Murine Gastrointestinal Tract Research Articles

Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tract.

Enteroendocrine cells (EECs) produce over 20 gut hormones which contribute to intestinal physiology, nutrient metabolism and the regulation of food intake. The objective of this study was to generate a comprehensive transcriptomic map of mouse EECs from the stomach to the rectum. EECs were purified by flow-cytometry from the stomach, upper small intestine, lower small intestine, caecum and large intestine of NeuroD1-Cre mice, and analysed by single cell RNA sequencing. Regional datasets were analysed bioinformatically and combined into a large cluster map. Findings were validated by L-cell calcium imaging and measurements of CCK secretion in vitro. 20,006 EECs across the full gastrointestinal tract could be subdivided based on their full transcriptome into 10 major clusters, each exhibiting a different pattern of gut hormone expression. EECs from the stomach were largely distinct from those found more distally, even when expressing the same hormone. Cell clustering was also observed when performed only using genes related to GPCR cell signalling, revealing GPCRs predominating in different EEC populations. Mc4r was expressed in 55% of Cck-expressing cells in the upper small intestine, where MC4R agonism was found to stimulate CCK release in primary cultures. Many individual EECs expressed more than one hormone as well as machinery for activation by multiple nutrients, which was supported by the finding that the majority of L-cells exhibited calcium responses to multiple stimuli. This comprehensive transcriptomic map of mouse EECs reveals patterns of GPCR and hormone co-expression that should be helpful in predicting the effects of nutritional and pharmacological stimuli on EECs from different regions of the gut. The finding that MC4R agonism stimulates CCK secretion adds to our understanding of the melanocortin system.

A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract.

Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2-3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3-4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.

Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon

Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.

Saccharomyces boulardii promoters for control of gene expression in vivo

BackgroundInterest in the use of engineered microbes to deliver therapeutic activities has increased in recent years. The probiotic yeast Saccharomyces boulardii has been investigated for production of therapeutics in the gastrointestinal tract. Well-characterised promoters are a prerequisite for robust therapeutic expression in the gut; however, S. boulardii promoters have not yet been thoroughly characterised in vitro and in vivo.ResultsWe present a thorough characterisation of the expression activities of 12 S. boulardii promoters in vitro in glucose, fructose, sucrose, inulin and acetate, under both aerobic and anaerobic conditions, as well as in the murine gastrointestinal tract. Green fluorescent protein was used to report on promoter activity. Promoter expression was found to be carbon-source dependent, with inulin emerging as a favourable carbon source. Furthermore, relative promoter expression in vivo was highly correlated with expression in sucrose (R = 0.99).ConclusionsThese findings provide insights into S. boulardii promoter activity and aid in promoter selection in future studies utilising S. boulardii to produce therapeutics in the gut.

Prophylactic oral application of resveratrol to alleviate acute campylobacteriosis in human gutmicrobiota associated IL-10-/- mice.

Human infections with the food-borne zoonotic enteropathogen Campylobacter jejuni are increasing globally. Since multi-drug resistant bacterial strains are further on the rise, antibiotic-independent measures are needed to fight campylobacteriosis. Given its anti-microbial and anti-inflammatory properties the polyphenolic compound resveratrol constitutes such a promising candidate molecule. In our present placebo-controlled intervention trial, synthetic resveratrol was applied perorally to human gut microbiota-associated (hma) IL-10-/- mice starting a week before oral C. jejuni infection. Our analyses revealed that the resveratrol prophylaxis did not interfere with the establishment of C. jejuni within the murine gastrointestinal tract on day 6 post-infection, but alleviated clinical signs of campylobacteriosis and resulted in less distinct colonic epithelial apoptosis. Furthermore, oral resveratrol dampened C.jejuni-induced colonic T and B cell responses as well as intestinal secretion of pro-inflammatory mediators including nitric oxide, IL-6, TNF-α, and IFN-γ to basal levels. Moreover, resveratrol application was not accompanied by significant shifts in the colonic commensal microbiota composition during campylobacteriosis in hma IL-10-/- mice. In conclusion, our placebo-controlled intervention study provides evidence that prophylactic oral application of resveratrol constitutes a promising strategy to alleviate acute campylobacteriosis and in consequence, to reduce the risk for post-infectious autoimmune sequelae.

