scholarly journals Separate Pathways for O Acetylation of Polymeric and Monomeric Sialic Acids and Identification of Sialyl O-Acetyl Esterase in Escherichia coli K1

2006 ◽  
Vol 188 (17) ◽  
pp. 6195-6206 ◽  
Author(s):  
Susan M. Steenbergen ◽  
Young-Choon Lee ◽  
Willie F. Vann ◽  
Justine Vionnet ◽  
Lori F. Wright ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Pasteurella Multocida ◽  
Vaccine Development ◽  
Polysialic Acid ◽  
Sialic Acids ◽  
Fluorescent Labeling ◽  
Phase Variable ◽  
Acetyl Esterase

ABSTRACT O acetylation at carbon positions 7 or 9 of the sialic acid residues in the polysialic acid capsule of Escherichia coli K1 is catalyzed by a phase-variable contingency locus, neuO, carried by the K1-specific prophage, CUS-3. Here we describe a novel method for analyzing polymeric sialic acid O acetylation that involves the release of surface sialic acids by endo-N-acetylneuraminidase digestion, followed by fluorescent labeling and detection of quinoxalinone derivatives by chromatography. The results indicated that NeuO is responsible for the majority of capsule modification that takes place in vivo. However, a minor neuO-independent O acetylation pathway was detected that is dependent on the bifunctional polypeptide encoded by neuD. This pathway involves O acetylation of monomeric sialic acid and is regulated by another bifunctional enzyme, NeuA, which includes N-terminal synthetase and C-terminal sialyl O-esterase domains. A homologue of the NeuA C-terminal domain (Pm1710) in Pasteurella multocida was also shown to be an esterase, suggesting that it functions in the catabolism of acetylated environmental sialic acids. Our combined results indicate a previously unexpected complexity in the synthesis and catabolism of microbial sialic and polysialic acids. These findings are key to understanding the biological functions of modified sialic acids in E. coli K1 and other species and may provide new targets for drug or vaccine development.

scholarly journals Unexpected Diversity of Escherichia coli SialateO-Acetyl Esterase NanS

2016 ◽  
Vol 198 (20) ◽  
pp. 2803-2809 ◽  
Author(s):  
Ariel Rangel ◽  
Susan M. Steenbergen ◽  
Eric R. Vimr
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Bacterial Species ◽  
Sialic Acids ◽  
Host Bacterium ◽  
Acetyl Esterase ◽  
Content Type ◽  
E Coli ◽  
Host Microbe Interactions ◽  
K 12

ABSTRACTThe sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, whereN-acetyl- orN-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations toN-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria withO-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity ofEscherichia coliNanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show thatE. colistrain O157:H7 Stx prophage or prophage remnants invariably include paralogs ofnanSoften located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialateO-acetyl esterases, as shown by complementation of anE. colistrain K-12nanSmutant and the unimpaired growth of anE. coliO157nanSmutant onO-acetylated sialic acid. We further demonstrate thatnanShomologs inStreptococcusspp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialateO-acetyl esterase.IMPORTANCEThe sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show thatnanShomologs exist in bacteria other thanEscherichia coli, as well as part of toxigenicE. coliprophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies ofnanSto include mucosal pathogens, prophage, and prophage remnants. This expansion of thenanSsuperfamily suggests important, although as-yet-unknown, functions in host-microbe interactions.

scholarly journals Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

2012 ◽  
Vol 19 (10) ◽  
pp. 1603-1608 ◽  
Author(s):  
Koushik Roy ◽  
David J. Hamilton ◽  
James M. Fleckenstein
Keyword(s):  
Escherichia Coli ◽  
Diarrheal Disease ◽  
Vaccine Development ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Colonization ◽  
Content Type ◽  
Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonizationin vivoand toxin delivery to epithelial cellsin vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development.

Effect of removing sialic acids from endothelium on the adherence of circulating platelets in arteries in vivo

1982 ◽  
Vol 214 (1197) ◽  
pp. 471-480 ◽  
Keyword(s):  
Sialic Acid ◽  
Proteolytic Activity ◽  
Negative Charge ◽  
Sialic Acids ◽  
Arterial Walls ◽  
Charge Excess ◽  
Flow Through ◽  
Adhesion Of Platelets ◽  
Prostacyclin Production

