A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors.

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

Diet-microbiome-immune interplay in multiple sclerosis: Understanding the impact of phytoestrogen metabolizing gut bacteria.

Multiple sclerosis (MS) is a chronic and progressive autoimmune disease of the central nervous system (CNS), with both genetic and environmental factors contributing to the pathobiology of the disease. Although HLA genes have emerged as the strongest genetic factor linked to MS, consensus on the environmental risk factors is lacking. Recently, the gut microbiota has garnered increasing attention as a potential environmental factor in MS, as mounting evidence suggests that individuals with MS exhibit microbial dysbiosis (changes in the gut microbiome). Thus, there has been a strong emphasis on understanding the role of the gut microbiome in the pathobiology of MS, specifically, factors regulating the gut microbiota and the mechanism(s) through which gut microbes may contribute to MS. Among all factors, diet has emerged to have the strongest influence on the composition and function of gut microbiota. As MS patients lack gut bacteria capable of metabolizing dietary phytoestrogen, we will specifically discuss the role of a phytoestrogen diet and phytoestrogen metabolizing gut bacteria in the pathobiology of MS. A better understanding of these mechanisms will help to harness the enormous potential of the gut microbiota as potential therapeutics to treat MS and other autoimmune diseases.

Competitiveness of reutericyclin producing and nonproducing Limosilactobacillus reuteri in food and intestinal ecosystems: a game of rock, paper, and scissors?

The ecological relationships among antimicrobial producing, resistant, and sensitive strains have been proposed to follow rock-paper-scissors dynamics, but evidence is mainly based on Gram-negative bacteriocins in vitro. The ecological relevance of antimicrobials in vivo or in situ has not been systematically studied. This study therefore aimed to analyze binary and ternary competitions among reutericyclin-producing strain Limosilactobacillus reuteri TMW1.656, its reutericyclin-resistant, nonproducing isogenic derivative L. reuteri TMW1.656∆rtcN, and the reutericyclin-sensitive, nonproducing L. reuteri TMW1.656∆rtcN∆rtcT in vitro (liquid culture and static plate), in situ (sourdough fermentation), and in vivo (gut of germ-free mice). In liquid culture, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Limosilactobacillus reuteri TMW1.656∆rtcN∆rtcT had a higher fitness than TMW1.656∆rtcN. On agar plates, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN∆rtcT. In situ, reutericyclin production and resistance had no influence on the fitness of the strains. In vivo, TMW1.656 had an advantage over TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Ternary competitions showed reutericyclin production was ecologically beneficial in all ecosystems. The findings support the ecological importance of reutericyclin in a variety of environments/niches, providing an explanation for the acquisition of the reutericyclin gene cluster in L. reuteri and its contribution to the ecological fitness of Streptococcus mutans.

Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes.

The community of microorganisms in the mammalian gastrointestinal tract, referred to as the gut microbiota, influences host physiology and immunity. The last decade of microbiome research has provided significant advancements for the field and highlighted the importance of gut microbes to states of both health and disease. Novel molecular techniques have unraveled the tremendous diversity of intestinal symbionts that potentially influence the host, many proof-of-concept studies have demonstrated causative roles of gut microbial communities in various pathologies, and microbiome-based approaches are beginning to be implemented in the clinic for diagnostic purposes or for personalized treatments. However, several challenges for the field remain: purely descriptive reports outnumbering mechanistic studies and slow translation of experimental results obtained in animal models into the clinics. Moreover, there is a dearth of knowledge regarding how gut microbes, including novel species that have yet to be identified, impact host immune responses. The sheer complexity of the gut microbial ecosystem makes it difficult, in part, to fully understand the microbiota-host networks that regulate immunity. In the present manuscript, we review key findings on the interactions between gut microbiota members and the immune system. Because culturing microbes allows performing functional studies, we have emphasized the impact of specific taxa or communities thereof. We also highlight underlying molecular mechanisms and discuss opportunities to implement minimal microbiome-based strategies.

Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract.

Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two ß-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway.IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Role of whole grains versus fruits and vegetables in reducing subclinical inflammation and promoting gastrointestinal health in individuals affected by overweight and obesity: a randomized controlled trial.

