Role of Rab5 early endosomes in regulating Drosophila gut antibacterial response

  1. Manish Joshi
  2. Annelise Viallat-Lieutaud
  3. Julien Royet

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Abstract

Interactions between prokaryotes and eukaryotes require a dialogue between MAMPs and PRRs. In Drosophila , bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, we show that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, we demonstrate that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-κB response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection

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  1. Version published to 10.1101/2022.05.04.490610v2 on bioRxiv
  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, Joshi et al investigate the intracellular behavior of PGRP-LE for the activation of the NF-kB pathway in Drosophila enterocytes undergoing pathogen infection. The authors identify that, upon enteric infection, PGRP-LE aggregates to form a microscopic structure of puncta, which colocalize with Rab5. The authors further analyze the role of Rab5 for the NF-kB pathway and suggest that Rab5-dependent pathway represents one of two distinct routes for the activation of the NF-kB pathway based on the observation that RNAi-mediated knockdown of Rab5 selectively downregulates PGRP-SC1, which results in systemic immune response. Generally, the manuscript provides convincing experimental results to support the authors' arguments raising an interesting cell biological aspect of PGRP-LE for the well-known immune pathway. However, in my opinion, there are some ambiguous points as well. I would like to have several suggestions to strengthen the manuscript.

    Major comments

    1. To identify the role of Rab5, the authors performed an RNAi-mediated knockdown experiment and found that the expression of PGRP-SC1 is downregulated but the expression of other target genes such as AttacinD are not affected. The authors concluded that the expression of PGRP-SC1 is under the control of a Rab5-dependent route while other targets are regulated by Rab5-independent route. However, an alternative interpretation would be that Rab5 is required for all target genes and the observed differential expression of the targets is due to residual activity of Rab5 after RNAi-mediated knockdown. If the authors show that RNAi-mediated Rab5 knockdown almost deplete Rab5 expression, it would be helpful for the authors' argument. Also, this alternative explanation is worth to be provided in the discussion section.
    2. The authors show that enterocytes with Rab5 knockdown still produce enlarged puncta without any further characterization. However, the identity of this subcellular structure would be an important piece of information to support the authors' argument concerned with a Rab5-independent route, which is largely a speculation at the moment. So, I would recommend to investigate whether the enlarged puncta colocalize with any known endosome and/or autophagosome markers. This information will enable to understand the Rab5-independent NF-kB activation pathway (e.g. by manipulating this pathway) in enterocytes.

    Minor comments

    It would be helpful for general readers to have an additional figure with a simple drawing of the authors' working model.

    Significance

    This paper showed for the first time a Rab5-dependent PGRP-LE aggregation that act as a signaling hub to finely modulate NF-kB pathway. As NF-kappaB is an evolutionarily conserved transcription factor that is essential for the immune activation from Drosophila to mammals, the present information would be of interest to a broad audience.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The manuscript presented by Joshi et al presents a body of results describing the aggregation of the peptidoglycan receptor PGRP-LE, which is an intracellular protein, in response to intestinal infection by oral ingestion in Drosophila. This study is based on the generation of two CRISPR/Cas9 mutant lines in which the PGRP-LE sequence has been fused to the V5 epitope (inserted into the PGRP domain) or the fluorescent protein eGFP (added at the C-terminal position). In each case, the "sensor protein" is expressed under the control of the endogenous promoter that ensures a physiological expression of the sensor.

    As expected from the literature, the authors show that the expression of each of the two PGRP-LE sensors is strongly induced in the digestive tract by the ingestion of the bacterium Erwinia carotorova carotorova (E.cc), which is known to produce a strong activation of the NF-kB signaling cascade under these infection conditions. In this study, the authors show that PGRP-LE-V5 sensors form clusters in the immunocompetent domains of the gut, particularly in the R4 domain where NF-kB activation is known to be primarily dependent on PGRP-LE. This clustering is not observed in clones with little or no expression of PGRP-LE due to RNAi-mediated knockdown of gene expression. The transcription of endogenous PGRP-LE or that of the PGRP-LE-V5 and eGFP sensors is not increased by the infection, allowing the authors to propose that the PGRP-LE protein pre-existing in the intestinal cells relocalizes into clusters or aggregates. These aggregates are also marked by the Rab5 protein, a marker of early endosomes, but not by the Rab7 marker, a marker of late endosomes. The expression of the antimicrobial peptide AttD is similar in the presence of the sensors as in control flies, which indicates that the immune response is not drastically affected by these sensors. Moreover, the kinetics of receptor aggregation parallels that of NF-kB pathway activation followed by AttD expression.

