A human milk oligosaccharide prevents intestinal inflammation in adulthood via modulating gut microbial metabolism.

Observational evidence suggests that human milk oligosaccharides (HMOs) promote the growth of commensal bacteria in early life and adulthood. However, the mechanisms by which HMOs benefit health through modulation of gut microbial homeostasis remain largely unknown. 2'-fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and contributes to the essential health benefits associated with human milk consumption. Here, we investigated how 2'-FL prevents colitis in adulthood through its effects on the gut microbial community. We found that the gut microbiota from adult mice that consumed 2'-FL exhibited an increase in abundance of several health-associated genera, including Bifidobacterium and Lactobacillus. The 2'-FL-modulated gut microbial community exerted preventive effects on colitis in adult mice. By using Bifidobacterium infantis as a 2'-FL-consuming bacterial model, exploratory metabolomics revealed novel 2'-FL-enriched secretory metabolites by Bifidobacterium infantis, including pantothenol. Importantly, pantothenate significantly protected the intestinal barrier against oxidative stress and mitigated colitis in adult mice. Furthermore, microbial metabolic pathway analysis identified 26 dysregulated metabolic pathways in fecal microbiota from patients with ulcerative colitis, which were significantly regulated by 2'-FL treatment in adult mice, indicating that 2'-FL has the potential to rectify dysregulated microbial metabolism in colitis. These findings support the contribution of the 2'-FL-shaped gut microbial community and bacterial metabolite production to the protection of intestinal integrity and prevention of intestinal inflammation in adulthood.IMPORTANCEAt present, neither basic research nor clinical studies have revealed the exact biological functions or mechanisms of action of individual oligosaccharides during development or in adulthood. Thus, it remains largely unknown whether human milk oligosaccharides could serve as effective therapeutics for gastrointestinal-related diseases. Results from the present study uncover 2'-FL-driven alterations in bacterial metabolism and identify novel B. infantis-secreted metabolites following the consumption of 2'-FL, including pantothenol. This work further demonstrates a previously unrecognized role of pantothenate in significantly protecting the intestinal barrier against oxidative stress and mitigating colitis in adult mice. Remarkably, 2'-FL-enhanced bacterial metabolic pathways are found to be dysregulated in the fecal microbiota of ulcerative colitis patients. These novel metabolic pathways underlying the bioactivities of 2'-FL may lay a foundation for applying individual oligosaccharides for prophylactic intervention for diseases associated with impaired intestinal homeostasis.

Total Synthesis of Ervaoffine J and K.

Herein, we describe the total synthesis of ervaoffine J & K from a central intermediate. Ervaoffine J was synthesized in eight steps in 14 % yield. Our strategy features an aerobic Winterfeldt oxidation to introduce the 4-quinolone moiety. Ervaoffine K was produced in ten steps and 10 % yield. The synthesis leveraged (bromodifluoromethyl)-trimethylsilane to induce a regioselective von Braun-type C-N bond fragmentation. This C-N bond cleavage unveiled the tetrasubstituted all-syn cyclohexane core of ervaoffine K and enabled the completion of its synthesis.

Environmental Toxicant Exposure Paralyzes Human Placental Macrophage Responses to Microbial Threat.

Exposure to environmental toxicants (such as dioxins) has been epidemiologically linked to adverse reproductive health outcomes, including placental inflammation and preterm birth. However, the molecular underpinnings that govern these outcomes in gravid reproductive tissues remain largely unclear. Placental macrophages (also known as Hofbauer cells) are crucial innate immune cells that defend the gravid reproductive tract and help promote maternal-fetal tolerance. We hypothesized that exposure to environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could alter placental macrophage responses to inflammatory insults such as infection. To test this, placental macrophages were cultured in the presence or absence of TCDD and then infected with the perinatal pathogen Group B Streptococcus (GBS). Our results indicate that TCDD is lethal to placental macrophages at and above a 5 nM concentration and that sublethal dioxin exposure inhibits phagocytosis and cytokine production. Taken together, these results indicate that TCDD paralyzes placental macrophage responses to bacterial infection.

The Utility of Human Milk Oligosaccharides against Group B Streptococcus Infections of Reproductive Tissues and Cognate Adverse Pregnancy Outcomes.

