can readily access the protein. These observations raise the question of whether it might, in the future, be possible to develop therapeutic co-drugs that increase the efficacy of conventional antibiotics. These could be particularly useful for treating chronic and recurrent infections.
Many other genes have previously been implicated in bacterial persistence, including toxin–antitoxin (TA) genes. Most type II TA genes encode inhibitors of translation — their expression might therefore contribute to the dormancy of persisters2,3. Indeed, deletion of several type II TA genes significantly reduces persistence in the bacterium Escherichia coli6 and in a subspecies of Salmonella enterica7. The small membrane proteins encoded by type I TA genes can also induce persistence, by depolarizing the membrane, thereby reducing cellular levels of the energy-carrying molecule ATP and thus contributing to dormancy8.
Expression of both type I and II TAs is induced stochastically by the signalling molecules guanosine tetra- and pentaphosphate, and so the two classes might contribute synergistically to dormancy by reducing ATP levels and protein synthesis, respectively. How could the stochastic variation of TolC levels observed by Pu et al. fit into the regulatory scheme that controls type I and II TAs? Expression of the tolC gene is regulated by several transcriptional activators that respond to chemical compounds, including antibiotics, but if expression is also induced stochastically before chemical stress, TolC might act in concert with type I and II TAs to increase the drug tolerance of persisters. This would be the first example of an active mechanism contributing to stochastically induced multiple-drug tolerance. More research is required to resolve this exciting, outstanding question. Footnote 1
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