Establishment of a knock-in mouse model expressing a hypomorphic variant of receptor guanylyl cyclase C

Abstract

Receptor guanylyl cyclase C (GC-C, gene GUCY2C) is expressed on the apical surface of the intestinal epithelial cells and gets activated by the endogenous ligands guanylin and uroguanylin. Heat-stable enterotoxin (ST) secreted by enterotoxigenic E. coli is a super-agonist of GC-C. Activated GC-C catalyses the conversion of guanosine 5’- triphosphate (GTP) to cyclic guanosine 3’,5’-monophosphate (cGMP). GC-C via cGMP plays a key role in several biological processes such as the maintenance of intestinal fluid-ion homeostasis, regulation of intestinal cell cytostasis and tumorigenesis, mediation of gastrointestinal inflammation and protection against enteric pathogens like Salmonella Typhimurium.

Several disease-causing mutations have been identified in the GUCY2C gene in humans. Gain-of-function mutations result in increased GC-C activity leading to chronic diarrhoea and intestinal inflammation and the loss-of-function mutations result in meconium ileus due to the inactivation of the receptor. This study addresses the physiological implications of reduced cGMP by the generation of a novel knock-in mouse model harbouring a hypomorphic mutation in the linker region of GC-C. Mice with the hypomorphic mutant of GC-C showed reduced GC-C binding to ST indicating misfolding of the receptors. Further, the mice displayed decreased levels of cGMP in the colonic epithelial cells. Initial phenotypic characterisation of the mice showed no difference in their feeding behaviour, gut morphology and histology. However, a faster total gut transit and higher colonic inflammation were observed in mutant mice suggesting subtle changes occurring at the molecular level due to reduced cGMP.

To further explore the effects of decreased cGMP on the regulation of GC-C mediated protection against enteric pathogens, mice were infected with Citrobacter rodentium (C.r), a murine model for enteropathogenic E. coli infections. Using a bioluminescent tagged strain of C.r and live imaging of animals, higher pathogen load in faeces and higher bioluminescence was observed in the gut, suggesting increased colonisation of the pathogen and/or a more oxidative environment in the gut of mutant mice. Increased tissue-associated bacterial burden as compared to the wild type mice was observed in mutant mice resulting in higher C.r induced colonic inflammation. Reduced expression of C.r virulence factors with respect to the total pathogen colonisation was observed indicating the presence of avirulent C. rodentium in the intestine of knock-in mice, which could be attributed to the microbial dysbiosis.

Taken together, the novel mouse model developed in this study mimics humans harbouring heterozygous loss-of-function mutants in the GUCY2C gene that do not cause visible phenotype but would result in low cGMP levels in the gut. Furthermore, the model provides novel insights into the role of cGMP in the mediation of protection against enteric infections. Therefore, the knock-in mice can be used as a pre-clinical model to understand the consequences of low cGMP levels on gut physiology and to develop potential therapeutic strategies for alleviation of the symptoms associated with GC-C/cGMP mediated diseases.