Toll-1-dependent immune evasion induced by fungal infection leads to cell loss in the Drosophila brain
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Abstract
Fungi can intervene in hosts’ brain function. In humans, they can drive neuroinflammation, neurodegenerative diseases and psychiatric disorders. However, how fungi alter the host brain is unknown. The mechanism underlying innate immunity to fungi is well-known and universally conserved downstream of shared Toll/TLR receptors, which via the adaptor MyD88 and the transcription factor Dif/NFκB, induce the expression of antimicrobial peptides (AMPs). However, in the brain, Toll-1 could also drive an alternative pathway via Sarm, which causes cell death instead. Sarm is the universal inhibitor of MyD88 and could drive immune evasion. Here, we show that exposure to the fungus Beauveria bassiana reduced fly life span, impaired locomotion and caused neurodegeneration. Beauveria bassiana entered the Drosophila brain and induced the up-regulation of AMPs , and the Toll adaptors wek and sarm , within the brain. RNAi knockdown of Toll-1, wek or sarm concomitantly with infection prevented B. bassiana- induced cell loss. By contrast, over-expression of wek or sarm was sufficient to cause neuronal loss in the absence of infection. Thus, B. bassiana caused cell loss in the host brain via Toll-1/Wek/Sarm signalling driving immune evasion. A similar activation of Sarm downstream of TLRs upon fungal infections could underlie psychiatric and neurodegenerative diseases in humans.
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Reply to the reviewers
The authors do not wish to post a response at this time. This is because this is not the submission of the revised version, which we have not completed yet. This is a preliminary revision together with a revision plan instead.
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, Singh et al. demonstrate that infection of D. melanogaster flies with B. bassiana fungus induces neurodegeneration via Toll/wek/sarm signalling. It is already known that fungal infection can be associated with neurodegeneration, but the exact mechanism is unclear. The authors demonstrate that the fungus enters the brain, causes hallmark symptoms of neurodegeneration, and requires Toll-1, Wek, and Sarm in order to do so. This is an important step forward as it demonstrates specific genes in the fly immune pathways that are involved in fungus-induced neurodegeneration, which could be informative for infections in …
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, Singh et al. demonstrate that infection of D. melanogaster flies with B. bassiana fungus induces neurodegeneration via Toll/wek/sarm signalling. It is already known that fungal infection can be associated with neurodegeneration, but the exact mechanism is unclear. The authors demonstrate that the fungus enters the brain, causes hallmark symptoms of neurodegeneration, and requires Toll-1, Wek, and Sarm in order to do so. This is an important step forward as it demonstrates specific genes in the fly immune pathways that are involved in fungus-induced neurodegeneration, which could be informative for infections in humans. Overall, the manuscript is thorough and well-written and the conclusions are broadly supported. A few mostly minor comments and questions are below, which could mostly be addressed by including additional details in the methods or discussion. The only major comments would be 1) that the control fly genotypes used in experiments were not always the most ideal controls (eg, compared WT genotypes to RNAi against a gene of interest; ideally would be RNAi against a control gene compared to RNAi against a gene of interest), and 2) negative controls of fluorescence microscopy imaging were not always included. It would be important to address these through clarification in the figures/methods, and/or discussion of the potential caveats, even though it is likely the conclusions would still hold. Notably, these comments are relatively easily addressed through edits in the text.
Major comments:
- For fluorescence imaging, were negative controls included (no infection or no gene expression etc.) for all stains (as with Figure 1H)? If so, it would help to include representative images as supplemental figures. Also, for all positive samples, was presence of the fungus noted in all samples?
- Figure 4: Here, it appears that the control fly genotypes are wildtype vs an RNAi line (similar for some other figures/assays as well, but using this one as an example). The best control would be RNAi against a control gene compared to RNAi against a gene of interest, rather than just a control WT genotype with no RNAi compared to RNAi against a gene of interest. This should be included as a caveat in the discussion since the experiments do not all account for the effect of RNAi (or other gene expression) on the phenotypes regardless of the gene.
