Toll-like receptor 4 contributes to colitis development but not to host defense during Citrobacter rodentium infection in mice

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are noninvasive bacterial pathogens that infect their hosts' intestinal epithelium, causing severe diarrheal disease. These infections also cause intestinal inflammation, although the mechanisms underlying the inflammatory response, as well as its potential role in host defense, are unclear. Since these bacteria are gram-negative, Toll-like receptor 4 (TLR4), the innate receptor for bacterial lipopolysaccharide may contribute to the host response; however, the role of TLR4 in the gastrointestinal tract is poorly understood, and its impact has yet to be tested against this family of enteric bacterial pathogens. Since EPEC and EHEC are human specific, we infected mice with Citrobacter rodentium, a mouse-adapted attaching and effacing (A/E) bacterium that infects colonic epithelial cells, causing colitis and epithelial hyperplasia, using a similar array of virulence proteins as EPEC and EHEC. We demonstrated that C. rodentium activates TLR4 and rapidly induced NF-kappaB nuclear translocation in host cells in a partially TLR4-dependent manner. Infection of TLR4-deficient mice revealed that TLR4-dependent responses mediate much of the inflammation and tissue pathology seen during infection, including the induction of the chemokines MIP-2 and MCP-1, as well as the recruitment of macrophages and neutrophils into the infected intestine. Surprisingly, spread of C. rodentium through the colon was delayed in TLR4-deficient mice, whereas the duration of the infection was unaffected, indicating that TLR4-mediated responses against this A/E pathogen are not host protective and are ultimately maladaptive to the host, contributing to both the morbidity and the pathology seen during infection.     

Epithelial phosphatidylinositol-3-kinase signaling is required for β-catenin activation and host defense against Citrobacter rodentium infection

Citrobacter rodentium infection of mice induces cell-mediated immune responses associated with crypt hyperplasia and epithelial β-catenin signaling. Recent data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin(552)). Our aim was to determine whether epithelial PI3K/Akt activation is required for β-catenin signaling and host defense against C. rodentium. C57BL/6 mice were infected with C. rodentium and treated with dimethyl sulfoxide (DMSO) (vehicle control) or with the PI3K inhibitor LY294002 or wortmannin. The effects of infection on PI3K activation and β-catenin signaling were analyzed by immunohistochemistry. The effects of PI3K inhibition on host defense were analyzed by the quantification of splenic and colon bacterial clearance, and adaptive immune responses were measured by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Increased numbers of P-β-catenin(552)-stained epithelial cells were found throughout expanded crypts in C. rodentium colitis. We show that the inhibition of PI3K signaling attenuates epithelial Akt activation, the Ser552 phosphorylation and activation of β-catenin, and epithelial cell proliferative responses during C. rodentium infection. PI3K inhibition impairs bacterial clearance despite having no impact on mucosal cytokine (gamma interferon [IFN-γ], tumor necrosis factor [TNF], interleukin-17 [IL-17], and IL-1β) or chemokine (CXCL1, CXCL5, CXCL9, and CXCL10) induction. The results suggest that the host defense against C. rodentium requires epithelial PI3K activation to induce Akt-mediated β-catenin signaling and the clearance of C. rodentium independent of adaptive immune responses.     

Local peroxynitrite formation contributes to early control of Cryptosporidium parvum infection

In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophil-derived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum. Infected piglets were treated daily with a selective iNOS inhibitor [L-N6-(1-iminoethyl)-lysine] or one of two peroxynitrite scavengers [5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) or uric acid] or received vehicle. At peak infection, peroxynitrite formation was restricted to sites of iNOS expression by parasitized epithelium and lamina propria of the apical villi. Peroxynitrite formation was dependent on iNOS activity and was inhibited by treatment with peroxynitrite scavengers. Scavengers increased the number of intracellular parasites and the number of infected epithelial cells present per villus and significantly exacerbated oocyst excretion. Recovery from infection was not delayed by ongoing treatment with scavenger. The present results are the first to demonstrate an in vivo role for peroxynitrite formation in acute mucosal defense against a noninvasive intestinal epithelial pathogen.     