Capsule production promotes Group B Streptococcus intestinal colonization.

Late-onset disease is the most common clinical presentation of Group B Streptococcus (GBS) infection during infancy, and gastrointestinal (GI) colonization is an important precursor. Previously, we described a murine model of postnatal GBS GI colonization that resulted in sustained colonization and progression to invasive disease. Capsular polysaccharide is an important GBS virulence factor. Vaccines based on a subset of capsular serotypes are in clinical trials. However, little is known regarding the role of specific GBS capsular serotypes in GI colonization. We examined the role of GBS capsule in GI colonization using capsule-producing and acapsular strains derived from GBS strain A909 (serotype Ia) in a murine model. Using isogenic GBS strains differing only in capsular serotypes, we explored the role of specific serotypes in GI colonization by determining competitive indices during cocolonization. We found that GBS A909 colonizes the murine GI tract without causing invasive disease. In monocolonization experiments, there was colonization persistence with the capsule-producing strain (100%) compared to the acapsular mutant strain (13%). In cocolonization experiments, the capsule-producing strain outcompeted its isogenic acapsular mutant, with a geometric mean competitive index of 8, 95% confidence interval (CI) [1.7, 38.9] in the colon at 7 days post-colonization. A909 expressing its native serotype Ia capsule outcompeted an isogenic mutant that expresses serotype III capsule, with a geometric mean competitive index of 2.5, 95% CI [1.2, 5.1] in the colon at 7 days post-colonization. Thus, polysaccharide capsule production enhances GBS GI colonization in vivo. In an A909 genetic background, the production of a serotype Ia capsule provides a competitive advantage over an isogenic strain producing type III capsule. The murine model is a valuable tool to understand the role of GBS capsule types in GI colonization. IMPORTANCE The establishment of GBS intestinal colonization is believed to be a critical precursor to late-onset disease in neonates, which has a significant impact on neurodevelopment outcomes in this population. Our prior work described a murine model of postnatal Group B Streptococcus (GBS) acquisition and invasive disease. Using this model, we explored the importance of GBS polysaccharide capsule production on gastrointestinal colonization. We found that the expression of capsule (compared to isogenic acapsular strains) provides an advantage in intestinal colonization and, importantly, that capsule type Ia has an advantage over capsule type III in a GBS A909 strain background. We speculate that specific serotypes may differ in colonization fitness, which may play a role in serotype distribution in neonatal disease.

Echinocandin Resistance in Candida auris Occurs in the Murine Gastrointestinal Tract Due to FKS1 Mutations.

Candida auris is resistant to multiple antifungal agents. This study investigated its antifungal susceptibility and explored FKS1 mutations across the isolates from mice enterically colonized with wild-type C. auris and treated with echinocandin. Resistant C. auris with FKS1 mutations, including S639F, S639Y, D642Y, R1354H, or R1354Y, were isolated and found to be micafungin- and caspofungin-resistant in vivo; however, the MICs of isolates with mutation in R1354 remained below the micafungin breakpoint in vitro.

Identification of Small-Molecule Inhibitors of the Salmonella FraB Deglycase Using a Live-Cell Assay.

