Molecular insights into phosphoethanolamine cellulose formation and secretion.

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, an associated periplasmic semicircle of hexameric BcsB, as well as the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a conserved periplasmic cellulase of unknown biological function. While events underlying the synthesis and translocation of cellulose by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three copies of BcsG near the nascent cellulose polymer. Further, the terminal subunit of the BcsB semicircle tethers the N-terminus of a single BcsC protein to establish a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of mislocalized cellulose. Lastly, we introduce pEtN modification of cellulose in orthogonal cellulose biosynthetic systems by protein engineering.

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.

Nordihydroguaiaretic Acid (NDGA) Inhibits CsgA Polymerization, Bacterial Amyloid Biogenesis, and Biofilm Formation.

Escherichia coli and other Enterobacteriaceae thrive in robust biofilm communities through the coproduction of curli amyloid fibers and phosphoethanolamine cellulose. Curli promote adhesion to abiotic surfaces and plant and human host tissues and are associated with pathogenesis in urinary tract infection and food-borne illness. The production of curli in the host has also been implicated in the pathogenesis of neurodegenerative diseases. We report that the natural product nordihydroguaiaretic acid (NDGA) is effective as a curlicide in E. coli. NDGA prevents CsgA polymerization in vitro in a dose-dependent manner. NDGA selectively inhibits cell-associated curli assembly and inhibits uropathogenic E. coli biofilm formation. More broadly, this work emphasizes the ability to evaluate and identify bioactive amyloid assembly inhibitors by using the powerful gene-directed amyloid biogenesis machinery in E. coli.

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.

The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System.

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.

Analysis of Antimicrobial and Antibiofilm Activity of Human Milk Lactoferrin Compared to Bovine Lactoferrin against Multidrug Resistant and Susceptible Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is an opportunistic bacterial pathogen that causes severe infections in immunocompromised patients. The emergence of multi- and pan-drug resistant strains of A. baumannii from clinical sources has confounded treatment and enhanced morbidity and mortality associated with these infections. One way that A. baumannii circumnavigates environmental and antimicrobial challenge is by forming tertiary architectural structures of cells known as biofilms. Biofilm-inhibiting molecules could be deployed as a potential chemotherapeutic strategy to inhibit or disrupt A. baumannii biofilms and mitigate adverse outcomes due to infection. Lactoferrin is an innate immune glycoprotein produced in high concentrations in both human and bovine milk which has previously been shown to have antibacterial and antibiofilm activities. We sought to test lactoferrin against a bank of clinical isolates of A. baumannii to determine changes in bacterial growth or biofilm formation. Our results indicate that human lactoferrin has slightly more potent antibacterial activities than bovine lactoferrin against certain strains of A. baumannii and that these effects are associated with anatomical site of isolation. Additionally, we have shown that both bovine and human lactoferrin can inhibit A. baumannii biofilm formation and that these effects are associated with anatomical site of isolation and whether the strain forms robust or weak biofilms.

Group B Streptococcus cpsE Is Required for Serotype V Capsule Production and Aids in Biofilm Formation and Ascending Infection of the Reproductive Tract during Pregnancy.

Group B Streptococcus (GBS) is an encapsulated Gram-positive pathogen that causes ascending infections of the reproductive tract during pregnancy. The capsule of this organism is a critical virulence factor that has been implicated in a variety of cellular processes to promote pathogenesis. Primarily comprised of carbohydrates, the GBS capsule and its synthesis is driven by the capsule polysaccharide synthesis (cps) operon. The cpsE gene within this operon encodes a putative glycosyltransferase that is responsible for the transfer of a Glc-1-P from UDP-Glc to an undecaprenyl lipid molecule. We hypothesized that the cpsE gene product is important for GBS virulence and ascending infection during pregnancy. Our work demonstrates that a GBS cpsE mutant secretes fewer carbohydrates, has a reduced capsule, and forms less biofilm than the wild-type parental strain. We show that, compared to the parental strain, the ΔcpsE deletion mutant is more readily taken up by human placental macrophages and has a significantly attenuated ability to invade and proliferate in the mouse reproductive tract. Taken together, these results demonstrate that the cpsE gene product is an important virulence factor that aids in GBS colonization and invasion of the gravid reproductive tract.

Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus.

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.

Analysis of virulence phenotypes and antibiotic resistance in clinical strains of Acinetobacter baumannii isolated in Nashville, Tennessee.