BACKGROUND: Whole grains (WG) and fruits and vegetables (FV) have been shown to reduce the risk of metabolic disease, possibly via modulation of the gut microbiota. The purpose of this study was to determine the impact of increasing intake of either WG or FV on inflammatory markers and gut microbiota composition. METHODS: A randomized parallel arm feeding trial was completed on forty-nine subjects with overweight or obesity and low intakes of FV and WG. Individuals were randomized into three groups (3 servings/d provided): WG, FV, and a control (refined grains). Stool and blood samples were collected at the beginning of the study and after 6 weeks. Inflammatory markers [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), lipopolysaccharide binding protein (LBP), and high sensitivity C-reactive protein (hs-CRP)] were measured. Stool sample analysis included short/branched chain fatty acids (S/BCFA) and microbiota composition. RESULTS: There was a significant decrease in LBP for participants on the WG (- 0.2 µg/mL, p = 0.02) and FV (- 0.2 µg/mL, p = 0.005) diets, with no change in those on the control diet (0.1 µg/mL, p = 0.08). The FV diet induced a significant change in IL-6 (- 1.5 pg/mL, p = 0.006), but no significant change was observed for the other treatments (control, - 0.009 pg/mL, p = 0.99; WG, - 0.29, p = 0.68). The WG diet resulted in a significant decrease in TNF-α (- 3.7 pg/mL; p < 0.001), whereas no significant effects were found for those on the other diets (control, - 0.6 pg/mL, p = 0.6; FV, - 1.4 pg/mL, p = 0.2). The treatments induced individualized changes in microbiota composition such that treatment group differences were not identified, except for a significant increase in α-diversity in the FV group. The proportions of Clostridiales (Firmicutes phylum) at baseline were correlated with the magnitude of change in LBP during the study. CONCLUSIONS: These data demonstrate that WG and FV intake can have positive effects on metabolic health; however, different markers of inflammation were reduced on each diet suggesting that the anti-inflammatory effects were facilitated via different mechanisms. The anti-inflammatory effects were not related to changes in gut microbiota composition during the intervention, but were correlated with microbiota composition at baseline. TRIAL REGISTRATION: ClinicalTrials.gov , NCT02602496 , Nov 4, 2017.

Characterization of the ecological role of genes mediating acid resistance in Lactobacillus reuteri during colonization of the gastrointestinal tract.

Rodent-derived strains of Lactobacillus reuteri densely colonize the forestomach of mice and possess several genes whose predicted functions constitute adaptations towards an acidic environment. The objective of this study was to systematically determine which genes of L. reuteri 100-23 contribute to tolerance towards host gastric acid secretion. Genes predicted to be involved in acid resistance were inactivated, and their contribution to survival under acidic conditions was confirmed in model gastric juice. Fitness of five mutants that showed impaired in vitro acid resistance were then compared through competition experiments in ex-germ-free mice that were either treated with omeprazole, a proton-pump inhibitor that suppresses acid secretion in the stomach, or left untreated. This analysis revealed that the urease cluster was the predominant factor in mediating resistance to gastric acid production. Population levels of the mutant, which were substantially decreased in untreated mice, were almost completely restored through omeprazole, demonstrating that urease production in L. reuteri is mainly devoted to overcome gastric acid. The findings provide novel information on the mechanisms by which L. reuteri colonizes its gastric niche and demonstrate that in silico gene predictions and in vitro tests have limitations for predicting the ecological functions of colonization factors in bacterial symbionts.

Prebiotics and synbiotics: dietary strategies for improving gut health.

PURPOSE OF REVIEW: A wide range of dietary carbohydrates, including prebiotic food ingredients, fermentable fibers, and milk oligosaccharides, are able to produce significant changes in the intestinal microbiota. These shifts in the microbial community are often characterized by increased levels of bifidobacteria and lactobacilli. More recent studies have revealed that species of Faecalibacterium, Akkermansia, and other less well studied members may also be enriched. We review the implications of these recent studies on future design of prebiotics and synbiotics to promote gastrointestinal health. RECENT FINDINGS: Investigations assessing the clinical outcomes associated with dietary modification of the gut microbiota have shown systemic as well as specific health benefits. Both prebiotic oligosaccharides comprised of a linear arrangement of simple sugars, as well as fiber-rich foods containing complex carbohydrates, have been used in these trials. However, individual variability and nonresponding study participants can make the outcome of dietary interventions less predictable. In contrast, synergistic synbiotics containing prebiotics that specifically stimulate a cognate probiotic provide additional options for personalized gut therapies. SUMMARY: This review describes recent research on how prebiotics and fermentable fibers can influence the gut microbiota and result in improvements to human health.

In vivo selection to identify bacterial strains with enhanced ecological performance in synbiotic applications.

