Targeted delivery of the probiotic Saccharomyces boulardii to the extracellular matrix enhances gut residence time and recovery in murine colitis.

Probiotic and engineered microbe-based therapeutics are an emerging class of pharmaceutical agents. They represent a promising strategy for treating various chronic and inflammatory conditions by interacting with the host immune system and/or delivering therapeutic molecules. Here, we engineered a targeted probiotic yeast platform wherein Saccharomyces boulardii is designed to bind to abundant extracellular matrix proteins found within inflammatory lesions of the gastrointestinal tract through tunable antibody surface display. This approach enabled an additional 24-48 h of probiotic gut residence time compared to controls and 100-fold increased probiotic concentrations within the colon in preclinical models of ulcerative colitis in female mice. As a result, pharmacodynamic parameters including colon length, colonic cytokine expression profiles, and histological inflammation scores were robustly improved and restored back to healthy levels. Overall, these studies highlight the potential for targeted microbial therapeutics as a potential oral dosage form for the treatment of inflammatory bowel diseases.

A consortia of clinical E. coli strains with distinct in vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

BACKGROUND: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. RESULTS: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. CONCLUSIONS: Our findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models. Video Abstract.

Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

Heterogeneity among Clinical Intestinal Escherichia coli Isolates upon Acquired Streptomycin Resistance.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.

A consortia of clinical E. coli strains with distinct in-vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

Background: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in-vitro definition fully predicts mucosal colonization in-vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. Results: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortia of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. Conclusions: Our findings establish the in-vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in-vivo colonization dynamics of patient-derived bacteria in murine models.

Microenvironmental Factors that Shape Bacterial Metabolites in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a significant global health problem that involves chronic intestinal inflammation and can involve severe comorbidities, including intestinal fibrosis and inflammation-associated colorectal cancer (CRC). Disease-associated alterations to the intestinal microbiota often include fecal enrichment of Enterobacteriaceae, which are strongly implicated in IBD development. This dysbiosis of intestinal flora accompanies changes in microbial metabolites, shaping host:microbe interactions and disease risk. While there have been numerous studies linking specific bacterial taxa with IBD development, our understanding of microbial function in the context of IBD is limited. Several classes of microbial metabolites have been directly implicated in IBD disease progression, including bacterial siderophores and genotoxins. Yet, our microbiota still harbors thousands of uncharacterized microbial products. In-depth discovery and characterization of disease-associated microbial metabolites is necessary to target these products in IBD treatment strategies. Towards improving our understanding of microbiota metabolites in IBD, it is important to recognize how host relevant factors influence microbiota function. For example, changes in host inflammation status, metal availability, interbacterial community structure, and xenobiotics all play an important role in shaping gut microbial ecology. In this minireview, we outline how each of these factors influences gut microbial function, with a specific focus on IBD-associated Enterobacteriaceae metabolites. Importantly, we discuss how altering the intestinal microenvironment could improve the treatment of intestinal inflammation and associated disorders, like intestinal fibrosis and CRC.

BET Protein Inhibition Regulates Macrophage Chromatin Accessibility and Microbiota-Dependent Colitis.

Introduction: In colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice. Methods: We performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10-/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization. Results: Treatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo resulted in a mild reduction in colitis severity as compared with vehicle-treated mice. Conclusion: We identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis.

Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue.

Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn's disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn's disease and non-Crohn's tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.

Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer.

Group 3 innate lymphoid cells (ILC3s) regulate immunity and inflammation, yet their role in cancer remains elusive. Here, we identify that colorectal cancer (CRC) manifests with altered ILC3s that are characterized by reduced frequencies, increased plasticity, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that a dialog between ILC3s and T cells via major histocompatibility complex class II (MHCII) is necessary to support colonization with microbiota that subsequently induce type-1 immunity in the intestine and tumor microenvironment. As a result, mice lacking ILC3-specific MHCII develop invasive CRC and resistance to anti-PD-1 immunotherapy. Finally, humans with dysregulated intestinal ILC3s harbor microbiota that fail to induce type-1 immunity and immunotherapy responsiveness when transferred to mice. Collectively, these data define a protective role for ILC3s in cancer and indicate that their inherent disruption in CRC drives dysfunctional adaptive immunity, tumor progression, and immunotherapy resistance.