Preterm birth affects nearly 10% of all pregnancies in the United States, with 40% of those due, in part, to infections. Streptococcus agalactiae (Group B Streptococcus, GBS) is one of the most common perinatal pathogens responsible for these infections. Current therapeutic techniques aimed to ameliorate invasive GBS infections are less than desirable and can result in complications in both the neonate and the mother. To this end, the need for novel therapeutic options is urgent. Human milk oligosaccharides (HMOs), an integral component of human breast milk, have been previously shown to possess antiadhesive and antimicrobial properties. To interrogate these characteristics, we examined HMO-mediated outcomes in both in vivo and ex vivo models of GBS infection utilizing a murine model of ascending GBS infection, an EpiVaginal human organoid tissue model, and ex vivo human gestational membranes. Supplementation of HMOs resulted in diminished adverse pregnancy outcomes, decreased GBS adherence to gestational tissues, decreased colonization within the reproductive tract, and reduced proinflammatory immune responses to GBS infection. Taken together, these results highlight the potential of HMOs as promising therapeutic interventions in perinatal health.

A Hitchhiker's Guide to Problem Selection in Carbohydrate Synthesis.

Oligosaccharides are ubiquitous in molecular biology and are used for functions ranging from governing protein folding to intercellular communication. Perhaps paradoxically, the exact role of the glycan in most of these settings is not well understood. One reason for this contradiction concerns the fact that carbohydrates often appear in heterogeneous form in nature. These mixtures complicate the isolation of pure material and characterization of structure-activity relationships. As a result, a major bottleneck in glycoscience research is the synthesis and modification of pure materials. While synthetic and chemoenzymatic methods have enabled access to homogeneous tool compounds, a central problem, particularly for newer synthetic chemists, is the matter of problem selection. This outlook aims to provide an entry level overview of fundamental principles in carbohydrate chemistry with an eye toward enabling solutions to frontier challenges.

Gram-Scale Total Synthesis of (±)-Ibogamine.

We describe the gram-scale total synthesis of (±)-ibogamine in nine steps and 24% overall yield. The approach features a Mitsunobu fragment coupling and macrocyclic Friedel-Crafts alkylation to establish the nitrogen-containing core of ibogamine. A regio- and diastereoselective hydroboration allows for simultaneous formation of the tetrahydroazepine and isoquinuclidine ring systems via sulfonamide deprotection and concomitant intramolecular cyclization.

Anti-biofilm Activity of Human Milk Oligosaccharides in Clinical Strains of Streptococcus agalactiae with Diverse Capsular and Sequence Types.

Group B Streptococcus (GBS) is an encapsulated Gram-positive bacterial pathogen that causes severe perinatal infections. Human milk oligosaccharides (HMOs) are short-chain sugars that have recently been shown to possess antimicrobial and anti-biofilm activity against a variety of bacterial pathogens, including GBS. We have expanded these studies to demonstrate that HMOs can inhibit and dismantle biofilm in both invasive and colonizing strains of GBS. A cohort of 30 diverse strains of GBS were analyzed for susceptibility to HMO-dependent biofilm inhibition or destruction. HMOs were significantly effective at inhibiting biofilm in capsular-type- and sequence-type-specific fashion, with significant efficacy in CpsIb, CpsII, CpsIII, CpsV, and CpsVI strains as well as ST-1, ST-12, ST-19, and ST-23 strains. Interestingly, CpsIa as well as ST-7 and ST-17 were not susceptible to the anti-biofilm activity of HMOs, underscoring the strain-specific effects of these important antimicrobial molecules against the perinatal pathogen Streptococcus agalactiae.

Streptococcus agalactiae npx Is Required for Survival in Human Placental Macrophages and Full Virulence in a Model of Ascending Vaginal Infection during Pregnancy.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx, which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δnpx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δnpx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host. IMPORTANCE This study sheds new light on the way that group B Streptococcus (GBS) defends itself against oxidative stress in the infected host. The enzyme encoded by the GBS gene npx is an NADH peroxidase that, our study reveals, provides defense against macrophage-derived reactive oxygen stress and facilitates infections of the uterus during pregnancy. This enzyme could represent a tractable target for future treatment strategies against invasive GBS infections.

Ameliorating adverse perinatal outcomes with Lactoferrin: An intriguing chemotherapeutic intervention.

Adverse pregnancy outcomes affect 54 million people globally per year, with at least 50% of these attributed to infection during gestation. These include inflammation of the membranes surrounding the growing fetus (chorioamnionitis), preterm prelabor rupture of membranes (PPROM), preterm birth (PTB), early-onset disease (EOD) and late-onset disease (LOD), neonatal and maternal sepsis, and maternal or fetal demise. Although universal screening and implementation of intrapartum antibiotic prophylaxis (IAP) has improved EOD outcomes, these interventions have not reduced the incidences of LOD or complications occurring early on during pregnancy such as PPROM and PTB. Thus, novel therapies are needed to prevent adverse pregnancy outcomes and to ameliorate disease risk in vulnerable populations. Lactoferrin has recently been explored as a potential therapeutic as it demonstrates strong antimicrobial and anti-biofilm activity. Lactoferrin is a glycoprotein capable of iron chelation found in a variety of human tissues and is produced in high concentrations in human breast milk. In recent studies, lactoferrin has shown promise inhibiting growth and biofilm formation of streptococcal species, including Group B Streptococcus (GBS), a prominent perinatal pathogen. Understanding the interactions between lactoferrin and GBS could elucidate a novel treatment strategy for adverse pregnancy outcomes caused by GBS infection.