Nontyphoidal salmonellosis is one of the most significant foodborne diseases in the United States and globally. There are no vaccines available for human use to prevent this disease, and only broad-spectrum antibiotics are available to treat complicated cases of the disease. However, antibiotic resistance is on the rise and new therapeutics are needed. We previously identified the Salmonella fraB gene, that mutation of causes attenuation of fitness in the murine gastrointestinal tract. The FraB gene product is encoded in an operon responsible for the uptake and utilization of fructose-asparagine (F-Asn), an Amadori product found in several human foods. Mutations in fraB cause an accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp), which is toxic to Salmonella. The F-Asn catabolic pathway is found only in the nontyphoidal Salmonella serovars, a few Citrobacter and Klebsiella isolates, and a few species of Clostridium; it is not found in humans. Thus, targeting FraB with novel antimicrobials is expected to be Salmonella specific, leaving the normal microbiota largely intact and having no effect on the host. We performed high-throughput screening (HTS) to identify small-molecule inhibitors of FraB using growth-based assays comparing a wild-type Salmonella and a Δfra island mutant control. We screened 224,009 compounds in duplicate. After hit triage and validation, we found three compounds that inhibit Salmonella in an fra-dependent manner, with 50% inhibitory concentration (IC50) values ranging from 89 to 150 μM. Testing these compounds with recombinant FraB and synthetic 6-P-F-Asp confirmed that they are uncompetitive inhibitors of FraB with Ki' (inhibitor constant) values ranging from 26 to 116 μM. IMPORTANCE Nontyphoidal salmonellosis is a serious threat in the United States and globally. We have recently identified an enzyme, FraB, that when mutated renders Salmonella growth defective in vitro and unfit in mouse models of gastroenteritis. FraB is quite rare in bacteria and is not found in humans or other animals. Here, we have identified small-molecule inhibitors of FraB that inhibit the growth of Salmonella. These could provide the foundation for a therapeutic to reduce the duration and severity of Salmonella infections.

P059 Klebicin as a novel therapy for Klebsiella infection management

Abstract Background Common infections are becoming almost untreatable, all because of the emergence and spread of drug-resistant pathogens. The highest risk is carried by rapidly spreading multi and pan-resistant bacteria that cause infections untreatable with existing antibiotics. Nowadays the biggest concern is Klebsiella bacteria which reportedly has resistance percentages of 25% or higher for third-generation antimicrobial medicine (WHO, 2022). Orally delivered recombinant bacteriocins, like klebicins, could be employed as oral antimicrobials to eradicate multidrug-resistant Klebsiella from the intestinal tract before hospitalization. Methods This study aimed to investigate the antimicrobial efficacy of orally delivered Eudragit - coated klebicin (KvarM) in a murine gastrointestinal tract model of K. pneumoniae infection. Biomodels were used (5 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarM: vehicle-only control group (K. pneumoniae, Eudragit coating) and experimental group with K. pneumoniae and KvarM coated to be released in small and large intestines. Faecal samples were used for the analysis of Klebsiella haemolysin gene (khe), which is highly specific for Klebsiella species, using RT-PCR. Results The gastrointestinal model of K. pneumoniae infection in mice was achieved per os without any antibiotic pretreatment following the introduction of coated klebicin KvarM therapy once per day for four days. In the first experimental group, the amounts of K. pneumoniae changed from 6,15 x 106 CFU/50 mg after K. pneumoniae administration to 4,68 x 106 CFU/50 mg after the last dose of therapy (23,9 percent efficacy) in the experimental group. Nonetheless, there was a significant difference between the vehicle-control group and the first group after klebicin administration (p=0,023). Conclusion Our study shows that the K. pneumoniae infection in the intestinal tract of mice was reduced by 23,9 percent and can be significantly lowered in bacterial counts using orally delivered klebicin.

Multimodal Imaging Mass Spectrometry of Murine Gastrointestinal Tract with Retained Luminal Content.

The gastrointestinal tract, including luminal content, harbors a complex mixture of microorganisms, host dietary content, and immune factors. Existing imaging approaches remove luminal content and only visualize small regions of the GI tract. Here, we demonstrate a workflow for multimodal imaging using matrix-assisted laser desorption/ionization imaging mass spectrometry, autofluorescence, and bright field microscopy for mapping intestinal tissue and luminal content. Results comparing tissue and luminal content in control murine tissue show both unique molecular and elemental distributions and abundances using multimodal protein, lipid, and elemental imaging. For instance, lipid PC(42:1) is 2× higher intensity in luminal content than tissue, while PC(32:0) is 80× higher intensity in tissue. Additionally, some ions such as the protein at m/z 3443 and the element manganese are only detected in luminal content, while the protein at m/z 8564 was only detected in tissue and phosphorus had 2× higher abundance in tissue. These data highlight the robust molecular information that can be gained from the gastrointestinal tract with the inclusion of luminal content.

