Citrobacter Rodentium Research Articles

Sialic acid plays a pivotal role in licensing Citrobacter rodentium’s transition from the intestinal lumen to a mucosal adherent niche

Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses and an established microbiota remain poorly defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy likely involves metabolic adaptation to the host's intestinal luminal environment, as a necessary precursor to reach and infect the mucosal surface. Suspecting this adaptation involved the intestinal mucus layer, we found that C. rodentium was able to catabolize sialic acid, a monosaccharide derived from mucins, and utilize it as its sole carbon source for growth. Moreover, C. rodentium also sensed and displayed chemotactic activity toward sialic acid. These activities were abolished when the nanT gene, encoding a sialic acid transporter, was deleted (ΔnanT). Correspondingly, the ΔnanT C. rodentium strain was significantly impaired in its ability to colonize the murine intestine. Intriguingly, sialic acid was also found to induce the secretion of two autotransporter proteins, Pic and EspC, which possess mucinolytic and host-adherent properties. As a result, sialic acid enhanced the ability of C. rodentium to degrade intestinal mucus (through Pic), as well as to adhere to intestinal epithelial cells (through EspC). We thus demonstrate that sialic acid, a monosaccharide constituent of the intestinal mucus layer, functions as an important nutrient and a key signal for an A/E bacterial pathogen to escape the colonic lumen and directly infect its host's intestinal mucosa.

IRF4 is required for migration of CD4+ T cells to the intestine but not for Th2 and Th17 cell maintenance.

The transcription factor Interferon Regulatory Factor 4 (IRF4) is central in control of T cell activation and differentiation. Deficiency of IRF4 results in severe immune deficiency and affects maturation and function of most if not all T cell subsets. Here we use mouse infection models for Citrobacter rodentium and Strongyloides ratti to analyze the function of IRF4 in T helper (Th) 17 and Th2 cell responses, respectively. IRF4 deficient mice were impaired in the control of both pathogens, failed to mount Th17 and Th2 cell responses and showed impaired recruitment of T helper cells to the intestine, the infection site of both pathogens. Compromised intestinal migration was associated with reduced expression of the intestinal homing receptors α4β7 integrin, CCR9 and GPR15. Identification of IRF4 binding sites in the gene loci of these receptors suggests a direct control of their expression by IRF4. Competitive T cell transfer assays further demonstrated that loss of one functional Irf4 allele already affected intestinal accumulation and Th2 and Th17 cell generation, indicating that lower IRF4 levels are of disadvantage for Th2 and Th17 cell differentiation as well as their migration to the intestine. Conversion of peripheral CD4+ T cells from an Irf4 wildtype to an Irf4 heterozygous or from an Irf4 heterozygous to a homozygous mutant genotype after C. rodentium or S. ratti infection did not reduce their capacity to produce Th17 or Th2 cytokines and only partially affected their persistence in the intestine, revealing that IRF4 is not essential for maintenance of the Th2 and Th17 phenotype and for survival of these T helper cells in the intestine. In conclusion, we demonstrate that the expression levels of IRF4 determine Th2 and Th17 cell differentiation and their intestinal accumulation but that IRF4 expression is not crucial for Th2 and Th17 cell survival.

Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.

Animals evolved two defense strategies to survive infections. Antagonistic strategies include immune resistance mechanisms that operate to kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here, we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Immune protection was dependent on systemic antibody responses and pathogen virulence behavior rather than the recognition of specific virulent antigens. Last, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in genes that are important for LPS structure. Our work reveals insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

Grimace scale assessment during Citrobacter rodentium inflammation and colitis development in laboratory mice.

