Abstract Background Clostridioides difficile is a common healthcare associated pathogen in U.S. hospitals, incurring billions of dollars in treatment costs each year. Microbiome analysis of C. difficile infected (CDI) patients have revealed alterations of the gut microbiota. It has been speculated that select members of this altered microbiota may influence C. difficile pathogenesis. C. difficile is known to reside in the intestinal mucus layer, but at present the interactions between C. difficile and other mucus-associated bacteria are poorly defined. To address these gaps in knowledge, we have focused on an entirely human-centered approach, employing human-derived MUC2, fecal bioreactors and patient samples. We hypothesized that select mucus-associated bacteria would promote C. difficile colonization and biofilm formation. Methods & Results To create a model of the human intestinal mucus layer and gut microbiota, we developed a bioreactor system with human MUC2-coated coverslips. Bioreactors were inoculated with healthy human feces, treated with clindamycin and infected with C. difficile to mimic CDI. C. difficile was found to colonize and form biofilms on MUC2-coated coverslips and 16S rRNA sequencing revealed a unique biofilm profile with substantial co-colonization with Fusobacterium. Consistent with our bioreactor data, publicly available datasets and patient stool samples revealed that a subset of patients with C. difficile infection harbored high levels of F. nucleatum OTUs. We also isolated microbes from adult patients and pediatric IBD patient stool who were positive for C. difficile and F. nucleatum and identified co-localization between these strains. RNAseq data revealed significant changes in C. difficile chemotaxis and surface adhesion genes following exposure to F. nucleatum metabolites. C. difficile was found to co-aggregate with F. nucleatum; an effect that was inhibited by blocking the Fusobacterial adhesin RadD and C. difficile flagella. Moreover, a ΔradD mutant of F. nucleatum lost the ability to aggregate with C. difficile. Conversely, removal of flagella from C. difficile significantly reduced the interaction between WT F. nucleatum and C. difficile. Addition of F. nucleatum also enhanced C. difficile biofilm formation, increasing the levels extracellular polysaccharide. Conclusions Collectively, these data demonstrate the unique role of mucus-associated bacteria such as F. nucleatum in facilitating colonization of the mucus layer by pathogenic C. difficile.
Candida albicans is an opportunistic human fungal pathogen that causes a variety of diseases, ranging from superficial mucosal to life-threatening systemic infections, the latter particularly in patients with defects in innate immune function. C. albicans cells phagocytosed by macrophages undergo a dramatic change in their metabolism in which amino acids are a key nutrient. We have shown that amino acid catabolism allows the cell to neutralize the phagolysosome and initiate hyphal growth. We show here that members of the 10-gene ATO family, which are induced by phagocytosis or the presence of amino acids in an Stp2-dependent manner and encode putative acetate or ammonia transporters, are important effectors of this pH change in vitro and in macrophages. When grown with amino acids as the sole carbon source, the deletion of ATO5 or the expression of a dominant-negative ATO1(G53D) allele results in a delay in alkalinization, a defect in hyphal formation, and a reduction in the amount of ammonia released from the cell. These strains also form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Furthermore, overexpression of many of the 10 ATO genes accelerates ammonia release, and an ato5Δ ATO1(G53D) double mutant strain has additive alkalinization and ammonia release defects. Taken together, these results indicate that the Ato protein family is a key mediator of the metabolic changes that allow C. albicans to overcome the macrophage innate immunity barrier.
Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit, the anti-inflammatory immune system, wound healing, normal social behavior in mice, and prevents bone reabsorption. Oxytocin impacts these functions and oxytocin signaling is required for L. reuteri-mediated wound healing and social behavior; however, the events in the gut leading to oxytocin stimulation and beneficial effects are unknown. Here we report evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We find that L. reuteri facilitates oxytocin secretion from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.
Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.
Observations that intestinal microbes can beneficially impact host physiology have prompted investigations into the therapeutic usage of such microbes in a range of diseases. For example, the human intestinal microbe
INTRODUCTION: Cronkhite Canada Syndrome (CCS) is a rare polyposis syndrome for which the cause remains unknown. Because the disease is so rare (1 in 1 million), no standard of care exists. The disease is most prevalent in males and has an average age of onset at 60 years of age. The case presented is unique as the patient is female and diagnosed at a younger age (38). CASE DESCRIPTION/METHODS: A 38-year-old woman of Asian (Laotian) descent presented in the gastroenterology clinic with fatigue, dizziness, alopecia, dysgeusia, and weight loss. Physical exam revealed onychodystrophy and hyperpigmentation of her face and hands. Laboratory testing confirmed iron deficiency anemia with hypoproteinemia and hypoalbuminemia. Anti-gliadin antibodies were negative. A computed tomography scan of her abdomen revealed no significant abnormalities. Endoscopy revealed dozens of erythematous pedunculated polyps in the stomach, duodenum, and ileum while the colon had close to a dozen large pedunculated polyps. Pathology noted no adenomas or dysplasias. However, pathology did note edema, chronic and acute inflammation and prominent eosinophil content. A diagnosis of CCS was made. In order to assess for intestinal microbial changes in the patient, a stool sample was obtained to perform total 16S sequencing of the stool flora. The 16S data is being compared to healthy sex and aged-matched controls at the species level. Additionally, human intestinal organoids were generated from her stomach, duodenum, ileum, and colon biopsies using published methods. To our knowledge, this is the first time that intestinal organoids have been made from a CCS patient. The CCS organoids grow faster, are larger, and have unique morphologies compared to organoids from other healthy donors. Cell type analysis also reveals an increased number of ChgA positive enteroendocrine cells in the colon and ileum. DISCUSSION: Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown. Currently the patient is maintained on azathioprine (dosing is 50 mg daily) and anti-histamines. Utilization of ex vivo organoid models will be an important tool for not only understanding more about the disease, but also as a way to advance precision medicine and evaluate potential therapies for this rare disease.
Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear.
ABSTRACT pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans , pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2 , a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1 , 2 (urea amidolyase), and ATO5 , a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis. IMPORTANCE Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease.
