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Glycomics Aims To Interpret the Third Molecular Language of Cells Some microbes exploit carbohydrate receptors to enter and disrupt particular host cells Alison A. Weiss and Suri S. Iyer eciphering the chemical codes used by cells provides valuable insights, many of which will help toward ameliorating diseases. In contrast to impressive progress decoding genomics and proteomics, efforts to crack the code for the third major class of biopolymers, carbohydrates, continue to prove frustrating. Glycomics, the systematic study of carbohydrate polymers and oligosaccharides, lags a distant third compared to genomics and proteomics— despite the ubiquitous presence of carbohydrates at cell surfaces, where cells engage in important molecular-based signaling. Glycolipids and glycoproteins play important but poorly understood roles in regulating processes in mammalian cells and maintaining human health. At the molecular level, pathogens exploit carbohydrate structures that decorate D Summary • The many ways in which nine common monosaccharide units are linked complicates efforts to describe—and to understand the biological impacts of—sugar-containing polymers. • Some viruses and bacterial toxins depend on carbohydrates that decorate mammalian cellsurface receptors to gain entry. • The preferential recognition by influenza virus of specific forms of sialic acid has significant implications for viral transmission and pathogenesis. • In each case, the interactions of botulinum neurotoxins, Shiga toxin, and pertussis toxin with particular carbohydrate receptors helps to explain tissue specificity and potency. mammalian cell surfaces, often using those structures as a means to infect such cells. By focusing on the glycolipids and glycoproteins that microbial pathogens target, we can begin to decipher the complex language of cell surface carbohydrates. Glycomics is providing us a more comprehensive approach to understanding how microbes interact with such carbohydrate receptors and could lead to the development of novel therapeutics and diagnostics. Nucleic Acids, Proteins, and Sugars Embody Distinctive Coding DNA, RNA, and proteins embody linear codes—arrays of four nucleotides in the case of nucleic acids, and 20 amino acids for proteins. In general, mammalian carbohydrates are composed of nine distinct types of monosaccharides along with some variations (Fig. 1). Moreover, the ways in which carbohydrates are linked further complicates efforts to describe sugar-containing polymers—let alone try to understand the biological effects of those different linkages. For example, any pair of six-carbon monosaccharides can be linked in 11 different ways (Fig. 2). Assuming that nine distinct types of monosaccharides are available, there are 9 ⫻ 9 ⫻ 11, or 891, 2-letter words for these sugars. In comparison, two amino acids can be joined in only one way, creating only 20 ⫻ 20, or 400, 2-letter words. With each additional unit, the number of potential combinations of sugars rapidly outpaces those for comparable peptides and proteins. For example, although six amino Alison A. Weiss is Professor in the Department of Biochemistry, Molecular Genetics and Microbiology and Suri S. Iyer is Assistant Professor in the Chemical and Biosensors group, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio. Volume 2, Number 10, 2007 / Microbe Y 489 containing materials serve disparate functions and some are remarkably multifunctional. For example, the carbohydrate-based molecule, hyaluronan, plays important roles in ovulation and fertilization, morphogenesis, tissue remodeling, atherosclerosis, restenosis, and other cellular processes. Some streptococcal strains also contain hyaluronan in their capsular material; its nonantigenic quality enables these microbes to avoid the host immune system. FIGURE 1 4 5 1 2 3 6 4 O HO HO OH OH OH 6 5 1 HO HO OH 2 1 2 3 NH O Glucose (Glc) O 5 HO OH 3 OH OH 6 4 O OH OH Galactose (Gal) N-acetyl glucosamine (GlcNAc) OH 4 OH 6 4 O 5 1 HO 2 3 OH O 5 HO HO 2 6 OH O O 5 HO HO OH 3 NH O 4 1 2 3 OH 1 Xylose (Xyl) OH Glycoproteins and Glycolipids OH Glucuronic acid (GlcA) N-acetyl galactosamine (GalNAc) HO OH 4 6 5 HO HO OH O 5 6 1 2 3 Mannose (Man) O 4 OH OH HO OH 3 1 OH 2 Fucose (Fuc) HO 9 8 1 COOH OH 7 6 O AcHN 4 5 HO 2 OH 3 N-acetylneuraminic acid or sialic acid (NeuAc) The nine common sugar “letters” of glycomics. The color symbol for each sugar is pictured next to the sugar name. acids can be combined to form 6.4 ⫻ 107 distinct hexapeptides, six monosaccharides theoretically yield 1.44 ⫻ 1015 distinct hexasaccharides. Positional isomerism, conformational changes, and hydrogen bonding can further alter oligosaccharide structures, and each variant has distinctive physicochemical and cellsignaling properties. While nucleic acids and proteins can be unambiguously represented through the use of oneletter code words, there is no comparably simple way to represent combinations of complex sugars. To deal with the greater complexity of carbohydrates, Ajit Varki of the University of California, San Diego, devised a way to represent glycan structures (see www.functionalglycomics .org/glycomics/molecule/jsp/carbohydrate/carb MoleculeHome.jsp). Each sugar type with the same mass uses the same symbol. Thus, for example, hexoses are circles; glucose is a blue circle, galactose a yellow circle, and mannose a green circle. In nature, some carbohydrate- 490 Y Microbe / Volume 2, Number 10, 2007 In many instances, specific oligosaccharides are attached to particular proteins, making them glycoproteins and adding further overall complexity. Carbohydrates typically are linked to proteins in one of two ways. If a protein is O-glycosylated, the hydroxyl of a serine, threonine, or hydroxyproline amino acid residue becomes linked to the anomeric carbon (carbon number 1 of the carbohydrate residues in Fig. 1) of N-acetylgalactosamine. If N-glycosylated, the sugar, typically N-acetylglucosamine, becomes directly linked to the amide nitrogen of an asparagine residue. These two types of glycosylation differ in several other ways. O-linked sugars typically are added one at a time to a protein, and generally contain only a few residues, typically no more than four. When a protein is N-glycosylated, however, oligosaccharide structures already containing as many as 14 residues are assembled separately before being linked to aspargine residues on the recipient protein. Once attached, the sugar unit can be further modified by enzymes that either add or delete sugar residues. There is more diversity in O-glycosylation than N-glycosylation because of the different biochemical pathways that lead to these products. The current consensus is that sugars promote proper folding of proteins by stabilizing particular conformations and, in some cases, protecting the protein portion of the glycoprotein against degradative enzymes. The semiflexible nature of the sugars in a glycoprotein allows for many more conformations than would their Weiss Studies both “Northern,” or Respiratory, and “Southern,” or Diarrheal Pathogens “I find it hard to accept a future where death can lurk in your spinach,” says microbiologist Alison Ann Weiss of the University of Cincinnati, who describes herself as an “avid gardener.” Doubtless her hands-on interest in food safety was a factor in her adding Shiga toxin to the set of bacterial toxins that she studies. One of Weiss’ current projects focuses on in vivo signals that promote Shiga toxin expression and its release from Escherichia coli O157:H7, as well as “why one antigenic form, Stx2, is 100 to 1,000 times more potent than Stx1,” she says. “What is especially interesting is that Stx1 is more toxic to cells in culture than Stx2, just the opposite of what is seen in intact animals.” Weiss, 54, was born in Milwaukee, the second of six children. Although mainly a “city kid,” early in life she learned firsthand about farming from her grandfather, who owned a dairy farm. “Sometimes we would graze a steer on his land, and once we butchered it in our basement,” she recalls. Her grandfather’s farm was at the top of a hill, and her family had a garden at the bottom that was “fertilized by the manure that gently decomposed on the way down. “Times have changed,” she points out. “Unlike people, cattle are not susceptible to Shiga toxin and can carry E. coli O157:H7 asymptomatically. Crops exposed to fecal runoff are a major source