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We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

Distinct pathways of adaptive evolution in Cryptococcus neoformans reveal a point mutation in adenylate cyclase with drastic tradeoffs for pathogenicity

Zoë A. Hilbert^1,3*, Krystal Y. Chung², Joseph M. Bednarek², Mara W. Schwiesow^1,3,4, Jessica C.S. Brown², Nels C. Elde^1,3*

doi: https://doi.org/10.1101/2022.09.27.509772   We will adhere to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

SUMMARY: The genetic diversity of pathogenic microorganisms is shaped by host-pathogen interactions. Rapid adaptation of viral and bacterial pathogens to new hosts is well documented but studies of environmentally derived pathogenic fungi are limited. Our understanding on how host-fungi interaction affects pathogenesis remains unclear. Here, Hilbert et al., investigated how the evolution of the environmentally derived pathogen Cryptococcus neoformans has been shaped by interactions with host cells. The authors evaluated the ability of 14 clinical and environmental isolates of C. neoformans to replicate in amoebae (Acanthamoeba castellanii) or J774A.1 cell lines (macrophage-like cells). Notably, the environmental isolate Ftc555-1 could replicate in macrophages but could not survive in cell culture media alone. The authors leveraged this phenotype to study the evolution of C. neoformans by host selective pressures in the absence of extracellular fungal growth. They performed serial in vitro passages of the C. neoformans Ftc555-1 strain in either amoeba or macrophages for a total of 12 passages to generate the adapted strains for each type of host cell. The authors noted that one of the adapted strains, known as M1, that was recovered from the serial passaging in macrophages showed a strong host-adapted phenotype with 5-fold increase in replication in macrophages compared to its parental strain. Whole genome sequencing of the M1 strain revealed that only a single nucleotide polymorphism in the adenylate cyclase gene CAC1 was acquired and swept to fixation in the final passage of the experimental evolution, which resulted in an Arg1227Pro substitution in the enzyme. The authors established that this substitution was sufficient for enhanced intracellular growth of M1. The authors showed that swapping the CAC1 allele in the parental Ftc555-1 strain with the mutated CAC1 allele (cac1-evo) from M1 strain was sufficient to induce M1-like phenotype. In addition, swapping the cac1-evo allele in the M1 strain with the parental CAC1 allele caused a complete loss of the M1 phenotype. Whole genome sequencing of intermediate stages in experimental evolution, known as the “fossil record”, showed that the cac1-evo allele arose very early during the serial passage and quickly spread through the population, which correlated with phenotypic changes in fungal growth in macrophages. This suggests that the cac1-evo allele confers a strong fitness advantage. Interestingly, the authors found that the Arg1227Pro substitution limited capsule formation by the M1 strain or cac1-evo allele-swapped Ftc555-1 strain. Finally, the authors demonstrated that the M1 strain failed to cause lethal disease in a mouse infection model, while the parental Ftc555-1 strain had a median time to endpoint of 34 days. This suggests a tradeoff between the in vivopathogenicity of M1 strain and its enhanced fitness in macrophages in vitro.

OVERALL ASSESSMENT: Experimental evolution studies often provide insights into host-pathogen interactions. Here, Hilbert et al, performed in vitro serial passaging of C. neoformans in different hosts to learn about fundamental mechanisms of host adaptation. The authors demonstrated that a non-synonymous mutation in CAC1 was sufficient to provide the M1 strain with an in vitro intracellular growth advantage in macrophages. The CAC1 mutation also affected fungal capsule thickness, which is known to be regulated through cAMP pathway. Overall, we considered the experimental evolution analysis of this study to be impressive and well structured. The main finding of this paper provided important insights into growth regulation of C. neoformans and highlighted the importance of cAMP pathway in fungal biogenesis. In our discussions, there was general agreement that the authors could improve the impact of their work by including mechanistic studies about the CAC1 mutation identified in the M1 strain. The final part of the study focused on characterization of the in vivo pathogenicity of the M1 strain. We were intrigued by the increase in vitrogrowth that did not correlate with lethality in mice. However, we were concerned about the completeness of this aspect of the study and encourage further exploration.

STRENGTHS: The main strength of the paper is the demonstration of rapid host adaptation of an environmental isolate of C. neoformans to a new host. This finding contributes to our understanding of the microevolution events of pathogenic fungi, which deserves more attention in the field. The strong correlation of the intracellular survival phenotype of the adapted M1 strain to the single amino acid substitution on CAC1 protein highlights the importance of the cAMP signaling pathway in controlling the survival and pathogenesis of C. neoformans.

