Selection on plastic adherence leads to hyper-multicellular strains and incidental virulence in the budding yeast
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Curated by eLife
Evaluation Summary:
The origin of virulence in pathogenic microbes is not understood for many microbial species. The concept of 'accidental virulence' was proposed as a mechanism by which a microbe could acquire the capacity for virulence through interaction with other microbial species, such as amoeba. This paper adds an important new dimension to that concept by showing that the capacity for virulence can emerge from abiotic interactions, such as adherence to plastic.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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- Evolutionary Biology (eLife)
Abstract
Many disease-causing microbes are not obligate pathogens; rather, they are environmental microbes taking advantage of an ecological opportunity. The existence of microbes whose life cycle does not require a host and are not normally pathogenic, yet are well-suited to host exploitation, is an evolutionary puzzle. One hypothesis posits that selection in the environment may favor traits that incidentally lead to pathogenicity and virulence, or serve as pre-adaptations for survival in a host. An example of such a trait is surface adherence. To experimentally test the idea of ‘accidental virulence’, replicate populations of Saccharomyces cerevisiae were evolved to attach to a plastic bead for hundreds of generations. Along with plastic adherence, two multicellular phenotypes— biofilm formation and flor formation— increased; another phenotype, pseudohyphal growth, responded to the nutrient limitation. Thus, experimental selection led to the evolution of highly-adherent, hyper-multicellular strains. Wax moth larvae injected with evolved hyper-multicellular strains were significantly more likely to die than those injected with evolved non-multicellular strains. Hence, selection on plastic adherence incidentally led to the evolution of enhanced multicellularity and increased virulence. Our results support the idea that selection for a trait beneficial in the open environment can inadvertently generate opportunistic, ‘accidental’ pathogens.
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Author Response
Reviewer 2 (Public Review):
Weaknesses: The paper is largely written within the 'accidental virulence' framework, ref [2]. This is a valuable framework, but it is worth noting that the ideas overlap with the earlier concept of 'coincidental selection for virulence', first developed by Bruce Levin and C Svanborg-Eden (1990) Parasitology 100, S103-S115 (more recent experimental work in this thread is reviewed by ref [1]).
We wholeheartedly agree. As noted above, the manuscript has been updated to reflect this omission.
Missing this thread leads to a number of statements that are just not supported by the literature. Some examples -
Summary: 'The existence of microbes that are not normally pathogenic, yet are well-suited to host exploitation, is an evolutionary paradox. ' - No, this is potentially explained by …
Author Response
Reviewer 2 (Public Review):
Weaknesses: The paper is largely written within the 'accidental virulence' framework, ref [2]. This is a valuable framework, but it is worth noting that the ideas overlap with the earlier concept of 'coincidental selection for virulence', first developed by Bruce Levin and C Svanborg-Eden (1990) Parasitology 100, S103-S115 (more recent experimental work in this thread is reviewed by ref [1]).
We wholeheartedly agree. As noted above, the manuscript has been updated to reflect this omission.
Missing this thread leads to a number of statements that are just not supported by the literature. Some examples -
Summary: 'The existence of microbes that are not normally pathogenic, yet are well-suited to host exploitation, is an evolutionary paradox. ' - No, this is potentially explained by coincidental selection for virulence, which has been documented in several studies
Summary: "Our results support the idea that selection in the environment for a trait unrelated to virulence can inadvertently generate opportunistic, "accidental" pathogens." - The trait is not unrelated to virulence, as they are correlated, likely shaped by adhesion behaviors. This correlation is not surprising as 'adhesins' are a classic category of virulence factor, presenting a potential common cause between sticking to a bead and killing an insect more rapidly.
Line 26 "hypothesis has not been directly tested experimentally". - this is probably the main concern, as the paper currently does not address related prior experimental work that has been developed within the co-incidental selection tradition. Please see refs that are cited by paper [1] for a start, and then follow forward for more recent work. One recent study comes to mind as it looks at correlated effects of in vitro bead attachment -- https://www.nature.com/articles/s41396020-0652-0 (virulence was not directly assayed, but of interest also noted a shift in antibiotic resistance following bead-attachment selection without drugs or a host).
We agree with these suggested edits and have updated the manuscript in the appropriate places.