Single cell genome sequencing of laboratory mouse microbiota improves taxonomic and functional resolution of this model microbial community.

Laboratory mice are widely studied as models of mammalian biology, including the microbiota. However, much of the taxonomic and functional diversity of the mouse gut microbiome is missed in current metagenomic studies, because genome databases have not achieved a balanced representation of the diverse members of this ecosystem. Towards solving this problem, we used flow cytometry and low-coverage sequencing to capture the genomes of 764 single cells from the stool of three laboratory mice. From these, we generated 298 high-coverage microbial genome assemblies, which we annotated for open reading frames and phylogenetic placement. These genomes increase the gene catalog and phylogenetic breadth of the mouse microbiota, adding 135 novel species with the greatest increase in diversity to the Muribaculaceae and Bacteroidaceae families. This new diversity also improves the read mapping rate, taxonomic classifier performance, and gene detection rate of mouse stool metagenomes. The novel microbial functions revealed through our single-cell genomes highlight previously invisible pathways that may be important for life in the murine gastrointestinal tract.

Semantic clustering analysis of E3-ubiquitin ligases in gastrointestinal tract defines genes ontology clusters with tissue expression patterns

BackgroundUbiquitin ligases (Ub-ligases) are essential intracellular enzymes responsible for the regulation of proteome homeostasis, signaling pathway crosstalk, cell differentiation and stress responses. Individual Ub-ligases exhibit their unique functions based on the nature of their substrates. They create a complex regulatory network with alternative and feedback pathways to maintain cell homeostasis, being thus important players in many physiological and pathological conditions. However, the functional classification of Ub-ligases needs to be revised and extended.MethodsIn the current study, we used a novel semantic biclustering technique for expression profiling of Ub-ligases and ubiquitination-related genes in the murine gastrointestinal tract (GIT). We accommodated a general framework of the algorithm for finding tissue-specific gene expression clusters in GIT. In order to test identified clusters in a biological system, we used a model of epithelial regeneration. For this purpose, a dextran sulfate sodium (DSS) mouse model, following with in situ hybridization, was used to expose genes with possible compensatory features. To determine cell-type specific distribution of Ub-ligases and ubiquitination-related genes, principal component analysis (PCA) and Uniform Manifold Approximation and Projection technique (UMAP) were used to analyze the Tabula Muris scRNA-seq data of murine colon followed by comparison with our clustering results.ResultsOur established clustering protocol, that incorporates the semantic biclustering algorithm, demonstrated the potential to reveal interesting expression patterns. In this manner, we statistically defined gene clusters consisting of the same genes involved in distinct regulatory pathways vs distinct genes playing roles in functionally similar signaling pathways. This allowed us to uncover the potentially redundant features of GIT-specific Ub-ligases and ubiquitination-related genes. Testing the statistically obtained results on the mouse model showed that genes clustered to the same ontology group simultaneously alter their expression pattern after induced epithelial damage, illustrating their complementary role during tissue regeneration.ConclusionsAn optimized semantic clustering protocol demonstrates the potential to reveal a readable and unique pattern in the expression profiling of GIT-specific Ub-ligases, exposing ontologically relevant gene clusters with potentially redundant features. This extends our knowledge of ontological relationships among Ub-ligases and ubiquitination-related genes, providing an alternative and more functional gene classification. In a similar way, semantic cluster analysis could be used for studding of other enzyme families, tissues and systems.

Type VI secretion systems of pathogenic and commensal bacteria mediate niche occupancy in the gut.

The type VI secretion system (T6SS) is a contractile nanomachine widely distributed among pathogenic and commensal Gram-negative bacteria. The T6SS is used for inter-bacterial competition to directly kill competing species; however, its importance during bacterial infection invivo remains poorly understood. We report that the murine pathogen Citrobacter rodentium, used as a model for human pathogenic Escherichia coli, harbors two functional T6SSs. C.rodentium employs its T6SS-1 to colonize the murine gastrointestinal tract by targeting commensal Enterobacteriaceae. We identify VgrG1 as a C.rodentium T6SS antibacterial effector, which exhibits toxicity in E.coli. Conversely, commensal prey species E.coli Mt1B1 employs two T6SSs of its own to counter C.rodentium colonization. Collectively, these data demonstrate that the T6SS is a potent weapon during bacterial competition and is used by both invading pathogens and resident microbiota to fight for a niche in the hostile gut environment.

