Heterogeneous efflux pump expression underpins phenotypic resistance to antimicrobial peptides

Reviewer #1 (Public Review):

Summary:

This work contributes several important and interesting observations regarding the heterotolerance of non-growing Escherichia coli and Pseudomonas aeruginosa to the antimicrobial peptide tachyplesin. The primary mechanism of action of tachyplesin is thought to be disruption of the bacterial cell envelope, leading to leakage of cellular contents after a threshold level of accumulation. Although the MIC for tachyplesin in exponentially growing E. coli is just 1 ug/ml, the authors observe that a substantial fraction of a stationary phase population of bacteria survive much higher concentrations, up to 64 ug/ml. By using a fluorescently-labelled analogue of tachyplesin, the authors show that the amount of per-cell intracellular accumulation of tachyplesin displays a bimodal distribution and that the fraction of "low accumulators" correlates with the fraction of survivors.

Using a microfluidic device, they show that low accumulators exclude propidium iodide, suggesting that their cell envelopes remain largely intact, while high accumulators of tachyplesin also stain with propidium iodide. They show that this phenomenon holds for several clinical isolates of E. coli with different genetic determinants of antibiotic resistance, and for a strain of Pseudomonas aeruginosa. However, the bimodal distribution does not occur in these organisms for several other antimicrobial peptides, or for tachyplesin in Klebsiella pneumoniae or Staphylococcus aureus, indicating some degree of specificity in the interaction between AMP and bacterial cell envelope. They next explore the dynamics of the fluorescent tachyplesin accumulation and show interestingly that a high degree of accumulation is initially seen in all cells, but that the "low accumulator" subpopulation manages to decrease the amount of intracellular fluorescence over time, while the "high accumulator" subpopulation continues to increase its intracellular fluorescence. Focusing on increased efflux as a hypothesised mechanism for the "low accumulator" phenotype, based on transcriptomic analysis of the two subpopulations, the authors screen putative efflux inhibitors to see if they can block the formation of the low accumulator subpopulation. They find that both the protonophore CCCP and the SSRI sertraline can block the formation of this subpopulation and that a combination of sertraline plus tachyplesin kills a greater fraction of the stationary phase cells than either agent alone, similar to the killing observed when growing cells are treated with tachyplesin.

Strengths:

This study provides new insight into the heterogeneous behaviours of non-growing bacteria when exposed to an antimicrobial peptide, and into the dynamics of their response. The single-cell analysis by FACS and microscopy is compelling. The results provide a much-needed single-cell perspective on the phenomenon of tolerance to AMPs and a good starting point for further exploration.

Weaknesses:

My main concerns surround the conclusions drawn about the physiological underpinnings of these behaviours, based in part on transcriptomic analysis and also on the observation of the dynamics. I think deeper consideration of the relative contributions of influx and efflux to the observed accumulation dynamics, and the slow/non-growing context of the observations would be helpful. In particular, these issues seem important:

(1) The initial high accumulation by all cells followed by the emergence of a sub-population that has reduced its intracellular levels of tachyplesin is a key observation and I agree with the authors' conclusion that this suggests an induced response to the AMP is important in facilitating the bimodal distribution. However, I think the conclusion that upregulated efflux is driving the reduction in signal in the "low accumulator" subpopulation is not fully supported. Steady-state amounts of intracellular fluorescent AMP are determined by the relative rates of influx and efflux and a decrease could be caused by decreasing influx (while efflux remained unchanged), increasing efflux (while influx remained unchanged), or both decreasing influx and increasing efflux. Given the transcriptomic data suggest possible changes in the expression of enzymes that could affect outer membrane permeability and outer membrane vesicle formation as well as efflux, it seems very possible that changes to both influx and efflux are important. The "efflux inhibitors" shown to block the formation of the low accumulator subpopulation have highly pleiotropic or incompletely characterised mechanisms of action so they also do not exclusively support a hypothesis of increased efflux.

