1,720,966 research outputs found
Defensins, lectins, mucins, and secretory immunoglobulin A: microbe-binding biomolecules that contribute to mucosal immunity in the human gut
No full textIn the intestine, the mucosal immune system plays essential roles in maintaining homeostasis between the host and microorganisms, and protecting the host from pathogenic invaders. Epithelial cells produce and release a variety of biomolecules into the mucosa and lumen that contribute to immunity. In this review, we focus on a subset of these remarkable host-defense factors–enteric α-defensins, select lectins, mucins, and secretory immunoglobulin A–that have the capacity to bind microbes and thereby contribute to barrier function in the human gut. We provide an overview of the intestinal epithelium, describe specialized secretory cells named Paneth cells, and summarize our current understanding of the biophysical and functional properties of these select microbe-binding biomolecules. We intend for this compilation to complement prior reviews on intestinal host-defense factors, highlight recent advances in the field, and motivate investigations that further illuminate molecular mechanisms as well as the interplay between these molecules and microbes.National Institutes of Health (U.S.) (Grant DP2OD007045
Targeting virulence: salmochelin modification tunes the antibacterial activity spectrum of β-lactams for pathogen-selective killing of Escherichia coli
No full textNew antibiotics are required to treat bacterial infections and counteract the emergence of antibiotic resistance. Pathogen-specific antibiotics have several advantages over broad-spectrum drugs, which include minimal perturbation to the commensal microbiota. We present a strategy for targeting antibiotics to bacterial pathogens that utilises the salmochelin-mediated iron uptake machinery of Gram-negative Escherichia coli. Salmochelins are C-glucosylated derivatives of the siderophore enterobactin. The biosynthesis and utilisation of salmochelins are important for virulence because these siderophores allow pathogens to acquire iron and evade the enterobactin-scavenging host-defense protein lipocalin-2. Inspired by the salmochelins, we report the design and chemoenzymatic preparation of glucosylated enterobactin–β-lactam conjugates that harbour the antibiotics ampicillin (Amp) and amoxicillin (Amx), hereafter GlcEnt–Amp/Amx. The GlcEnt scaffolds are based on mono- and diglucosylated Ent where one catechol moiety is functionalized at the C5 position for antibiotic attachment. We demonstrate that GlcEnt–Amp/Amx provide up to 1000-fold enhanced antimicrobial activity against uropathogenic E. coli relative to the parent β-lactams. Moreover, GlcEnt–Amp/Amx based on a diglucosylated Ent (DGE) platform selectively kill uropathogenic E. coli that express the salmochelin receptor IroN in the presence of non-pathogenic E. coli and other bacterial strains that include the commensal microbe Lactobacillus rhamnosus GG. Moreover, GlcEnt–Amp/Amx evade the host-defense protein lipocalin-2, and exhibit low toxicity to mammalian cells. Our work establishes that siderophore–antibiotic conjugates provide a strategy for targeting virulence, narrowing the activity spectrum of antibiotics in clinical use, and achieving selective delivery of antibacterial cargos to pathogenic bacteria on the basis of siderophore receptor expression.Massachusetts Institute of Technology. Department of ChemistryNational Institutes of Health (U.S.) (Pacific Southwest Research Center of Excellence for Biodefense and Emerging Infectious Disease)Kinship Foundation. Searle Scholars ProgramRoyal Thai Government (RTG) (Scholarship program
Human α-Defensin 6: A Small Peptide That Self-Assembles and Protects the Host by Entangling Microbes
Get PDFConspectus Human α-defensin 6 (HD6) is a 32-residue cysteine-rich peptide that contributes to innate immunity by protecting the host at mucosal sites. This peptide is produced in small intestinal Paneth cells, stored as an 81-residue precursor peptide named proHD6 in granules, and released into the lumen. One unusual feature of HD6 is that it lacks the broad-spectrum antimicrobial activity observed for other human α-defensins, including the Paneth cell peptide human α-defensin 5 (HD5). HD6 exhibits unprecedented self-assembly properties, which confer an unusual host-defense function. HD6 monomers self-assemble into higher-order oligomers termed “nanonets”, which entrap microbes and prevent invasive gastrointestinal pathogens such as Salmonella enterica serovar Typhimurium and Listeria monocytogenes from entering host cells. One possible advantage of this host-defense mechanism is that HD6 helps to keep microbes in the lumen such that they can be excreted or attacked by other components of the immune system, such as recruited neutrophils. In this Account, we report our current understanding of HD6 and focus on work published since 2012 when Bevins and co-workers described the discovery of HD6 nanonets in the literature. First, we present