Turning to experimental choices, the use of a 'no bead' experimental control is an important point of comparison, to ensure that the evolutionary effects of interest are particular to the presence of the bead. But if there were no beads, how are you measuring 'cells on bead' (y-axis in Figure 1)? I assume this is an oversight and you're measuring cells per x volume.
This is a good question. To be clear, Figure 1 does not represent measurements taken throughout the course of the experiment; rather, it represents a large phenotyping effort after the experiment ended. Ancestors and evolved populations from multiple timepoints (including the control populations) were started from cryopreserved stocks, then challenged to grow in the presence of a bead. As can be seen in the figure, both ancestors had some plastic adherence ability, which was maintained in the control populations.
Moving into the key virulence assays, I was expecting a similar and simple design: compare the virulence of ancestor versus 'evolved with bead' versus 'evolved without bead'. This would allow answers to the key question of whether bead attachment leads to the evolution of increased virulence, with appropriate controls for adaptation to the general passaging environment. Why not use this simple and standard design?
Instead, we get a more complex design, contrasting isolates that are filtered on the adhesionrelated traits (biofilm, etc), but sampled across timepoints. This does establish that less adhesive and less biofilmy isolates are less virulent so this remains useful information, but the motivation for only using this design is not well spelled out. In principle, you could do this purely on standing variation and not require an experimental evolution step.
We understand and respect this criticism. Please see the response above (in the section responding to the editor’s summary).
As for the question of using standing variation, it is true that a large part of the evolution observed in this experiment is from the sorting of standing genetic variation. We did not anticipate the evolved phenotypes we observed. Perhaps if we had known they were possible, we could have searched for them in the standing genetic variation in F1 offspring/segregants. Related to this idea, we have investigated 350+ segregants from each of these clinical backgrounds (that were generated in order to map the genetic basis of plastic adherence for a manuscript in preparation). The evolved populations occupy different phenotypic space than the mapping population, although there is obviously overlap. Thus, in order to get to the hypermulticellular phenotype observed in the experiment, either multiple rounds of recombination were required to get many high alleles into one background, or new mutations were required.
Concerning the role of plastic, I would encourage caution in the interpretation, given the experimental design. Consider this line from the discussion "In this experiment, favoring the ability to adhere to plastic, a surface that is alarmingly common in industrial, medical, and domestic settings [69], led to a suite of aggregative phenotypes and increased virulence." - by bringing up applied consequences of plastic exposure, this really raises the stakes. At present, the data does not separate the role of bead attachment from the specific role of plastic as a material. What would happen if you repeated with glass beads? I suspect a similar pattern, again driven by adhesin changes. The data at present does not resolve this issue.
We respect this note of caution, which is in opposition to Reviewer #1, who thinks we should add more information about the increase in microplastics. We agree that the results are likely due to selection for adherence, rather than specifically adherence to plastic. That being said, the experiment does show that plastic is a surface on which these yeast can be selected to adhere. And it is also true that this surface is increasingly common. As a compromise, we took out the word alarming and added references to the effect of microplastics on other microbes.
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Evaluation Summary:
The origin of virulence in pathogenic microbes is not understood for many microbial species. The concept of 'accidental virulence' was proposed as a mechanism by which a microbe could acquire the capacity for virulence through interaction with other microbial species, such as amoeba. This paper adds an important new dimension to that concept by showing that the capacity for virulence can emerge from abiotic interactions, such as adherence to plastic.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
While some microbes have evolved to need an animal host for completion of the life cycle or survival, termed "obligate pathogens" (i.e. Plasmodium falciparum, viruses, Neisseria gonorrhoeae), there are many bacteria and fungi that can cause disease in insect or mammalian hosts, but can survive okay in the environment and do not need a host in order to survive, persist, and replicate. These microbes are often described as "accidental pathogens," where they evolved in the absence of a host to survive durable conditions in the environment, and only caused disease when they are accidentally exposed to a host (i.e. breathing in spores or traumatic inoculation). In this work, the authors set out to determine whether selection for Saccharomyces cerevisiae's ability to bind plastic in the absence of a host resulted …
Reviewer #1 (Public Review):
While some microbes have evolved to need an animal host for completion of the life cycle or survival, termed "obligate pathogens" (i.e. Plasmodium falciparum, viruses, Neisseria gonorrhoeae), there are many bacteria and fungi that can cause disease in insect or mammalian hosts, but can survive okay in the environment and do not need a host in order to survive, persist, and replicate. These microbes are often described as "accidental pathogens," where they evolved in the absence of a host to survive durable conditions in the environment, and only caused disease when they are accidentally exposed to a host (i.e. breathing in spores or traumatic inoculation). In this work, the authors set out to determine whether selection for Saccharomyces cerevisiae's ability to bind plastic in the absence of a host resulted in enhanced virulence when the yeast was exposed to a host. In doing so, they would be experimentally showing that adaptation to environmental conditions without a host, can "accidentally" produce pathogens. In this work, the authors indeed demonstrated that S. cerevisiae which had been selected for its ability to adhere to plastics evolved multicellular phenotypes and enhanced virulence in the Galleria mellonella wax moth host. In doing so, they effectively show experimental proof of the accidental virulence hypothesis, which can serve as the basis for future studies to better understand existing non-obligate microbes, and how new changes/exposures in the environment can cause the adaptation of microbes, which may result in the emergence of new "accidental" human pathogens. The work also investigated the evolutionary relationship between the different forms of multicellularity and uncovered that there was a strong correlation between the emergence of multiple multicellular phenotypes over cycles of plastic adhesion selection, which is a new finding compared to previous reports that these phenotypes in the environment or in laboratory/mutant strains are independent.
