Competition between lysogenic and sensitive bacteria is determined by the fitness costs of the different emerging phage-resistance strategies
Curation statements for this article:-
Curated by eLife
eLife assessment
The overarching question of the manuscript is important and the findings inform the patterns and mechanisms of phage-mediated bacterial competition, with implications for microbial evolution and antimicrobial resistance. The evidence in the manuscript is, however, still incomplete and some of the conclusions made are not supported by the data. This manuscript would additionally be strengthened by a clearer narrative, to enable readers to more easily extract the key message this paper wants to convey.
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- Evaluated articles (eLife)
- Evolutionary Biology (eLife)
Abstract
Many bacterial genomes carry prophages whose induction can eliminate competitors. In response, bacteria may become resistant by modifying surface receptors, by lysogenization, or by other poorly known processes. All these mechanisms affect bacterial fitness and population dynamics. To understand the evolution of phage resistance, we co-cultivated a phage-sensitive strain (BJ1) and a polylysogenic Klebsiella pneumoniae strain (ST14) under different phage pressures. The population yield remained stable after 30 days. Surprisingly, the initially sensitive strain remained in all populations and its frequency was highest when phage pressure was strongest. Resistance to phages in these populations emerged initially through mutations preventing capsule biosynthesis. Protection through lysogeny was rarely observed because the lysogens have increased death rates due to prophage induction. Unexpectedly, the adaptation process changed at longer time scales: the frequency of capsulated cells in BJ1 populations increased again because the production of the capsule was fine-tuned, reducing the ability of phage to absorb. Contrary to the lysogens, these capsulated-resistant clones are pan-resistant to a large panel of phages. Intriguingly, some clones exhibited transient non-genetic resistance to phages, suggesting an important role of phenotypic resistance in coevolving populations. Our results show that interactions between lysogens and sensitive strains are shaped by antagonistic co-evolution between phages and bacteria. These processes may involve key physiological traits, such as the capsule, and depend on the time frame of the evolutionary process. At short time scales, simple and costly inactivating mutations are adaptive, but in the long term, changes drawing more favorable trade-offs between resistance to phages and cell fitness become prevalent.
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Authoor Response
Reviewer #1 (Public Review):
This manuscript investigates the question of how polylysogeny impacts competition with a sensitive non-lysogen, and how this is shaped by phage resistance. This is an important and timely question, as lysogeny can be a strategy to invade new niches, and prophages are important vehicles for the acquisition of a range of virulence factors by pathogens including Klebsiella. The authors use a polylysogenic Klebsiella clone in competition with a non-lysogen that is sensitive to at least some of the prophages produced by the polylysogen. They compete these strains over a 30-day period and measure host population dynamics and evolution of phage resistance and lysogenic conversion in the (initially) sensitive competitor. Overall, the experiment shows that lysogen formation is relatively rare and …
Authoor Response
Reviewer #1 (Public Review):
This manuscript investigates the question of how polylysogeny impacts competition with a sensitive non-lysogen, and how this is shaped by phage resistance. This is an important and timely question, as lysogeny can be a strategy to invade new niches, and prophages are important vehicles for the acquisition of a range of virulence factors by pathogens including Klebsiella. The authors use a polylysogenic Klebsiella clone in competition with a non-lysogen that is sensitive to at least some of the prophages produced by the polylysogen. They compete these strains over a 30-day period and measure host population dynamics and evolution of phage resistance and lysogenic conversion in the (initially) sensitive competitor. Overall, the experiment shows that lysogen formation is relatively rare and short-lived. Instead, phage resistance through complete loss of the capsule is the primary mechanism evolving, but other resistant capsule mutants, with more subtle mutations affecting capsule expression, emerge as well. The authors have collected a very impressive amount of data and made some very interesting observations.
