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

Clayton M. Carey; Sarah E. Apple; Zoe A. Hilbert; Michael S. Kay; Nels C. Elde

Bats are known to harbor many types of viruses, including several human pathogens 14 . This widespread association with viruses is attributed to high density population structures and the ability of long-distance flight 15 . Notably, frequent infection with coronaviruses likely sparked high levels of sequence diversification in the viral entry receptor ACE-2 among bats 16 , illustrating the intensity of selection pathogens place on these species. In addition to viruses, bats can carry bacteria that act as human pathogens, including genera that encode STa 17 . Furthermore, STa variants have been detected in DNA isolated from the feces of wild bats 18 . Our detection of signals of intense positive selection in the GC-C ligand-binding domain of bats suggests that diarrheal pathogens may have profoundly impacted the evolution of bats, similar to their established coevolutionary relationship with viruses ( Figure 1B ). To test if rapid evolution of GC-C ligand-binding domains result in functional differences in STa susceptibility, we generated cell lines stably expressing GC-C from seven primate and five bat species. Functional diversity in these GC-C variants was assessed by measuring intracellular cGMP production in response to chemically synthesized uroguanylin and STa variants from four strains of pathogenic bacteria. HEK293T cells expressing human, chimpanzee, gorilla, gibbon and African green monkey GC-C generated similar levels of cGMP upon stimulation with STa variants from pathogenic E. coli (STp and STh), Yersinia enterocolitica (Y-ST), and Vibrio mimicus (V-ST) (Figure 2A , S5). In contrast, cells expressing GC-C from orangutan and rhesus macaque were significantly more susceptible to STp, showing a large increase in cGMP production relative to other species (Figure 2A ). Dose-response curves comparing maximal activation of human and orangutan GC-C to each toxin revealed similar maximal responses for human GC-C, while orangutan GC-C exhibited increases in relative vulnerability to STp and STh toxin variants compared to Y-ST and V-ST ( Figure 2B , 2C). Thus, variation in GC-C among primates results in differing levels of susceptibility to diverse toxins, providing evidence that ancient STa-like peptides influenced the course of GC-C evolution.