Conflicts with diarrheal pathogens trigger rapid evolution of an intestinal signaling axis

Clayton M. Carey, Sarah E. Apple, Zoё A. Hilbert, Michael S. Kay, Nels C. Elde

Preprint posted on 30 March 2020

Article now published in Cell Host & Microbe at

Conflicts in the Gut – Diarrheal Pathogens Driving Host Evolution

Selected by Connor Rosen


Pathogens represent a danger for all organisms necessitating the evolution of molecular defenses, from bacterial immune systems such as CRISPR and restriction enzymes to the evolution of adaptive immune systems in vertebrate animals. Beyond the development of specialized immune systems, the effect of pathogens on particular biological processes can be identified from genomic signatures. The co-evolution of pathogens with particular host proteins can be tracked with evidence of adaptive evolution and natural selection. In particular, the more rapid change in proteins targeted by particular pathogens, often measured by the ratio dN/dS (the ratio of non-synonymous to synonymous nucleotide changes between homologous proteins across species), is strong evidence of an evolutionary pressure from the pathogen. The back-and-forth evolution of pathogens to target the host, the host mutating to avoid or neutralize pathogen activity, and subsequent re-adaptation of the pathogen can result in an “arms-race” dynamic. While individual investigations have revealed signatures of host-pathogen co-evolution and “arms-races” in a number of immune proteins [Daugherty and Malik, 2012], the extent of the evolutionary pressures that pathogens represent on a broader range of physiological pathways has remained unclear. In this preprint, Carey et al identify signatures of rapid evolution at mammalian Guanylate Cyclase-C (GC-C), a regulator of intestinal osmotic balance, that appear to be driven by pressures from pathogen-derived toxins.


Key findings:

  • Rapid evolution in GC-C and response to bacterial enterotoxins

GC-C is targeted by diarrhea-inducing enterotoxins from multiple pathogens. Primate GC-C sequences showed evidence of positive selection in the extracellular region, where toxins bind, and divergent binding towards enterotoxins derived from different pathogens. Among other mammalian lineages, bats showed extremely widespread positive selection in GC-C, again located exclusively in the extracellular region, and divergent susceptibility towards toxins from particular pathogens. These data suggest that the evasion of pathogen toxins is a sufficient evolutionary pressure to drive adaptive evolution in the toxin target, a regulator of a basic physiological process.

  • Co-evolution of uroguanylin with GC-C in bats

The rapid evolution of GC-C in bats raises an evolutionary conundrum – GC-C responds to endogenous ligands (guanylin and uroguanylin) in order to carry out the basic function of intestinal water regulation. Uroguanylin sequences in bats show more variability across species than primates, consistent with the increased burden of GC-C mutations. Uroguanylin peptides showed species-specificity in their activating potential towards bat GC-C receptors, suggesting co-evolution to maintain binding in the presence of the “third party” pressure from the toxins. Interestingly, one species of bat showed reduced activity towards all uroguanylin peptides, including its own, suggesting it exists in an evolutionary “intermediate state” where it has decreased sensitivity to both toxins and uroguanylin.



This preprint presents strong evidence for the pathogen-driven evolution of GC-C in primates and bats, and the co-evolution of uroguanylin in bats. This shows the immense selective pressure individual pathogens can exert on a broad range of pathways in host physiology – driving evolution not only of classical “immune” genes, but demonstrating that bacterial enterotoxins are sufficient to shape basic intestinal physiology over evolutionary timescales. Additionally, this study reveals the rapid evolution of bats in response to bacterial pathogens, including the ability to partially “neglect” basic GC-C regulation. This is of particular interest given that the range of unique adaptations in bat antiviral immunity may factor into their role as reservoirs of zoonotic viruses (Xie et al 2018, Ahn et al 2019, Lu and Liu et al 2019 as non-exhaustive recent examples), and raises the question of whether they might harbor similar reservoirs of zoonotic bacteria.


Moving Forward / Questions for Authors:

  • Why might bats be so sensitive to diarrheal pathogens? The strong evidence of positive selection contrasts with a lack of positive selection in other lineages with evidence of bacterial enterotoxins, such as Bovidae. One obvious difference is a flying lifestyle, but are there other aspects of bat physiology that might explain the need to evolve so rapidly as to reach those “intermediate states” of impaired endogenous uroguanylin sensitivity?
  • Are the patterns of evolution and toxin evasion sufficient to guess at the original selective pressures driving differences in bats? For example, it appears that the major difference between lucifugus and P. vampyrus is in sensitivity to Yersinia enterotoxin, with minor differences in sensitivity to the other toxins. How would the authors predict this would occur from, for example, a single plague outbreak leading to a bottlenecking event in an ancestor of P. vampyrus, versus geographic isolation and differential pathogen exposure leading to gradual selection during speciation?



  • Ahn M et al, “Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host”, Nature Microbiology (2019) 4:789-799
  • Daugherty MD and Malik HS, “Rules of Engagement: Molecular Insights from Host-Virus Arms Races”, Annual Reviews of Genetics (2012) 46:677-700
  • Lu D and Liu K et al, “Peptide presentation by bat MHC class I provides new insight into the antiviral immunity of bats”, PLOS Biology (2019) doi:10.1371/journal.pbio.3000436
  • Xie J et al, “Dampened STING-Dependent Interferon Activation in Bats”, Cell Host and Microbe (2018) 23:297-301


Posted on: 17 April 2020


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Author's response

Clay Carey shared

Hello, this is Clay Carey. I am the first author of the study highlighted here. Thank you for your great write-up, I am grateful for your interest in our work!

We observed very striking levels of divergence in GC-C among bats and substitution patterns consistent with strong selective pressure to modulate toxin interactions. Why bats might be subject to such intense selection relative to other mammals is an interesting question. As you noted, they have the unique ability of flight among mammals, which allows them to cover large geographic distances, providing more opportunities to encounter pathogens. What I think is probably a more important lifestyle factor is that all the bat species we sampled in our study live in high-density colonies with thousands or even millions of individuals sharing confined spaces and communal resources. We know conditions like these are conducive to the spread of diarrheal diseases in humans. It would be interesting to know whether these evolutionary patterns are different in bat species that live a more solitary lifestyle. An additional factor to consider is a heightened metabolic rate in bats might make water an even more precious resource, so losing the ability to fine tune water secretion via uroguanylin signaling might be more tolerable. I hope that our work can spark more interest in these hypotheses and the community can begin to experimentally test the links between host behavior and physiology and susceptibility to diarrheal disease.

We did find significant differences in susceptibility to toxin from Yersinia enterocolitica between different bat species, to the point where the giant fruit bat Pteropus vampyrus seems to be nearly immune to the effects of the toxin compared to the more susceptible vesper bat Myotis lucifugus. While it is tempting to speculate that there was a bottleneck event during an outbreak of Yersinia enterocolitica in an ancestor of the modern old world fruit bats that resulted in fixation of a resistant allele, we really can’t know if that is the case. Given the evidence of intense selection in GC-C even in the deep branches of the chiropteran tree, it is likely that bats have encountered many different STa-like peptides over the course of millions of years of evolution. One thing to consider is that our study has an anthropocentric bias in terms of the toxins we tested. This is out of necessity, because we only really know about the toxin variants found in human pathogens! I think we have only scratched the surface in terms of bacterial diversity of these heat-stable enterotoxins that target GC-C. I think it will be fascinating to learn about the sequences and activities of these toxins that are circulating in bat populations. Stay tuned to the Elde lab for more on this in the future!

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