Role of inflammasomes in host defense against Citrobacter rodentium infection

Citrobacter rodentium is an enteric bacterial pathogen of the mouse intestinal tract that triggers inflammatory responses resembling those of humans infected with enteropathogenic and enterohemorrhagic Escherichia coli. Inflammasome signaling is emerging as a central regulator of inflammatory and host responses to several pathogens, but the in vivo role of inflammasome signaling in host defense against C. rodentium has not been characterized. Here, we show that mice lacking the inflammasome components Nlrp3, Nlrc4, and caspase-1 were hypersusceptible to C. rodentium-induced gastrointestinal inflammation. This was due to defective interleukin (IL)-1β and IL-18 production given that il-1β(-/-) and il-18(-/-) mice also suffered from increased bacterial burdens and exacerbated histopathology. C. rodentium specifically activated the Nlrp3 inflammasome in in vitro-infected macrophages independently of a functional bacterial type III secretion system. Thus, production of IL-1β and IL-18 downstream of the Nlrp3 and Nlrc4 inflammasomes plays a critical role in host defense against enteric infections caused by C. rodentium.     

Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium

Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.     

Influence of human gastrointestinal tract bacterial pathogens on host cell apoptosis

Several pathogenic bacteria are able to trigger apoptosis in the host cell, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Induction and/or blockage of programmed cell death upon infection is a result of complex interaction of bacterial proteins with cellular proteins involved in signal transduction and apoptosis. In this review we focus on pro/anti-apoptotic activities exhibited by two enteric pathogens Salmonella enterica, Yersinia spp. and gastric pathogen Helicobacter pylori. We present current knowledge on how interaction between mammalian and bacterial cell relates to the molecular pathways of apoptosis, and what is the role of apoptosis in pathogenesis.     

Functional properties of free and encapsulated Lactobacillus reuteri DPC16 during and after passage through a simulated gastrointestinal tract

Lactobacillus reuteri DPC16 is a probiotic bacterium that has strong antimicrobial activities on pathogens, mainly due to its ability to produce reuterin, an antimicrobial compound. The objective of this study was to examine the ability of an encapsulation technique to protect the functional properties of cells of L. reuteri DPC16 during passage through a simulated GI tract. The functional properties of the cells were studied before and after passage through the tract. An alginate-skim milk encapsulation system was used to deliver the probiotic bacterium through the simulated GI tract, allowing for the release of the cells into the simulated colonic fluid. The cells were then isolated and cultured. The recovered cells showed no diminution in functional properties, including their growth kinetics, ability to adhere to epithelial cells and ability to inhibit the adhesion of E. coli to epithelial cells. The bacteriostatic and bactericidal properties of the recovered cells against some pathogens were significantly greater (P < 0.05) than those of the original cells. Production of reuterin by the recovered cells was significantly greater than that of the original cells when cultured in MRS medium in the absence of its metabolic precursor, glycerol. The results demonstrate significant consequences for the application of the encapsulation technique to protect and/or enhance the functional properties of the probiotic cells.     

Citrobacter rodentium virulence in mice associates with bacterial load and the type III effector NleE

Severe disease caused by Shiga toxin-producing Escherichia coli (STEC) has been associated with a pathogenicity island, O-Island 122, which encodes the type III secretion system-effector NleE. Here we show that full virulence of the related attaching and effacing mouse pathogen Citrobacter rodentium requires NleE. Relative to wild-type bacteria, nleE-mutant C. rodentium are attenuated for colonisation in mice in both single and mixed infections. Examination of the ability of nleE-mutant bacteria to induce pathologic change in vivo revealed that nleE-mutant bacteria induce significantly less pathologic change than wild-type bacteria in susceptible mice. Consistent with these results, mice infected with nleE-mutant bacteria exhibit delayed mortality. These results suggested that pathologic change during attaching and effacing pathogen infection may associate with the degree of pathogen colonisation. Using mutants of 23 type III secretion genes, including the type III effectors nleC, nleD, nleE and nleF, the association of pathologic change with the ability of these mutants to colonise mice was examined. The induction of in vivo disease correlates strongly with the degree of colonisation, suggesting that the colonisation advantage type III secretion genes afford the bacteria, contribute to, and are required for, full virulence.     