Dynamics of plasmid-mediated niche invasion, immunity to invasion, and pheromone-inducible conjugation in the murine gastrointestinal tract

Microbial communities provide protection to their hosts by resisting pathogenic invasion. Microbial residents of a host often exclude subsequent colonizers, but this protection is not well understood. The Enterococcus faecalis plasmid pCF10, whose conjugative transfer functions are induced by a peptide pheromone, efficiently transfers in the intestinal tract of mice. Here we show that an invading donor strain established in the gastrointestinal tract of mice harboring resident recipients, resulting in a stable, mixed population comprised of approximately 10% donors and 90% recipients. We also show that the plasmid-encoded surface protein PrgB (Aggregation Substance), enhanced donor invasion of resident recipients, and resistance of resident donors to invasion by recipients. Imaging of the gastrointestinal mucosa of mice infected with differentially labeled recipients and donors revealed pheromone induction within microcolonies harboring both strains in close proximity, suggesting that adherent microcolonies on the mucosal surface of the intestine comprise an important niche for cell-cell signaling and plasmid transfer.

Identification of MrgprD expression in mouse enteric neurons.

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract.

A199 UTILIZATION OF MUCUS-DERIVED O-GLYCAN SUGARS BY THE PATHOGEN C. RODENTIUM FACILITATE ITS COLONIZATION OF THE MURINE GUT

BackgroundThe ability of enteric pathogens to colonize and expand within the mammalian gastrointestinal (GI) tract is determined by several factors, including the ability to find and acquire nutrients. The thick mucus layer that lines the inner surface of the large intestine is rich in sugars that can serve as nutrient sources for several members of the microbiota. Whether these sugars can also be used by invading bacterial pathogens to colonize the GI tract is still unclear, in particular for the family of attaching and effacing (A/E) bacterial pathogens, including the human diarrheal pathogens EHEC and EPEC.AimsTo investigate the ability of the murine A/E pathogen Citrobacter rodentium to use mucin-derived sugars as a nutrient source, and the importance of these sugars in the virulence of C. rodentium during in-vivo infection.MethodsTo identify which sugar(s) are required for C. rodentium to colonize and grow in the murine GI tract, we generated mutants lacking single or multiple genes involved in the uptake and catabolism of mucin-derived O-glycan sugars. This was followed by in-vitro growth assays in minimal media supplemented with mucin sugars to investigate the growth properties of C. rodentium and the generated mutants on mucin sugars.ResultsWe determined that C. rodentium was able to use three mucin O-glycan sugars: sialic acid, galactose, and N-acetylglucosamine (GlcNAc) as both carbon and nitrogen sources for in-vitro growth. C. rodentium exhibited the maximal growth rate and density on GlcNAc, followed by sialic acid, and finally galactose. A mutant C. rodentium strain carrying a deletion in the nagA gene was unable to grow on both GlcNAc and sialic acid, confirming that the breakdown pathways for these two sugars merge and are processed by shared suite of enzymes. As for galactose, combined deletions in the genes mglB and galP were required to abolish growth on this sugar. Notably, a mutant strain carrying simultaneous deletions in nagA, mglB, and galP was unable to grow on all three mucin sugars, as well as on purified mucin.ConclusionsOur results demonstrate that intestinal mucin sugars serve as potential nutrient sources for C. rodentium and that C. rodentium can catabolize three of these sugars. Future work will examine whether these sugar pathways contribute to C. rodentium colonization of the murine GI tract.Funding AgenciesCCC, CIHRCH.I.L.D. Fdn

Isolation and Characterization of a Novel Temperate Escherichia coli Bacteriophage, Kapi1, Which Modifies the O-Antigen and Contributes to the Competitiveness of Its Host during Colonization of the Murine Gastrointestinal Tract.