The conclusions drawn in this paper are well supported by the data, and the experiments are well-designed and straightforward and presented in a way that is generally able to be understood. This study is of great interest to both microbiologists who study how microbes adapt to the environment and those who study microbes in the context of infectious disease. This study experimentally proves the tenet of the accidental virulence hypothesis that adaptation/evolutionary selection to environmental factors may incidentally enhance the ability to survive within hosts.
Additionally, the authors conducted the experiments in a robust, well-controlled manner, and systematically analyzed the multicellular phenotypes. They assayed the multicellular phenotypes in a clearly defined manner and were able to characterize the traits of a large number of individual clones grown encompassing different timepoints, sexual and asexual reproduction, strains, and selection. Additionally, their virulence studies in G. mellonella show an extraordinary amount of work, which allowed them to see the increase in virulence (~30%) in the hyper-multicellular phenotype after performing infections using 40 different isolates from the experiment.
The authors also sought to find a genetic basis for the increase of multicellular phenotypes and virulence following plastic adherence selection. To do so, they studied the length of the FLO11 gene in their S. cerevisiae isolates. FLO11 length had been previously implicated in fungal adhesion and fungal virulence, which could offer a reason for the enhanced virulence phenotype following selection for plastic adherence. While their findings show the ancestral and control isolates did not have increased FLO11 length while many of the plastic-selected isolates did, there was not a definitive correlation between the multicellular phenotypes and FLO11 length. The authors did not pursue an additional investigation into the genetic basis of these adaptations.
Two aspects the reader must consider are the host and the microbe used in these virulence experiments. S. cerevisiae is an interesting choice to use because it is not considered to be a "pathogenic" microbe, although there are rare cases in which it can cause disease in humans. However, previous studies have shown S. cerevisiae has been shown to cause disease in G. mellonella, which can also be seen in the survival curves presented in this paper. Therefore, using G. mellonella in this study shows that the selection process resulted in increased virulence in the host. To strongly show the accidental virulence/pathogen hypothesis, a host-microbe pair where disease does not normally/typically occur could be used. In the context of this study, it could be done using a mouse model infected with S.cerevisiae. In doing so, it could show that environmental selection in the absence of a host has the ability to turn a "non-disease-causing microbe" into a disease-causing one, rather than a disease-causing microbe into a more disease-causing microbe. This could also make a more applicable leap to human infectious diseases.
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Reviewer #2 (Public Review):
The authors experimentally evolve yeast in with and without selection for attachment to plastic beads. The bead selected lines show an increase in surface attachment, and in a set of correlated traits including biofilm complexity and virulence in an insect model. The results support the conclusion that increased virulence can be selected in a non-host environment.
Strengths: The experimental evolution of virulence literature skews disproportionately towards bacteria, so it is very welcome to see a eukaryotic pathogen take a turn.
Overall, the results show a pattern that experimental evolution on bead attachment can drive adhesion-related traits (biofilm, flors, virulence) towards higher levels, compared to the ancestor.