The contribution of the net negative charge excess due to sialic acids on endothelium in preventing adhesion of circulating platelets in vivo was investigated in anaesthetized rabbits. Platelets in the rabbit’s circulation were selectively labelled with radioactive 5-hydroxytryptamine in vivo . Segments of carotid arteries temporarily isolated from the circulation were perfused with one or other of two commercial preparations of neuraminidase; the opposite carotid artery was perfused similarly without the enzyme, as control. A neuraminidase preparation from Behringwerke free of proteolytic activity released sialic acid into the perfusate with a peak concentration after 10–15 min which decreased gradually later. A neuraminidase preparation from Sigma that contained demonstrable proteolytic activity released sialic acid similarly during the first hour and thereafter more sialic acid in a second peak. After blood flow through the carotids had been restored the adhesion of labelled platelets in the artery perfused with neuraminidase was compared with that in the artery perfused without the enzyme. The radioactivities were significantly higher in carotids that had been perfused with neuraminidase than in those that had been perfused without the enzyme. Neuraminidase perfusion had no effect on the production of prostacyclin by the carotids. Perfusion with acetylsalicylic acid before neuraminidase increased the adhesion of platelets significantly. It is concluded that diminution in electrostatic repulsion between circulating platelets and vascular endothelium from which the net negative charge excess due to sialic acids has been removed increases the adhesion of circulating platelets, irrespective of the production of prostacyclin by the arterial walls, and that inhibition of prostacyclin production augments this adhesion of platelets.

scholarly journals Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 2665-2679 ◽  
Author(s):  
Manohar John ◽  
Indira T. Kudva ◽  
Robert W. Griffin ◽  
Allen W. Dodson ◽  
Bethany McManus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Human Infection ◽  
Escherichia Coli O157 ◽  
Standard Laboratory ◽  
E Coli ◽  
Phase Culture ◽  
Stool Specimens

ABSTRACT Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

scholarly journals Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

2014 ◽  
Vol 70 (7) ◽  
pp. 1801-1811 ◽  
Author(s):  
Thanuja Gangi Setty ◽  
Christine Cho ◽  
Sowmya Govindappa ◽  
Michael A. Apicella ◽  
S. Ramaswamy
Keyword(s):  
Sialic Acid ◽  
Binding Site ◽  
Pathogenic Bacteria ◽  
Pasteurella Multocida ◽  
Binding Proteins ◽  
Molecular Mimicry ◽  
Binding Pocket ◽  
Sialic Acids ◽  
Host Immune System ◽  
Binding Affinities

Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteriaFusobacterium nucleatum,Pasteurella multocidaandVibrio choleraeand their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of theHaemophilus influenzaeprotein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Concomitant release of sialic acids and calcium by neuraminidase from rat aorta in situ

1988 ◽  
Vol 235 (1279) ◽  
pp. 139-144 ◽  
Keyword(s):  
Sialic Acid ◽  
Wistar Rats ◽  
Rat Aorta ◽  
Sialic Acids ◽  
Molar Ratio ◽  
Male Wistar Rats

Male Wistar rats were heparinized and killed with pentobarbital. The upper and lower ends of the aortae were cannulated and the blood was washed out with saline until the washings contained calcium and sialic-acid-reacting material at minimal concentrations. The aortae were perfused with neuraminidase for 15 min. This caused the appearance of calcium as well as of sialic acids in the perfusate in total amounts of about 5.3 nmol and about 3.6 nmol per aorta respectively. The molar ratio of about 1.5 is sufficiently close to that determined for the association of calcium with sialic acids in vitro to suggest that their association is similar in vivo .

scholarly journals B cell antigen receptor signal strength and peripheral B cell development are regulated by a 9-O-acetyl sialic acid esterase

2008 ◽  
Vol 206 (1) ◽  
pp. 125-138 ◽  
Author(s):  
Annaiah Cariappa ◽  
Hiromu Takematsu ◽  
Haoyuan Liu ◽  
Sandra Diaz ◽  
Khaleda Haider ◽  
...  
Keyword(s):  
Sialic Acid ◽  
B Cell ◽  
Cell Receptor ◽  
Immunological Tolerance ◽  
Cell Development ◽  
B Cell Development ◽  
Acetyl Esterase ◽  
B Cell Signaling ◽  
Inhibitory Signaling

We show that the enzymatic acetylation and deacetylation of a cell surface carbohydrate controls B cell development, signaling, and immunological tolerance. Mice with a mutation in sialate:O-acetyl esterase, an enzyme that specifically removes acetyl moieties from the 9-OH position of α2–6-linked sialic acid, exhibit enhanced B cell receptor (BCR) activation, defects in peripheral B cell development, and spontaneously develop antichromatin autoantibodies and glomerular immune complex deposits. The 9-O-acetylation state of sialic acid regulates the function of CD22, a Siglec that functions in vivo as an inhibitor of BCR signaling. These results describe a novel catalytic regulator of B cell signaling and underscore the crucial role of inhibitory signaling in the maintenance of immunological tolerance in the B lineage.

scholarly journals Host-Like Carbohydrates Promote Bloodstream Survival of Vibrio vulnificusIn Vivo

