Gastrointestinal dysfunction in patients and mice expressing the autism-associated R451C mutation in neuroligin-3

Gastrointestinal (GI) problems constitute an important comorbidity in many patients with autism. Multiple mutations in the neuroligin family of synaptic adhesion molecules are implicated in autism, however whether they are expressed and impact GI function via changes in the enteric nervous system is unknown. We report the GI symptoms of two brothers with autism and an R451C mutation in Nlgn3 encoding the synaptic adhesion protein, neuroligin-3. We confirm the presence of an array of synaptic genes in the murine GI tract and investigate the impact of impaired synaptic protein expression in mice carrying the human neuroligin-3 R451C missense mutation (NL3R451C ). Assessing in vivo gut dysfunction, we report faster small intestinal transit in NL3R451C compared to wild-type mice. Using an ex vivo colonic motility assay, we show increased sensitivity to GABAA receptor modulation in NL3R451C mice, a well-established Central Nervous System (CNS) feature associated with this mutation. We further show increased numbers of small intestine myenteric neurons in NL3R451C mice. Although we observed altered sensitivity to GABAA receptor modulators in the colon, there was no change in colonic neuronal numbers including the number of GABA-immunoreactive myenteric neurons. We further identified altered fecal microbial communities in NL3R451C mice. These results suggest that the R451C mutation affects small intestinal and colonic function and alter neuronal numbers in the small intestine as well as impact fecal microbes. Our findings identify a novel GI phenotype associated with the R451C mutation and highlight NL3R451C mice as a useful preclinical model of GI dysfunction in autism. LAY SUMMARY: People with autism commonly experience gastrointestinal problems, however the cause is unknown. We report gut symptoms in patients with the autism-associated R451C mutation encoding the neuroligin-3 protein. We show that many of the genes implicated in autism are expressed in mouse gut. The neuroligin-3 R451C mutation alters the enteric nervous system, causes gastrointestinal dysfunction, and disrupts gut microbe populations in mice. Gut dysfunction in autism could be due to mutations that affect neuronal communication.This work was supported by an Idea Development Award from the United States Department of Defense’s Congressionally Directed Medical Research Programs (CDMRP) Autism Research Program (AR110134) to E.L.H.-Y. and J.C.B.; the Victorian Government through the Operational Infrastructure Scheme, National Health and Medical Research Council (NHMRC) project grants (APP566642 to J.C.B. and APP1047674 to E.L.H.-Y.) and the Royal Melbourne Hospital Neuroscience Foundation. E.L.H.-Y. also received an ARC Future Fellowship (FT160100126) and an RMIT Vice Chancellor’s Senior Research Fellowship which supported G.K.B. and S.H. T.S., P.U., and N.Y. were funded by grants RO1AI100914, P30-DK56338, and U01-AI24290 awards to Baylor College of Medicine funded from the National Institute of Allergy and Infectious Diseases and National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health (T.C.S.)

Epithelial Invasion by Escherichia coli Bearing Dr Fimbriae Is Controlled by Nitric Oxide-Regulated Expression of CD55

We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr(+)). Epithelial binding and invasion by Dr(+) E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr(+) E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr(+) E. coli and a Dr(−) E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr(+) E. coli. Invasion by Dr(+) E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection

Structure-Function Analysis of Decay-Accelerating Factor: Identification of Residues Important for Binding of the Escherichia coli Dr Adhesin and Complement Regulation

Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a(−)); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids—predominantly those in close proximity to Ser165 to alanine—and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart

Hydrophilic Domain II of Escherichia coli Dr Fimbriae Facilitates Cell Invasion

Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decay-accelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated

High resolution studies of the Afa/Dr adhesin DraE and its interaction with chloramphenicol.

Pathogenic Escherichia coli expressing Afa/Dr adhesins are able to cause both urinary tract and diarrheal infections. The Afa/Dr adhesins confer adherence to epithelial cells via interactions with the human complement regulating protein, decay accelerating factor (DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other by only three residues but are reported to have several different properties. One such difference is disruption of the interaction between DraE and CD55 by chloramphenicol, whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer in complex with chloramphenicol, as well as NMR data supporting the binding position of chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that in contrast to other chloramphenicol-protein complexes, drug binding is mediated via recognition of the chlorine "tail" rather than via intercalation of the benzene rings into a hydrophobic pocket