    Ingestion of E. coli or commensal bacteria or PGN, which do not induce a significant immune response according to the literature and data reproduced here by the authors, do not induce receptor aggregation either. Surprinsingly, heat-killed E.cc bacteria, which induce no or a very slight expression of AttD cause more but smaller aggregates of PGRP-LE. Moreover, these aggregates are not labeled by the Rab5 protein. The authors show that this aggregation of PGRP-LE is not affected by the down-regulation of the HH pathway, and is correctly induced by a uracil auxotrophic Ecc mutant. The expression of RNAi directed against the dFADD protein, an adaptor of the PGRP-LC membrane receptor contributing to the activation of the Imd/NF-kB pathway, does not alter this aggregation either. Finally, the authors observed that a set of genes whose expression in response to E.cc is dependent on PGRP-LE shows a differential dependence on Rab5 expression: while PGRP-SC1 expression is affected by Rab5 silencing, this is not the case for PGRP-LB or PGRP-SC2 expression. Furthermore, directed Rab5 knock-down in the adult gut induces an exacerbated immune response in the fat body. The combined action of PGRP-LE and Rab5 would therefore be necessary for the activation of PGRP-SC1 but not of PGRP-LB or PGRP-SC2. From these results the authors propose the existence of two pathways of activation of NF-kB target genes downstream of PGRP-LE, depending or not on an endosomal Rab5 signaling platform. The authors also propose that the amount of PGN may control the choice of Rab5-dependent or Rab5-independent pathway activation.

    Major comments:

    The authors have constructed beautiful genetic tools (PGRP-LE sensors). They present a set of convincing results concerning the formation of PGRP-LE protein aggregates in response to E.cc infection under different infection conditions or genetic backgrounds. Nevertheless, the study remains essentially descriptive and based on immunofluorescence and expression studies of a small set of genes responsive to the NF-kB pathway. To better support the hypotheses and conclusions, deep sequencing studies would be very powerful to reveal whether the differential expression observed for the target genes PGRP-SC1 versus PGRP-SC2 and PGRP-LB is also true for a large set of genes of the immune response, which would make the results more accurate. It would also be interesting to study more genetic conditions, e.g. affecting the endocytic pathway, proteasomal degradation or autophagy in order to determine the fate of aggregates and the mechanisms of their removal/resolution. Furthermore, biochemical studies, such as immunoblots, would allow following the fate of PGRP-LE at the protein level. The authors indeed show that the expression of PGRP-LE gene is not induced by E.cc but one can wonder if the protein is stabilized. They propose that PGRP-LE is not recycled because it does not colocalize with Rab7, but it might be also degraded by the lysosomal pathway rather than recycled. It would be interesting to test if aggregates are removed by the lysosomal pathway or by autophagy. Moreover, a recycling via Rab7 is maybe not expected for a protein that is not localized on the plasma membrane. A kinetic study including co-staining with Rab7 would better support the conclusion that there is no colocalization with Rab7. Otherwise, they may miss the right timing to observe this colocalization. Similarly, the absence of colocalization with Lamp1 at a given time does not allow concluding with certainty that PGRP-LE is not degraded by the lysosomal pathway. The 24h staining (Fig2A) sounds similar to a Lamp1 profile. One should therefore be more cautious in drawing conclusions about these co-staining experiments. Moreover, Rab7 and Lamp-1 staining are faint and miss RNAi controls to show the specificity of the staining.

    In conclusion, a corpus of additional experiments would be necessary to significantly advance the field and demonstrates the existence of a Rab5 signalization platform causing differential expression of target genes of the immune response. The expression of a large set of genes could be tested, some of the RNAi lines used needs to be better characterized, complementary genetic and biochemistry experiments would help to understand the fate of PGRP-LE, the effect of the Imd pathway could be more documented with other RNAi than FADD... The role of other components of the endocytic pathway tan Rab5 could be assayed with other RNAi (Rab7, ESCRT, ... ) to block the endocytic pathway and observe if it interferes with the aggregates. The authors could also possibly test the proposed hypothesis on the amount of PGN/bacteria that would be at the origin of a differential response.