BACKGROUND: Acinetobacter baumannii is a gram-negative bacterium which causes opportunistic infections in immunocompromised hosts. Genome plasticity has given rise to a wide range of strain variation with respect to antimicrobial resistance profiles and expression of virulence factors which lead to altered phenotypes associated with pathogenesis. The purpose of this study was to analyze clinical strains of A. baumannii for phenotypic variation that might correlate with virulence phenotypes, antimicrobial resistance patterns, or strain isolation source. We hypothesized that individual strain virulence phenotypes might be associated with anatomical site of isolation or alterations in susceptibility to antimicrobial interventions. METHODOLOGY: A cohort of 17 clinical isolates of A. baumannii isolated from diverse anatomical sites were evaluated to ascertain phenotypic patterns including biofilm formation, hemolysis, motility, and antimicrobial resistance. Antibiotic susceptibility/resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin was determined. RESULTS: Antibiotic resistance was prevalent in many strains including resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin. All strains tested induced hemolysis on agar plate detection assays. Wound-isolated strains of A. baumannii exhibited higher motility than strains isolated from blood, urine or Foley catheter, or sputum/bronchial wash. A. baumannii strains isolated from patient blood samples formed significantly more biofilm than isolates from wounds, sputum or bronchial wash samples. An inverse relationship between motility and biofilm formation was observed in the cohort of 17 clinical isolates of A. baumannii tested in this study. Motility was also inversely correlated with induction of hemolysis. An inverse correlation was observed between hemolysis and resistance to ticarcillin-k clavulanate, meropenem, and piperacillin. An inverse correlation was also observed between motility and resistance to ampicillin-sulbactam, ceftriaxone, ceftoxamine, ceftazidime, ciprofloxacin, or levofloxacin. CONCLUSIONS: Strain dependent variations in biofilm and motility are associated with anatomical site of isolation. Biofilm and hemolysis production both have an inverse association with motility in the cohort of strains utilized in this study, and motility and hemolysis were inversely correlated with resistance to numerous antibiotics.

Vitamin D and Streptococci: The Interface of Nutrition, Host Immune Response, and Antimicrobial Activity in Response to Infection.

Streptococcus species are common causes of human infection. These Gram-positive, encapsulated bacterial pathogens infect diverse anatomic spaces, leading to infections including skin and soft tissue infection, endocarditis, pneumonia, meningitis, sinusitis, otitis media, chorioamnionitis, sepsis, and even death. Risk for streptococcal infection is highest in low- and middle-income countries where micronutrient deficiency is common. Epidemiological data reveal that vitamin D deficiency is associated with enhanced risk of streptococcal infection and cognate disease outcomes. Additionally, vitamin D improves antibacterial defenses by stimulating innate immune processes such as phagocytosis and enhancing production of reactive oxygen species (oxidative burst) and antimicrobial peptides (including cathelicidin and lactoferrin), which are important for efficient killing of bacteria. This review presents the most recent published work that studies interactions between the micronutrient vitamin D, the host immune system, and pathogenic streptococci as well as comparisons with other relevant infection models.

Leveraging Stereoelectronic Effects in Biofilm Eradication: Synthetic ß-Amino Human Milk Oligosaccharides Impede Microbial Adhesion As Observed by Scanning Electron Microscopy.

Alongside Edward, Lemieux was among the earliest researchers studying negative hyperconjugation (i.e., the anomeric effect) or the preference for gauche conformations about the C1-O5 bond in carbohydrates. Lemieux also studied an esoteric, if not controversial, theory known as the reverse anomeric effect (RAE). This theory is used to rationalize scenarios where predicted anomeric stabilization does not occur. One such example is the Kochetkov amination where reducing end amines exist solely as the ß-anomer. Herein, we provide a brief account of Lemieux's contributions to the field of stereoelectronics and apply this knowledge toward the synthesis of ß-amino human milk oligosaccharides (ßΑ-HMOs). These molecules were evaluated for their ability to inhibit growth and biofilm production in Group B Streptococcus (GBS) and Staphylococcus aureus. While the parent HMOs lacked antimicrobial and antibiofilm activity, their ß-amino derivatives significantly inhibited biofilm formation in both species. Field emission gun-scanning single electron microscopy (FEG-SEM) revealed that treatment with ß-amino HMOs significantly inhibits bacterial adherence and eliminates the ability of both microbes to form biofilms.

Two-step conversion of unprotected oligosaccharides to generate bioorthogonal oligosaccharide tool compounds.