Minor comments:
- It would help to have line numbers throughout
- Figure 1- what are the arrows in panels D-G?
- Methods: A few details are unclear:
- Was only one fly sex used or were both used for the various assays? If both were used, were they statistically assessed for differences? Sex is only mentioned in a couple of the methods sections.
- How old were the flies at the start of the experiments? A few experiments noted age, but it was not clear for all
- For longevity, was the fungal culture ever replaced during the experiment?
- For the climbing assay when the flies were initially flipped, how much time was there between flips?
- Figures 2A, 2D, 2E, 3E, & 3H: If multiple replicates or samples are represented in the data, it would help to be able to see the data points underlying these bars. If so, please add them to the graphs to see the spread of data points.
- Figure 3F- what do arrows indicate?
- It is interesting that Wek-RNAi with infection not only rescues loss caused by the infection alone, but also increases YFP cells beyond the uninfected controls (Figure 5C). The same is true with toll-1 RNAi (Figure 4C). Why might this be?
- It would be ideal if data underlying data points and full statistical models and outputs could be included through a public repository such as Dryad. This would be ideal for full assessment of statistical approaches
Very Minor comments:
- Check italicizing throughout- missed a few "Drosophila" or "B. bassiana" in main text or figures
- Looks like no space between C. and elegans in C. elegans in a few cases
- Word missing: "No effect was seen after three days exposure to B. bassiana, but seven days exposure impaired climbing"... seven days of exposure?
- Toll-1 misspelled pg 6 last paragraph
Referee Cross-Commenting
Regarding the major comments, I agree with Reviewer 1 that more thorough proof of spores entering the brain (and what proportion of exposed flies this happens to) would be beneficial. I also agree with Reviewer 2 that a rescue experiment for the climbing assay and my earlier suggestion for more controls in the microscopy could help address this concern, at least in part. Other responses or experiments may also be appropriate to address some of the major concerns- maybe additional assay(s) of brain function other than climbing?
Reviewer 1 also brought up the point that flies with advanced infection were used for the experiments- it would be helpful to know if earlier time points were ever checked for BBB damage, loss of brain cells, or presence of fungus etc. This would clarify if the same phenotypes are present in flies that die early, along with other concerns from Reviewer 1.
However, whether directly or indirectly, several later figures show loss of brain cells with infection followed by rescue with RNAi against genes of interest. This does lend support to the conclusions that fungal infection negatively impacts brain cells and the fungus requires these host genes to do so.
Other concerns Reviewer 2 and I raised about the fly genetic controls being unclear should also be addressed. What is the full genotype of the flies in each case? What is considered "+" in each case? Were these driver background strains, WT (like Oregon R), or RNAi against control genes (best controls)?
Significance
General assessment: The manuscript by Singh et al. is a thorough investigation into the fungus-host interactions in the brain, demonstrating that the common insect fungal pathogen B. bassiana requires the host genes Toll, wek, and sarm to induce negative phenotypes in the brain. The strengths are in the multi-pronged approaches that use several independent techniques (fly behavior assays, gene expression, microscopy, etc.) and multiple genes, conducted with many replicates, that all show clear and consistent trends supporting the conclusions of the authors. The weaknesses include some cases where controls are either not completely clear or not the most ideal controls. This weakness could be addressed with either edits to the text, where appropriate, or addition of supplemental figures. However, the conclusions are still broadly supported.
Advance: Although it is known that fungal infections can impair brain function, it is not fully understood how this happens. This manuscript identifies Toll-associated molecules that are required for fungus-mediated neurodegeneration, which is a critical first step to understanding the process and for future development of therapies.
Audience: This finding would be of broad interest to scientists in immunology, microbiology, neuroscience, and other areas.