Bacterial infections and chronic intestinal inflammations triggered by genetic susceptibility, environment or an imbalance in the intestinal microbiome are usually long-lasting and painful diseases in which the development and maintenance of these various intestinal inflammations is not yet fully understood, research is still needed. This still requires the use of animal models and is subject to the refinement principle of the 3Rs, to minimize suffering or pain perceived by the animals. With regard to this, the present study aimed at the recognition of pain using the mouse grimace scale (MGS) during chronic intestinal colitis due to dextran sodium sulfate (DSS) treatment or after infection with Citrobacter rodentium. In this study 56 animals were included which were divided into 2 experimental groups: 1. chronic intestinal inflammation (n = 9) and 2. acute intestinal inflammation (with (n = 23) and without (n = 24) C. rodentium infection). Before the induction of intestinal inflammation in one of the animal models, mice underwent an abdominal surgery and the live MGS from the cage side and a clinical score were assessed before (bsl) and after 2, 4, 6, 8, 24, and 48 hours. The highest clinical score as well as the highest live MGS was detected 2 hours after surgery and almost no sign of pain or severity were detected after 24 and 48 hours. Eight weeks after abdominal surgery B6-Il4/Il10-/- mice were treated with DSS to trigger chronic intestinal colitis. During the acute phase as well as the chronic phase of the experiment, the live MGS and a clinical score were evaluated. The clinical score increased after DSS administration due to weight loss of the animals but no change of the live MGS was observed. In the second C57BL/6J mouse model, after infection with C. rodentium the clinical score increased but again, no increased score values in the live MGS was detectable. In conclusion, the live MGS detected post-operative pain, but indicated no pain during DSS-induced colitis or C. rodentium infection. In contrast, clinical scoring and here especially the weight loss revealed a decreased wellbeing due to surgery and intestinal inflammation.

TRAF6 signaling in dendritic cells plays protective role against infectious colitis by limiting C. rodentium infection through the induction of Th1 and Th17 responses

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role in the induction of inflammatory responses not only in innate immune cells but also in non-immune cells, leading to the activation of adaptive immunity. Signal transduction mediated by TRAF6, along with its upstream molecule MyD88 in intestinal epithelial cells (IECs) is crucial for the maintenance of mucosal homeostasis following inflammatory insult. The IEC-specific TRAF6-deficient (TRAF6ΔIEC) and MyD88-deficient (MyD88ΔIEC) mice exhibit increased susceptibility to DSS-induced colitis, emphasizing the critical role of this pathway. Moreover, MyD88 also plays a protective role in Citrobacter rodentium (C. rodentium) infection-induced colitis. However, its pathological role of TRAF6 in infectious colitis remains unclear. To investigate the site-specific roles of TRAF6 in response to enteric bacterial pathogens, we infected TRAF6ΔIEC and dendritic cell (DC)-specific TRAF6-deficient (TRAF6ΔDC) mice with C. rodentium and found that the pathology of infectious colitis was exacerbated with significantly decreased survival rates in TRAF6ΔDC mice, but not in TRAF6ΔIEC mice, compared to those in control mice. TRAF6ΔDC mice showed increased bacterial burdens, marked disruption of epithelial and mucosal structures with increased infiltration of neutrophils and macrophages, and elevated cytokine levels in the colon at the late stages of infection. The frequencies of IFN-γ producing Th1 cells and IL-17A producing Th17 cells in the colonic lamina propria were significantly reduced in TRAF6ΔDC mice. Finally, we demonstrated that TRAF6-deficient DCs failed to produce IL-12 and IL-23 in response to C. rodentium stimulation, and to induce both Th1 and Th17 cells in vitro. Thus, TRAF6 signaling in DCs, but not in IECs, protects against colitis induced by C. rodentium infection by producing IL-12 and IL-23 that induce Th1 and Th17 responses in the gut.

Helicobacter spp. are prevalent in wild mice and protect from lethal Citrobacter rodentium infection in the absence of adaptive immunity

Transfer of the gut microbiota from wild to laboratory mice alters the host's immune status and enhances resistance to infectious and metabolic diseases, but understanding of which microbes and how they promote host fitness is only emerging. Our analysis of metagenomic sequencing data reveals that Helicobacter spp. are enriched in wild compared with specific-pathogen-free (SPF) and conventionally housed mice, with multiple species commonly co-colonizing their hosts. We create laboratory mice harboring three non-SPF Helicobacter spp. to evaluate their effect on mucosal immunity and colonization resistance to the enteropathogen Citrobacter rodentium. Our experiments reveal that Helicobacter spp. interfere with C.rodentium colonization and attenuate C.rodentium-induced gut inflammation in wild-type (WT) mice, even preventing lethal infection in Rag2-/- SPF mice. Further analyses suggest that Helicobacter spp. interfere with tissue attachment of C.rodentium, putatively by reducing the availability of mucus-derived sugars. These results unveil pivotal protective functions of wild mouse microbiota constituents against intestinal infection.

MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T-cell responses

Although intestinal epithelial cells (IECs) can express major histocompatibility complex class II (MHC II), especially during intestinal inflammation, it remains unclear if antigen presentation by IECs favors pro- or anti-inflammatory CD4+ T-cell responses. Using selective gene ablation of MHC II in IECs and IEC organoid cultures, we assessed the impact of MHC II expression by IECs on CD4+ T-cell responses and disease outcomes in response to enteric bacterial pathogens. We found that intestinal bacterial infections elicit inflammatory cues that greatly increase expression of MHC II processing and presentation molecules in colonic IECs. Whilst IEC MHC II expression had little impact on disease severity following Citrobacter rodentium or Helicobacter hepaticus infection, using a colonic IEC organoid-CD4+ T cell co-culture system, we demonstrate that IECs can activate antigen-specific CD4+ T cells in an MHC II-dependent manner, modulating both regulatory and effector Th cell subsets. Furthermore, we assessed adoptively transferred H. hepaticus-specific CD4+ T cells during intestinal inflammation in vivo and report that IEC MHC II expression dampens pro-inflammatory effector Th cells. Our findings indicate that IECs can function as non-conventional antigen-presenting cells and that IEC MHC II expression fine-tunes local effector CD4+ T-cell responses during intestinal inflammation.

Multiparametric Sensing of Outer Membrane Vesicle-Derived Supported Lipid Bilayers Demonstrates the Specificity of Bacteriophage Interactions.

The use of bacteriophages, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phages of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria can be used to make supported lipid bilayers (SLBs) and therefore in vitro membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we employed Escherichia coli OMV derived SLBs and use both fluorescent imaging and mechanical sensing techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalized with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phages with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from Citrobacter rodentium, which is resistant to T4 phage infection, and identify their lack of interaction with the phage. The work presented here shows how interactions occurring between the phages and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phages that work against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.

Oral vaccination with live attenuated pathogenic bacteria protects immunocompromised hosts from lethal colitis

Abstract Enteropathogenic Escherichia coli(EPEC) and enterohemorrhagic E. coli(EHEC) are significant causative agents for diarrheal diseases globally. Vaccination has been deemed as the most effective strategy for preventing EPEC and EHEC caused foodborne illnesses; however, FDA has not yet licensed any vaccine against EPEC or EHEC. Of note, live attenuated bacterial vaccine strategy has been scarcely investigated for against EPEC or EHEC infection, in particular in immunocompromised populations where the infections are associated with severe symptoms and outcomes. Infection with Citrobacter rodentium(CR), the murine equivalent of EPEC and EHEC, in mice has been widely used to develop novel vaccine strategies and evaluate vaccination efficacy. CR infection in immunocompromised mice such as interleukin-22 knockout (Il22−/−) mice leads to severe symptoms and mortality, which mirrors the severe outcomes of EPEC and EHEC infections in immunocompromised populations. We explored whether oral inoculation with live CR mutants with rationally attenuated virulence could protect lethal infection in susceptible Il22−/−mice. We showed that oral administration of live attenuated CR strains elicited efficient systemic and humoral immunity against variant CR virulence proteins, hence protecting otherwise lethal CR infection, in Il22−/−mice. Our findings provide proof of concept evidence for live attenuated vaccination strategy to prevent EPEC/EHEC infections in immunocompromised hosts associated with more severe symptoms and lethality, and reveal novel CR-specific antibody subtypes and virulence determinants that are critical for live attenuated vaccination offered immune protection.

RORγt-Raftlin1 complex regulates the pathogenicity of Th17 cells and intestinal inflammation.