for human infection.” Weiss grew up during the Vietnam War era “when young people challenged authority and con- ventional wisdom at every level.” So it was in her household. “My father and I loved each other, but we also loved to argue, a trait he also shared with his father,” she says. “We would have at it over ‘conventional wisdom’ every night at the dinner table. Later, I learned our arguments gave my siblings indigestion.” In those days, she continues, “Women were not encouraged to pursue careers in science. When I told my mother I didn’t want to go to college, she was pragmatic and told me to take typing courses, which she knew would provide exactly the motivation I needed.” Weiss received her A.B. in 1975 from Washington University in St. Louis and her M.S. in 1981 from the University of Washington in Seattle. She received her Ph.D. in 1983 from Stanford University, where her thesis advisor was Stanley Falkow. “Stanley has the extraordinary ability to inspire everyone to perform beyond expectations,” she says. Falkow’s wife, Stanford University microbiologist Lucy Tompkins, “once told me she attributed the massive entry of women into science to the Vietnam War,” Weiss says. “Drafteligible men could get a deferment for undergraduate education, but not a deferment to pursue graduate studies. Suddenly, women comprised the bulk of the pool of applicants to postgraduate programs, likely due to challenges to the idea that women shouldn’t pursue higher education—and a reduction in the pool of male applicants.” Weiss wryly refers to bacterial pathogens that cause diarrhea as “‘Southern hemisphere’ agents . . . that hit you ‘below the belt,’” and to pathogens that cause respiratory diseases as “‘Northern hemisphere,’ agents.” In Falkow’s lab, “mostly everyone was working with a below-the-belt agent,” she says. “Whether it was psychosomatic or not, everyone seemed to get a bit ‘loose’ immediately after they started working in Stanley’s lab. “I bucked the trend and worked with a Northern hemisphere agent, the respiratory pathogen Bordetella pertussis,” she says. This pathogen, which causes pertussis, or whooping cough, makes a toxin that is “essential for virulence.” For example, it “disrupts cellular communication mediated by GTP-binding proteins, and has a profound influence on the ability to mount an effective immune response,” she points out. “Currently we are looking at how pertussis toxin alters T cells. While I haven’t stopped working with B. pertussis, I feel like my studies with E. coli O157:H7 are returning me to my ‘Southern hemisphere’ roots.” Weiss and her husband, an immunologist, have two teenage children, a 17-year-old son and a 14-year-old daughter. “My son has been doing graphic arts for me; he generated schematics for this piece, and he made the logo for the Midwest Microbial Pathogenesis Conference,” she says. Marlene Cimons Marlene Cimons is a freelance writer in Bethesda, Md. Volume 2, Number 10, 2007 / Microbe Y 491 Some Pathogens Key in on Complex Carbohydrates on Host Cells FIGURE 2 OH OH O OH OH HO OH O O HO HO OH HO HO OH O HO HO HO HO HO O HO OH OH OH OH OH HO OH HO OH HO OH HO OH OH OH HO O OH OH HO OH OH OH OH OH OH OH HO OH OH OH O O HO O OH OH O OH OH OH O O OH OH O HO OH O HO HO O HO O O OH O O HO O OH OH O OH OH O O O OH HO OH O OH HO OH HO OH OH HO O O O OH OH O OH O OH HO HO O O O OH OH OH OH OH OH OH O HO OH OH Eleven possible disaccharide combinations using one monosaccharide. purely protein amino-acid counterparts. One of the biggest challenges in glycobiology is the lack of analytic means for delineating the effects of individual molecular components on downstream signaling mechanisms. Glycolipids bring in another set of complexities. One or more of the hydrophilic sugars (Fig. 1) can be directly attached to hydrophobic lipids embedded in the outer leaflet of lipid bilayers in mammalian membranes. Glycolipids can form clusters within such membranes, called lipid rafts. Such rafts consist of glycosphingolipid microdomains that are enriched in cholesterol. These domains facilitate protein-protein and lipid-protein interactions at cell surfaces and play a critical role in communications between cells. An important class of glycolipids includes the gangliosides, which are composed of a glycosphingolipid (oligosaccharide and ceramide) containing one or more sialic acids residues on the sugar chain. Gangliosides are abundantly present on neurons, making them a target for toxins. 