WEAKNESSES: We identified the following weaknesses:

1.     The experimental evaluation in this paper resulted in a C. neoformans strain with a strong intracellular growth advantage. The authors pinpointed the specific mutation that is responsible for the strong phenotype but did not extend their work into studies of the molecular mechanisms that drive the phenotype.

2.     Some aspects of data presentation were incomplete. For example, microscopy images of the fungal capsule were not included in the paper, even though it would have helped the reader (only quantitative data shown). Furthermore, the animal data was incomplete, as the authors could have shared data like body weight changes and appearance that are routinely collected during mouse studies to help bolster their conclusions about pathogenesis.

3.     Based on the information given in the Introduction, the amoeba is a potential source of environmental selective pressure for virulence factors of microorganisms. This is due to the similarities between amoeba and macrophages. It justifies the use of amoebae in this paper as the environmental host for C. neoformans. However, the amoeba studies were underdeveloped and would benefit from further mechanistic elaboration. Furthermore, the fact that the macrophage adapted strains did not show growth advantage in amoeba also did not align with claims made in the Introduction.

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.  Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2

● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.

The general comment we have about the figures is the way that statistical significance was presented. We found that indicating significance (* or NS) on top of the bar did not clearly indicate which two values were being compared. For example, in Figure 1B, it was unclear if each value was compared to H99O or compared to the neighboring value. In addition, it was unclear if the significance (* or NS) on top of each strain reflected values of both media and co-culture groups or for only one of these groups. We suggest that the statistical analysis could be presented more clearly.

Figure 3: This figure provides key supporting data to validate that the intracellular growth is being selected and improved in the M1 strain. We think that the fluconazole data in Supplemental Figure 3 provides important support and that the reader would benefit from including it as part of Figure 3.  

Figure 5: Here, the authors comment on the “striking difference in the pathogenicity of the serially passaged M1 strain when compared to the parental Ftc555-1 strain”. To support this statement, the authors should compare the level of fungal infection in mice between the M1 strain and the parental Ftc555-1 strain. We really think it is important to include the CFU in the lung and brain of the mice infected with Ftc555-1 parental strain as a critical control, which also serves as a base line of the C. neoformans infection in mice.

●     Are specific analyses performed using methods that are consistent with answering the specific question?

The difference of the survival rate between the mice infected with M1 and parental strain was striking. It is indeed a strong implication for a reduced pathogenicity of the M1 strain. In our opinion, there are many inter-related criteria that affects the pathogenicity of any given pathogenic microorganisms, such as their ability to cause disease, the severity of the disease, their ability to establish an infection, and their ability to replicate in the host. We appreciate the discussion of those criteria in the paper, but we believe more data from the animal infection experiment is required to give better support of the pathogenicity difference between M1 and parental strain. We think it would be helpful to show the mouse body weight that was measured during the infection. Especially, it would be interesting to see how the body weight changes during the first 20 days post infection.

We understand that the in vivo study is expensive and time consuming, but growth conditions are known to affect the capsule. Thus, we think that if the authors could perform a capsule assay post-infection it will forge a stronger link between the in vitro and in vivophenotypes of M1. It would also be interesting to measure the intracellular levels of M1 and the parental strain in the liver macrophages of the infected mice, as the liver macrophages are known to play an important role in C. neoformans infection.  

●     Is there appropriate technical expertise in the collection and analysis of data presented?

Yes.

●     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

We think that the one-way ANOVA tests used throughout are generally acceptable. However, we would like to address our concerns about the stringency of using one-way ANOVA with multiple comparison in Figure 1C, which only has two biological replicates. We suggest either using t-test to compare the growth difference between each isolated strain to the control strain or completing the dataset with third biological replicate, to enable ANOVA analysis.

In addition, we were confused by the use of both t-tests and ANOVA in Figure 5A. It created confusion about the significance of the results by using two different statistical tests with overlapping functions for the same dataset. The comparison between the M1 and FTc555-1 cac1 mutant strain can be done with multiple comparison tests that normally run alongside of one-way ANOVA.

●     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. N/A

Adequate.

2.  Quality: Completeness (1–3 scale) SCORE =1.5

●     Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

We think the title and abstract are generally well supported by the data presented in the paper. However, we want to address our concerns about the animal infection data that was used to support the reduced pathogenicity of M1 strain as a tradeoff for its intracellular growth advantage in vitro. As mentioned in the previous section of this review, only showing the mice survival data is not sufficient to establish the conclusion about pathogenicity of the M1 strain. Data from the parental strain is needed in all assays to show the base line of the C. neoformans infection. Other aspects of the infection should also be evaluated, such as the animal weight changes, capsule thickness of both strains post-infection, and the growth of both strains in infected mouse macrophages. It is also important to have biological replications for the animal experiments to show the stringency of the results.