Flagellin-dependent and -independent inflammatory responses following infection by enteropathogenic Escherichia coli and Citrobacter rodentium

Enteropathogenic Escherichia coli (EPEC) and the murine pathogen Citrobacter rodentium belong to the attaching and effacing (A/E) family of bacterial pathogens. These noninvasive bacteria infect intestinal enterocytes using a type 3 secretion system (T3SS), leading to diarrheal disease and intestinal inflammation. While flagellin, the secreted product of the EPEC fliC gene, causes the release of interleukin 8 (IL-8) from epithelial cells, it is unclear whether A/E bacteria also trigger epithelial inflammatory responses that are FliC independent. The aims of this study were to characterize the FliC dependence or independence of epithelial inflammatory responses to direct infection by EPEC or C. rodentium. Following infection of Caco-2 intestinal epithelial cells by wild-type and DeltafliC EPEC, a rapid activation of several proinflammatory genes, including those encoding IL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 3alpha (MIP3alpha), and beta-defensin 2, occurred in a FliC-dependent manner. These responses were accompanied by mitogen-activated protein kinase activation, as well as the Toll-like receptor 5 (TLR5)-dependent activation of NF-kappaB. At later infection time points, a subset of these proinflammatory genes (IL-8 and MIP3alpha) was also induced in cells infected with DeltafliC EPEC. The nonmotile A/E pathogen C. rodentium also triggered similar innate responses through a TLR5-independent but partially NF-kappaB-dependent mechanism. Moreover, the EPEC FliC-independent responses were increased in the absence of the locus of enterocyte effacement-encoded T3SS, suggesting that translocated bacterial effectors suppress rather than cause the FliC-independent inflammatory response. Thus, we demonstrate that infection of intestinal epithelial cells by A/E pathogens can trigger an array of proinflammatory responses from epithelial cells through both FliC-dependent and -independent pathways, expanding our understanding of the innate epithelial response to infection by these pathogens.     

Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection

The bacterial pathogen Citrobacter rodentium belongs to a family of gastrointestinal pathogens that includes enteropathogenic and enterohemorrhagic Escherichia coli and is the causative agent of transmissible colonic hyperplasia in mice. The molecular mechanisms used by these pathogens to colonize host epithelial surfaces and form attaching and effacing (A/E) lesions have undergone intense study. In contrast, little is known about the host's immune response to these infections and its importance in tissue pathology and bacterial clearance. To address these issues, wild-type mice and mice lacking T and B lymphocytes (RAG1 knockout [KO]) were infected with C. rodentium. By day 10 postinfection (p.i.), both wild-type and RAG1 KO mice developed colitis and crypt hyperplasia, and these responses became more exaggerated in wild-type mice over the next 2 weeks, as they cleared the infection. By day 24 p.i., bacterial clearance was complete, and the colitis had subsided; however, crypt heights remained increased. In contrast, inflammatory and crypt hyperplastic responses in the RAG1 KO mice were transient, subsiding after 2 weeks. By day 24 p.i., RAG1 KO mice showed no signs of bacterial clearance and infection was often fatal. Surprisingly, despite remaining heavily infected, tissues from RAG1 KO mice surviving the acute colitis showed few signs of disease. These results thus emphasize the important contribution of the host immune response during infection by A/E bacterial pathogens. While T and/or B lymphocytes are essential for host defense against C. rodentium, they also mediate much of the tissue pathology and disease symptoms that occur during infection.     

Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium

Many studies have shown that genetic susceptibility plays a key role in determining whether bacterial pathogens successfully infect and cause disease in potential hosts. Surprisingly, whether host genetics influence the pathogenesis of attaching and effacing (A/E) bacteria such as enteropathogenic and enterohemorrhagic Escherichia coli has not been examined. To address this issue, we infected various mouse strains with Citrobacter rodentium, a member of the A/E pathogen family. Of the strains tested, the lipopolysaccharide (LPS) nonresponder C3H/HeJ mouse strain experienced more rapid and extensive bacterial colonization than did other strains. Moreover, the high bacterial load in these mice was associated with accelerated crypt hyperplasia, mucosal ulceration, and bleeding, together with very high mortality rates. Interestingly, the basis for the increased susceptibility was not due to LPS hyporesponsiveness, as the genetically related but LPS-responsive C3H/HeOuJ and C3H/HeN mouse strains were also susceptible to infection. Analysis of the intestinal pathology in these susceptible strains revealed significant crypt epithelial cell apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining) as well as bacterial translocation to the mesenteric lymph nodes. Further studies with infection of SCID (T- and B-lymphocyte-deficient) C3H/HeJ mice demonstrated that loss of lymphocytes had no effect on bacterial numbers but did reduce crypt cell apoptosis and delayed mortality. These studies thus identify the adaptive immune system, crypt cell apoptosis, and bacterial translocation but not LPS responsiveness as contributing to the tissue pathology and mortality seen during C. rodentium infection of highly susceptible mouse strains. Determining the basis for these strains' susceptibility to intestinal colonization by an A/E pathogen will be the focus of future studies.     