492 Y Microbe / Volume 2, Number 10, 2007 OH Not only eukaryotes, but also bacteria make both O-linked and N-linked glycoproteins. For example, Campylobacter jejuni and Campylobacter coli coat their flagella with acetamidinosubstituted analogs of N-acetyl neuraminic acid, and this coating is required for cell motility. Even though bacterial pathogens apparently do not use sugars to communicate among themselves, microbial cells and their toxins impinge on the carbohydrates that decorate mammalian cell surfaces—for example, gaining entry into mammalian cells by binding to different sugar-containing receptors. Researchers are beginning to identify the broad specificities of some pathogen-carbohydrate interactions and toxin-carbohydrate interactions (see table). Prominent examples include influenza virus, and bacterial toxins such as botulinum toxin, Shiga toxin, and pertussis toxin. Each example illustrates different facets of the biology of sugars on mammalian host cells and the roles that they can play in infectious diseases. Carbohydrates Affect Tissue Tropism, Virulence, Transmissibility of Influenza Virus Understanding the crosstalk between host carbohydrates and pathogens such as influenza virus could prove especially valuable from a public health standpoint. With highly pathogenic strains of avian influenza now circulating, one major concern is whether strains will emerge that can transmit freely and efficiently among humans. This concern is further amplified because some strains of avian influenza are resistant to available antiviral drugs. Influenza viruses, including the highly pathogenic avian H5N1 species, carry two glycoproteins on their surfaces— hemagglutinin (HA) and neuraminidase (NA) that bind to N-acetyl neuraminic acid (sialic acid) residues on host cells. These interactions with sialic acid are es- sential for influenza viruses to enter FIGURE 3 mammalian cells. Indeed, sialic aciddeficient cells are impervious to this virus. Influence of viral tropism on transmission and virulence of influenza Each influenza viral particle possesses approximately 500 copies of HA HO COOH OH 1 HO trimers and 100 copies of NA tetramers 7 6 8 9 2 on its surface. The HA trimers mediate O AcHN 4 5 cell recognition and entry. Viral host 3 OH O HO range strongly depends on the ability of 6 4 O 5 HA to recognize specific structural fea1 HO tures of sialic acid derivatives and also 2 3 OH the density and presentation of these sugar residues on host cells (see table). For example, influenza C virus infects animal cells that display 9-O-acetyl sialic acids on their glycoproteins and glycolipids, while avian influenza strains preferentially target sialic acids linked to the 3 position, and influenza strains that infect humans preferentially target sialic acids linked to the 6 positions of galactose or lactose. This preferential recognition of specific forms of sialic acid has significant OH OH OH 6 implications for viral transmission and HOOC OH 4 O 1 HO 5 7 6 pathogenesis. For instance, the upper 8 1 9 O 2 O 2 AcHN respiratory tract of humans is rich in 3 4 OH 5 3 ␣-2,6 sialic acid-linked glycans (Fig. 3, HO denoted in blue), whereas cells in the lower respiratory tract are rich in termiInfluence of viral tropism on transmission and virulence of influenza. nal ␣-2,3 linkages (Fig. 3, in green). When influenza infects the upper respiratory tract, high concentrations of viral partiwith birds, and human to human transmission is cles in respiratory secretions enhance human to rare. However, mortality is extremely high. The human transmission via coughing and sneezing. deadly 1918 influenza pandemic is believed to In contrast, viral replication in the lower respihave been due to an avian virus that mutated ratory tract hinders transmission. This greater from an ␣-2,3 sialic acid binding preference to efficiency of transmission helps to explain why ␣-2,6, while retaining its high pathogenic poteninfluenza strains that preferentially bind ␣-2,6 tial, making this