studies that address the biosynthesis, storage, and maturation of HD6, which demonstrate that nature uses a propeptide strategy to spatially and temporally control the formation of HD6 nanonets in the small intestine. The propeptide is stored in Paneth cell granules, and proteolysis occurs during or following release into the lumen, which affords the 32-residue mature peptide that self-assembles. We subsequently highlight structure-function studies that provide a foundation for understanding the molecular basis for why HD6 exhibits unusual self-assembly properties compared with other characterized defensins. The disposition of hydrophobic residues in the HD6 primary structure differs from that of other human α-defensins and is an important structural determinant for oligomerization. Lastly, we consider functional studies that illuminate how HD6 contributes to mucosal immunity. We recently discovered that in addition to blocking bacterial invasion into host epithelial cells by Gram-negative and Gram-positive gastrointestinal pathogens, HD6 suppresses virulence traits displayed by the opportunistic human fungal pathogen Candida albicans. In particular, we found that C. albicans biofilm formation, which causes complications in the treatment of candidiasis, is inhibited by HD6. This observation suggests that HD6 may contribute to intestinal homeostasis by helping to keep C. albicans in its commensal state. We intend for this Account to inspire further biochemical, biophysical, and biological investigations that will advance our understanding of HD6 in mucosal immunity and the host-microbe interaction.National Institutes of Health (U.S.) (Grant DP2OD007045
Molecular Basis for Self-Assembly of a Human Host-Defense Peptide That Entraps Bacterial Pathogens
No full textHuman α-defensin 6 (HD6) is a 32-aa cysteine-rich peptide of the innate immune system. Although HD6 is a member of an antimicrobial peptide family, it exhibits negligible antibacterial activity in vitro. Rather, HD6 possesses a unique innate immune mechanism whereby it self-assembles into oligomers that capture pathogens to prevent microbial invasion of the intestinal epithelium and subsequent dissemination. Molecular-level understanding for why HD6 functions differently from other human defensins remains unclear. To further elucidate the HD6 self-assembly process and its biological activity, we developed robust protocols for obtaining native and mutant HD6 in high purity from overexpression in Escherichia coli. We combined biophysical characterization with biological assays to probe HD6 structure and function. We report that native HD6 readily self-assembles into elongated fibrils observable by transmission electron microscopy, agglutinates both Gram-negative and -positive bacteria, and prevents the human gastrointestinal pathogen Listeria monocytogenes from invading cultured mammalian cells. Mutation of hydrophobic residues (F2A, I22T, V25T, F29A) perturbs self-assembly and results in attenuated biological activity. In particular, the F2A and F29A mutants do not form fibrils under our experimental conditions and neither agglutinate bacteria nor prevent L. monocytogenes invasion. In total, our results demonstrate that the hydrophobic effect is essential for promoting HD6 self-assembly and innate immune function, and indicate that HD6 may provide host defense against Listeria in the gut. This investigation provides a timely description of how variations in amino acid sequence confer diverse physiological functions to members of the defensin family.United States. National Institutes of Health (DP2OD007045
Proteolysis triggers self-assembly and unmasks innate immune function of a human α-defensin peptide
Get PDFHuman α-defensin 6 (HD6) is a unique peptide of the defensin family that provides innate immunity in the intestine by self-assembling to form higher-order oligomers that entrap bacteria and prevent host cell invasion. Here, we report critical steps in the self-assembly pathway of HD6. We demonstrate that HD6 is localized in secretory granules of small intestinal Paneth cells. HD6 is stored in these granules as an 81-residue propeptide (proHD6), and is recovered from ileal lumen as a 32-residue mature peptide. The propeptide neither forms higher-order oligomers, nor agglutinates bacteria, nor prevents Listeria monocytogenes invasion into epithelial cells. The Paneth cell granules also contain the protease trypsin, and trypsin-catalyzed hydrolysis of proHD6 liberates mature HD6, unmasking its latent activities. This work illustrates a remarkable example of how nature utilizes a propeptide strategy to spatially and temporally control peptide self-assembly, and thereby initiates innate immune function in the human intestine.National Institutes of Health (U.S.) (Office of the Director, (NIH Grant 1DP2OD007045)Thailand (Royal Thai Government Fellowship)National Institutes of Health (U.S.) (NIH Grant T32AI060555)National Institutes of Health (U.S.) (grant AI032738)National Institutes of Health (U.S.) (grant AI099519