    In the figure and figures legends and methods, the authors describe the aggregates as oligomers, but no experiment support this assumption. In the text, the authors stick with the nomenclature as clusters or aggregates which is more appropriate.

    Minor comments:

    • The abstract would benefit from being rewritten: the first half provides general information that is not strictly necessary, which prevents a more thorough description of the results. I disagree or misunderstand the statement "little is known about the subcellular events required to translate these early steps into downstream target gene transcription" because extensive studies of the fly immune response have been done.
    • Two spellings in the intro: PeptidoGlycaN or PeptidoGlycan. I suggest peptidoglycan
    • "the innate immune response that might otherwise be obscured by the action of the adaptive immune response": this is a rather archaic way of thinking because it is clear that the two responses are complex and intimately intertwined.
    • "to visualize PGN detection by PGRP": correct "by PGRP-LE". -avoid "to our surprise". -"locus-directed": I suggest "tissue directed" or "in a localized manner in the digestive tract".
    • Describe the purpose and procedure of smurf methodology.
    • As noted above, do not describe clusters as oligomers in the methods and figures and figure legends. -"PGRP-LE recruits Rab5 protein": do the authors suggest a direct interaction between the two products? If so, it would be interesting to test this with co-IP experiments. However, it is possible that the aggregates are internalized in the endosomal compartment, independently of any Rab5/PGRP-LC interaction. Therefore, the term "recruits" is confusing here. -To make the results accessible to a broader audience, the authors may clarify the drosophila-specific genetic tools used in this study (Flpout clones, Gal80ts conditional expression...)
    • In some cases, statistical analysis of RT-qPCR data are performed using a one-way ANOVA (fig 1H, 5A) whereas in others (fig 2H and L, 5B) a non-parametric Kruskal-Wallis test is used. The rationale for these discrepancies should be explained. Moreover, in all these experiments the data are compared to a control that is set to 100% and has no standard deviation. This violates some of the ANOVA assumptions (normality of the data points). To be correct, an outside control should be used to normalize the data (including the control to which the other genotypes are compared)
    • Could the authors better explain the rationale for using PGRP-LE::V5 in some experiments and PGRP-LE::GFP in others? -Fig 1H: in this experiment, according to the legend, all the genotypes are infected. So it's not clear how the authors conclude that infection does not activate PGRP-LE expression in the absence of a non-infected control. We may have missed some points. Furthermore, as stated above, the authors could also perform a Western blot to ensure that PGRP-LE translation is not activated, or the protein stabilized, following infection.
    • Fig 2A: The PGRP-LE aggregates a 24 hpi look different from the previous time points. It would be interesting to make a double staining with a Lamp1 antibody to check for colocalization at this late time points.
    • Fig 2H: attD induction by hk E.cc is indicated as not significantly different from uninfected control and presumably not from E. coli and PGN. So the statement "hk E.cc which induced a weak AttD transcription" in the text is not correct.
    • Fig 3: The RNAi lines used in this figure have no effect on PGRP-LE aggregation. To safely conclude that the corresponding proteins do not play a role in this process, the efficiency of the RNAi lines against their respective targets should be shown.
    • Fig 3A,B : why no quantification of the aggregates are presented in this particular figure?
    • Fig 4D: the pictures are too small, use the same magnification as in A and C

    Significance

    The studies presented in this manuscript are interesting and well done but remain mainly descriptive without sufficient data to support what could be a conceptual advance. Further work is needed to demonstrate that PGRP-LE would signal via two different pathways, dependent or not on Rab5 and the endocytic machinery. Further genetic and biochemical studies would allow to better describe these two putative signaling pathways leading to differential immune response genes expression, and/or the nature (oligomeric or not) and fate of PGRP-LE aggregates (endocytic-, lysosomal-, autophagic-patways,...). Such endosomal signaling platform has been described for the activation of the Toll pathway. Exacerbated immune response in the fat body following inactivation of Rab5, Fab1, and ESCRT components has been described earlier suggesting that accurate termination of IMD signaling also requires the endocytic machinery.