Bacillus subtilis-mediated protection from Citrobacter rodentium-associated enteric disease requires espH and functional flagella

Commensals limit disease caused by invading pathogens; however, the mechanisms and genes utilized by beneficial microbes to inhibit pathogenesis are poorly understood. The attaching and effacing mouse pathogen Citrobacter rodentium associates intimately with the intestinal epithelium, and infections result in acute colitis. C. rodentium is used to model the human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. To confirm that Bacillus subtilis, a spore-forming bacterium found in the gut of mammals, could reduce C. rodentium-associated disease, mice received wild-type B. subtilis spores and 24 h later were infected by oral gavage with pathogenic C. rodentium. Disease was assessed by determining the extent of colonic epithelial hyperplasia, goblet cell loss, diarrhea, and pathogen colonization. Mice that received wild-type B. subtilis prior to enteric infection were protected from disease even though C. rodentium colonization was not inhibited. In contrast, espH and hag mutants, defective in exopolysaccharides and flagellum production, respectively, did not protect mice from C. rodentium-associated disease. A motAB mutant also failed to protect mice from disease, suggesting that B. subtilis-mediated protection requires functional flagella. By expanding our current mechanistic knowledge of bacterial protection, we can better utilize beneficial microbes to prevent intestinal disease caused by pathogenic bacteria, ultimately reducing human disease. Our data demonstrate that wild-type B. subtilis reduced disease caused by C. rodentium infection through a mechanism that required espH and functional flagella.     

IL-17RE is the functional receptor for IL-17C and mediates mucosal immunity to infection with intestinal pathogens

Interleukin 17 receptor E (IL-17RE) is an orphan receptor of the IL-17 receptor family. Here we show that IL-17RE is a receptor specific to IL-17C and has an essential role in host mucosal defense against infection. IL-17C activated downstream signaling through IL-17RE-IL-17RA complex for the induction of genes encoding antibacterial peptides as well as proinflammatory molecules. IL-17C was upregulated in colon epithelial cells during infection with Citrobacter rodentium and acted in synergy with IL-22 to induce the expression of antibacterial peptides in colon epithelial cells. Loss of IL-17C-mediated signaling in IL-17RE-deficient mice led to lower expression of genes encoding antibacterial molecules, greater bacterial burden and early mortality during infection. Together our data identify IL-17RE as a receptor of IL-17C that regulates early innate immunity to intestinal pathogens.     

Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-κB and p38 mitogen-activated protein kinase activation

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are noninvasive attaching and effacing (A/E) bacterial pathogens that cause intestinal inflammation and severe diarrheal disease. These pathogens utilize a type III secretion system to deliver effector proteins into host epithelial cells, modulating diverse cellular functions, including the release of the chemokine interleukin-8 (IL-8). While studies have implicated the effectors NleE (non-locus of enterocyte effacement [LEE]-encoded effector E) and NleH1 in suppressing IL-8 release, by preventing NF-κB nuclear translocation, the impact of these effectors only partially replicates the immunosuppressive actions of wild-type EPEC, suggesting another effector or effectors are involved. Testing an array of EPEC mutants, we identified the non-LEE-encoded effector C (NleC) as also suppressing IL-8 release. Infection by ΔnleC EPEC led to exaggerated IL-8 release from infected Caco-2 and HT-29 epithelial cells. NleC localized to EPEC-induced pedestals, with signaling studies revealing NleC inhibits both NF-κB and p38 mitogen-activated protein kinase (MAPK) activation. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that ΔnleC and wild-type C. rodentium-infected mice carried similar pathogen burdens, yet ΔnleC strain infection led to worsened colitis. Similarly, infection with ΔnleC C. rodentium in a cecal loop model induced significantly greater chemokine responses than infection with wild-type bacteria. These studies thus advance our understanding of how A/E pathogens subvert host inflammatory responses.     

Expression of intimin gamma from enterohemorrhagic Escherichia coli in Citrobacter rodentium

The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain. At least five different types of Int280, designated alpha, beta, gamma, delta, and epsilon, have been described based on sequence variation in this region. Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium, which normally displays intimin beta, to express intimin gamma from enterohemorrhagic Escherichia coli. We show that intimin gamma binds to the translocated intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing intimin gamma could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.     