virus both highly contagious sialic acids of the upper respiratory tract are and highly virulent. Whether the currently cirpredominant among humans. culating strains of avian influenza will mutate to In contrast, the preference of avian influenza recognize ␣-2,3 and ␣-2,6 linkages to become for ␣-2,3 sialic acids suits the H5N1 virus for highly transmissible remains an open concern. infecting the intestinal tract of birds, but makes Understanding these molecular details involvthem not so effective with humans. However, ing sialic acids is crucial for the development of when H5N1 viruses reach the lower respiratory antiviral drugs. Viral neuraminidase cleaves tract, they tend to cause severe, life-threatening sialic acid residues from infected cells, releasing infections, particularly because gas exchange viral progeny that infect new cells. Influenza becomes compromised. drugs, such as Relenza威 and Tamiflu威, are anaThis ␣-2,3 sialic acid binding preference helps logs that bind and inactivate neuraminidase. to explain what happens when humans become However, some avian influenza strains now are infected with avian influenza. Thus, infection resistant to Tamiflu威, making it important to rates are low, usually requiring close contact pursue alternative means for inhibiting these OH OH Volume 2, Number 10, 2007 / Microbe Y 493 Saccharide specificities for select bacterial and viral pathogens and toxins Bacterial pathogen, virus, or toxin Escherichia coli Type 1 pili P S CFA/1 K99 Shigella dysenteriae Neisseria gonorrheae Bordetella pertussis Viruses Foot and mouth virus Avian influenza virus Human influenza Influenza C Bacterial toxins Botulinum neurotoxins Shiga toxin 1 and 2 Broad saccharide specificity Man(␣1,3)Man(␣1,6)Man Gal (␣1,4)gal NeuAc(␣2,3)Gal(1,3)GalNAc NeuAc(␣2,8)NeuAc NeuGc(␣2,3) Gal(1,4)Glc GM1, asialo GM1 Gal(1,4)GalNAc Gal(1,4)Glc-ceramide Heparin sulfate 2,3 linked sialic acids 2,6 linked sialic acids 9-O-acetyl sialic acids Pertussis toxin Cholera toxin and LT-I E. coli enterotoxin, LT-IIa gangliosides GD1a, GD1b, GT1a Gal (␣1,4) Gal(1,4)Glc-ceramide (Gb3) and analogues Sialic acid, Gal(1,4)Glc-ceramide, gangliosides Ganglioside GM1 Gangliosides GD1b, GD1a, GM1 viruses, including by blocking their binding to sialic acid residues, as potential new drugs. Bacterial Toxins Such as Botulinum Toxin Bind Carbohydrate Receptors Although many bacterial toxins enter cells via protein receptors, other toxins, including several on the biothreat list such as the botulinum neurotoxins and Shiga toxin, depend partly or fully on carbohydrate receptors (see table). Even though protein receptors confer exquisite selectivity, cells generally express few molecules of a particular protein, whereas carbohydrate residues typically are abundant. Moreover, the same glycosylation pattern may decorate many different proteins on a single cell type, while also decorating each protein at several sites. Thus, the extremely high potency of some biothreat toxins may reflect their binding to high-density carbohydrate receptors. The family of seven botulinum neurotoxins (BoNT) produced by Clostridium botulinum, a gram-positive, spore-forming bacterium, are among the most toxic substances found in na- 494 Y Microbe / Volume 2, Number 10, 2007 ture. The A subunit of BoNT is a protease that cleaves components of the secretory machinery in mammalian nerve cells. Cleavage then prevents acetylcholine-containing vesicles from fusing with the terminal membranes of motor neurons, leading to flaccid muscle paralysis and, ultimately, death due to respiratory failure. One gram of aerosolized BoNT serotype A is enough to cause more than 1 million deaths. A single lethal dose of about 1010 molecules, or 10 ng, happens to be the same as the number of neurons in a human. BoNT gains entry into cells via two distinct types of receptors— ganglioside carbohydrate receptors, such as GT1b or GD1a, and a protein receptor, synaptotagamin. The ganglioside coreceptors are highly expressed at nerve terminals, and aid in defining such cells as targets for this toxin. The gangliosides provide a high-density, low-affinity receptor— enabling high concentrates of BoNT to build on nerve cell surfaces, increasing the probability