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Non-LEE-encoded Effector A (NleA) is a type III secreted effector protein of enterohaemorrhagic and enteropathogenic Escherichia coli as well as the related mouse pathogen Citrobacter rodentium . NleA translocation into host cells is essential for virulence. We previously published several lines of evidence indicating that NleA is modified by host-mediated mucin-type O-linked glycosylation, the first example of a bacterial effector protein modified in this way. In this study, we use lectins to provide direct evidence for the modification of NleA by O-linked glycosylation and determine that the interaction of NleA with the COPII complex is necessary for this modification to occur.

There is a growing appreciation that the interaction between diet, the gut microbiota and the immune system contribute to the development and progression of inflammatory bowel disease (IBD). A mounting body of scientific evidence suggests that high-fat diets exacerbate IBD; however, there is a lack of information on how specific types of fat impact colitis. The Mediterranean diet (MD) is considered a health-promoting diet containing approximately 40% total fat. It is not known if the blend of fats found in the MD contributes to its beneficial protective effects., Mice deficient in the mucin 2 gene (Muc 2 -/- ) were weaned to 40% fat, isocaloric, isonitrogenous diets. We compared the MD fat blend (high monounsaturated, 2:1 n-6:n-3 polyunsaturated and moderate saturated fat) to diets composed of corn oil (CO, n-6 polyunsaturated-rich), olive oil (monounsaturated-rich) or milk fat (MF, saturated-rich) on spontaneous colitis development in Muc2 -/- mice. The MD resulted in lower clinical and histopathological scores and induced tolerogenic CD103+ CD11b+ dendritic, Th22 and IL-17+ IL-22+ cells necessary for intestinal barrier repair. The MD was associated with beneficial microbes and associated with higher cecal acetic acid levels negatively correlated with colitogenic microbes like Akkermansia muciniphila . In contrast, CO showed a higher prevalence of mucin-degraders including A. muciniphila and Enterobacteriaceae, which have been associated with colitis., A dietary blend of fats mimicking the MD, reduces disease activity, inflammation-related biomarkers and improves metabolic parameters in the Muc2 -/- mouse model. Our findings suggest that the MD fat blend could be incorporated into a maintenance diet for colitis.

Parkinson's disease (PD) is a neurodegenerative disorder that has been shown to be influenced by the intestinal milieu. The gut microbiota is altered in PD patients, and murine studies have begun suggesting a causative role for the gut microbiota in progression of PD. We have previously shown that repeated infection with the intestinal murine pathogen Citrobacter rodentium resulted in the development of PD-like pathology in Pink1 -/- mice compared to wild-type littermates. This addendum aims to expand this work by characterizing the gut microbiota during C. rodentium infection in our Pink1 -/- PD model. We observed little disturbance to the fecal microbiota diversity both between infection timepoints and between Pink1 -/- and wild-type control littermates. However, the level of short-chain fatty acids appeared to be altered over the course of infection with butyric acid significantly increasing in Pink1 -/- mice and isobutyric acid increasing in wild-type mice.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric human pathogens that colonize the large and small intestines, respectively. To establish infection EHEC and EPEC must overcome innate host defenses, such as antimicrobial peptides (AMPs) produced by the intestinal epithelium. Gram-negative pathogens have evolved different mechanisms to resist AMPs, including outer-membrane proteases that degrade AMPs. We showed that the protease OmpT degrades the human AMP LL-37 more rapidly in EHEC than in EPEC. Promoter-swap experiments showed that this is due to differences in the promoters of the two genes, leading to greater ompT expression and subsequently greater levels of OmpT in EHEC. Here, we propose that the different ompT expression in EHEC and EPEC reflects the varying levels of LL-37 throughout the human intestinal tract. These data suggest that EHEC and EPEC adapted to their specific niches by developing distinct AMP-specific resistance mechanisms.