Expertise of reviewer: Drosophila, fly genetics, invertebrate immunology, insect-fungal interactions
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Referee #2
Evidence, reproducibility and clarity
Summary
The authors describe a role for Toll signaling in detrimental neuronal loss associated with B. bassiana fungal infection in Drosophila melanogaster model. They show that this effect is mediated by wek/sarm as silencing either of them prevents neuronal loss after the infection. Similar results are obtained with Toll-1 RNAi, suggesting that the response is dependent on the activation of Toll signaling by B. bassiana. The study is well executed,main conclusions are backed up by the data presented and experiments are conducted with adequate numbers of replications and individuals. Below I give some comments that I think would …
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Referee #2
Evidence, reproducibility and clarity
Summary
The authors describe a role for Toll signaling in detrimental neuronal loss associated with B. bassiana fungal infection in Drosophila melanogaster model. They show that this effect is mediated by wek/sarm as silencing either of them prevents neuronal loss after the infection. Similar results are obtained with Toll-1 RNAi, suggesting that the response is dependent on the activation of Toll signaling by B. bassiana. The study is well executed,main conclusions are backed up by the data presented and experiments are conducted with adequate numbers of replications and individuals. Below I give some comments that I think would help in further improving the manuscript.
Major comments
As the initial experiments (including the effect on survival and climbing assay) have been performed using OR/CantonS, it would be interesting to investigate if the same is seen with a more similar background to that what is used in the genetic experiments. In addition, I'd suggest an experiment to see if the Toll (or wek/sarm) RNAi in the brain rescues the climbing defect caused by the fungal infection.
It is somewhat unclear what are the controls in the genetic experiments. For example, in Figure 2, the control is UAS-TrpA1/+. Does this mean that the UAS-TrpA1 flies have been crossed to something (like the driver background strain) or used as it is? In Figure 4, controls are ">+". Again, are MyD88>histoneYFP;tubulinGal80ts flies crossed to something (in this case, maybe the w1118 background of the KK library RNAi strains) or used as homozygous? And same for the subsequent figures. I'd ask the authors to clarify these points in the manuscript.
Could the authors please explain why they opted for MyD88-GAL4 in the experiments in Figures 5-7? What is the overall expression pattern of MyD88-GAL4? Is there a possibility that some of the effects seen could arise from the Toll/sarm/wek knockdown elsewhere in the fly? How do the flies survive the infection with Toll knockdown in MyD88-epressing cells (expressed at least in all immunogenic tissues)? A bit more explanation would clarify the situation.
Minor comments
Page 3: Full species name should be given here (Drosophila melanogaster)
A short description of the FM4-64 dye (what it stains etc) would be useful for the readers unfamiliar with it.
Page 6: Please explain shortly why TrpA1 overexpression was used to activate the neurons.
Figure 2E: What is the genotype of the flies? mtk is lacking statistics
Page 8: third row refers to Figure 2 but should be Figure 3.
Although antibody stainings are performed using "standard methods", a short overview on the process should be presented also in the current manuscript. Also, I imagine fungal spores are all over the flies retrieved from the infection chamber. I'd like to know (and this could also be described in the materials) how the flies (and the brains) were treated/washed prior to preparing brains for immunostaining and imaging?
Some typos and inconsistencies at various places. For example, at some occasions B. bassiana written without a space in between "B" and "bassiana" and not in italics (both in figures and in text); on page 5, first line: "mimicked" misspelled
Referee Cross-Commenting
As the fungal infiltration into the brain is central to the conclusions made in the manuscript, I agree that care should be taken in making this argument solid. I believe this can be achieved adding controls as reviewer #3 suggests together with additional experiment(s) verifying that Toll/wek/sarm in the brain is mediating the neuronal loss caused by the fungal infection (rescue experiments). Of note, I wonder if, similarly to mammalian macrophages, hemocytes could be responsible for delivering the fungal cells into the brain?