Recent advances on human milk oligosaccharide antimicrobial activity.

Over the past century, human health has been enhanced by antimicrobial development. Following the deployment of the first antibiotics in the 1940s, bacterial resistance evolved and has increasingly outmaneuvered even the most promising antimicrobial agents. Accordingly, increased interest has been placed on alternative methods to circumvent antimicrobial resistance evolution. In the enclosed short review, we discuss the antimicrobial properties of human breast milk with a special emphasis on human milk oligosaccharides (HMOs). We recount studies across gram-negative and gram-positive pathogens, highlighting the usage of HMOs in promoting human health and wellness.

Streptococcus agalactiae cadD alleviates metal stress and promotes intracellular survival in macrophages and ascending infection during pregnancy.

Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.

Synthesis of the Aeromonas veronii strain Bs8 disaccharide repeating unit.

Presented herein is the synthesis of the Aeromonas veronii disaccharide repeating unit which has been achieved in 11 steps starting from d-fucose and d-galactosamine.

Galacto-Oligosaccharide Supplementation Modulates Pathogen-Commensal Competition between Streptococcus agalactiae and Streptococcus salivarius.

The members of the infant microbiome are governed by feeding method (breastmilk vs. formula). Regardless of the source of nutrition, a competitive growth advantage can be provided to commensals through prebiotics - either human milk oligosaccharides (HMOs) or plant oligosaccharides that are supplemented into formula. To characterize how prebiotics modulate commensal - pathogen interactions, we have designed and studied a minimal microbiome where a pathogen, Streptococcus agalactiae engages with a commensal, Streptococcus salivarius. We discovered that while S. agalactiae suppresses the growth of S. salivarius via increased lactic acid production, galacto-oligosaccharides (GOS) supplementation reverses the effect. This result has major implications in characterizing how single species survive in the gut, what niche they occupy, and how they engage with other community members.

2'-Fucosyllactose Ameliorates Chemotherapy-Induced Intestinal Mucositis by Protecting Intestinal Epithelial Cells Against Apoptosis.

BACKGROUND & AIMS: Intestinal mucositis, a severe complication of antineoplastic therapeutics, is characterized by mucosal injury and inflammation in the small intestine. Therapies for the prevention and treatment of this disease are needed. We investigated whether 2'-fucosyllactose (2'-FL), an abundant oligosaccharide in human milk, protects intestinal integrity and ameliorates intestinal mucositis. METHODS: A mouse small intestinal epithelial (MSIE) cell line, mouse enteroid cultures, and human gastrointestinal tumor cell lines (AGS and HT29) were co-treated with the chemotherapy agent 5-fluorouracil (5-FU) and 2'-FL. Mice were injected intraperitoneally with 5-FU to induce intestinal mucositis. 2'-FL was administered in the drinking water to mice before (pretreatment) or concurrently with 5-FU injection. Body weight and pathologic changes were analyzed. RESULTS: 2'-FL alleviated 5-FU inhibition of cell growth in MSIE cells, but not in AGS and HT29 cells. The 5-FU-induced apoptosis in MSIE cells and enteroids was suppressed by 2'-FL. Compared with 5-FU treatment alone, 2'-FL pretreatment protected against body weight loss, and ameliorated inflammation scores, proinflammatory cytokine production, shortening of villi, epithelial cell apoptosis, goblet cell loss, and tight junctional complex disruption in the small intestine. 2'-FL concurrent treatment had less of an effect on intestinal mucositis than 2'-FL pretreatment. Interestingly, no effect of 2'-FL was observed on 5-FU-induced S-phase arrest in MSIE, AGS, and HT29 cells. Neither pretreatment nor concurrent treatment with 2'-FL affected 5-FU-induced inhibition of proliferation in MSIE cells. CONCLUSIONS: This study shows a novel direct effect of 2'-FL in protecting small intestinal epithelial cells against apoptosis stimulated by 5-FU, which may contribute to prevention of 5-FU-induced intestinal mucositis.

The Diverse Antimicrobial Activities of Human Milk Oligosaccharides against Group B Streptococcus.