    This study concerns fly scientists interested in the fine understanding of the signaling mechanisms of the innate immune response and may have a wider audience in the community of scientists interested in the molecular mechanisms of cell signaling in eukaryotic cells in response to external stimuli, and the role of endocytic trafficking in this response. Our expertise (reviewer and co-reviewer) covers the NF-kB-dependent immune response and some aspects of intracellular trafficking.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors used CRISPR engineering to drop a V5 or GFP tag into teh PGRP-LE locus (protein fusions) to monitor the behavior of this intracellular peptidoglycan sensing receptor in the drosophila midgut. They show that upon immune stimulation with Ecc that PGRP-LE forms some sort of aggregate or punctae that is dynamic during the 24 hour of infection monitored. A similar response is not seen with live E. coli but a week and smaller response is observed with heat killed E. coli, for unclear reasons. These punctae appear to form independent of the classic IMD signaling components, suggesting it is upstream event in the pathway which is consistent with early studies showing the PGRP-LE multimerizes (infinitely) upon binding PGN and also that it forms amyloid fibrils doing signaling. The Ecc punctae tightly colocalize with Rab5 but not Rab7 or other early endosome markers, but in the absence of Rab5 the PGRP-LE punctae are greatly enlarged. Rab5 was found to critical for induction of PGRP-SC1 but not the classic IMD pathway AMP, Attain.

    While the conclusions of the report are intriguing and the development of these tools is very exciting, the conclusions are not fully convincing. To start, the author wish to conclude that PGRP-LE localization is altered with Ecc infection but they have not excluded that the expression of the protein is sharply upregulated. I.e. in the uninfected animals there is not really any PGRP-LE observed (1D). The try to tackle this by looking at mRNA expression, but this data lacks the unaffected control. [In fact, the uninfected control is missing on most of the gene expression data, which is a troubling omission and makes it hard to really understand what the data shows.]. Moreover, the mRNA levels do not necessarily corresponding to the protein levels, i.e. there could be post translation control. So, overall, the authors need to provide more compelling evidence that PGRP-LE is relocalized upon Ecc challenge rather than upregulated.

    Moreover, the paper contains some seeming contradictory findings that the authors make little effort explain. For example, they conclude "These results suggest that although smaller PGRP-LE aggregates can form normally in the absence of Rab5, the latter is required for proper bigger E.cc mediated PGRP-LE aggregates" because E. coli induced PGRP-LE clusters don't colocalize with Rab5, yet in the absence of Rab5, the Ecc cluster are super-enlarged (4F). This makes no sense with the conclusions.

    Finally, the interaction and function of the Rab5 interaction is underdeveloped and lacks insight. For example, why is Rab5 required for the induction of one target gene but not another? And, why not characterize this more completely? Why is there not Rab5 vesicle with E. coli feeding or even uninfected? The cell biology requires more in-depth consideration. From 4E, the authors wish to conclude that the Rab5 vesicle are induced by Ecc (even in the absence of PGRP-LE) yet the uninfected control is not shown. IN a simple world, would not one would expect Rab5 endosomes in all cells, at least to some level?

    And, focusing on the big picture, the authors claim that it is "not easily testible" if the PGRP-LE aggregates are amyloidal, as suggested by earlier publications. This could actually be tested by staining with amyloid specific dies and/or suitable mutants engineered int he RHIM domain. This would be very informative if the authors could extend this work to examine this question.

    Minor comments:

    All the colocalization data should be quantified as in 4B. It is not true that DAP = Gram negative. Gram-positive bacilli also have DAP PGN. The wording in the Introduction should be adjusted. The text needs a careful proofreading.

    Referees cross-commenting

    I think the comments from #2 and myself are aligned. Working is interesting, tools are especially exciting, but the studies are descriptive and under-developed. I will further add, I found the absence of uninfected controls for many assays a major problem.

    Significance

    The significance of this work lies in the development of powerful tools to track an intracellular innate immune receptor in an intact animals. The connection to Rab5 is curious and likely an important advance in our understanding of the cell biology of this pathway, but is under-developed. The significance is this difficult to know for certain. The Drosophila immunity field, and the insect immunity field more broadly, will be keenly interested in this study. The wider NF-κB/innate immune field will also be interested in these findings, given teh similarity between this pathway and NOD1/NOD2 immune sensing in mammals.

    My area of expertise is the Drosophila immune response and this manuscript is very much in my wheelhouse.

  6. Version published to 10.1101/2022.05.04.490610v1 on bioRxiv