(2) A conclusion of the transcriptomic analysis is that the lower accumulating subpopulation was exhibiting "a less translationally and metabolically active state" based on less upregulation of a cluster of genes including those involved in transcription and translation. This conclusion seems to borrow from well-described relationships referred to as bacterial growth laws in which the expression of genes involved in ribosome production and translation is directly related to the bacterial growth (and metabolic) rate. However, the assumptions that allow the formulation of the bacterial growth laws (balanced, steady state, exponential growth) do not hold in growth arrest. A non-growing cell could express no genes at all or could express ribosomal genes at a very low level, or efflux pumps at a high level. The distribution of transcripts among the functional classes of genes does not reveal anything about metabolic rates within the context of growth arrest - it only allows insight into metabolic rates when the constraint of exponential growth can be assumed. Efflux pumps can be highly metabolically costly; for example, Tn-Seq experiments have repeatedly shown that mutants for efflux pump gene transcriptional repressors have strong fitness disadvantages in energy-limited conditions. There are no data presented here to disprove a hypothesis that the low accumulators have high metabolic rates but allocate all of their metabolic resources to fortifying their outer membranes and upregulating efflux. This could be an important distinction for understanding the vulnerabilities of this subpopulation. Metabolic rates can be more directly estimated for single cells using respiratory dyes or pulsed metabolic labelling, for example, and these data could allow deeper insight into the metabolic rates of the two subpopulations.

The observation that adding nutrients to the stationary phase cultures pushes most of the cells to the "high accumulator" state is presented as support of the hypothesis that the high accumulator state is a higher metabolism/higher translational activity state. However, it is important to note that adding nutrients will cause most or all of the cells in the population to start to grow, thus re-entering the familiar regime in which bacterial growth laws apply. This is evident in the slightly larger cell sizes seen in the nutrient-amended condition. In contrast to stationary phase cells, growing cells largely do not exhibit the bimodal distribution, and they are much more sensitive to tachyplesin, as demonstrated clearly in the supplement. Growing cells are not necessarily the same as the high-accumulating subpopulation of non-growing cells.

It might also be worth adding some additional context around the potential to employ efflux inhibitors as therapeutics. It is very clear that obtaining sufficient antimicrobial drug accumulation within Gram-negative bacteria is a substantial barrier to effective treatments, and large concerted efforts to find and develop therapeutic efflux pump inhibitors have been undertaken repeatedly over the last 25 years. Sufficiently selective inhibitors of bacterial efflux pumps with appropriate drug-like properties have been challenging to find and none have entered clinical trials. Multiple psychoactive drugs have been shown to impact efflux in bacteria but usually using concentrations in the 10-100 uM range (as here). Meanwhile, the Ki values for their human targets are usually in the sub- to low-nanomolar range. The authors rightly note that the concentration of sertraline they have used is higher than that achieved in patients, but this is by many orders of magnitude, and it might be worth expanding a bit on the substantial challenge of finding efflux inhibitors that would be specific and non-toxic enough to be used therapeutically. Many advances in structural biology, molecular dynamics, and medicinal chemistry may make the quest for therapeutic efflux inhibitors more fruitful than it has been in the past but it is likely to remain a substantial challenge.

Reviewer #2 (Public Review):

Summary:

This study reports on the existence of subpopulations of isogenic E. coli and P. aeruginosa cells that are tolerant to the antimicrobial peptide tachyplesin and are characterized by the accumulation of low levels of a fluorescent tachyplesin-NBD conjugate. The authors then set out to address the molecular mechanisms, providing interesting insights even though the mechanism remains incompletely defined: The work suggests that amongst others changes in membrane lipid composition and increased drug efflux may cause this phenotype and it demonstrates that pharmacological manipulation can prevent generation of tolerance. The authors are cautious in their interpretation and the claims made are largely justified by the data.

