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  1. Evaluation Summary:

    Despite the broad impact of microsporidia on diverse animals, host factors involved in their invasion have not been characterized. The present study identifies a previously uncharacterized intestinal factor (AAIM-1) that is necessary for efficient infection of early larval stages of C. elegans. The secreted factor has a minor effect mediating resistance to pathogenic bacteria, suggesting more general impact on the susceptibility of the worm to diverse pathogens.

    (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. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    The manuscript by Reinke and colleagues describes the identification and characterization of a host factor, aaim-1, which promotes microsporidia infection of C. elegans. The authors conduct a genetic screen that results in the identification of aaim-1 and confirm the identity through rescue experiments and the characterization of additional alleles. Evidence for intestinal expression is also provided through tissue-specific expression experiments though endogenous expression is challenging to observe. A potential mechanism is inferred through careful characterization of intestinal lumen uptake and invasion in mutant host animals, and the authors suggest that the aaim-1 promotes the orientation of microsporidia spores that promotes successful invasion of the C. elegans intestine. The authors then show a modest increase in susceptibility to the bacterial pathogen Pseudomonas aeruginosa in aaim-1 mutant animals. Further characterization in competitive advantage assays and coinfection assays are also carried out.

    Little is known regarding the host factors required for microsporidia invasion, and thus the successful forward genetic identification of a previously uncharacterized host factor is noteworthy and represents an elegant use of the experimental system in which microsporidia infect C. elegans. The authors' data point to a functional role for secretion of AAIM-1 into the host lumen. As is often the case in trying to define mechanism of action of previously uncharacterized genes identified from genetic screens, defining a molecular mechanism is challenging. The model in which AAIM-1 may function to promote the proper orientation of spores to the intestinal lumen represent an appealing hypothesis, but the relative low-resolution images and unclear implications of the orientation leave considerable doubt. Perhaps higher-resolution microscopy (and more three-dimensional definition) and further discussion of the evidence of the significance of the angle with regard to successful infection would be helpful. Nevertheless, the authors have conducted a straightforward dissection the possible steps of infection where AAIM-1 may act and have arrived at a plausible hypothesis.

    Whereas the characterization of AAIM-1 as a host factor required for microsporidia invasion is novel and convincing, the characterization of its phenotype in bacterial resistance is less compelling. The magnitude of the effects observed with regard to Pseudomonas are modest at best. Competitive fitness assays do little more than corroborate the resistance phenotypes. The manuscript argues for the novelty and significance of a factor that promotes microsporidia invasion but limits bacterial infection (including emphasis in the title of the manuscript), yet the relative strengths of the phenotypes (microsporidia-STRONG, SOLID; bacteria-WEAK, MARGINAL) are such that this narrative is not compelling. I think the more straightforward message of this work, which focuses on the identification of a novel host factor required for microsporidia invasion out of an elegant C. elegans genetic screen, is well-supported by the data and will be of interest to the community of investigators studying the interaction between animal hosts and eukaryotic pathogens.

  3. Reviewer #2 (Public Review):

    Using a forward genetics screen, the authors discovered a gene, aaim-1, to be a regulator of invasion of C. elegans intestine by microsporidia, a pathogen present in its natural habitat. This gene was known to be induced by infection with pathogenic bacterium P. aeruginosa. The authors go on to show that the phenotype is L1 larva stage-specific although the reason for this specificity is not entirely clear. The study proposes that the orientation of the spore in the intestinal lumen is somewhat altered in the aaim-1 mutant. They go on to show that aaim-1 mutant has better fitness during microsporidia infection but lower fitness during P. aeruginosa infection. The study is incomplete in its present form and a close review of the data indicates that the conclusions are not entirely supported by the data.

    Strengths: The strength of the study lies in the use of a forward genetics approach to uncover a new regulator for microsporidia invasion. I commend the authors for taking the approach. All 4 strains identified in the screen map to the same locus, for aaim-1. The phenotype of these alleles is phenocopied by a deletion allele created using CRISPR. The authors show some interesting evidence for alteration in microsporidia and P. aeruginosa load in aaim-1 mutants suggesting that AAIM-1 may regulate responses to these two classes of pathogens differentially.

    The study appears to be incomplete in its current form. The major weakness lies in the lack of data on the possible functions of AAIM-1.

    The authors have missed taking note of the observation that microsporidia invasion causes larval arrest along with brood size defects (Fig 1). Both these can also result from starvation, caloric restriction, and in the case of certain infections [Van Gilst 2005; Garsin 2003; Dasgupta 2020]. It is quite possible that AAIM-1 mutation relieves the block caused by infection (microsporidia or other) which may impinge upon C. elegans metabolism (specifically lipid metabolism). Several papers in the recent past have indicated that lipid metabolism is altered during infection with both Gram-negative and Gram-positive bacteria. Does the same happen in microsporidia invasion?

    The significance of spore orientation relative to the epithelium is not clear. Is there published literature, from authors or others, to show that spore orientation is causal or correlated with the firing of polar tubes and subsequent invasion? Authors should present evidence for this. I suggest that the authors find some means (perhaps the use of microfluidic channels with adhesive molecules), to show how spore orientation is linked to the presence of AAIM-1 protein. In absence of this information, the conclusion that AAIM-1 regulates spore orientation and firing is misleading.

    An alternate explanation for altered spore orientation is that adhesion molecules expressed on intestinal epithelia in the aaim-1 mutant might be different from those in wild type/reference strain of worms. This can be studied at the level of transcriptome and proteome and specifically for proteins with carbohydrate modifications.

    Microvilli are known to be lost in intestinal epithelial during S. aureus infection [Irazoqui 2010], is there any evidence of whether microvilli are altered in the aaim-1 mutant which may affect adhesion or orientation of spores?

    The effect of aaim-1 mutation on worm's survival during PA14 infection is very mild. Did authors find a known regulator of the innate immune pathway (p38 MAPK, XBP-1, ATF-7, ZIP-2, ELT-2, etc) or their transcriptional targets to be altered?

    Along the same lines, one would predict that aaim-1 mutation would affect fitness on other pathogenic bacteria found in the same habitat as microsporidia [Troemel 2008; Schulenberg 2016]. If AAIM-1 provides a trade-off between protection from pathogenic bacteria and invasion by microsporidia, it should become apparent in other cases as well.

    The statement that AAIM-1 is exploited by microsporidia for successful invasion is not satisfactorily supported by data. To support this claim, authors can test whether AAIM-1 binds to or decorates spores which would suggest a possible impact on the binding of spores to worm's intestinal epithelium? Alternatively, authors can examine expression of lectins in aaim-1 mutant (Lectins are altered widely in C. elegans during infection with various pathogens and are known to affect adhesion in various contexts).