Abstract Th17 cells that produce IL-17 are pathogenic in many human diseases, including inflammatory bowel disease (IBD), but are, paradoxically, essential for maintaining the integrity of the intestinal barrier in a non-inflammatory state. However, the intracellular mechanisms that regulate distinct transcriptional profiles and functional diversity of Th17 cells remain unclear. We have found that Raftlin1, a lipid raft protein, is specifically upregulated in and forms a complex with RORγt via its conserved LLNSL motif in pathogenic Th17 cells. Disruption of the RORγt-Raftlin1 complex in Raftlin1 ΔLLNSLknock-in mice resulted in attenuated pathogenic Th17 cells elicited by Citrobacter rodentium; however, there was no effect on nonpathogenic Th17 cells induced by commensal segmented filamentous bacteria (SFB). Mechanistically, we found that Raftlin1 recruits distinct phospholipids to RORγt and promotes the pathogenicity of Th17 cells. Thus, we have identified a fundamental mechanism that drives the pathogenic function of Th17 cells, which could provide a platform for new therapeutic strategies to dampen Th17-mediated inflammatory diseases. This work was supported by funds from the National Institutes of Health (R01-DK115668, R01-AI155786) and Cancer Prevention Research Institute of Texas (RP190527) and a stimulus grant from the Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center.

A vanishing family of commensal bacteria and its impact on host health and disease

Abstract The gut microbiome plays a critical role in intestinal structure, immune homeostasis, and inflammatory diseases. The rise of these diseases in industrialized countries has been correlated with the loss of microbial diversity in the gut. One bacterial family, Muribaculaceae (Mb), is becoming a lost member in industrialized human microbiomes and many studies have correlated low Mb abundance with different disease states. Our goal is to determine how Mb depletion may be connected to disease, if introducing Mb back can ameliorate the impact of disease, and uncover immune mechanisms necessary for colonization of Mb. To study this, we developed two mouse models with complex microbiomes; one containing Mb (Mb positive), and one without Mb (Mb naïve). We evaluated intestinal structure with confocal microscopy, measured the production of short-chain fatty acids (SCFAs), and compared these mice in a Citrobacter rodentium (C. rodentium) infection model. Mice without Mb lost colonic mucus striation and had a significant drop in the production of SCFAs. Additionally, Mb positive mice had reduced C. rodentium colonization compared to mice lacking Mb. Lastly, we introduced 8 characterized Mb isolates to the Mb naïve mice to quantify immune responses by flow cytometry. We found that Mb is capable of establishing in Mb naïve mice, resulting in robust germinal centre B cell responses, yet no subsequent plasmablast accumulation in the mLN or lamina propria. Determining the mechanisms involved in disease amelioration by Mb and its impact on host immune responses is a novel research direction that could increase our understanding of colonization dynamics and highlights the potential of this bacteria as a model for microbiota therapies. Supported by grants from The W. Garfield Weston Foundation

Highly-conserved regulatory activity of the ANR family in the virulence of diarrheagenic bacteria through interaction with master and global regulators

ANR (AraC negative regulators) are a novel class of small regulatory proteins commonly found in enteric pathogens. Aar (AggR-activated regulator), the best-characterized member of the ANR family, regulates the master transcriptional regulator of virulence AggR and the global regulator HNS in enteroaggregative Escherichia coli (EAEC) by protein–protein interactions. On the other hand, Rnr (RegA-negative regulator) is an ANR homolog identified in attaching and effacing (AE) pathogens, including Citrobacter rodentium and enteropathogenic Escherichia coli (EPEC), sharing only 25% identity with Aar. We previously found that C. rodentium lacking Rnr exhibits prolonged shedding and increased gut colonization in mice compared to the parental strain. To gain mechanistic insights into this phenomenon, we characterized the regulatory role of Rnr in the virulence of prototype EPEC strain E2348/69 by genetic, biochemical, and human organoid-based approaches. Accordingly, RNA-seq analysis revealed more than 500 genes differentially regulated by Rnr, including the type-3 secretion system (T3SS). The abundance of EspA and EspB in whole cells and bacterial supernatants confirmed the negative regulatory activity of Rnr on T3SS effectors. We found that besides HNS and Ler, twenty-six other transcriptional regulators were also under Rnr control. Most importantly, the deletion of aar in EAEC or rnr in EPEC increases the adherence of these pathogens to human intestinal organoids. In contrast, the overexpression of ANR drastically reduces bacterial adherence and the formation of AE lesions in the intestine. Our study suggests a conserved regulatory mechanism and a central role of ANR in modulating intestinal colonization by these enteropathogens despite the fact that EAEC and EPEC evolved with utterly different virulence programs.

