Introduction: Clinical research to develop treatment for inflammatory bowel disease (IBD) is focusing on a nutritional regimen restricting certain carbohydrates while incorporating the use of an optimal diet that includes pre- and probiotic foods. Current assessments are not able to measure elements of this nutritional regimen, thus we developed a food frequency questionnaire (FFQ). This FFQ will be utilized in a prospective study of IBD patients following an anti-inflammatory diet (IBD-AID) developed by us and used clinically at UMASS. We will track the bacterial communities inhabiting the microbiome of patients to determine diet-dependent changes, and their relation with patient wellbeing. Objectives: 1) Develop an FFQ capable of identifying dietary components important to IBD: prebiotics, probiotics, beneficial nutrient intake, and avoidance of certain foods. 2) Determine diet-dependent changes of the gut microbiome. Hypothesis: This study will show the microbiome of patients adopting the IBD-AID converge to one or more healthy 'enterotype' signatures, as compared to a non-IBD-AID control group. Experimental design: Patients record daily FFQ. Foods and food groups (270) are categorized and grouped according to criteria of interest. Each food has a referent by which the patient can compare their own consumption. A scoring system satisfying dietary guidelines and components of the IBD-AID will be utilized. Twice per week patients collect stool samples for microbiome analysis. Microbiome composition and ecological metrics are compared to identify components influenced by the IBD-AID, and to separate bacterial 'enterotype' signatures of patients before, during and after diet intervention. We are currently recruiting patients.

Enterohemorrhagic E. coli (EHEC) is a major cause of food borne diarrheal illness worldwide. While disease symptoms are usually self-resolving and limited to severe gastroenteritis with bloody diarrhea, EHEC infection can lead to a life threatening complication known as Hemolytic Uremic Syndrome (HUS), which strikes children disproportionately and is the leading cause of kidney failure in children. Upon infection of gut epithelia, EHEC produces characteristic lesions called actin pedestals. These striking formations involve dramatic rearrangement of host cytoskeletal proteins. EHEC hijacks mammalian signaling pathways to cause destruction of microvilli and rebuilds the actin cytoskeleton underneath sites of bacterial attachment. Here, we present a brief study on a host factor, Calpain, involved in microvilli effacement, and an in depth investigation on a bacterial factor, EspFU, required for actin pedestal formation in intestinal cell models. Calpain is activated by both EHEC and the related pathogen, enteropathogenic E. coli (EPEC), during infection and facilitates microvilli disassembly by cleavage of a key membrane-cytoskeleton anchoring substrate, Ezrin. Actin pedestal formation is facilitated by the injection of two bacterial effectors, Tir and EspFU, into host cells, which work in concert to manipulate the host actin nucleators N-WASP and Arp2/3. EspFU hijacks key host signaling proteins N-WASP and IRTKS by mimetic displacement and has evolved to outcompete mammalian host ligands. Multiple repeats of key functional domains of EspFU are essential for actin pedestal activity through proper localization and competition against the an abundant host factor Eps8 for binding to IRTKS.

Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions.

Pathogenic Escherichia coli are genetically diverse and encompass a broad variety of pathotypes, such as enteroaggregative E. coli (EAEC) or enterohemorrhagic E. coli (EHEC), which cause distinct clinical syndromes. The historically large 2011 German outbreak of hemolytic uremic syndrome (HUS), caused by a Shiga-toxin producing E. coli (STEC) of the serotype O104:H4, illustrated the emerging importance of non-O157 STEC. STEC O104:H4, with features characteristic of both enteroaggregative E. coli and enterohemorrhagic E. coli, represents a unique and highly virulent pathotype. The German outbreak both allowed for the evaluation of several potential therapeutic approaches to STEC-induced HUS and emphasizes the importance of early and specific detection of both O157 and non-O157 STEC.

A member of the attaching and effacing (AE) family of pathogens, enterohemorrhagic Escherichia coli (EHEC) induces dramatic changes to the intestinal cell cytoskeleton, including effacement of microvilli. Effacement by the related pathogen enteropathogenic E. coli (EPEC) requires the activity of the Ca(+2)-dependent host protease, calpain, which participates in a variety of cellular processes, including cell adhesion and motility. We found that EHEC infection results in an increase in epithelial (CaCo-2a) cell calpain activity and that EHEC-induced microvillar effacement was blocked by ectopic expression of calpastatin, an endogenous calpain inhibitor, or by pretreatment of intestinal cells with a cell-penetrating version of calpastatin. In addition, ezrin, a known calpain substrate that links the plasma membrane to axial actin filaments in microvilli, was cleaved in a calpain-dependent manner during EHEC infection and lost from its normal locale within microvilli. Calpain may be a central conduit through which EHEC and other AE pathogens induce enterocyte cytoskeletal remodeling and exert their pathogenic effects.