Sirtuin 6 inhibits group 3 innate lymphoid cell function and gut immunity by suppressing IL-22 production.

Introduction: Group 3 innate lymphoid cells (ILC3s) are enriched in the intestinal mucosa and play important roles in host defense against infection and inflammatory diseases. Sirtuin 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD+)- dependent deacetylase and has been shown to control intestinal epithelial cell differentiation and survival. However, the role of SIRT6 in ILC3s remains unknown. Methods: To investigate the role of SIRT6 in gut ILC3s, we generated SIRT6 conditional knockout mice by crossing Rorccre and Sirt6flox/flox mice. Cell number and cytokine production was examined using flow cytometry. Citrobacter rodentium infection and dextran sodium sulfate-induced colitis models were used to determine the role of SIRT6 in gut defense. RT-qPCR, flow cytometry and immunohistochemistry were used to assess the intestinal inflammatory responses. Results: Here we show that SIRT6 inhibits IL-22 expression in intestinal ILC3s in a cell-intrinsic manner. Deletion of SIRT6 in ILC3s does not affect the cell numbers of total ILC3s and subsets, but results in increased IL-22 production. Furthermore, ablation of SIRT6 in ILC3s protects mice against Citrobacter rodentium infection and dextran sodium sulfate-induced colitis. Our results suggest that SIRT6 may play a role in ILC3 function by regulating gut immune responses against bacterial infection and inflammation. Discussion: Our finding provided insight into the relation of epigenetic regulators with IL-22 production and supplied a new perspective for a potential strategy against inflammatory bowel disease.

Both pathogen and host dynamically adapt pH responses along the intestinal tract during enteric bacterial infection.

Enteric pathogens navigate distinct regional microenvironments within the intestine that cue important adaptive behaviors. We investigated the response of Citrobacter rodentium, a model of human pathogenic Escherichia coli infection in mice, to regional gastrointestinal pH. We found that small intestinal pH (4.4-4.8) triggered virulence gene expression and altered cell morphology, supporting initial intestinal attachment, while higher pH, representative of C. rodentium's replicative niches further along the murine intestine, supported pathogen growth. Gastric pH, a key barrier to intestinal colonization, caused significant accumulation of intra-bacterial reactive oxygen species (ROS), inhibiting growth of C. rodentium and related human pathogens. Within-host adaptation increased gastric acid survival, which may be due to a robust acid tolerance response (ATR) induced at colonic pH. However, the intestinal environment changes throughout the course of infection. We found that murine gastric pH decreases postinfection, corresponding to increased serum gastrin levels and altered host expression of acid secretion-related genes. Similar responses following Salmonella infection may indicate a protective host response to limit further pathogen ingestion. Together, we highlight interlinked bacterial and host adaptive pH responses as an important component of host-pathogen coevolution.

Context-dependent role of group 3 innate lymphoid cells in mucosal protection.

How group 3 innate lymphoid cells (ILC3s) regulate mucosal protection in the presence of T cells remains poorly understood. Here, we examined ILC3 function in intestinal immunity using ILC3-deficient mice that maintain endogenous T cells, T helper 17 (TH17) cells, and secondary lymphoid organs. ILC3s were dispensable for generation of TH17 and TH22 cell responses to commensal and pathogenic bacteria, and absence of ILC3s did not affect IL-22 production by CD4 T cells before or during infection. However, despite the presence of IL-22-producing T cells, ILC3s and ILC3-derived IL-22 were required for maintaining homeostatic functions of the intestinal epithelium. T cell-sufficient, ILC3-deficient mice were capable of pathogen clearance and survived infection with a low dose of Citrobacter rodentium. However, ILC3s promoted pathogen tolerance at early time points of infection by activating tissue-protective immune pathways. Consequently, ILC3s were indispensable for survival after high-dose infection. Our results demonstrate a context-dependent role for ILC3s in immune-sufficient animals and provide a blueprint for uncoupling of ILC3 and TH17 cell functions.

A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation.

The intestinal tract generates significant reactive oxygen species (ROS), but the role of T cell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C. rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.

Ovomucin and its hydrolysates differentially influenced colitis severity in Citrobacter rodentium-infected mice.