that the toxins will bind synaptotagamin, its low-density, highaffinity receptor. Synaptotagamin, which is part of the neurotransmitter secretory machinery, further defines the cell as a BoNT target. Synaptotagamin is incorporated into secretory vesicles and is expressed transiently on cell surfaces immediately after secretory vesicles fuse at presynaptic nerve terminals and before endocytosis recycles membrane components. Molecules of BoNT bind to gangliosides at the cell surface, then bind to molecules of synaptotagamin once they become surface exposed. As endocytosis internalizes synaptotagamin, any BoNT that is bound to those molecules also is internalized. The seven serotypes (A-G) of botulinum neurotoxin differ in toxicity. Most (52%) human botulism cases result from BoNT/B, followed by BoNT/A at 34%, and BoNT/E at 12%. The other serotypes do not appear to be toxic for humans. It appears that affinity for the ganglioside receptor dictates susceptibility, and minor structural differences can determine the difference between life and death following exposure to these toxins. For example, GT1b and GD1b, gangliosides that differentially bind BoNT/B, differ from one another by a single sialic acid moiety. The differences are even more subtle between GD1a, which binds BoNT/B with lower efficiency, and GD1b. GD1b and GD1a differ only in position of one sialic acid present in both gangliosides. More detailed information about the effects of structure on activity might help medicinal chemists develop better inhibitors and diagnostics for this deadly toxin. FIGURE 4 A Site 1 Site 2 B Site 3 Multivalency of Shiga Toxin Ensures Affinity for Receptor Shiga toxin is a major virulence factor of several types of gram-negative bacteC D ria, including Escherichia coli O157: SA1 S1 H7. Shiga toxin belongs to the AB5 family of toxins (Fig. 4). The A subunit S2 TYR is an N-glycosidase, which cleaves sinSA2 SA3 gle adenine residues from 28S riboS3 S2 S3 somal RNA molecules, rendering the LAC GAN ribosome incapable of synthesizing proSA2 LAC SA3 GAN teins. Curiously, this same activity is Structures of two diverse AB5 toxins, Shiga toxin and pertussis toxin. (A,B) Structure of associated with the select agent from Shiga toxin 1. Stx1A subunit, orange; and Stx2 B-pentamer, blue. Amino acid regions castor bean plants, ricin, which preferassociated with binding to the mammalian receptor, Gb3, shown in shades of red; Site entially binds to both galactose and N1 (red), Site 2 (magenta), and Site 3 (salmon). A, Bottom view of the B-pentamer; B, Side view. (C,D) Structure of Pertussis Toxin. S1, orange; S2, blue; S3, green, S4, white; and acetylgalactose. However, the B, or S5, gray. Amino acid regions associated with binding to mammalian cells are highlighted binding subunit of Shiga toxin, binds to in red; SA1-SA3, sialic acid binding sites 1–3; LAC, lactosylceramide binding site; GAN, terminal sugars on globotriaosylceramganglioside binding site; and TYR, tyrosine-associated binding site. C, Bottom view of the B-pentamer; D, Side view. ide (Gb3), delivering the A catalytic subunit to its cytoplasmic target. The B subunit of Shiga toxin consists geting enables B subunits to activate Src family of five identical polypeptides, each with multiple kinases within minutes of binding to cells, probinding sites for Gb3 (Fig. 4A). While the bindmoting rapid toxin entry. Furthermore, this siging affinity for individual molecules of Gb3 is naling may determine the fate of the toxin, very low, the affinity of the toxin for its receptor which is not toxic to all cells. For example, increases more than 1,000-fold when that recepalthough Shiga toxin enters the cytoplasm and tor is embedded in a membrane in the form of a damages ribosomes in susceptible cells, toxinlipid raft on mammalian cell surfaces. The sugar resistant bovine cells shuttle Shiga toxin to lysoresidues in such membranes are tightly clustered somes to be degraded, allowing cattle to carry E. and thus can simultaneously engage multiple coli O157:H7 without developing symptoms. binding sites on the toxin. The low-affinity interactions are synergistic, amounting to binding forces that rival antibody affinities in strength. Furthermore, variations