Siderophore-based immunization strategy to inhibit growth of enteric pathogens
No full textInfections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB-siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and whenmice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophoretargeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections. Keyword: siderophore; immunization; iron; Salmonella; microbiot
Self-assembly of human defensin 6 and its role in innate immunity and siderophore-based strategies to target Gram-Negative bacteria
No full textThesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2016.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.The first part of this thesis focuses on studies of structural and functional properties of human defensin 6 (HD6). This peptide is produced and secreted by intestinal Paneth cells as a part of innate immune response to invading microbes. Instead of killing microbes, HD6 self-assembles into higher-order oligomers to entrap and prevent microbes from invading into the host cells. This activity of HD6 is unusual among defensins. Herein, we employed biophysical and biological techniques to decipher the molecular details of the unusual behavior of HD6. We demonstrate that the self-assembly of HD6 is driven by hydrophobicity and this work highlights how variable amino acid sequences among defensins afford different biological function. We further elucidated how HD6 is stored in Paneth cells such that its self-assembly is suppressed. Similar to zymogens, HD6 utilizes a pro region to control its self-assembly and upon secretion, trypsin cleaves the propeptide to unleash mature HD6 and trigger host-defense function. We also discovered that HD6 suppresses virulence traits of Candida albicans, an opportunistic fungal pathogen. This study expands the scope of the broad-spectrum function of HD6. In the second part of this thesis, we present two siderophore-based strategies that target Gram-negative bacteria. Siderophores are small-molecule iron chelators that bacteria employ to sequester iron from the environment. Our approaches focus on enterobactin (Ent) and its glycosylated derivatives (GIcEnt), which are virulence factors of certain enteric pathogens, such as Salmonella spp. The first approach relies on the use of GlcEnt-[beta]-lactam conjugates to target these pathogens. In addition to enhanced uptake efficiency of the drug into bacteria, GIcEnt specifically delivers the antibiotic to pathogens and leave commensal bacteria unaffected. In the second approach, we aim to use Ent/GlcEnt-specific antibodies to inhibit bacterial iron acquisition, and thereby prevent the bacteria from colonizing in the host. We demonstrate that antibodies against Ent/GlcEnt are produced in the mice immunized with a protein-siderophore conjugate. These mice exhibit reduced intestinal colonization, reduced systemic dissemination of S. Typhimurium, and increased resistance against the challenge of this pathogen.by Phoom Chairatana.Ph. D. in Biological Chemistr
Visualizing Attack of Escherichia coli by the Antimicrobial Peptide Human Defensin 5
No full textHuman α-defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and contributes to innate immunity in the human gut and other organ systems. Despite many years of investigation, its antimicrobial mechanism of action remains unclear. In this work, we report that HD5[subscript ox], the oxidized form of this peptide that exhibits three regiospecific disulfide bonds, causes distinct morphological changes to Escherichia coli and other Gram-negative microbes. These morphologies include bleb formation, cellular elongation, and clumping. The blebs are up to ∼1 μm wide and typically form at the site of cell division or cell poles. Studies with E. coli expressing cytoplasmic GFP reveal that HD5[subscript ox] treatment causes GFP emission to localize in the bleb. To probe the cellular uptake of HD5[subscript ox] and subsequent localization, we describe the design and characterization of a fluorophore–HD5 conjugate family. By employing these peptides, we demonstrate that fluorophore–HD5[subscript ox] conjugates harboring the rhodamine and coumarin fluorophores enter the E. coli cytoplasm. On the basis of the fluorescence profiles, each of these fluorophore–HD5[subscript ox] conjugates localizes to the site of cell division and cell poles. These studies support the notion that HD5[subscript ox'], at least in part, exerts its antibacterial activity against E. coli and other Gram-negative microbes in the cytoplasm.United States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)National Science Foundation (U.S.) (Grant 007031)Massachusetts Institute of Technology (MIT UROP Program funds)Royal Thai Government (RTG) (Fellowship)Massachusetts Institute of Technology (2014 Richard R. Schrock summer graduate fellowship)National Institutes of Health (U.S.) (NIH Office of the Director, grant DP2OD007045
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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