Oligosaccharides have valuable effects in biological systems, but their complex nature makes them difficult to convert into chemical tools. We have developed a two-step conversion of unprotected oligosaccharides that is highly amenable to generating a variety of complex oligosaccharide tool compounds. This sequence features an optimized Kochetkov amination procedure and subsequent amide coupling. With these simple synthetic conversions, the creation of novel bioorthogonal carbohydrate probes becomes easily accessible and new avenues for chemical biology may be opened.•Optimized Kochetkov amination procedure for complex oligosaccharides.•Highly versatile amide coupling for facile probe synthesis.

Like mother, like microbe: human milk oligosaccharide mediated microbiome symbiosis.

Starting shortly after parturition, and continuing throughout our lifetime, the gut microbiota coevolves with our metabolic and neurological programming. This symbiosis is regulated by a complex interplay between the host and environmental factors, including diet and lifestyle. Not surprisingly, the development of this microbial community is of critical importance to health and wellness. In this targeted review, we examine the gut microbiome from birth to 2 years of age to characterize the role human milk oligosaccharides play in early formation of microbial flora.

Lactoferrin: A Critical Mediator of Both Host Immune Response and Antimicrobial Activity in Response to Streptococcal Infections.

Streptococcal species are Gram-positive bacteria responsible for a variety of disease outcomes including pneumonia, meningitis, endocarditis, erysipelas, necrotizing fasciitis, periodontitis, skin and soft tissue infections, chorioamnionitis, premature rupture of membranes, preterm birth, and neonatal sepsis. In response to streptococcal infections, the host innate immune system deploys a repertoire of antimicrobial and immune modulating molecules. One important molecule that is produced in response to streptococcal infections is lactoferrin. Lactoferrin has antimicrobial properties including the ability to bind iron with high affinity and sequester this important nutrient from an invading pathogen. Additionally, lactoferrin has the capacity to alter the host inflammatory response and contribute to disease outcome. This Review presents the most recent published work that studies the interaction between the host innate immune protein lactoferrin and the invading pathogen, Streptococcus.

A Solution to Antifolate Resistance in Group B Streptococcus: Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim.

Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs' mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs.IMPORTANCE Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.

Synthetic Ellagic Acid Glycosides Inhibit Early Stage Adhesion of Streptococcus agalactiae Biofilms as Observed by Scanning Electron Microscopy.

Ellagic acid derivatives possess antimicrobial and antibiofilm properties across a wide-range of microbial pathogens. Due to their poor solubility and ambident reactivity it is challenging to synthesize, purify, and characterize the activity of ellagic acid glycosides. In this study, we have synthesized three ellagic acid glycoconjugates and evaluated their antimicrobial and antibiofilm activity in Streptococcus agalactiae (Group B Streptococcus, GBS). Their significant impacts on biofilm formation were examined via SEM to reveal early-stage inhibition of cellular adhesion. Additionally, the synthetic glycosides were evaluated against five of the six ESKAPE pathogens and two fungal pathogens. These studies reveal that the ellagic acid glycosides possess inhibitory effects on the growth of gram-negative pathogens.

Bioorthogonal human milk oligosaccharide probes for antimicrobial target identification within Streptococcus agalactiae.

Human milk oligosaccharides (HMOs) are a structurally diverse class of carbohydrates that possess strong antibacterial activity against Streptococcus agalactiae (Group B Strep, GBS). This work highlights the design, synthesis, and retained biological activity of several HMO bioorthogonal probes within GBS, a first in class advance. The use of such probes will assist in identifying HMO-protein interactions within GBS and may be broadly applicable in researching HMO cellular targets within a variety of biological systems. Finally, this strategy is highly amenable to other oligosaccharide scaffolds, requiring minimal synthetic transformations and chemical perturbation.

The DARK Side of Total Synthesis: Strategies and Tactics in Psychoactive Drug Production.

Humankind has used and abused psychoactive drugs for millennia. Formally, a psychoactive drug is any agent that alters cognition and mood. The term "psychotropic drug" is neutral and describes the entire class of substrates, licit and illicit, of interest to governmental drug policy. While these drugs are prescribed for issues ranging from pain management to anxiety, they are also used recreationally. In fact, the current opioid epidemic is the deadliest drug crisis in American history. While the topic is highly politicized with racial, gender, and socioeconomic elements, there is no denying the toll drug mis- and overuse is taking on this country. Overdose, fueled by opioids, is the leading cause of death for Americans under 50 years of age, killing ca. 64,000 people in 2016. From a chemistry standpoint, the question is in what ways, if any, did organic chemists contribute to this problem? In this targeted review, we provide brief historical accounts of the main classes of psychoactive drugs and discuss several foundational total syntheses that ultimately provide the groundwork for producing these molecules in academic, industrial, and clandestine settings.