One strategy for enhancing the establishment of probiotic bacteria in the human intestinal tract is via the parallel administration of a prebiotic, which is referred to as a synbiotic. Here we present a novel method that allows a rational selection of putative probiotic strains to be used in synbiotic applications: in vivo selection (IVS). This method consists of isolating candidate probiotic strains from fecal samples following enrichment with the respective prebiotic. To test the potential of IVS, we isolated bifidobacteria from human subjects who consumed increasing doses of galactooligosaccharides (GOS) for 9 weeks. A retrospective analysis of the fecal microbiota of one subject revealed an 8-fold enrichment in Bifidobacterium adolescentis strain IVS-1 during GOS administration. The functionality of GOS to support the establishment of IVS-1 in the gastrointestinal tract was then evaluated in rats administered the bacterial strain alone, the prebiotic alone, or the synbiotic combination. Strain-specific quantitative real-time PCR showed that the addition of GOS increased B. adolescentis IVS-1 abundance in the distal intestine by nearly 2 logs compared to rats receiving only the probiotic. Illumina 16S rRNA sequencing not only confirmed the increased establishment of IVS-1 in the intestine but also revealed that the strain was able to outcompete the resident Bifidobacterium population when provided with GOS. In conclusion, this study demonstrated that IVS can be used to successfully formulate a synergistic synbiotic that can substantially enhance the establishment and competitiveness of a putative probiotic strain in the gastrointestinal tract.

Resistant starches for the management of metabolic diseases.

PURPOSE OF REVIEW: Recent clinical trials and animal studies indicate that resistant starches may be beneficial therapeutic tools for the management of metabolic diseases. The purpose of this review is to summarize these findings and discuss the established and proposed mechanisms by which resistant starches exert their benefits. We also examine open questions regarding how resistant starches improve metabolism and propose future research directions for the field. RECENT FINDINGS: Data from both humans and animal models clearly support a role for resistant starches in improving a variety of metabolic features; however, discrepancies do exist regarding specific effects. Concomitant improvements in both insulin levels and body fat depots are often reported in rodents fed resistant starches, whereas resistant starch feeding in humans improves insulin sensitivity without having a major impact on fat mass. These differences could be explained by the coexistence of several mechanisms (both gut microbiota-dependent and gut microbiota-independent) underpinning the metabolic benefits of resistant starches. SUMMARY: Together, the studies presented in this review offer new insights into the potential pathways by which resistant starches enhance metabolic health, including modulation of the gut microbiota, gut peptides, circulating inflammatory mediators, innate immune cells, and the bile acid cycle.

Pulmonary biodistribution and cellular uptake of intranasally administered monodisperse particles.

PURPOSE: For the rational design of nanovaccines against respiratory pathogens, careful selection of optimal particle size and chemistry is paramount. This work investigates the impact of these properties on the deposition, biodistribution, and cellular interactions of nanoparticles within the lungs. METHOD: In this work, biodegradable poly(sebacic anhydride) (poly(SA)) nanoparticles of multiple sizes were synthesized with narrow particle size distributions. The lung deposition and retention as well as the internalization by phagocytic cells of these particles were compared to that of non-degradable monodisperse polystyrene nanoparticles of similar sizes. RESULTS: The initial deposition of intranasally administered particles in the lungs was dependent on primary particle size, with maximal deposition occurring for the 360-470 nm particles, regardless of chemistry. Over time, both particle size and chemistry affected the frequency of particle-positive cells and the specific cell types taking up particles. The biodegradable poly(SA) particles associated more closely with phagocytic cells and the dynamics of this association impacted the clearance of these particles from the lung. CONCLUSIONS: The findings reported herein indicate that both size and chemistry control the fate of intranasally administered particles and that the dynamics of particle association with phagocytic cells in the lungs provide important insights for the rational design of pulmonary vaccine delivery vehicles.

High-fat diet feeding exacerbates HIV-1 rectal transmission.