My main problem with this paper is that the manuscript lacks a clear narrative, making it very hard to extract the key message this paper wants to convey. Related to this, (some of) the conclusions that the authors make do not appear to be well supported by the data. For example, the authors conclude that selection favours more subtle capsule mutations because they are less costly than capsule-loss mutants (lines 497-500). However, there are no data to support this conclusion, as fitness costs of the various resistance phenotypes analysed were not measured. Apart from the genotypes, the data that are presented in this show that these subtle mutants have more subtle decreases in capsule production compared to the mutants that show a complete loss of capsule. But this does not tell us their relative cost. It also doesn’t tell us how the emergence of these different mutants relates to phage pressure, because whilst bacterial population dynamics data are monitored meticulously, phage dynamics data are missing (I have not found them in the supplemental information either). This makes it impossible to directly relate the emergence of the various resistance mechanisms to phage infection pressure during the coevolution experiment, even though this appears to be a hypothesis the authors wish to test.
Overall I think the overarching question of the manuscript is important and the model system is a very relevant one to study this question, but in my view, the current data don’t support the conclusions of the paper. Apart from these criticisms, the manuscript is very well written and the figures are overall easy to interpret.
We thank the reviewer for the critical assessment of our work and the time invested in the process. We have modified our manuscript following the recommendations, provided new data and we are convinced that our main results are now fully supported by the data.
Reviewer #2 (Public Review):
This manuscript presents data on multiple experiments regarding the co-evolution of poly-lysogenic and phage-susceptible Klebsiella pneumoniae strains. In particular, the manuscript aimed to determine the mechanisms of resistance that would shape bacterial competition over co-evolutionary timescales. The major finding is that the potential for lysogenization as a phage resistance mechanism is narrow and only likely to occur given certain circumstances. Moreover, the manuscript again reinforces the importance of receptor changes -initially loss, but modification in structure or expression over longer time scales- as a major mechanism of phage resistance that influences bacterial competition.
Strengths
A major strength of this manuscript is the care in designing experiments and conducting follow-up experiments to isolate the essential elements to support each of the conclusions. This includes using orthogonal methods such as sequencing and modeling to support or expand the findings from culturing and experimental evolution. The study features results that were beautifully replicated (e.g. Figure 3) lending confidence to the findings.
Weaknesses
Two weaknesses of the manuscript in its current form are: 1) a need to discuss other studies that also have found context-dependent results and 2) more focus on delivering the key overall "message" of the paper to the reader. Finally, not a weakness, but a (necessary) limitation is the study system, but this manuscript sets a bar for other groups to test in their systems to probe the generality of the findings.
The support for the conclusions is compelling. The findings were counter to the initial expectation (lysogenization as a major feature) and the manuscript does an admirable job of supporting the unexpected conclusion with thorough experimental work, supplemented with modeling.
This manuscript will be of great significance in microbial evolution, both for its implications in limiting the scope of lysogenization as a viable phage resistance mechanism in the long term and for its significant experimental rigor, particularly with regard to the co-evolutionary timescale studied. The study has very important implications for the evolution of antimicrobial resistance and phage therapy.
We thank the reviewer for the time spent and enthusiasm towards our experimental set-up.
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eLife assessment
The overarching question of the manuscript is important and the findings inform the patterns and mechanisms of phage-mediated bacterial competition, with implications for microbial evolution and antimicrobial resistance. The evidence in the manuscript is, however, still incomplete and some of the conclusions made are not supported by the data. This manuscript would additionally be strengthened by a clearer narrative, to enable readers to more easily extract the key message this paper wants to convey.
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Reviewer #1 (Public Review):
This manuscript investigates the question of how polylysogeny impacts competition with a sensitive non-lysogen, and how this is shaped by phage resistance. This is an important and timely question, as lysogeny can be a strategy to invade new niches, and prophages are important vehicles for the acquisition of a range of virulence factors by pathogens including Klebsiella. The authors use a polylysogenic Klebsiella clone in competition with a non-lysogen that is sensitive to at least some of the prophages produced by the polylysogen. They compete these strains over a 30-day period and measure host population dynamics and evolution of phage resistance and lysogenic conversion in the (initially) sensitive competitor. Overall, the experiment shows that lysogen formation is relatively rare and short-lived. …
Reviewer #1 (Public Review):
This manuscript investigates the question of how polylysogeny impacts competition with a sensitive non-lysogen, and how this is shaped by phage resistance. This is an important and timely question, as lysogeny can be a strategy to invade new niches, and prophages are important vehicles for the acquisition of a range of virulence factors by pathogens including Klebsiella. The authors use a polylysogenic Klebsiella clone in competition with a non-lysogen that is sensitive to at least some of the prophages produced by the polylysogen. They compete these strains over a 30-day period and measure host population dynamics and evolution of phage resistance and lysogenic conversion in the (initially) sensitive competitor. Overall, the experiment shows that lysogen formation is relatively rare and short-lived. Instead, phage resistance through complete loss of the capsule is the primary mechanism evolving, but other resistant capsule mutants, with more subtle mutations affecting capsule expression, emerge as well. The authors have collected a very impressive amount of data and made some very interesting observations.