Evolution of Polymyxin Resistance Regulates Colibactin Production in Escherichia coli.

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

A nadA Mutation Confers Nicotinic Acid Auxotrophy in Pro-carcinogenic Intestinal Escherichia coli NC101.

Inflammatory bowel diseases (IBDs) and inflammation-associated colorectal cancer (CRC) are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.

Microbiota Effects on Carcinogenesis: Initiation, Promotion, and Progression.

Carcinogenesis is a multistep process by which normal cells acquire genetic and epigenetic changes that result in cancer. In combination with host genetic susceptibility and environmental exposures, a prominent procarcinogenic role for the microbiota has recently emerged. In colorectal cancer (CRC), three nefarious microbes have been consistently linked to cancer development: (a) Colibactin-producing Escherichia coli initiates carcinogenic DNA damage, (b) enterotoxigenic Bacteroides fragilis promotes tumorigenesis via toxin-induced cell proliferation and tumor-promoting inflammation, and (c) Fusobacterium nucleatum enhances CRC progression through two adhesins, Fap2 and FadA, that promote proliferation and antitumor immune evasion and may contribute to metastases. Herein, we use these three prominent microbes to discuss the experimental evidence linking microbial activities to carcinogenesis and the specific mechanisms driving this stepwise process. Precisely defining mechanisms by which the microbiota impacts carcinogenesis at each stage is essential for developing microbiota-targeted strategies for the diagnosis, prognosis, and treatment of cancer.

Dietary iron variably modulates assembly of the intestinal microbiota in colitis-resistant and colitis-susceptible mice.

Iron deficiency, a common comorbidity of gastrointestinal inflammatory disorders such as inflammatory bowel diseases (IBD), is often treated with oral iron supplementation. However, the safety of oral iron supplementation remains controversial because of its association with exacerbated disease activity in a subset of IBD patients. Because iron modulates bacterial growth and function, one possible mechanism by which iron may exacerbate inflammation in susceptible hosts is by modulating the intestinal microbiota. We, therefore, investigated the impact of dietary iron on the intestinal microbiota, utilizing the conventionalization of germ-free mice as a model of a microbial community in compositional flux to recapitulate the instability of the IBD-associated intestinal microbiota. Our findings demonstrate that altering intestinal iron availability during community assembly modulated the microbiota in non-inflamed wild type (WT) and colitis-susceptible interleukin-10-deficient (Il10-/-) mice. Depletion of luminal iron availability promoted luminal compositional changes associated with dysbiotic states irrespective of host genotype, including an expansion of Enterobacteriaceae such as Escherichia coli. Mechanistic in vitro growth competitions confirmed that high-affinity iron acquisition systems in E. coli enhance its abundance over other bacteria in iron-restricted conditions, thereby enabling pathobiont iron scavenging during dietary iron restriction. In contrast, distinct luminal community assembly was observed with dietary iron supplementation in WT versus Il10-/- mice, suggesting that the effects of increased iron on the microbiota differ with host inflammation status. Taken together, shifts in dietary iron intake during community assembly modulate the ecological structure of the intestinal microbiota and is dependent on host genotype and inflammation status.

Yersiniabactin-Producing Adherent/Invasive Escherichia coli Promotes Inflammation-Associated Fibrosis in Gnotobiotic Il10-/- Mice.