Egg white protein ovomucin and its hydrolysates were previously reported to exhibit anti-inflammatory and anti-adhesive activities. However, their potential to regulate pathogen colonization and disease severity has not been fully characterized. To investigate the effects of ovomucin (OVM) and its hydrolysates including ovomucin-Protex 26L (OP) and -pepsin/pancreatin (OPP) on host resistance to pathogen infection, a well-documented colitis model in mice for attaching and effacing E. coli pathogens, Citrobacter rodentium, was used in the current study. C57Bl/6J female mice were fed on a basal diet supplemented with OVM or its hydrolysates for 3 weeks prior to the C. rodentium challenge, with the dietary treatments continued for seven days. Body weight was not affected throughout the experimental period. OP supplementation resulted in lower (P < 0.05) pathogen loads at 7 dpi. Attenuated colitis severity was observed in mice that received OVM and OP, as indicated by reduced colonic pathological scores and pro-inflammatory responses compared with the infected control group. In contrast, OPP consumption resulted in enhanced C. rodentium colonization and disease severity. Notably, reduced microbial diversity indices of the gut microbiota were observed in the OPP-supplemented mice compared with the OVM- and OP-supplemented groups. This study showed the potential of OVM and OP to alleviate the severity of colitis induced by infection while also suggesting the opposite outcome of OPP in mitigating enteric infection.

Histone demethylase JMJD2D protects against enteric bacterial infection via up-regulating colonic IL-17F to induce ß-defensin expression.

Histone demethylase JMJD2D (also known as KDM4D) can specifically demethylate H3K9me2/3 to activate its target gene expression. Our previous study has demonstrated that JMJD2D can protect intestine from dextran sulfate sodium (DSS)-induced colitis by activating Hedgehog signaling; however, its involvement in host defense against enteric attaching and effacing bacterial infection remains unclear. The present study was aimed to investigate the role of JMJD2D in host defense against enteric bacteria and its underlying mechanisms. The enteric pathogen Citrobacter rodentium (C. rodentium) model was used to mimic clinical colonic infection. The responses of wild-type and JMJD2D-/- mice to oral infection of C. rodentium were investigated. Bone marrow chimeric mice were infected with C. rodentium. JMJD2D expression was knocked down in CMT93 cells by using small hairpin RNAs, and Western blot and real-time PCR assays were performed in these cells. The relationship between JMJD2D and STAT3 was studied by co-immunoprecipitation and chromatin immunoprecipitation. JMJD2D was significantly up-regulated in colonic epithelial cells of mice in response to Citrobacter rodentium infection. JMJD2D-/- mice displayed an impaired clearance of C. rodentium, more body weight loss, and more severe colonic tissue pathology compared with wild-type mice. JMJD2D-/- mice exhibited an impaired expression of IL-17F in the colonic epithelial cells, which restricts C. rodentium infection by inducing the expression of antimicrobial peptides. Accordingly, JMJD2D-/- mice showed a decreased expression of ß-defensin-1, ß-defensin-3, and ß-defensin-4 in the colonic epithelial cells. Mechanistically, JMJD2D activated STAT3 signaling by inducing STAT3 phosphorylation and cooperated with STAT3 to induce IL-17F expression by interacting with STAT3 and been recruited to the IL-17F promoter to demethylate H3K9me3. Our study demonstrates that JMJD2D contributes to host defense against enteric bacteria through up-regulating IL-17F to induce ß-defensin expression.

Citrobacter rodentium infection impairs dopamine metabolism and exacerbates the pathology of Parkinson's disease in mice.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder with indistinct etiology and ill-defined pathophysiology. Intestinal inflammation involved in the pathogenesis of PD, but the underlying mechanism is not fully understood. Citrobacter rodentium (C.R) is a gram-negative bacterium that can be used to induce human inflammatory bowel disease in mice. Here, we investigated whether the proinflammatory effects caused by C.R infection initiate PD-like injury and/or exacerbate PD pathology and extensively studied the underlying mechanism. Mice were gavaged once with C.R and monitored for several pathological features at 9 days post infection. The results showed that C.R delivery in mice induced IBD-like symptoms, including significant weight loss, increased fecal water content, an impaired intestinal barrier, intestinal hyperpermeability and inflammation, and intestinal microbiota disturbances. Notably, C.R infection modified dopamine (DA) metabolism in the brains of both male and female mice. Subsequently, a single high dose of MPTP or normal saline was administered at 6 days post infection. At 3 days after MPTP administration, the feces were collected for 16 S rRNA analysis, and PD-like phenotypes and mechanisms were systemically analyzed. Compared with C.R or MPTP injection alone, the injection of C.R and MPTP combined worsened behavioral performance. Moreover, such combination triggered more severe dopaminergic degeneration and glial cell overactivation in the nigrostriatal pathway of mice. Mechanistically, the combination of C.R and MPTP increased the expression of TLR4 and NF-κB p65 in the colon and striatum and upregulated proinflammatory cytokine expression. Therefore, C.R infection-induced intestinal inflammation can impair dopamine metabolism and exacerbate PD pathological processes.

