The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as IL-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPEC∆nleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentium∆nleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11 deficient mice. Thus, IECs play a key role in modulating early innate immune responses in the gut via a caspase-4/11 - IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

Infection of mice with Citrobacter rodentium is a robust model to study bacterial pathogenesis, mucosal immunology, the health benefits of probiotics and the role of the microbiota during infection. C. rodentium was first isolated by Barthold from an outbreak of mouse diarrhea in Yale University in 1972 and was 'rediscovered' by Falkow and Schauer in 1993. Since then the use of the model has proliferated, and it is now the gold standard for studying virulence of the closely related human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Here we provide a detailed protocol for various applications of the model, including bacterial growth, site-directed mutagenesis, mouse inoculation (from cultured cells and after cohabitation), monitoring of bacterial colonization, tissue extraction and analysis, immune responses, probiotic treatment and microbiota analysis. The main protocol, from mouse infection to clearance and analysis of tissues and host responses, takes ∼5 weeks to complete.

The lung is colonized by commensal bacteria, some of which are associated with asthma exacerbations. Using the intranasal house-dust mite-sensitized mouse model of allergic airway disease, we show an imbalance in novel antibacterial pathways that culminates in a reduction in neutrophil recruitment to the airspaces and leads to bacterial invasion and dissemination. The expression of TREM (Triggering Receptor Expressed on Myeloid cells)-1 that amplifies Toll-like receptor (TLR) signaling and TREM-2 that inhibits this process is reversed. Furthermore, endogenous TLR inhibitors (A20, Tollip, SOCS1, and IRAK-M) and proteins involved in receptor recycling (TRIAD3) are raised. Consequently, the production of neutrophil chemoattractants is reduced. Intranasal administration of either chemokine restores the ability to recruit neutrophils, which prevents bacterial invasion. A background of allergic airway disease therefore exacerbates bacterial infection by altering key antibacterial innate immune pathways that are amenable to therapeutic intervention.

The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection. Enteropathogenic Escherichia coli (EPEC) strains are important human pathogens causing infantile diarrhea in low-income countries (1) Recently, the Global Enteric Multicenter Study (GEMS), designed to detect the cause of pediatric diarrheal disease in sub-Saharan Africa and south Asia, found that infection with typical EPEC is associated with increased risk of fatality in infants aged 0 to 11 months (2). Citrobacter rodentium is a mouse-specific pathogen, the etiological agent of transmissible colonic hyperplasia, and a model EPEC microorganism, as both pathogens share an infection strategy and virulence factors (3, 4). Host resistance to C. rodentium infection is mediated by diverse T cell effector responses, including T cell production of interferon gamma (IFN-␥) (5, 6), interleukin 17A (IL-17A) (7,8),. Expression of the proinflammatory cytokine IL-17A leads to recruitment of neutrophils (10), and the anti-inflammatory cytokine IL-22 upregulates expression of antimicrobial peptides (such as REGIII␤ and REGIII␥) in enterocytes (9, 11).While colonizing the gut mucosa, EPEC and C. rodentium induce attaching and effacing (A/E) lesions. These are characterized by extensive remodeling of the gut epithelium leading to elongation and effacement of the brush border (BB) microvilli, intimate bacterial attachment to the enterocyte apical...

Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.