Citrobacter rodentium is a natural pathogen of mice that causes intestinal hyperplasia and colitis. Resistant strains such as C57BL/6J (B6) experience a self-limiting disease that peaks between one and two weeks post infection, followed by a clearing of the infection and complete recovery. However, the inbred mouse strains C3H/HeJ (C3), C3H/HeOuJ (C3Ou) and FVB/N (FVB) are highly susceptible to C. rodentium infection and develop more severe symptoms of disease leading to high rates of mortality during infection. We have recently demonstrated through a systematic genetics approach that a single locus on proximal chromosome 15 is responsible for the susceptibility of both C3 and C3Ou mice to C. rodentium infection. We have named the locus Citrobacter rodentium infection 1 (Cri1). Here we show that Cri1 also controls susceptibility to C. rodentium in FVB mice, using a targeted method of genotyping to stratify (B6 x FVB)F2 mice according to their genotype at Cri1. Mice that inherit two copies of the resistant B6 allele have 97% cumulative survival at day 30 post-infection, whereas those that inherit one or two copies of Cri1 from the FVB parent have significantly lower rates of survival (35% and 42%, respectively). These results provide evidence for a common genetic cause of fatal infectious colitis in C3, C3Ou and FVB mice following infection with Citrobacter rodentium.

Autophagy mediates the degradation of cytoplasmic components in eukaryotic cells and plays a key role in immunity. The mechanism of autophagosome formation is not clear. Here we examined two potential membrane sources for antibacterial autophagy: the ER and mitochondria. DFCP1, a marker of specialized ER domains known as 'omegasomes,' associated with Salmonella-containing autophagosomes via its PtdIns(3)P and ER-binding domains, while a mitochondrial marker (cytochrome b5-GFP) did not. Rab1 also localized to autophagosomes, and its activity was required for autophagosome formation, clearance of protein aggregates and peroxisomes, and autophagy of Salmonella. Overexpression of Rab1 enhanced antibacterial autophagy. The role of Rab1 in antibacterial autophagy was independent of its role in ER-to-Golgi transport. Our data suggest that antibacterial autophagy occurs at omegasomes and reveal that the Rab1 GTPase plays a crucial role in mammalian autophagy.

Enteropathogenic Escherichia coli (EPEC) is an enteric pathogen able to cause severe diarrhea. Once adhered to the small intestine, EPEC disrupts tight junctions that are important for intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Recently we have shown that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. NleA has a predicted PDZ-binding domain at its C-terminus which is proposed to be involved in protein interactions with PDZ domain containing proteins. Since several PDZ-domain-containing proteins localize to tight junctions, we hypothesized that the PDZ-binding domain of NleA might be important for its role in tight junction disruption. However, here we show that a molecular variant of NleA lacking the PDZ-binding domain behaves indistinguishably from the wild-type protein with respect to disruption of tight junctions.

We have analyzed the means by which the Nck family of adaptor proteins couples adhesion proteins to actin reorganization. The nephrin adhesion protein is essential for the formation of actin-based foot processes in glomerular podocytes. The clustering of nephrin induces its tyrosine phosphorylation, Nck recruitment, and sustained localized actin polymerization. Any one of three phosphorylated (p)YDXV motifs on nephrin is sufficient to recruit Nck through its Src homology 2 (SH2) domain and induce localized actin polymerization at these clusters. Similarly, Nck SH3 mutants in which only the second or third SH3 domain is functional can mediate nephrin-induced actin polymerization. However, combining such nephrin and Nck mutants attenuates actin polymerization at nephrin-Nck clusters. We propose that the multiple Nck SH2-binding motifs on nephrin and the multiple SH3 domains of Nck act cooperatively to recruit the high local concentration of effectors at sites of nephrin activation that is required to initiate and maintain actin polymerization in vivo. We also find that YDXV motifs in the Tir protein of enteropathogenic Escherichia coli and nephrin are functionally interchangeable, indicating that Tir reorganizes the actin cytoskeleton by molecular mimicry of nephrin-like signaling. Together, these data identify pYDXV/Nck signaling as a potent and portable mechanism for physiological and pathological actin regulation.