Infection leaves a genetic and functional mark on the gut population of a commensal bacterium

Gastrointestinal infection changes microbiome composition and gene expression. In this study, we demonstrate that enteric infection also promotes rapid genetic adaptation in a gut commensal. Measurements of Bacteroides thetaiotaomicron population dynamics within gnotobiotic mice reveal that these populations are relatively stable in the absence of infection, and the introduction of the enteropathogen Citrobacter rodentium reproducibly promotes rapid selection for a single-nucleotide variant with increased fitness. This mutation promotes resistance to oxidative stress by altering the sequence of a protein, IctA, that is essential for fitness during infection. We identified commensals from multiple phyla that attenuate the selection of this variant during infection. These species increase the levels of vitamin B6 in the gut lumen. Direct administration of this vitamin is sufficient to significantly reduce variant expansion in infected mice. Our work demonstrates that a self-limited enteric infection can leave a stable mark on resident commensal populations that increase fitness during infection.

SRC2 controls CD4+ T cell activation via stimulating c-Myc-mediated upregulation of amino acid transporter Slc7a5

Tcell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ Tcell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in Tcells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C.rodentium)infection. Adoptive transfer of naive CD4+ Tcells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/recipients. Further, CD4+ Tcells from SRC2fl/fl/CD4Cre mice display defective Tcell proliferation, cytokine production, and differentiation both invitro and invivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for Tcell activation. Slc7a5 fails to be up-regulated in CD4+ Tcells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ Tcells to induce EAE. Therefore, SRC2 is essential for CD4+ Tcell activation and, thus, a potential drug target for controlling CD4+ Tcell-mediated autoimmunity.

Ginkgo biloba Extract Preventively Intervenes in Citrobacter Rodentium-Induced Colitis in Mice.

Inflammatory bowel disease (IBD) represents a highly recurrent gastrointestinal disorder and global public health issue. However, it lacks effective and safe strategies for its control. Although Ginkgo biloba extract (GBE) has been suggested to exhibit preventive and therapeutic activity for the control of IBD, whether its activity is associated with its ability to modulate intestinal microbiota remains to be addressed. To investigate the effect of GBE on controlling IBD, a Citrobacter Rodentium (CR)-induced mouse colitis model was used, and then histopathological examinations, biochemical assays, immunohistochemistry, and immunoblotting were performed to detect histological changes, cytokines, and tight junction (TJ) proteins in the intestine samples. We also studied 16s rRNA to detect changes in intestinal microbiota and used GC-MS to determine the microbiota-related metabolites short chain fatty acids (SCFAs). The results of our studies revealed that pre-treatment with GBE was sufficient for protecting the animals from CR-induced colitis. As a mechanism for GBE activity, GBE treatment was able to modulate the intestinal microbiota and increase the SCFAs capable of decreasing the pro-inflammatory factors and up-regulating the anti-inflammatory factors while elevating the intestinal-barrier-associated proteins to maintain the integrity of the intestines. Accordingly, our results led to a strong suggestion that GBE should be seriously considered in the preventive control of CR-induced colitis and in the development of effective and safe therapeutic strategies for controlling IBD.

Gasdermin D independent canonical inflammasome responses cooperate with caspase-8 to establish host defense against gastrointestinal Citrobacter rodentium infection