High-fat diet (HFD) is well known to impact various aspects of gut health and has been associated with many diseases and inflammation. However, the impact of HFD feeding on HIV-1 rectal transmission has not yet been well addressed. With an increasing threat of HIV-1 infection in men who have sex with men (MSM), where the rectal route is the primary mode of infection, it is imperative to understand the impact of HFD on gut microbiota and inflammation and consequently, its effect on HIV-1 rectal transmission. Here, we utilized our double humanized bone marrow, liver, thymus (dHu-BLT) mouse model to assess the impact of HFD feeding on the host's susceptibility to HIV-1 rectal transmission. We found that feeding an HFD successfully altered the gut microbial composition within 3 weeks in the dHu-BLT mouse model. In addition, levels of inflammatory mediators, specifically IL-12p70, IP-10, ICAM-1, and fecal calprotectin, were significantly higher in HFD-fed mice compared to control mice on a regular chow diet. We also observed that significantly different inflammatory markers (IL-12p70 and ICAM-1) were negatively correlated with the number of observed ASVs, Shannon diversity, and Faith's diversity in the HFD-fed group. Notably, when repeatedly challenged with a low dose of HIV-1 via a rectal route, mice receiving an HFD were significantly more susceptible to HIV-1 rectal infection than control mice. Together, these results underscore the impact of HFD feeding on the gut microbiota and inflammation and suggest the significance of diet-induced gut microbial dysbiosis and inflammation in promoting viral infection.IMPORTANCEHFD induces gut microbial dysbiosis and inflammation and has been associated with many infections and disease progression; however, its impact on HIV-1 rectal transmission is largely unknown. Given the increasing threat of HIV-1 incidence in men who have sex with men (MSM), it has become crucial to comprehend the impact of factors associated with gut health, like HFD consumption, on host susceptibility to HIV-1 rectal transmission. This is particularly important since anal intercourse remains the primary mode of HIV transmission within the MSM group. In this study, utilizing our unique mouse model, featuring both the human immune system and gut microbiota, we showed that HFD feeding led to gut microbial dysbiosis, induced inflammation, and increased HIV-1 rectal transmission. Collectively, our study highlights the significant impact of HFD on gut microbiota and inflammation and suggests an HFD consumption as a potential risk factor for promoting HIV-1 rectal susceptibility.

Loss of claudin-3 expression increases colitis risk by promoting Gut Dysbiosis.

Dysregulation of both the gut barrier and microbiota (dysbiosis) promotes susceptibility to and severity of Inflammatory Bowel Diseases (IBD). Leaky gut and dysbiosis often coexist; however, potential interdependence and molecular regulation are not well understood. Robust expression of claudin-3 (CLDN3) characterizes the gut epithelium, and studies have demonstrated a positive association between CLDN3 expression and gut barrier maturity and integrity, including in response to probiotics. However, the exact status and causal role of CLDN3 in IBD and regulation of gut dysbiosis remain unknown. Analysis of mouse and human IBD cohorts helped examine CLDN3 expression in IBD. The causal role was determined by modeling CLDN3 loss of expression during experimental colitis. 16S sequencing and in silico analysis helped examine gut microbiota diversity between Cldn3KO and WT mice and potential host metabolic responses. Fecal microbiota transplant (FMT) studies were performed to assess the role of gut dysbiosis in the increased susceptibility of Cldn3KO mice to colitis. A significant decrease in CLDN3 expression characterized IBD and CLDN3 loss of expression promoted colitis. 16S sequencing analysis suggested gut microbiota changes in Cldn3KO mice that were capable of modulating fatty acid metabolism and oxidative stress response. FMT from naïve Cldn3KO mice promoted colitis susceptibility in recipient germ-free mice (GFM) compared with GFM-receiving microbiota from WT mice. Our data demonstrate a critical role of CLDN3 in maintaining normal gut microbiota and inflammatory responses, which can be harnessed to develop novel therapeutic opportunities for patients with IBD.

The diet-microbiota axis: a key regulator of intestinal permeability in human health and disease.

The intestinal barrier orchestrates selective permeability to nutrients and metabolites while excluding noxious stimuli. Recent scientific advances establishing a causal role for the gut microbiota in human health outcomes have generated a resurgent interest toward intestinal permeability. Considering the well-established role of the gut barrier in protection against foreign antigens, there is mounting evidence for a causal link between gut permeability and the microbiome in regulating human health. However, an understanding of the dynamic host-microbiota interactions that govern intestinal barrier functions remains poorly defined. Furthermore, the system-level mechanisms by which microbiome-targeted therapies, such as probiotics and prebiotics, simultaneously promote intestinal barrier function and host health remain an area of active investigation. This review summarizes the recent advances in understanding the dynamics of intestinal permeability in human health and its integration with gut microbiota. We further summarize mechanisms by which probiotics/prebiotics influence the gut microbiota and intestinal barrier functions.

The waxy mutation in sorghum and other cereal grains reshapes the gut microbiome by reducing levels of multiple beneficial species.