I agree with the reviewer #1 that the climbing defect could be because of multiple reasons other than the fungal spores in the brain causing neuronal loss (for instance flies being generally weak at this point, ). However, the authors do show convincingly that there is neuronal loss in fungal-infected flies.
Significance
Fungal infections are understudied in any research model considering the threat they pose to humans and other animals alike. Due to the high conservation of the signaling components studied here, the results provide a good basis for future research, extending to mammalian models. I think these results will be of interest to a wider audience because of the reasons stated above.
My fields of expertise are Drosophila melanogaster, innate immunity, cell-mediated immunity, blood cell homeostasis
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Referee #1
Evidence, reproducibility and clarity
Singh et al. report that, after exposure to the entomopathogenic fungus Beauveria bassiana, the Drosophila adults impaired fly locomotion and died within two weeks. During which time, the authors designed experiments and showed the decline of brain cells via a Toll-1/Wek/Sarm pathway, mimicking the neurodegenerative diseases in humans in association with fungal infections. Providing that the rather solid genetic evidence was shown for the pathway in mediating fly brain cell losses, critical issues of experiment design/setup and conclusion validity were concerned.
Specific comments:
The fungus-exposed flies died within two weeks were …
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Referee #1
Evidence, reproducibility and clarity
Singh et al. report that, after exposure to the entomopathogenic fungus Beauveria bassiana, the Drosophila adults impaired fly locomotion and died within two weeks. During which time, the authors designed experiments and showed the decline of brain cells via a Toll-1/Wek/Sarm pathway, mimicking the neurodegenerative diseases in humans in association with fungal infections. Providing that the rather solid genetic evidence was shown for the pathway in mediating fly brain cell losses, critical issues of experiment design/setup and conclusion validity were concerned.
Specific comments:
The fungus-exposed flies died within two weeks were largely typical. However, it was unclear how those flies could be uniformly contaminated with fungal spores in the infection chamber shown in Fig. 1A, by landing on fungal "carpet"? It is publicly known that entomopathogenic fungi (EPF) like B. bassiana infect insects via spore germination on cuticle and then penetration of cuticles by fungal hyphae/mycelia (e.g., Trends Microbiol. 2024. 32, 302-316).
It is typical that EPF killed and mycosed insects within 5-14 days after topical infection by immersion in or spraying spore suspensions, or dusting on the sporulated plates. Fungal spores can be ingested by insects, largely those with chewing mouthparts. However, fungal spores can barely survive the highly-alkaline foreguts. It is questionable that flies could ingest spores and spores "infiltrated" the brains.
Regarding the detection of fungal cells in fly brains, on the one hand, the authors argued that detection of fungal SPORES in fly brain THREE days post exposure (page 5) by infiltration. It would be impossible that, even fungus could breach the blood brain barrier (BBB), it might be the fungal hyphae/mycelia but not the spores. One the other hand, the authors provided the evidence of the damaged BBB SEVEN days post exposure, a few days LATER than the detection of fungal spores in brains (THREE days) post treatment mentioned above. Did "spore infiltration" (even impossible) occur before BBB damage?
The authors stated that "by day seven more than half of the flies had died" (Fig. 1B). It is questionable therefore that the "other half" of the diseased and dying insects were used for the following experiments. There would be no wonder that the climbing of these diseased and dying flies was impaired, however, which could be due to muscle damage, hemocyte number decline and reduction of energy production etc. apart from brain cell loss. The brain function of dying animals could be compromised by multiple direct or indirect factors.
Issue of Fig. 2D labelling.
Referee Cross-Commenting
I agree with that Reviewers 2 and 3 that rather solid evidence of fly brain loss was shown in this work, however, at most in association with exposure to fungal cultures (volatiles could not be excluded etc.). "Spores" entry into fly brains were suspicious or impossible. If the dying flies had been used for these neurological experiments, the reliability of conclusions would be highly concerned.
Significance
Since there are critical concerns of experiment designs/setup in this work, it is questionable that fly brain cell loss was caused by fungal entry into brains.
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