●     Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. N/A

3. Quality: Reproducibility (1–3 scale) SCORE = 2

●     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

Most experiments have at least 3 biological replicates, with two exceptions.

1.     The experiment testing the replication of C. neoformans strains in co-incubation experiments with amoeba cells (Figure 1C) only has two biological replicates. We believe either including a few sentences to justify the reason for having only two replicates or adding an extra biological replicate of the experiment would increase the stringency of the results.

2.     According to the methods, the C. neoformans mice infection has only been done once. We believe that at least three biological replicates are required for the mice infection model to show the reproducibility of the results. In this way, the results of Figure 5C will better support the “tradeoff” phenotype of M1 strain, as the authors concluded. If there were more than one biological replicate for the animal experiments, we suggest including the information clearly in the Methods and Figure caption.

●     Is there sufficient raw data presented to assess rigor of the analysis?

The raw data provided is generally sufficient. However, we found that only violin plots were included in the paper to show the effects of CAC1 mutation on the capsule thickness of C. neoformans. We suggest including the microscopy images that the measurements were performed on in addition to the violin plot. We believe including the images will help the audience to better visualize the difference of the capsule size/thickness between different strains. This will provide better support for the statement “the point mutation in CAC1 gene affects the C. neoformans capsule production”.

●     Are methods for experimentation and analysis adequately outlined to permit reproducibility?

Yes.

●     If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?

N/A

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE =1.5

●     Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

OK

●     Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

               OK

●     Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions.

We think there are few points in the paper that could be improved by making small changes. This includes the following:

1.     In Figure 3, the legend for the solid and striped bar was missing. There was typo in the figure caption for Figure 3A “Solid barsindicate competitive indices in media only, solid bars indicate competitive indices in the macrophage”.

2.     We think that including cAMP pathway diagram in Figure 4A raised questions about the mechanisms of how cac1 mutation affected the cAMP pathway, which was not investigated in depth the manuscript. Therefore, we suggest removing Figure 4A if cAMP pathway genetic and physical interactions are not going to be studied in detail.

3.     We were not sure the exact massage the authors want to deliver with the sweeping statement [Regardless of exact mechanism, the complex impact of a single nucleotide change in the genome of this pathogenic fungus highlights the long reach of simple adaptive changes to fundamentally change the course of evolution]. We were specifically confused about the meaning of “fundamentally change the course of evolution”. We suggest that expanding this statement could help the audience understand how the preprint supports these conclusions.  

4.     We noticed that the y-axis scale of Figure 5A only has 0, 5 and 10, which makes it difficult to discern the intermediate values.

5.     We also noticed that Figure 5B was covered before Figure 5A in the body text of the Results. It would be easier for the audience to follow the paper by discussing the results in the same order they were presented in the figure.

MORE SUBJECTIVE CRITERIA (IMPACT)

1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2.5

A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.]

●     How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

The novelty of the study is that it demonstrates rapid host adaptation of C. neoformans through experimental evolution experiments. Amazingly, the authors were able to pinpoint a specific mutation in the CAC1 gene that drives the adaptive phenotype of the M1 strain. However, molecular studies of the single amino acid substitution in the CAC1 protein were underdeveloped. Was the phenotype the result of the loss of arginine at a key position, or the gain of a proline? How might enzyme activity be affected and is this an allosteric site? How might other substitutions at this arginine affect CAC1 enzyme activity? Do other mutations arise in the pathway during experimental evolution, either upstream or downstream?

          The authors hypothesized in the discussion that the Arg1227Pro mutation might affect interactions between CAC1 and its regulatory proteins. Could comparative structural analysis of the parental and mutant CAC1 proteins provide some insight into these interactions? We would love to see more attention paid to protein structure in the study, especially considering the available crystal structures and recent revolution in structural prediction capability. This would undoubtedly boost the impact of the study.

          As the authors stated, the identification of the mutation on CAC1 gene in M1 strain agrees with established literature that the cAMP signaling pathway could be a hotspot for genetic variation. However, only the M1 strain and its corresponding fossil record were sequenced. The fossil record is a great tool to trace the arise and frequency changes of gene mutations along the serial passaging. It would be interesting to investigate if there were any mutations that had occurred in the cAMP signaling pathway in the M3 strain, which also showed enhanced replication in macrophages.

Impact: Extensibility (1–4 or N/A scale) SCORE = N/A

●     Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? N/A

●     This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). N/A

Competing interests

The author declares that they have no competing interests.