2015 ◽  
Vol 83 (8) ◽  
pp. 3126-3136 ◽  
Author(s):  
Jean-Bernard Lubin ◽  
Warren G. Lewis ◽  
Nicole M. Gilbert ◽  
Cory M. Weimer ◽  
Salvador Almagro-Moreno ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
The United States ◽  
Sialic Acids ◽  
Natural Resistance ◽  
Bacterial Survival ◽  
Content Type ◽  
Life Threatening

Sialic acids are found on all vertebrate cell surfaces and are part of a larger class of molecules known as nonulosonic acids. Many bacterial pathogens synthesize related nine-carbon backbone sugars; however, the role(s) of these non-sialic acid molecules in host-pathogen interactions is poorly understood.Vibrio vulnificusis the leading cause of seafood-related death in the United States due to its ability to quickly access the host bloodstream, which it can accomplish through gastrointestinal or wound infection. However, little is known about how this organism persists systemically. Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide ofV. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B. Further experiments in a murine model of intravenousV. vulnificusinfection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissuesin vivo. In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed byV. vulnificusisolates. Taken together, these results suggest that molecules similar to sialic acids evolved to facilitate the aquatic lifestyle ofV. vulnificusbut that their emergence also resulted in a gain of function with life-threatening potential in the human host.

scholarly journals A versatile human intestinal organoid-derived epithelial monolayer model for the study of enteric pathogens

2020 ◽  
Author(s):  
Kourtney P. Nickerson ◽  
Alejandro Llanos-Chea ◽  
Laura Ingano ◽  
Gloria Serena ◽  
Alba Miranda-Ribera ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Terminal Ileum ◽  
Vaccine Development ◽  
Enteric Pathogens ◽  
Cell Populations ◽  
Tissue Tropism ◽  
M Cells ◽  
Therapeutic Development ◽  
Human Specific

AbstractGastrointestinal infections cause significant morbidity and mortality worldwide. The complexity of human biology and limited insights into host-specific infection mechanisms are key barriers to current therapeutic development. Here, we demonstrate that two-dimensional epithelial monolayers derived from human intestinal organoids, combined with in vivo-like bacterial culturing conditions, provide significant advancements for the study of enteropathogens. Monolayers from the terminal ileum, cecum, and ascending colon recapitulated the composition of the gastrointestinal epithelium, in which several techniques were used to detect the presence of enterocytes, mucus-producing goblet cells, and other cell types following differentiation. Importantly, the addition of receptor activator of nuclear factor kappa-B ligand (RANKL) increased the presence of M cells, critical antigen-sampling cells often exploited by enteric pathogens. For infections, bacteria were grown under in vivo-like conditions known to induce virulence. Overall, interesting patterns of tissue tropism and clinical manifestations were observed. Shigella flexneri adhered efficiently to the cecum and colon; however, invasion in the colon was best following RANKL treatment. Both Salmonella Typhi and Typhimurium serovars displayed different infection patterns, with S. Typhimurium causing more destruction of the terminal ileum and S. Typhi infecting the cecum more efficiently than the ileum, particularly with regards to adherence. Finally, various pathovars of Escherichia coli validated the model by confirming only adherence was observed with these strains. This work demonstrates that the combination of human-derived tissue with targeted bacterial growth conditions enables powerful analyses of human-specific infections that could lead to important insights into pathogenesis and accelerate future vaccine development.ImportanceWhile traditional laboratory techniques and animal models have provided valuable knowledge in discerning virulence mechanisms of enteric pathogens, the complexity of the human gastrointestinal tract has hindered our understanding of physiologically relevant, human-specific interactions; and thus, has significantly delayed successful vaccine development. The human intestinal organoid-derived epithelial monolayer (HIODEM) model closely recapitulates the diverse cell populations of the intestine, allowing for the study of human-specific infections. Differentiation conditions permit the expansion of various cell populations, including M cells that are vital to immune recognition and the establishment of infection by some bacteria. We provide details of reproducible culture methods and infection conditions for the analyses of Shigella, Salmonella, and pathogenic Escherichia coli in which tissue tropism and pathogen-specific infection patterns were detected. This system will be vital for future studies that explore infection conditions, health status, or epigenetic differences; and will serve as a novel screening platform for therapeutic development.

scholarly journals Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells

2016 ◽  
Vol 113 (12) ◽  
pp. 3329-3334 ◽  
Author(s):  
Maurizio Perdicchio ◽  
Juan M. Ilarregui ◽  
Marleen I. Verstege ◽  
Lenneke A. M. Cornelissen ◽  
Sjoerd T. T. Schetters ◽  
...  
Keyword(s):  
T Cells ◽  
Sialic Acid ◽  
T Cell ◽  
De Novo ◽  
Ex Vivo ◽  
Treg Cells ◽  
Sialic Acids ◽  
Antigen Uptake

Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.

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