Long-term Selenium Deficiency Increases the Pathogenicity of a Citrobacter rodentium Infection in Mice

Citrobacter rodentium is a mouse pathogen that causes infectious colitis and shares characteristics with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, including the ability to cause attaching and effacing lesions in the colon and serves as a useful model to study the pathogenicity of these bacteria. In this study, mice were fed a selenium-deficient diet for 5 or 20?weeks and then infected with C. rodentium. Colonization of the colon by C. rodentium was similar in mice fed adequate or selenium-deficient diets, but total bacterial colonization of the spleen was elevated in mice fed selenium-deficient diet for 20?weeks. Infection-induced changes to the colon included inflammatory cell infiltration, gross changes in crypt architecture, and ulceration and denuding of the epithelial layer that were greatest in mice fed a selenium-deficient diet for 20?weeks. Expression of pro-inflammatory genes was significantly higher 12-days post-infection in mice fed the selenium-deficient diet for 20?weeks compared to mice fed a selenium-adequate diet or selenium-deficient diet for 5?weeks. Diarrhea was prevalent in mice fed the selenium-deficient diet for 20?weeks but not 5?weeks, and this was associated with decreased expression of solute carrier family 26a3 and carbonic anhydrase IV, genes involved in ion transport. These results indicated that selenium played an important role in resistance to the pathological effects of a C. rodentium infection, and therefore, selenium status may be important in the expression of human disease caused by common food-borne bacteria.     

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecal samples after passage through the gastrointestinal tract of mice

AIM: This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. METHODS AND RESULTS: Nine Lact. plantarum ivi gene replacement mutants were constructed, focussing on ivi genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation. The in vitro growth characteristics of the mutants appeared identical to those observed for the wild-type strain, which agrees with the recombination-based in vivo expression technology suggestion that these genes are not transcribed in the laboratory. Quantitative PCR experiments demonstrated differences in the relative population dynamics of the Lact. plantarum ivi mutants in faecal samples after passage through the GI tract of mice. CONCLUSIONS: The in situ competition experiments revealed a 100- to 1000-fold reduction of the relative abundance of three of the ivi gene mutants, harbouring deletions of genes predicted to encode a copper transporter, an orphan IIC cellobiose PTS and a cell wall anchored extracellular protein. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments clearly establish that the proteins encoded by these three genes play a key role in Lact. plantarum performance during passage of the GI tract.     

Influence of different storage methods on the predatory capacity of the fungi Arthrobotrys robusta and Monacrosporium thaumasium after passage through the bovine gastrointestinal tract

The biological control of helminth parasites of bovines by nematophagous fungi is an alternative to the use of drugs with the principal objective of reducing the source of infection available on pastureland. The maintenance of predatory activity of the fungal isolates is one of the basic prerequisites to ensure the success of this form of control. In this study behaviour of the isolates I31 of Arthrobotrys robusta and NF34a of Monacrosporium thaumasium was investigated following three storage methods: stored at 4 °C, cryopreserved with or without cryoprotectants or preserved in silica gel. All samples were subsequently passed through the gastrointestinal tract of calves. The latter involved the administration of 20 g of mycelia to the animals. This quantity was sufficient to recover fungal material from the faeces. The peak reduction in the number of infective larvae in the faeces occurred 24 h after administration of the samples (P < 0.05). The storage at 4 °C was the treatment that produced the greatest reduction in larvae for NF34a (81.3%) and I31 (65.1%) isolates. Nf34a isolate was responsible for the highest percentage reduction of larval helminth populations (P < 0.05). Cryopreservation appears to be an efficient method of preserving isolates, although diminished predatory capacity compared to storage at 4 °C was seen only for isolate NF34a (73.2%). Cryopreservation did not interfere in predatory activity of I31 isolate (P < 0.05). Maintenance of isolates in silica gel showed the lowest reduction throughout the experiment (P < 0.05).     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Folding of a bacterial outer membrane protein during passage through the periplasm.

The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.     

Breathing life into pathogens: the influence of oxygen on bacterial virulence and host responses in the gastrointestinal tract

The gastrointestinal tract provides a variety of environmental challenges to any bacterium seeking to successfully colonize or cause disease in a host. A major obstacle is the varied oxygen concentrations encountered at different sites in the intestine. Here we review the mechanisms bacterial pathogens utilize to sense oxygen within the gastrointestinal tract, and recent insights into how this acts as a signal to trigger virulence and to modulate host responses.     

Survival of lactococci during passage through mouse digestive tract

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.