Streptococcus agalactiae or Group B Streptococcus (GBS) is a Gram-positive bacterial pathobiont that is the etiological cause of severe perinatal infections. GBS can colonize the vagina of pregnant patients and invade tissues causing ascending infections of the gravid reproductive tract that lead to adverse outcomes including preterm birth, neonatal sepsis, and maternal or fetal demise. Additionally, transmission of GBS during labor or breastfeeding can also cause invasive infections of neonates and infants. However, human milk has also been shown to have protective effects against infection; a characteristic that is likely derived from antimicrobial and immunomodulatory properties of molecules that comprise human milk. Recent evidence suggests that human milk oligosaccharides (HMOs), short-chain sugars that comprise 8-20 % of breast milk, have antimicrobial and anti-biofilm activity against GBS and other bacterial pathogens. Additionally, HMOs have been shown to potentiate the activity of antibiotics against GBS. This review presents the most recent published work that studies the interaction between HMOs and GBS.

Antibiofilm Activity of Human Milk Oligosaccharides against Multidrug Resistant and Susceptible Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is a serious threat to human health, per the Centers for Disease Control and Prevention's latest threat assessment. A. baumannii is a Gram-negative opportunistic bacterial pathogen that causes severe community and nosocomial infections in immunocompromised patients. Treatment of these infections is confounded by the emergence of multi- and pan-drug resistant strains of A. baumannii. A. baumannii colonizes abiotic and biotic surfaces and evades antimicrobial challenges by forming biofilms, which are three-dimensional architectural structures of cells adhered to a substrate and encased in an extracellular matrix comprised of polymeric substances such as polysaccharides, proteins, and DNA. Biofilm-inhibiting compounds have recently gained attention as a chemotherapeutic strategy to prevent or disperse A. baumannii biofilms and restore the utility of traditional antimicrobial strategies. Recent work indicates that human milk oligosaccharides (HMOs) have potent antibacterial and biofilm-inhibiting properties. We sought to test the utility of HMOs against a bank of clinical isolates of A. baumannii to ascertain changes in bacterial growth or biofilm formation. Our results indicate that out of 18 strains tested, 14 were susceptible to the antibiofilm activities of HMOs, and that the potent antibiofilm activity was observed in strains isolated from diverse anatomical sites, disease manifestations, and across antibiotic-resistant and susceptible strains.

Analysis of Susceptibility to the Antimicrobial and Anti-Biofilm Activity of Human Milk Lactoferrin in Clinical Strains of Streptococcus agalactiae With Diverse Capsular and Sequence Types.

Group B Streptococcus (GBS) is one of the leading infection-related causes of adverse maternal and neonatal outcomes. This includes chorioamnionitis, which leads to preterm ruptures of membranes and can ultimately result in preterm or stillbirth. Infection can also lead to maternal and neonatal sepsis that may contribute to mortality. Currently, treatment for GBS infection include a bolus of intrapartum antibiotic prophylaxis to mothers testing positive for GBS colonization during late pregnancy. Lactoferrin is an antimicrobial peptide expressed in human breast milk, mucosal epithelia, and secondary granules of neutrophils. We previously demonstrated that lactoferrin possesses antimicrobial and antibiofilm properties against several strains of GBS. This is largely due to the ability of lactoferrin to bind and sequester iron. We expanded upon that study by assessing the effects of purified human breast milk lactoferrin against a panel of phenotypically and genetically diverse isolates of GBS. Of the 25 GBS isolates screened, lactoferrin reduced bacterial growth in 14 and biofilm formation in 21 strains. Stratifying the data, we observed that colonizing strains were more susceptible to the growth inhibition activity of lactoferrin than invasive isolates at lactoferrin concentrations between 250-750 µg/mL. Treatment with 750 µg/mL of lactoferrin resulted in differences in bacterial growth and biofilm formation between discrete sequence types. Differences in bacterial growth were also observed between capsular serotypes 1a and III. Maternally isolated strains were more susceptible to lactoferrin with respect to bacterial growth, but not biofilm formation, compared to neonatal sepsis isolates. Finally, high biofilm forming GBS strains were more impacted by lactoferrin across all isolates tested. Taken together, this study demonstrates that lactoferrin possesses antimicrobial and antibiofilm properties against a wide range of GBS isolates, with maternally isolated colonizing strains being the most susceptible.

Synthesis and Cytotoxic Evaluation of Arimetamycin A and Its Daunorubicin and Doxorubicin Hybrids.

The arimetamycin A glycan governs the compound's cytotoxicity (IC50). To study this branched, deoxy-amino disaccharide, we designed and synthesized a modified acyl donor that underwent glycosylation with three anthracycline aglycones: steffimycinone, daunorubicinone, and doxorubicinone. The result of the approach was a synthesis of arimetamycin A and two novel hybrid anthracyclines. Each molecule exhibited enhanced cytotoxicity in comparison to the parent anthracyclines, steffimycin B, daunorubicin, and doxorubicin. An orienting mechanistic evaluation revealed that the daunorubicin hybrid inhibits the ability of human topoisomerase IIα to relax negatively and positively supercoiled DNA.