These are useful results, but concerns remain over some of the experimental and analysis …
Reviewer #2 (Public Review):
The authors experimentally evolve yeast in with and without selection for attachment to plastic beads. The bead selected lines show an increase in surface attachment, and in a set of correlated traits including biofilm complexity and virulence in an insect model. The results support the conclusion that increased virulence can be selected in a non-host environment.
Strengths: The experimental evolution of virulence literature skews disproportionately towards bacteria, so it is very welcome to see a eukaryotic pathogen take a turn.
Overall, the results show a pattern that experimental evolution on bead attachment can drive adhesion-related traits (biofilm, flors, virulence) towards higher levels, compared to the ancestor.
These are useful results, but concerns remain over some of the experimental and analysis choices and the integration of the results into the literature.
Weaknesses: The paper is largely written within the 'accidental virulence' framework, ref [2]. This is a valuable framework, but it is worth noting that the ideas overlap with the earlier concept of 'coincidental selection for virulence', first developed by Bruce Levin and C Svanborg-Eden (1990) Parasitology 100, S103-S115 (more recent experimental work in this thread is reviewed by ref [1]).
Missing this thread leads to a number of statements that are just not supported by the literature. Some examples -
Summary: 'The existence of microbes that are not normally pathogenic, yet are well-suited to host exploitation, is an evolutionary paradox. ' - No, this is potentially explained by coincidental selection for virulence, which has been documented in several studies
Summary: "Our results support the idea that selection in the environment for a trait unrelated to virulence can inadvertently generate opportunistic, "accidental" pathogens." - The trait is not unrelated to virulence, as they are correlated, likely shaped by adhesion behaviors. This correlation is not surprising as 'adhesins' are a classic category of virulence factor, presenting a potential common cause between sticking to a bead and killing an insect more rapidly.
Line 26 "hypothesis has not been directly tested experimentally". - this is probably the main concern, as the paper currently does not address related prior experimental work that has been developed within the co-incidental selection tradition. Please see refs that are cited by paper [1] for a start, and then follow forward for more recent work. One recent study comes to mind as it looks at correlated effects of in vitro bead attachment -- https://www.nature.com/articles/s41396-020-0652-0 (virulence was not directly assayed, but of interest also noted a shift in antibiotic resistance following bead-attachment selection without drugs or a host).
Line 15 "Intricate co-evolution is a requirement of microbial pathogenesis and virulence". This is outside of the scope of coincidental selection but I would note that while there are models of virulence that invoke co-evolution, this is definitely not a requirement. It is not even the mainstream of virulence evolution theory (most models posit evolution in the pathogen alone, eg virulence/transmission tradeoff models, shortsighted evolution models, etc).
Turning to experimental choices, the use of a 'no bead' experimental control is an important point of comparison, to ensure that the evolutionary effects of interest are particular to the presence of the bead. But if there were no beads, how are you measuring 'cells on bead' (y-axis in Figure 1)? I assume this is an oversight and you're measuring cells per x volume.
Moving into the key virulence assays, I was expecting a similar and simple design: compare the virulence of ancestor versus 'evolved with bead' versus 'evolved without bead'. This would allow answers to the key question of whether bead attachment leads to the evolution of increased virulence, with appropriate controls for adaptation to the general passaging environment. Why not use this simple and standard design?
Instead, we get a more complex design, contrasting isolates that are filtered on the adhesion-related traits (biofilm, etc), but sampled across timepoints. This does establish that less adhesive and less biofilmy isolates are less virulent so this remains useful information, but the motivation for only using this design is not well spelled out. In principle, you could do this purely on standing variation and not require an experimental evolution step.
Concerning the role of plastic, I would encourage caution in the interpretation, given the experimental design. Consider this line from the discussion "In this experiment, favoring the ability to adhere to plastic, a surface that is alarmingly common in industrial, medical, and domestic settings [69], led to a suite of aggregative phenotypes and increased virulence." - by bringing up applied consequences of plastic exposure, this really raises the stakes. At present, the data does not separate the role of bead attachment from the specific role of plastic as a material. What would happen if you repeated with glass beads? I suspect a similar pattern, again driven by adhesin changes. The data at present does not resolve this issue.
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Author Response
We appreciate the thoughtful and thorough critique provided by the two reviewers, and generally agree with their assessment. The revised submission will address the issues they raise. In particular, we agree that the framework of the paper should be broadened to include bacteria and the deep literature associated with coincidental selection.
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