Waxy starches from cereal grains contain >90% amylopectin due to naturally occurring mutations that block amylose biosynthesis. Waxy starches have unique organoleptic characteristics (e.g. sticky rice) as well as desirable physicochemical properties for food processing. Using isogenic pairs of wild type sorghum lines and their waxy derivatives, we studied the effects of waxy starches in the whole grain context on the human gut microbiome. In vitro fermentations with human stool microbiomes show that beneficial taxonomic and metabolic signatures driven by grain from wild type parental lines are lost in fermentations of grain from the waxy derivatives and the beneficial signatures can be restored by addition of resistant starch. These undesirable effects are conserved in fermentations of waxy maize, wheat, rice and millet. We also demonstrate that humanized gnotobiotic mice fed low fiber diets supplemented with 20% grain from isogenic pairs of waxy vs. wild type parental sorghum have significant differences in microbiome composition and show increased weight gain. We conclude that the benefits of waxy starches on food functionality can have unintended tradeoff effects on the gut microbiome and host physiology that could be particularly relevant in human populations consuming large amounts of waxy grains.

Human Alcohol-Microbiota Mice have Increased Susceptibility to Bacterial Pneumonia.

Preclinical studies have shown that chronic alcohol abuse leads to alterations in the gastrointestinal microbiota that are associated with behavior changes, physiological alterations, and immunological effects. However, such studies have been limited in their ability to evaluate the direct effects of alcohol-associated dysbiosis. To address this, we developed a humanized alcohol-microbiota mouse model to systematically evaluate the immunological effects of chronic alcohol abuse mediated by intestinal dysbiosis. Germ-free mice were colonized with human fecal microbiota from individuals with high and low Alcohol Use Disorders Identification Test (AUDIT) scores and bred to produce human alcohol-associated microbiota or human control-microbiota F1 progenies. F1 offspring colonized with fecal microbiota from individuals with high AUDIT scores had increased susceptibility to Klebsiella pneumoniae and Streptococcus pneumoniae pneumonia, as determined by increased mortality rates, pulmonary bacterial burden, and post-infection lung damage. These findings highlight the importance of considering both the direct effects of alcohol and alcohol-induced dysbiosis when investigating the mechanisms behind alcohol-related disorders and treatment strategies.

The Inherited Intestinal Microbiota from Myeloid-Specific ZIP8KO Mice Impairs Pulmonary Host Defense against Pneumococcal Pneumonia.

Intestinal dysbiosis increases susceptibility to infection through the alteration of metabolic profiles, which increases morbidity. Zinc (Zn) homeostasis in mammals is tightly regulated by 24 Zn transporters. ZIP8 is unique in that it is required by myeloid cells to maintain proper host defense against bacterial pneumonia. In addition, a frequently occurring ZIP8 defective variant (SLC39A8 rs13107325) is strongly associated with inflammation-based disorders and bacterial infection. In this study, we developed a novel model to study the effects of ZIP8-mediated intestinal dysbiosis on pulmonary host defense independent of the genetic effects. Cecal microbial communities from a myeloid-specific Zip8 knockout mouse model were transplanted into germ-free mice. Conventionalized ZIP8KO-microbiota mice were then bred to produce F1 and F2 generations of ZIP8KO-microbiota mice. F1 ZIP8KO-microbiota mice were also infected with S. pneumoniae, and pulmonary host defense was assessed. Strikingly, the instillation of pneumococcus into the lung of F1 ZIP8KO-microbiota mice resulted in a significant increase in weight loss, inflammation, and mortality when compared to F1 wild-type (WT)-microbiota recipients. Similar defects in pulmonary host defense were observed in both genders, although consistently greater in females. From these results, we conclude that myeloid Zn homeostasis is not only critical for myeloid function but also plays a significant role in the maintenance and control of gut microbiota composition. Further, these data demonstrate that the intestinal microbiota, independent of host genetics, play a critical role in governing host defense in the lung against infection. Finally, these data strongly support future microbiome-based interventional studies, given the high incidence of zinc deficiency and the rs13107325 allele in humans.

Home-site advantage for host species-specific gut microbiota.

Mammalian species harbor compositionally distinct gut microbial communities, but the mechanisms that maintain specificity of symbionts to host species remain unclear. Here, we show that natural selection within house mice (Mus musculus domesticus) drives deterministic assembly of the house-mouse gut microbiota from mixtures of native and non-native microbiotas. Competing microbiotas from wild-derived lines of house mice and other mouse species (Mus and Peromyscus spp.) within germ-free wild-type (WT) and Rag1-knockout (Rag1-/-) house mice revealed widespread fitness advantages for native gut bacteria. Native bacterial lineages significantly outcompeted non-native lineages in both WT and Rag1-/- mice, indicating home-site advantage for native microbiota independent of host adaptive immunity. However, a minority of native Bacteriodetes and Firmicutes favored by selection in WT hosts were not favored or disfavored in Rag1-/- hosts, indicating that Rag1 mediates fitness advantages of these strains. This study demonstrates home-site advantage for native gut bacteria, consistent with local adaptation of gut microbiota to their mammalian species.

Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.