Fibrosis is a significant complication of intestinal disorders associated with microbial dysbiosis and pathobiont expansion, notably Crohn's disease (CD). Mechanisms that favor fibrosis are not well understood, and therapeutic strategies are limited. Here we demonstrate that colitis-susceptible Il10-deficient mice develop inflammation-associated fibrosis when monoassociated with adherent/invasive Escherichia coli (AIEC) that harbors the yersiniabactin (Ybt) pathogenicity island. Inactivation of Ybt siderophore production in AIEC nearly abrogated fibrosis development in inflamed mice. In contrast, inactivation of Ybt import through its cognate receptor FyuA enhanced fibrosis severity. This corresponded with increased colonic expression of profibrogenic genes prior to the development of histological disease, therefore suggesting causality. fyuA-deficient AIEC also exhibited greater localization within subepithelial tissues and fibrotic lesions that was dependent on Ybt biosynthesis and corresponded with increased fibroblast activation in vitro Together, these findings suggest that Ybt establishes a profibrotic environment in the host in the absence of binding to its cognate receptor and indicate a direct link between intestinal AIEC and the induction of inflammation-associated fibrosis.

The influence of the microbiota on immune development, chronic inflammation, and cancer in the context of aging.

From birth, the microbiota plays an essential role in human development by educating host immune responses. Proper maturation of the immune system perturbs chronic inflammation and the pathogenesis of disease by preventing inappropriate immune responses. While many have detailed the roles of specific microbial groups in immune development and human disease, it remains to be elucidated how the microbiota influences the immune system during aging. Furthermore, it is not yet understood how age-related changes to the microbiota and immune system influence the development of age-related diseases. In this review, we outline the role of the microbiota in immune system development as well as functional changes that occur to immune cell populations during immunosenescence. In addition, we highlight how commensal microbes influence the pathogenesis of cancer, a prominent disease of aging. The information provided herein suggests that age-related changes to the microbiota and immune system should be considered in disease treatment and prevention strategies.

The Azoxymethane/Il10 -/- Model of Colitis-Associated Cancer (CAC).

Mouse models have proved essential for generating a mechanistic understanding of human disease processes. The azoxymethane/interleukin-10 knockout (AOM/Il10-/-) model is a powerful tool for assessing the effects of intestinal microbiota and inflammation on colon tumorigenesis. This model of colitis-associated colorectal cancer (CAC) is particularly relevant to inflammatory bowel disease (IBD)-associated colon cancer and recapitulates many of the molecular effects underlying inflammation's influence on human colorectal cancer. The model utilizes inflammation-susceptible Il10-/- mice injected intraperitoneally (i.p.) with the colon-specific carcinogen AOM. AOM and its metabolites cause mutagenesis and colorectal tumorigenesis in an inflammation-dependent manner, which in Il10-/- mice is driven by the presence and composition of the intestinal microbiota. Here we describe bacterial colonization with the pathobiont Escherichia coli strain NC101, cancer initiation with AOM, bacterial quantification, and histologic assessment of inflammation and cancer.

Siderophore-mediated iron acquisition and modulation of host-bacterial interactions.

Iron is an essential micronutrient for most life forms including the majority of resident bacteria of the microbiota and their mammalian hosts. Bacteria have evolved numerous mechanisms to competitively acquire iron within host environments, such as the secretion of small molecules known as siderophores that can solubilize iron for bacterial use. However, siderophore biosynthesis and acquisition is not a capability equally harbored by all resident bacteria. Moreover, the structural diversity of siderophores creates variability in the susceptibility to host mechanisms that serve to counteract siderophore-mediated iron acquisition and limit bacterial growth. As a result, the differential capabilities to acquire iron among members of a complex microbial community carry important implications for the growth and function of resident bacteria. Siderophores can also directly influence host function by modulating cellular iron homeostasis, further providing a mechanism by which resident bacteria may influence their local environment at the host-microbial interface. This review will explore the putative mechanisms by which siderophore production by resident bacteria in the intestines may influence microbial community dynamics and host-bacterial interactions with important implications for pathogen- and microbiota-driven diseases including infection, inflammatory bowel diseases and colorectal cancer.