Clearance of Citrobacter rodentium requires B cells but not secretory immunoglobulin A (IgA) or IgM antibodies

Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and cecum and causes crypt hyperplasia and mucosal inflammation. Mice infected with C. rodentium develop a secretory immunoglobulin A (IgA) response, but the role of B cells or secretory antibodies in host defense is unknown. To address this question, we conducted oral C. rodentium infections in mice lacking B cells, IgA, secreted IgM, polymeric Ig receptor (pIgR), or J chain. Normal mice showed peak bacterial numbers in colon and feces at 1 week and bacterial eradication after 3 to 4 weeks. B-cell-deficient mice were equally susceptible initially but could not control infection subsequently. Tissue responses showed marked differences, as infection of normal mice was accompanied by transient crypt hyperplasia and mucosal inflammation in the colon and cecum at 2 but not 6 weeks, whereas B-cell-deficient mice had few mucosal changes at 2 weeks but severe epithelial hyperplasia with ulcerations and mucosal inflammation at 6 weeks. The functions of B cells were not mediated by secretory antibodies, since mice lacking IgA or secreted IgM or proteins required for their transport into the lumen, pIgR or J chain, cleared C. rodentium normally. Nonetheless, systemic administration of immune sera reduced bacterial numbers significantly in normal and pIgR-deficient mice, and depletion of IgG abrogated this effect. These results indicate that host defense against C. rodentium depends on B cells and IgG antibodies but does not require production or transepithelial transport of IgA or secreted IgM.     

Toxoplasma gondii prevents neuron degeneration by interferon-gamma-activated microglia in a mechanism involving inhibition of inducible nitric oxide synthase and transforming growth factor-beta1 production by infected microglia

Interferon (IFN)-gamma, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-gamma-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-gamma-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-gamma-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-gamma-activated microglia. In the presence of transforming growth factor (TGF)-beta1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-gamma-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-beta1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-beta1 secretion by infected microglia.     

Intimin from enteropathogenic Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium (formerly C. freundii biotype 4280)-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC (Int(EPEC)) and C. rodentium (Int(CR)). A secreted protein, EspB (formally EaeB), is also necessary for A/E-lesion formation. Here we report that expression of a cloned Int(EPEC), encoded by plasmid pCVD438, restores murine virulence to an intimin-deficient mutant of C. rodentium DBS255. Replacement of Cys937 with Ala abolished the ability of the cloned EPEC intimin to complement the deletion mutation in DBS255. Ultrastructural examination of tissues from wild-type C. rodentium and DBS255(pCVD438)-infected mice revealed multiple A/E lesion on infected cells and loss of contact between enterocytes and basement membrane. Histological investigation showed that although both wild-type C. rodentium and DBS255(pCVD438) colonized the descending colon and induced colonic hyperplasia in orally infected 21-day-old mice, the latter strain adhered to epithelial cells located deeper within crypts. Nonetheless, infection with the wild-type strain was consistently more virulent, as indicated by a higher mortality rate. All the surviving mice, challenged with either wild-type C. rodentium or DBS255(pCVD438), developed a mucosal immunoglobulin A response to intimin and EspB. These results show that C. rodentium infection provides a relevant, simple, and economic model to investigate the role of EPEC proteins in the formation of A/E lesions in vivo and in intestinal disease.     

A metronidazole-resistant isolate of Blastocystis spp. is susceptible to nitric oxide and downregulates intestinal epithelial inducible nitric oxide synthase by a novel parasite survival mechanism

Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response.     

Mucosal T cells regulate Paneth and intermediate cell numbers in the small intestine of T. spiralis-infected mice.

Secretions of Paneth, intermediate and goblet cells have been implicated in innate intestinal host defense. We have investigated the role of T cells in effecting alterations in small intestinal epithelial cell populations induced by infection with the nematode Trichinella spiralis. Small intestinal tissue sections from euthymic and athymic (nude) mice, and mice with combined deficiency in T-cell receptor beta and delta genes [TCR(beta/delta)-/-] infected orally with T. spiralis larvae, were examined by electron microscopy and after histochemical and lineage-specific immunohistochemical staining. Compared with uninfected controls, Paneth and intermediate cell numbers increased significantly in infected euthymic and nude mice but not infected TCR(beta/delta)-/- mice. Transfer of mesenteric lymph node cells before infection led to an increase in Paneth and intermediate cells in TCR(beta/delta)-/- mice. In infected euthymic mice, Paneth cells and intermediate cells expressed cryptdins (alpha-defensins) but not intestinal trefoil factor (ITF), and goblet cells expressed ITF but not cryptdins. In conclusion, a unique, likely thymic-independent population of mucosal T cells modulates innate small intestinal host defense in mice by increasing the number of Paneth and intermediate cells in response to T. spiralis infection.     