in the structure of the glycolipid can lead to selectivity between serotypes, and small structural changes in the host receptor can lead to high specificity between closely related Shiga toxins. In targeting glycolipids in lipid rafts, Shiga toxin gains another advantage. Because lipid rafts play a critical role in cell-signaling processes and Gb3 associates with lipid rafts, bound toxin molecules do not depend merely on receptor turnover to carry them into cells. This tar- Pertussis Toxin: Lectin Activity and a Second Layer of Toxicity Pertussis toxin is the major virulence factor of Bordetella pertussis, which infects humans and causes whooping cough. Whooping cough has the dubious distinction of being the only vaccine-preventable disease that is increasing in incidence in the United States. Its toxin is the most complex bacterial toxin described to date. Like Shiga toxin, pertussis toxin is an AB5 toxin, with an enzymatically active A subunit and a 5-member B (binding) subunit (Fig. 4). Volume 2, Number 10, 2007 / Microbe Y 495 The A subunit catalyzes transfers of the ADPribosyl group of nicotine adenine dinucleotide (NAD) to regulatory GTP-binding proteins of mammalian cells, ultimately disrupting G-protein-mediated intracellular signaling. Unlike Shiga toxin, the B subunits of pertussis toxin are not identical. Instead the pertussis toxin B-pentamer (Ptx-B) is composed of four different subunits termed S2, S3, S4, and S5 in a 1:1:2:1 ratio (Fig. 4C-D). The B subunit not only transports A subunits into the cytoplasm of target cells, but also possesses a toxicity of its own. Thus, Ptx-B binds to and promotes signaling through diverse receptors on different classes of mammalian cells, including glycoprotein 1B expressed by platelets, Mac-1 (CD11b/CD18) expressed on monoblasts, CD14 expressed by macrophages, TLR-4 expressed by dendritic cells, and the T-cell receptor expressed by T cells. This ability to affect so many different receptors on such a diverse array of cells suggests that these cells and receptors share some property. In fact, all these receptors are glycosylated, and all of them are sensitive to activation by clustering. Specifically, Ptx-B activates these receptors by clustering them, and Ptx-B clusters them by binding to their sugars. This activity of Ptx-B is analogous to that of plant lectins, which contain two or more carbohydrate binding domains and activate cells by clustering receptors expressing appropriate binding sugars. For instance, concanavalin A (ConA) and Phaseolus vulgaris lectin, PHA, activate T-cells of the immune system. While lectins typically possess identical binding sites, PTX-B has five binding sites that recognize different sugars, including three sialic acid binding sites, a lactosylceramide-binding site, and a ganglioside-binding site. Ptx-B targets so many different cell types and receptors because it binds such diverse sugars. While Ptx-B is a mammalian cell toxin, it also has limited antiviral activity. It can prevent replication of HIV in peripheral blood leukocytes in vitro, in activated primary T-cells, monocyte- derived macrophages, and in ex vivo cultures of human lymphoid tissues. In vivo, Ptx-B has activity against simian immunodeficiency virus (SIV) in chronically infected macaques, and against HIV in SCID mice reconstituted with human peripheral blood leukocytes. Thus, this lectin-like activity of pertussis toxin could lead to development of novel therapies for treating HIV. Concluding Remarks Several novel methods have been developed to probe the interactions of cell-surface carbohydrates with their receptors. Metabolic engineering allows for the creation and study of cells deficient in glycosylation or directed to display unnatural sugar patterns on the cell surface. Glycomicroarrays have been generated, but are limited by the lack of pure preparations of complex sugars. Recent advances in synthetic tools such as enzymatic, programmable one-pot and solid-phase synthesis have alleviated the bottleneck for some compounds. However, there are still challenges to be met. Finally, the study of glycolipids and glycoproteins targeted by microbial pathogens provides yet another research tool to investigate the basic biology of