Evaluation of the protective effect of the intranasal vaccines adjuvanted with bacterium-like particles against intestinal infection.

Mucosal vaccination presents a promising complement to parenteral vaccination. Bacterium-like particles (BLPs), peptidoglycan structures prepared from lactic acid bacteria, are explored as potential nasal vaccine adjuvants for respiratory infections. To date, studies on BLP-adjuvanted nasal vaccines against intestinal infections have remained limited. In this study, we demonstrated the efficacy of intranasal BLP-adjuvanted vaccination in controlling intestinal infections using the Citrobacter rodentium (C. rodentium) model in C57BL/6 mice. Intranasal vaccination of Intimin, an adhesin critical for intimate bacterial adhesion to colonic epithelial cells, combined with BLP (BLP+I) elicited robust Intimin-specific intestinal secretory IgA production, reduced bacterial load in feces and almost completely inhibited colonic hyperplasia, a characteristic symptom of C. rodentium infection in mice. Conversely, parenteral vaccination with Alhydrogel-adjuvanted Intimin failed to induce intestinal Intimin-specific IgA production, resulting in poor protection against C. rodentium infection. This underscores the pivotal role of mucosal IgA responses elicited by intranasal immunization in its protective efficacy. As this study did not delineate the precise protective mechanism conferred by BLP+I intranasal immunization against C. rodentium infection, further elucidation of the mechanisms underlying intranasal BLP+I immunization is required.

Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

Transcription factor Tox2 is required for metabolic adaptation and tissue residency of ILC3 in the gut.

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

Possible link between colonization of the gastrointestinal tract by Citrobacter rodentium in C57BL/6 mice and microbiota composition.

Colonization resistance, conferred by the host's microbiota through both direct and indirect protective actions, serves to protect the host from enteric infections. Here, we identified the specific members of the gut microbiota that impact gastrointestinal colonization by Citrobacter rodentium, a murine pathogen causing colonic crypt hyperplasia. The gut colonization levels of C. rodentium in C57BL/6 mice varied among breeding facilities, probably due to differences in microbiota composition. A comprehensive analysis of the microbiota revealed that specific members of the microbiota may influence gut colonization by C. rodentium, thus providing a potential link between the two.

Deficiency of IL-22-binding protein enhances the ability of the gut microbiota to protect against enteric pathogens.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

IL-22-dependent responses and their role during Citrobacter rodentium infection.

The mouse pathogen Citrobacter rodentium is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and to elucidate mechanisms of mucosal immunity. In response to C. rodentium infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from C. rodentium infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during C. rodentium infection. In line with previous research, mice lacking the IL-22 gene (Il22-/- mice) were highly susceptible to C. rodentium infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: S100A9-/- (calprotectin), Lcn2-/- (lipocalin-2), Reg3b-/- (Reg3ß), Reg3g-/- (Reg3γ), and C3-/- (C3). All knockout mice tested displayed a considerable level of resistance to C. rodentium infection, and none phenocopied the lethality observed in Il22-/- mice. By investigating another arm of the IL-22 response, we observed that C. rodentium-infected Il22-/- mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal C. rodentium infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.

Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence.

Interleukin 22 (IL-22) has a non-redundant role in immune defence of the intestinal barrier1-3. T cells, but not innate lymphoid cells, have an indispensable role in sustaining the IL-22 signalling that is required for the protection of colonic crypts against invasion during infection by the enteropathogen Citrobacter rodentium4 (Cr). However, the intestinal epithelial cell (IEC) subsets targeted by T cell-derived IL-22, and how T cell-derived IL-22 sustains activation in IECs, remain undefined. Here we identify a subset of absorptive IECs in the mid-distal colon that are specifically targeted by Cr and are differentially responsive to IL-22 signalling. Major histocompatibility complex class II (MHCII) expression by these colonocytes was required to elicit sustained IL-22 signalling from Cr-specific T cells, which was required to restrain Cr invasion. Our findings explain the basis for the regionalization of the host response to Cr and demonstrate that epithelial cells must elicit MHCII-dependent help from IL-22-producing T cells to orchestrate immune protection in the intestine.