Effects of nitric oxide on Pseudomonas aeruginosa infection of epithelial cells from a human respiratory cell line derived from a patient with cystic fibrosis

Cystic fibrosis (CF) is characterized by airway inflammation and chronic bacterial lung infection, most commonly with Pseudomonas aeruginosa, an opportunistic human pathogen. Despite the persistent airway inflammation observed in patients with CF, although phagocyte inducible nitric oxide synthase (iNOS) production is upregulated, expression of iNOS in the respiratory epithelium is markedly reduced. Given the antimicrobial action of NO, this may contribute to the chronic airway infection of this disease. To define the role of epithelium-derived NO in airway defense against P. aeruginosa, we infected differentiated human bronchial epithelial cells derived from a patient with CF (CFBE41o- cells) with different strains of this pathogen at low multiplicities of infection. Using cells transfected with human iNOS cDNA, we studied the effect of NO on P. aeruginosa replication, adherence, and internalization. P. aeruginosa adherence to iNOS-expressing cells was reduced by 44 to 72% (P = 0.02) compared with control values. Absolute P. aeruginosa uptake into these cells was reduced by 44%, but uptake expressed as a percentage of adherent bacteria did not differ from the control uptake. Survival of P. aeruginosa within iNOS-expressing cells was reduced at late times postinfection (P = 0.034). NO production did not alter host cell viability. NO production reduced P. aeruginosa adherence to human bronchial epithelial cells and enhanced killing of internalized bacteria, suggesting that a lack of epithelial iNOS in patients with CF may contribute to P. aeruginosa infection and colonization.     

The pathogenicity of an enteric Citrobacter rodentium Infection is enhanced by deficiencies in the antioxidants selenium and vitamin E

The pathogenesis of a Citrobacter rodentium infection was evaluated in mice fed diets with a single deficiency in either selenium or vitamin E or with a double deficiency in both selenium and vitamin E compared to mice on nutritionally adequate diets. Mice fed the selenium- and vitamin E-deficient diet for 6 weeks had increased loads of C. rodentium in the colon and spleen, which were not observed in mice fed either of the singly deficient diets or the adequate diet. Infected mice fed the doubly deficient diet had increased colon crypt hyperplasia and an influx of infiltrating cells along with gross changes to crypt architecture, including ulceration and denuding of the epithelial layer. Cytokine and chemokine mRNA levels in the colon were measured by real-time PCR. Expression of proinflammatory cytokines and chemokines was upregulated on day 12 after infection with C. rodentium in mice fed the doubly deficient diet compared to mice fed the control diet. Heme oxygenase 1, an enzyme upregulated by oxidative stress, also was more highly induced in infected mice fed the doubly deficient diet. Production of C. rodentium antigen-specific IgM and IgG antibodies was not affected by feeding the doubly deficient diet. The results indicated that selenium and vitamin E play an important role in host resistance and in the pathology induced by C. rodentium, an infection that mimics disease caused by common food-borne bacterial pathogens in humans.     

Modulation of host cytoskeleton function by the enteropathogenic Escherichia coli and Citrobacter rodentium effector protein EspG

EspG is a conserved protein encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli and Citrobacter rodentium. EspG is delivered into infected host cells by a type III secretion system. The role of EspG in virulence has not yet been defined. Here we describe experiments that probe the virulence characteristics and biological activities of EspG in vitro and in vivo. A C. rodentium espG mutant displayed a significantly reduced ability to colonize C57BL/6 mice and to cause colonic hyperplasia. Epitope-tagged EspG was detected in the apical regions of infected colonic epithelial cells in infected mice, partially localizing with another LEE-encoded effector protein, Tir. EspG was found to interact with mammalian tubulin in both genetic screens and gel overlay assays. Binding to tubulin by EspG caused localized microtubule depolymerization, resulting in actin stress fiber formation through an undefined mechanism. Heterologous expression of EspG in yeast resulted in loss of cytoplasmic microtubule structure and function, preventing coordination between bud development and nuclear division. Yeast expressing EspG were also unable to control cortical actin polarity. We suggest that EspG contributes to the ability of A/E pathogens to establish infection through a modulation of the host cytoskeleton involving transient microtubule destruction and actin polymerization in a manner akin to the Shigella flexneri VirA protein.     

The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohn's disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.