Strengths:

Going beyond the commonly used bulk techniques for studying susceptibility to AMPs , Lee et al. used fluorescent antibiotic conjugates in combination with flow cytometry analysis to study variability in drug accumulation at the single-cell level. This powerful approach enabled the authors to expose bimodal drug accumulation patterns that were condition-dependent, but conserved across a variety of E. coli clinical isolates. Using cell sorting in combination with colony-forming unit assays as well as quantitative fluorescence microscopic analysis in a microfluidics setup the authors compellingly demonstrate that low accumulators (where the fluorescence signal is mostly restricted to the membrane), can survive antibiotic treatment, whereas high accumulators (with high intracellular fluorescence) were killed. Comparative transcriptomics analysis of sorted ´low accumulator´ and ´high accumulator´ subpopulations suggest that changes in the lipid composition, increased efflux, and other mechanisms may contribute to tachyplesin-tolerance in this subpopulation. Lipidomics analysis of bulk untreated vs. tachyplesin-NBD treated cells confirmed changes in the lipid composition in accordance with the transcriptomics data. Intriguingly, a time-course experiment on tachyplesin-NBD accumulation revealed that all cells initially were high accumulators, before a subpopulation of cells subsequently managed to reduce the signal intensity (most likely through efflux), demonstrating that the ´low accumulator´ phenotype is an induced response and not a pre-existing property.

Finally, the demonstration that treatment with efflux pump inhibitors (although some caution needs to be taken regarding the selectivity of these inhibitors, see comment on weaknesses below) prevents the generation of low accumulators and enhances tachyplesin-based killing is an important basis for developing combination therapies.

The study convincingly illustrates how susceptibility to tachoplesin adaptively changes in a heterogeneous way dependent on the growth phases/ environments and availability of nutrients. This is highly relevant also beyond the presented example of tachyplesin and similar subpopulation-based adaptive changes to the susceptibility towards antimicrobial peptides or other drugs that may occur during infections in vivo and they would likely be missed out by standardized in vitro susceptibility testing.

Weaknesses:

Some questions regarding the mechanism remain. One shortcoming of the setup of the transcriptomics experiment is that the tachyplesin-NBD probe itself has antibiotic efficacy and induces phenotypes (and eventually cell death) in the ´high accumulator´cells. This makes it challenging to interpret whether any differences seen between the two groups are causative for the observed accumulation pattern or if they are a consequence of differential accumulation and downstream phenotypic effects. The role of efflux systems is further supported by the finding that efflux pump inhibitors sensitize E. coli to tachyplesin and prevent the occurrence of the tolerant ´low accumulator´ subpopulations. In principle, this is a great way of validating the role of efflux pumps, but the limited selectivity of these inhibitors (CCCP is an uncoupling agent, and for sertraline direct antimicrobial effects on E. coli have been reported by Bohnert et al.) leaves some ambiguity as to whether the synergistic effect is truly mediated via efflux pump inhibition. It would be relevant to test and report the MIC of sertraline for the strain tested, particularly since in Figure 4G an initial reduction in CFUs is observed for sertraline treatment, which suggests the existence of biological effects in addition to efflux inhibition.

Reviewer #3 (Public Review):

Summary:

The study tests the phenotypic response of bacteria (mainly E. coli) to antimicrobial peptides (AMPs) such as tachyplesin. The resistance mechanisms to AMPs differ from those to classical antibiotics in that AMP resistance involves more non-genetic mechanisms, which are largely unknown but are important to understand. This work aims to elucidate the mechanism of such phenotypic resistance.

Strengths:

The experiments unambiguously reveal that the cells respond to stress heterogeneously, with two distinct subpopulations - one with better survival than the other. This primary phenotype is convincingly shown across various E. coli strains, including clinical isolates.

Weaknesses:

The authors' claims about high efflux being the main mechanism of survival are unconvincing, given the current data. There can be several alternative hypotheses that could explain their results, such as lower binding of the AMP, lower rate of internalization, metabolic inactivity, etc. It is unclear how efflux can be important for survival against a peptide that the authors claim binds externally to the cell. The addition of efflux assays would be beneficial for clear interpretations. Further genetic experiments are necessary to test whether efflux genes are involved at all.