Citrobacter rodentium is an enteropathogen that causes intestinal inflammatory responses in mice reminiscent of the pathology provoked by enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. C. rodentium expresses various virulence factors that target specific signaling proteins involved in executing apoptotic, necroptotic and pyroptotic cell death, suggesting that each of these distinct cell death modes performs essential host defense functions that the pathogen aims to disturb. However, the relative contributions of apoptosis, necroptosis and pyroptosis in protecting the host against C. rodentium have not been elucidated. Here we used mice with single or combined deficiencies in essential signaling proteins controlling apoptotic, necroptotic or pyroptotic cell death to reveal the roles of these cell death modes in host defense against C. rodentium. Gastrointestinal C. rodentium infections in mice lacking GSDMD and/or MLKL showed that both pyroptosis and necroptosis were dispensable for pathogen clearance. In contrast, while RIPK3-deficient mice showed normal C. rodentium clearance, mice with combined caspase-8 and RIPK3 deficiencies failed to clear intestinal pathogen loads. Although this demonstrated a crucial role for caspase-8 signaling in establishing intestinal host defense, Casp8–/–Ripk3–/– mice remained capable of preventing systemic pathogen persistence. This systemic host defense relied on inflammasome signaling, as Casp8–/–Ripk3–/– mice with combined caspase-1 and -11 deletion succumbed to C. rodentium infection. Interestingly, although it is known that C. rodentium can activate the non-canonical caspase-11 inflammasome, selectively disabling canonical inflammasome signaling by single caspase-1 deletion sufficed to render Casp8–/–Ripk3–/– mice vulnerable to C. rodentium-induced lethality. Moreover, Casp8–/–Ripk3–/– mice lacking GSDMD survived a C. rodentium infection, suggesting that pyroptosis was not crucial for the protective functions of canonical inflammasomes in these mice. Taken together, our mouse genetic experiments revealed an essential cooperation between caspase-8 signaling and GSDMD-independent canonical inflammasome signaling to establish intestinal and systemic host defense against gastrointestinal C. rodentium infection.

Barley Leaf Ameliorates Citrobacter-rodentium-Induced Colitis through Arginine Enrichment.

Inflammatory bowel disease (IBD) has become a global public health challenge. Our previous study showed that barley leaf (BL) significantly reduces Citrobacter-rodentium (CR)-induced colitis, but its mechanism remains elusive. Thus, in this study, we used non-targeted metabolomics techniques to search for potentially effective metabolites. Our results demonstrated that dietary supplementation with BL significantly enriched arginine and that arginine intervention significantly ameliorated CR-induced colitis symptoms such as reduced body weight, shortened colon, wrinkled cecum, and swollen colon wall in mice; in addition, arginine intervention dramatically ameliorated CR-induced histopathological damage to the colon. The gut microbial diversity analysis showed that arginine intervention significantly decreased the relative abundance of CR and significantly increased the relative abundance of Akkermansia, Blautia, Enterorhabdus, and Lachnospiraceae, which modified the CR-induced intestinal flora disorder. Notably, arginine showed a dose-dependent effect on the improvement of colitis caused by CR.

Duodenal macrophages control dietary iron absorption via local degradation of transferrin

AbstractIron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.

ACONITATE DECARBOXYLASE 1 IS A KEY REGULATOR OF COLITIS

Aconitate decarboxylase 1 (ACOD1; also known as IRG1) is highly expressed in activated macrophages and converts cis-aconitate to itaconate, which contributes to the antimicrobial activity of macrophages. The role of ACOD1 in intestinal inflammation remains unknown. Therefore, we investigated the role of ACOD1 during colitis induced by Citrobacter rodentium (C. rodentium), a murine bacterial pathogen used to model the pathogenesis of enteropathogenic Escherichia coli, and dextran sulfate sodium (DSS) a model of epithelial injury-mediated inflammation.

Reprogramming Targeted-Antibacterial-Plasmids (TAPs) to achieve broad-host range antibacterial activity

The emergence and spread of antimicrobial resistance results in antibiotic inefficiency against multidrug resistant bacterial strains. Alternative treatment to antibiotics must be investigated to fight bacterial infections and limit this global public health problem. We recently developed an innovative strategy based on mobilizable Targeted-Antibacterial-Plasmids (TAPs) that deliver CRISPR/Cas systems with strain-specific antibacterial activity, using the F plasmid conjugation machinery for transfer into the targeted strains. These TAPs were shown to specifically kill a variety of Enterobacteriaceae strains, including E. coli K12 and the pathogen strains EPEC, Enterobacter cloacae and Citrobacter rodentium. Here, we extend the host-range of TAPs using the RP4 plasmid conjugation system for their mobilization, thus allowing the targeting of E. coli but also phylogenetically distant species, including Salmonella enterica Thyphimurium, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. This work demonstrates the versatility of the TAP strategy and represents a significant step toward the development of non-antibiotic strain-specific antimicrobial treatments.