cellsurface carbohydrates. The Consortium for Functional Glycomics (CFG—http://www.functionalglycomics.org/ static/index.shtml) is a large research initiative composed of more than 300 Participating Investigators and seven scientific core laboratories. Funded by the National Institute of General Medical Sciences in 2001 to elucidate the roles of carbohydrate-protein interactions in cell communication at the cell surface, the CFG has made enormous progress towards its goals. The scientific cores produce a variety of resources and services for investigators performing experiments that address the goals of the CFG. Data produced by the scientific cores are accessible from their website. Specialty databases for glycan-binding proteins, glycan structures, and glycosyltransferases are also available. ACKNOWLEDGMENTS This work was supported by NIH/NIAID R01 AI064893 and AI23695 (AAW), and the Center for Biosensors, Department of Chemistry, University of Cincinnati (SSI), the University of Cincinnati Research Council (AAW and SSI). The authors gratefully acknowledge the artistic contributions of Peter Monaco, Scott Millen, Michael Flagler, and David Friedmann. 496 Y Microbe / Volume 2, Number 10, 2007 SUGGESTED READING Katagiri, Y. U., T. Mori, H. Nakajima, C. Katagiri, T. Taguchi, T. Takeda, N. Kiyokawa, and J. Fujimoto. 1999. Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergentinsoluble microdomains. J. Biol. Chem. 274:35278 –35282. Manes, S., G. del Real, and A. C. Martinez. 2003. Pathogens: raft hijackers. Nature Rev. Immunol. 3:557–568. Paton, J. C., and A. W. Paton. 2006. Shiga toxin “goes retro” in human primary kidney cells. Kidney Int. 70:2049 –2051. Paulson, J. C., O. Blixt, and B. E. Collins. 2006. Sweet spots in functional glycomics. Nature Chem. Biol. 2:238 –248. Rizzi, C., M. P. Crippa, R. E. Jeeninga, B. Berkhout, F. Blasi, G. Poli, and M. Alfano. 2006. Pertussis toxin B-oligomer suppresses IL-6 induced HIV-1 and chemokine expression in chronically infected U1 cells via inhibition of activator protein 1. J. Immunol. 176:999 –1006. Rossetto, O., and C. Montecucco. 2007. Peculiar binding of botulinum neurotoxins. A.C.S. Chem. Biol. 2:96 –98. Schneider, O. D., A. A. Weiss, and W. E. Miller. 2007. Pertussis toxin utilizes proximal components of the T-cell receptor complex to initiate signal transduction events in T cells. Infect. Immun. 75:760 –765. Seeberger, P. H., and D. B. Werz. 2007. Synthesis and medical applications of oligosaccharides. Nature 446:1046 –1051. Shinya, K., M. Ebina, S. Yamada, M. Ono, N. Kasai, and Y. Kawaoka. 2006. Avian flu: influenza virus receptors in the human airway. Nature 440:435– 436. Varki, A., R. Cummings, J. Esko, H. Freeze, G. Hart, and J. Marth (ed.). 2002. Essentials of glycobiology, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Q: When does 4 x $17 = $48? Postdoctoral Membership Recognizing the unique status of scientists during the postdoctoral training phase of their careers, ASM now offers Postdoctoral Membership. Now postdoctoral trainees can receive all the privileges* of Full Membership for $37 a year, for 4 consecutive years – two years longer than the Transitional Member rate offers. The 2007 membership fee will be $37 ($35 for those who join or renew online). Any microbiologist who has earned a doctorate within the past 12 months is eligible. The Postdoctoral rate may not be combined with the Transitional rate to allow 6 years of reduced rate membership. Contact Membership Services at service@asmusa. org for more information, or join online at estore.asm.org today! 1752 N Street, NW Washington, DC 20036 http://www.asm.org [email protected] *except the right to vote or hold office A: When Professors, Mentors and University Department Chairs purchase 4 or more student memberships for their undergraduate and graduate scholars! Buy 4 Student Memberships and receive a discount of $5 per membership! To take advantage of this offer or to get more information contact Membership Services at [email protected]. You can also go to the Membership Menu to get started http://www.asm.org/Membership/index.asp?bid+363. 1752 N Street, NW Washington, DC 20036 http://www.asm.org [email protected] Volume 2, Number 10, 2007 / Microbe Y 497