ABSTRACTIn this study, we describe the isolation and characterization of novel bacteriophage vB_EcoP_Kapi1 (Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the seroconverting phages of Shigella flexneri and clusters taxonomically with P22-like phages. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its host’s O-antigen in multiple ways. Kapi1 displays unstable lysogeny, and we find that the lysogenic state is more stable during growth in simulated intestinal fluid. Furthermore, Kapi1 lysogens have a competitive advantage over their nonlysogenic counterparts both in vitro and in vivo, suggesting a role for Kapi1 during colonization. We thus report the use of MP1 and Kapi1 as a model system to explore the molecular mechanisms of mammalian colonization by E. coli to ask what the role(s) of prophages in this context might be.

P105 Treatment of the lower gastrointestinal tract Klebsiella infections by recombinant bacteriocin KvarIa

Abstract Background Hospital-acquired infections (HAI) are one of the leading worldwide health care issues. The most prevalent HAI are the Klebsiella species. K. quasipneumoniae strains tend to colonize the gastrointestinal tract (GI) and show high rates of resistance against antibiotics. Orally delivered recombinant bacteriocins, like klebicins, could be employed as oral antimicrobials to eradicate multidrug-resistant Klebsiella from the intestinal tract prior to/during hospitalization. Methods This study aimed to investigate the antimicrobial efficacy of orally delivered Eudragit - coated klebicin (KvarIa) in a murine lower gastrointestinal tract model of K. quasipneumoniae infection. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy, and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated KvarIa. Faecal and tissue samples were used for Klebsiella haemolysin gene (khe) analysis using RT-PCR. Results The gastrointestinal K. quasipneumoniae colonization was achieved only in the antibiotic-treated mice groups ((i) penicillin, streptomycin or (ii) penicillin, streptomycin, and metronidazole) followed by the introduction of K. quasipneumoniae. Analysis of the antimicrobial efficacy of Kvarla showed that coated-KvarIa significantly reduced colonization of K. quasipneumoniae in both mice groups compared to the control group. The amounts of K. quasipneumoniae changed from 6.3 x 107 CFU/50mg on the 18th day to 3.9 x 105 CFU/50mg on the 22nd day (p = 0.01) in the Eudragit S100-coated KvarIa 100 µg group and from 4.0 x 107 CFU/50mg on the 18th day to 1.6 x 105 CFU/50mg on 22nd day (p = 0.009) in the Eudragit S100-coated KvarIa 1000 µg group. Conclusion Our study demonstrates that colonization of mice intestinal tract by K. quasipneumoniae can be achieved after eradication of resident gut microbiota with antibiotics. Furthermore, using this murine model, we showed that orally delivered encapsulated klebicins substantially reduce K. quasipneumoniae colonization in the lower gastrointestinal tract.

Escherichia coli Promoters with Consistent Expression throughout the Murine Gut.

Advanced microbial therapeutics have great potential as a novel modality to diagnose and treat a wide range of diseases. Yet, to realize this potential, robust parts for regulating gene expression and consequent therapeutic activity in situ are needed. In this study, we characterized the expression level of more than 8000 variants of the Escherichia coli sigma factor 70 (σ70) promoter in a range of different environmental conditions and growth states using fluorescence-activated cell sorting and deep sequencing. Sampled conditions include aerobic and anaerobic culture in the laboratory as well as growth in several locations of the murine gastrointestinal tract. We found that σ70 promoters in E. coli generally maintain consistent expression levels across the murine gut (R2: 0.55-0.85, p value < 1 × 10-5), suggesting a limited environmental influence but a higher variability between in vitro and in vivo expression levels, highlighting the challenges of translating in vitro promoter activity to in vivo applications. Based on these data, we design the Schantzetta library, composed of eight promoters spanning a wide expression range and displaying a high degree of robustness in both laboratory and in vivo conditions (R2 = 0.98, p = 0.000827). This study provides a systematic assessment of the σ70 promoter activity in E. coli as it transits the murine gut leading to the definition of robust expression cassettes that could be a valuable tool for reliable engineering and development of advanced microbial therapeutics.

IRE1α-Driven Inflammation Promotes Clearance of Citrobacter rodentium Infection.

ABSTRACTEndoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.