Genomic deletion of Bcl6 differentially affects conventional dendritic cell subsets and compromises Tfh/Tfr/Th17 cell responses.

Conventional dendritic cells (cDC) play key roles in immune induction, but what drives their heterogeneity and functional specialization is still ill-defined. Here we show that cDC-specific deletion of the transcriptional repressor Bcl6 in mice alters the phenotype and transcriptome of cDC1 and cDC2, while their lineage identity is preserved. Bcl6-deficient cDC1 are diminished in the periphery but maintain their ability to cross-present antigen to CD8+ T cells, confirming general maintenance of this subset. Surprisingly, the absence of Bcl6 in cDC causes a complete loss of Notch2-dependent cDC2 in the spleen and intestinal lamina propria. DC-targeted Bcl6-deficient mice induced fewer T follicular helper cells despite a profound impact on T follicular regulatory cells in response to immunization and mounted diminished Th17 immunity to Citrobacter rodentium in the colon. Our findings establish Bcl6 as an essential transcription factor for subsets of cDC and add to our understanding of the transcriptional landscape underlying cDC heterogeneity.

Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Infection and antibiotic-associated changes in the fecal microbiota of C. rodentium ϕstx2dact-infected C57BL/6 mice.

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.

Citrobacter rodentium possesses a functional type II secretion system necessary for successful host infection.

Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens. To establish successful host infections these pathogens employ a plethora of virulence factors necessary to compete with the resident microbiota, and evade and subvert the host defenses. The type II secretion system (T2SS) is one such conserved molecular machine that allows for the delivery of effector proteins into the extracellular milieu. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of human intestinal disease caused by pathogenic Escherichia coli such as Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC). In this study, we determined that the C. rodentium genome encodes one T2SS and 22 potential T2SS-secreted protein effectors, as predicted via sequence homology. We demonstrated that this system was functional in vitro, identifying a role in intestinal mucin degradation allowing for its utilization as a carbon source, and promoting C. rodentium attachment to a mucus-producing colon cell line. During host infection, loss of the T2SS or associated effectors led to a significant colonization defect and lack of systemic spread. In mice susceptible to lethal infection, T2SS-deficient C. rodentium was strongly attenuated, resulting in reduced morbidity and mortality in infected hosts. Together these data highlight the important role of the T2SS and its effector repertoire during C. rodentium pathogenesis, aiding in successful host mucosal colonization.

Effects of myeloperoxidase on inflammatory responses with hypoxia in Citrobacter rodentium-infectious mice.

PURPOSE: Myeloperoxidase (MPO) has been identified as a mediator in various inflammatory diseases. Bacterial infection of the intestine and hypoxia can both lead to inflammatory responses, but the role of MPO in these phenomena remains unclear. METHODS: By building the MPO-/- mice, we evaluated relevant inflammatory factors and tissue damage in mice with intestinal Citrobacter rodentium infection and hypoxia. The body weight and excreted microorganisms were monitored. Intestinal tissues were collected 7 days after bacterial infection under hypoxia to undergo haematoxylin-eosin staining and assess the degree of pathological damage. ELISA assays were performed to quantify the serum levels of TNF-α, IFN-γ, IL-6, and IL-1ß inflammatory cytokines. PCR, WB, and IF assays were conducted to determine the expression of chemokines MCP1, MIP2, and KC in the colon and spleen. RESULTS: The C. rodentium infection and hypoxia caused weight loss, intestinal colitis, and splenic inflammatory cells active proliferation in wild-type mice. MPO deficiency alleviated this phenomenon. MPO-/- mice also displayed a significant decline in bacteria clearing ability. The level of TNF-α in the serum and spleen was both lower in MPO-/- hypoxia C. rodentium-infected mice than that in wild-type mice. The chemokines expression levels of MIP2, KC, and MCP1 in the spleen and colon of each bacterial infected group were significantly increased (p < .05), while in hypoxia, the factors in the spleen and colon were decreased (p < .05). MPO deficiency was found to lower the levels of these chemokines compared with wild-type mice. CONCLUSION: MPO plays an important role of the inflammatory responses in infectious enteritis and hypoxia in mice, and the loss of MPO may greatly reduce the body's inflammatory responses to fight diseases.