Colonization of Intestinal Pathogen Changes the Gut Microbiota

Enterohemorrhagic Escherechia coli is a serious human pathogen causing bloody diarrhea and hemolytic uremic syndrome. It is difficult to study in animal models, but pathogenesis may be modeled in mice with the similar murine pathogen, Citrobacter rodentium. C. rodentium does not cause disease in streptomycin-treated mice, suggesting that it is competition with other facultative anaerobes that triggers pathogenesis. Streptomycin-treated mice were co-colonized with C. rodentium and a commensal E. coli strain. The intestinal microbiota of each group was observed over a 15-day period using quantitative PCR. Colon weights were also measured over the same period. Results indicate that the disease caused by competition is not similar to normal C. rodentium pathogenesis. Further research is necessary to determine the precise mechanism of pathogenesis in this experimental model. The outcome may provide new insight into enterohemorrhagic E. coli prevention and treatment

Reactive oxygen species regulate caspase-11 expression and activation of the non-canonical NLRP3 inflammasome during enteric pathogen infection

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3-and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway

Model of host-pathogen Interaction dynamics links In vivo optical imaging and immune responses

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

T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium.

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses

Intestinal Acid Sphingomyelinase Protects From Severe Pathogen-Driven Colitis

Inflammatory diseases of the gastrointestinal tract are emerging as a global problem with increased evidence and prevalence in numerous countries. A dysregulated sphingolipid metabolism occurs in patients with ulcerative colitis and is discussed to contribute to its pathogenesis. In the present study, we determined the impact of acid sphingomyelinase (Asm), which catalyzes the hydrolysis of sphingomyelin to ceramide, on the course of Citrobacter (C.) rodentium-driven colitis. C. rodentium is an enteric pathogen and induces colonic inflammation very similar to the pathology in patients with ulcerative colitis. We found that mice with Asm deficiency or Asm inhibition were strongly susceptible to C. rodentium infection. These mice showed increased levels of C. rodentium in the feces and were prone to bacterial spreading to the systemic organs. In addition, mice lacking Asm activity showed an uncontrolled inflammatory Th1 and Th17 response, which was accompanied by a stronger colonic pathology compared to infected wild type mice. These findings identified Asm as an essential regulator of mucosal immunity to the enteric pathogen C. rodentium

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targetsSpanish Ministerio de Economía, Industria y Competitividad , Agencia Estatal de Investigación, and the European Regional Development Fund, grant number SAF2016‐75365‐REuropean Union’s Horizon 2020 e Marie Skłodowska‐Curie grant agreement No 84262

From ingestion to colonization: the influence of the host environment on regulation of the LEE encoded type III secretion system in enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) binds to host tissue and intimately attaches to intestinal cells using a dedicated type III secretion system (T3SS). This complex multi-protein organelle is encoded within a large pathogenicity island called the locus of enterocyte effacement (LEE), which is subject to extensive regulatory control. Over the past 15 years we have gained a wealth of knowledge concerning how the LEE is regulated transcriptionally by specific, global and phage encoded regulators. More recently, significant advances have been made in our understanding of how specific signals, including host or microbiota derived metabolic products and various nutrient sources, can affect how the LEE-encoded T3SS is regulated. In this review we discuss regulation of the LEE, focusing on how these physiologically relevant signals are sensed and how they affect the expression of this major virulence factor. The implications for understanding the disease process by specific regulatory mechanisms are also discussed

A Murine Model of Shigellosis: Pathophysiology of Shiga Toxin-2 Secreting Citrobacter Rodentium

Graduate Basi

Influence of NleH effector expression, host genetics, and inflammation on Citrobacter rodentium colonization of mice

The Escherichia coli NleH1 and NleH2 virulence proteins differentially regulate host transcription of innate immunity genes. The mouse pathogen Citrobacter rodentium encodes one NleH protein, which functions equivalently to E. coli NleH1. We examined the impact of host genetics and intestinal inflammation on the contribution of NleH to C. rodentium colonization of mice differing in LPS responsiveness. NleH expression was detrimental to C. rodentium in C57BL/10ScNJ mice, which do not mount LPS-induced inflammatory responses. This phenotype was reversed if inflammation was induced by chemical means. C. rodentium that expressed both E. coli NleH1 and NleH2 was hypervirulent in C3H/HeJ mice