My main problem with this paper is that the manuscript lacks a clear narrative, making it very hard to extract the key message this paper wants to convey. Related to this, (some of) the conclusions that the authors make do not appear to be well supported by the data. For example, the authors conclude that selection favours more subtle capsule mutations because they are less costly than capsule-loss mutants (lines 497-500). However, there are no data to support this conclusion, as fitness costs of the various resistance phenotypes analysed were not measured. Apart from the genotypes, the data that are presented in this show that these subtle mutants have more subtle decreases in capsule production compared to the mutants that show a complete loss of capsule. But this does not tell us their relative cost. It also doesn't tell us how the emergence of these different mutants relates to phage pressure, because whilst bacterial population dynamics data are monitored meticulously, phage dynamics data are missing (I have not found them in the supplemental information either). This makes it impossible to directly relate the emergence of the various resistance mechanisms to phage infection pressure during the coevolution experiment, even though this appears to be a hypothesis the authors wish to test.
Overall I think the overarching question of the manuscript is important and the model system is a very relevant one to study this question, but in my view, the current data don't support the conclusions of the paper. Apart from these criticisms, the manuscript is very well written and the figures are overall easy to interpret.
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Reviewer #2 (Public Review):
This manuscript presents data on multiple experiments regarding the co-evolution of poly-lysogenic and phage-susceptible Klebsiella pneumoniae strains. In particular, the manuscript aimed to determine the mechanisms of resistance that would shape bacterial competition over co-evolutionary timescales. The major finding is that the potential for lysogenization as a phage resistance mechanism is narrow and only likely to occur given certain circumstances. Moreover, the manuscript again reinforces the importance of receptor changes -initially loss, but modification in structure or expression over longer time scales- as a major mechanism of phage resistance that influences bacterial competition.
Strengths
A major strength of this manuscript is the care in designing experiments and conducting follow-up experiments …Reviewer #2 (Public Review):
This manuscript presents data on multiple experiments regarding the co-evolution of poly-lysogenic and phage-susceptible Klebsiella pneumoniae strains. In particular, the manuscript aimed to determine the mechanisms of resistance that would shape bacterial competition over co-evolutionary timescales. The major finding is that the potential for lysogenization as a phage resistance mechanism is narrow and only likely to occur given certain circumstances. Moreover, the manuscript again reinforces the importance of receptor changes -initially loss, but modification in structure or expression over longer time scales- as a major mechanism of phage resistance that influences bacterial competition.
Strengths
A major strength of this manuscript is the care in designing experiments and conducting follow-up experiments to isolate the essential elements to support each of the conclusions. This includes using orthogonal methods such as sequencing and modeling to support or expand the findings from culturing and experimental evolution. The study features results that were beautifully replicated (e.g. Figure 3) lending confidence to the findings.Weaknesses
Two weaknesses of the manuscript in its current form are: 1) a need to discuss other studies that also have found context-dependent results and 2) more focus on delivering the key overall "message" of the paper to the reader. Finally, not a weakness, but a (necessary) limitation is the study system, but this manuscript sets a bar for other groups to test in their systems to probe the generality of the findings.The support for the conclusions is compelling. The findings were counter to the initial expectation (lysogenization as a major feature) and the manuscript does an admirable job of supporting the unexpected conclusion with thorough experimental work, supplemented with modeling.
This manuscript will be of great significance in microbial evolution, both for its implications in limiting the scope of lysogenization as a viable phage resistance mechanism in the long term and for its significant experimental rigor, particularly with regard to the co-evolutionary timescale studied. The study has very important implications for the evolution of antimicrobial resistance and phage therapy.
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