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  1. Reviewer #3 (Public Review):

    In this manuscript, Filipowicz and Aballay present a nice story that characterizes a new learned behavioral phenotype prompted by intestinal distention during infection with the bacterial pathogen E. faecalis. The authors show that distention of the anterior portion of the intestine by E. faecalis induces an aversive behavioral response. Importantly, the authors show that this aversive learning response is controlled by multiple sets of neurons, including some that express the GPCR NPR-1 and others that express the ion channels TAX-2/4. The authors nicely showed that TAX-2 expression in ASE neurons was sufficient for pathogen avoidance, but not other chemosensory neurons. Next the authors examined the mechanism of aversive learning following ingestion of E. faecalis, showing that AWB and AWC neurons are required. Finally, the authors show that two proteins that could be mechanoreceptors in the intestine (GON2 and GTL-2) are required for pathogen avoidance. Together these data characterize important mechanisms of pathogen avoidance and an aversive learning response.

    I have one issue for the authors to consider. The title of the manuscript emphasizes the role of TRPM channels in mediating the learned pathogen avoidance response. Demonstrating that the site of action of the TRPM channels is the intestine could further strengthen this exciting finding.

  2. Reviewer #2 (Public Review):

    Work in the nematode C. elegans has shown that these worms learn to avoid pathogens like Pseudomonas aeruginosa after consumption and infection over a period of 12 or more hours. Here, the authors confirm and expand upon earlier observations that - in contrast to P. aeruginosa - avoidance of Gram-positive pathogens such as E. faecalis, E. faecium and S. aureus occurs rapidly on a timescale as short as even several minutes. Consistent with this more rapid response, they present evidence that behavioral avoidance occurs via distinct molecular, neuronal and phenotypic mechanisms from those of P. aeruginosa.

    The first major finding that the authors describe is that behavioral avoidance of E. faecalis occurs as a consequence of rapid intestinal distension and not through immune responses or other pathways. They show that anterior intestinal distension occurs rapidly - as early as 1 hr, which is a striking finding and is consistent with rapid behavioral effects. They show that neither E. faecalis bacterial RNA, nor bacterial virulence are necessary for behavioral avoidance and that immune response genes are induced only after distension. These data are consistent with a model in which intestinal distension underlies behavioral avoidance, but this assertion could be strengthened by showing that bloating is necessary for behavioral avoidance, that it occurs prior to observable behavioral avoidance, and by more definitively ruling out a role for immune responses.

    Next, the authors show that behavioral avoidance in laboratory conditions requires intact neuropeptide signaling via the npr-1 receptor and this is because worms tend to avoid high oxygen conditions outside of bacterial lawns that typically exists in the lab. At lower oxygen concentrations, npr-1 is dispensable for avoidance. This is consistent with previous work implicating this neuropeptide pathway in lawn avoidance and is convincingly demonstrated.

    The second major finding presented in this manuscript is that rapid behavioral avoidance of Gram-positive bacteria occurs via a learning process involving both gustatory and olfactory neurons. This suggests that worms may rapidly learn to avoid the taste and smell of these bacteria. They show that lawn avoidance of E. faecalis occurs in minutes and coincides with changes in lawn leaving and re-entry rates. They identify sensory neurons involved in lawn avoidance through genetic ablation and cell-specific rescue of signal transduction in the ASE, AWC and AWB neurons. A role for ASE in avoidance is specific to E. faecalis and is a new finding. The authors also show that after a 4hr training exposure to E. faecalis, worms switch from their naïve preference for E. faecalis odors to preferring E. coli odors. This switch in olfactory preference appears to require the AWC and AWB neurons, but not the ASE neurons. While the authors show a clear change in olfactory preference with these data, it is currently unclear whether this reflects associative learning as opposed to non-associative olfactory plasticity resulting from, for example, intestinal distension. Previous work from this group showed that longer-term bloating from bacteria could induce avoidance of different bacteria, arguing against a strictly associative learning role for previously described bloating phenotypes. It is also not currently clear from the authors' data whether ASE plays a role in training-dependent changes in food preference, how this training process relates to the timecourse of intestinal distension, and what role nutrient status might play here.

    Lastly, the authors present the intriguing hypothesis that TRPM family channels may sense bloating either directly or indirectly to mediate this colonization-dependent aversive behavior. Mutations in TRPM channels gon-2 and gtl-2 block lawn aversion that occurs after intestinal distension elicited by E. faecalis colonization or through interference with the defecation motor program. The authors convincingly show that these channels, which are expressed in the intestine but also play known roles in the germline, do not act via the germline in this context. The hypothesis that these channels act in the intestine to sense bloating is an exciting and particularly important one; however, both of these channels are known to be expressed in multiple tissues, and there is no data demonstrating a sensory function for these receptors in the intestine as opposed to other roles.

  3. Reviewer #1 (Public Review):

    In this work, the authors set out to better understand the mechanisms by which the nematode C. elegans responds to bacterial pathogens.

    Using behavioral assays and genetic manipulations, the authors find that C. elegans can rapidly learn to avoid the pathogen E. faecalis (E.f.). While recent studies from other groups have shown that small RNAs (sRNAs) produced by some pathogenic bacteria can trigger aversive learning, the authors find that this seems not to be the case for E. faecalis. Instead, they provide evidence that E. faecalis causes abdominal distention, and that this may provide the trigger for learning. Because the evidence for this is largely correlative, alternative explanations may still be possible. Further, the authors identify two TRPM-class ion channels whose function appears to be necessary for learned avoidance of E.f. The authors propose that one or both of these may mediate detection of abdominal distention, an interesting idea that merits further study. While the paper's title indicates that these channels "mediate" this function, this remains speculative.

    The authors also find that wild-type C. elegans prefer olfactory stimuli from E.f. to those of their regular diet, E. coli, but that this pattern is reversed after exposure to E.f. This plasticity involves the function of the chemosensory neurons ASE, AWC, and AWB, as well as the cyclic-nucleotide-gated channel TAX-2/TAX-4. This finding provides important insight into the nature of the changes in neural circuit function that are triggered by pathogen exposure, leading to pathogen avoidance.

    The paper also examines a role for the neuropeptide receptor npr-1 in learned E.f. avoidance. Animals lacking npr-1 function are known to strongly avoid high (ambient) oxygen concentrations, and instead prefer the lower-oxygen environment of a bacterial lawn. The authors find that this oxygen avoidance overcomes any avoidance of E.f.; thus, npr-1 mutants do not avoid E.f. when tested with ambient oxygen, but they do avoid it in a low-oxygen environment. This indicates that npr-1 is not required for pathogen avoidance per se. Although the authors suggest that npr-1 may be a target of the learning process, this is not well justified by the data and it may be more likely that oxygen avoidance and pathogen avoidance are separate processes.

    Together, these findings demonstrate that the mechanisms underlying learned pathogen avoidance in C. elegans differ substantially depending on the nature of the pathogen, and that worms likely use a combination of strategies to deal with these threats in the wild.

  4. Evaluation Summary:

    In this manuscript, the authors aim to address an important and interesting question: when an animal's intestine is colonized by pathogenic bacteria, how can it sense these bacteria and learn to avoid consuming them? Here the authors suggest that in C. elegans, sensing of intestinal distension or bloating caused by Gram-positive bacteria via intestinal ion channels may drive rapid behavioral responses through a process involving associative learning. While these findings are of broad interest to both the microbiology and neurobiology community, some of their conclusions are not currently fully supported by their data, and reasonable alternative interpretations exist.

    (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 and Reviewer #2 agreed to share their names with the authors.)