Streptococcus pyogenes is an important human pathogen that selectively interacts with proteins involved in the humoral defense system, such as immunoglobulins and complement factors. In this report we show that S.pyogenes has the ability to hydrolyze the chitobiose core of the asparagine-linked glycan on immuno globulin G (IgG) when bacteria are grown in the presence of human plasma. This activity is associated with the secretion of a novel 108 kDa protein denoted EndoS. EndoS has endoglycosidase activity on purified soluble IgG as well as IgG bound to the bacterial surface. EndoS is required for the activity on IgG, as an isogenic EndoS mutant could not hydrolyze the glycan on IgG. In addition, we show that the secreted streptococcal cysteine proteinase SpeB cleaves IgG in the hinge region in a papain-like manner. This is the first example of an endoglycosidase produced by a bacterial pathogen that selectively hydrolyzes human IgG, and reveals a novel mechanism which may contribute to S.pyogenes pathogenesis.

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EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG

Enzyme immunoassays are attaining increased usage for the direct detection of microbial antigens in body fluids. Advantages of enzyme immunoassays include a high degree of sensitivity resulting from the inherent magnification of the enzyme-substrate reaction and the use of objective end points without the need for radioactivity. Enzyme immunoassays have been developed for the reliable detection of several important microbial antigens in body fluids, including antigens of rotavirus, hepatitis B virus, and Haemophilus influenzae type b. However, standard enzyme immunoassay techniques are not sufficiently sensitive for the measurement of some antigens from other viruses, bacteria, and parasites in concentrations that commonly occur in body fluids during the course of infectious diseases. This review examines some of the limitations of currently available enzyme immunoassay technology and discusses approaches to increasing the sensitivity and specificity of enzyme immunoassay systems. Methods for improving these assay systems include the use of monoclonal antibodies, improved methods of enzyme-immunoreactant conjugation, more sensitive substrate systems, improved methods of antigen-antibody access, and the direct measurement of microbial enzymes. The use of such techniques should lead to the development of efficient enzyme immunoassay systems for the direct detection of a wide range of bacterial, viral, and parasitic infections.

Enzyme Immunoassays for the Detection of Infectious Antigens in Body Fluids: Current Limitations and Future Prospects

1 Chemistry and Analysis of Sialic Acid.- I. Historical Background.- II. Natural Occurrence of Sialic Acids.- III. Isolation and Purification.- IV. Chemistry of Sialic Acids.- A. Basic Structures.- B. Stereochemistry.- C. Chemical Reactions and Derivatives.- V. Synthesis.- VI. Quantification of Sialic Acids.- A. Colorimetric and Fluorometric Assays.- B. Enzymatic Assay.- C. Gas-Liquid Chromatography.- VII. References.- 2 The Natural Occurrence of Sialic Acids.- I. Introduction.- II. The Natural Occurrence of Sialic Acids.- A. Viruses.- B. Bacteria.- C. Plants.- D. Invertebrates.- E. Primitive Chordates.- F. Vertebrates.- III. Evolution of Sialic Acids.- IV. References.- 3 The Distribution of Sialic Acids Within the Eukaryotic Cell.- I. Introduction.- II. Extracellular Sialic Acids.- III. Distribution within the Cell.- A. The Plasma Membrane.- B. Endoplasmic Reticulum.- C. Mitochondria.- D. Nuclei.- E. Other Fractions.- IV. Conclusions.- V. References.- 4 Anabolic Reactions Involving Sialic Acids.- I. Introduction: Perspective and Directions.- II. Biosynthesis of the Sialic Acids.- A. Glucose to Sialic Acid.- B. Activation.- C. Regulatory Problems.- D. Other Derivatizations.- III. Biosynthesis of Polymers, Glycoproteins, Mucins, and Glycolipids Containing Sialic Acid.- A. Colominic Acid Synthesis.- B. CMP-Sialic Acid: Lactose (?-Galactosyl) Sialyltransferase.- C. CMP-Sialic Acid: Glycoprotein (?-Galactosyl) Sialyltransferases.- D. CMP-Sialic Acid: Mucin (?-N-Acetylgalactosaminyl) Sialyltransferase.- E. CMP-Sialic Acid: Ganglioside (Glycolipid) Sialyltransferases.- IV. Thoughts on Physiological Function of Sialic Acids.- V. References.- 5 Catabolism of Sialyl Compounds in Nature.- I. Introduction.- II. Pathways of Degradation.- A. Degradation of Gangliosides.- B. Degradation of Glycoproteins.- III. Cellular Mechanism of Degradation.- A. Lysosomes.- B. Uptake and Disposition of Substrates.- IV. Functional Implications.- V. Concluding Remarks.- VI. References.- 6 Disorders of Ganglioside Catabolism.- I. Introduction-The Catabolism of Gangliosides.- II. Tay-Sachs Disease (Type I GM2-Gangliosidosis).- A. Clinical Aspects.- B. Pathology.- C. Chemistry of the Storage Material.- D. Nature of the Metabolic Defect.- E. Enzymology of Type I GM2-Gangliosidosis.- F. Prenatal Diagnosis and Treatment.- III. Type II GM2-Gangliosidosis.- A. Clinical and Pathological Aspects.- B. Chemistry of the Storage Material.- C. Metabolic Defect-Diagnosis and Treatment.- IV. Other Variant Forms.- A. Type III GM2-Gangliosidosis.- B. Hexosaminidase-A-Deficient Adults.- V. Generalized Gangliosidosis (GM1-Gangliosidosis).- A. Clinical Aspects.- B. Pathology.- C. Chemistry of the Stored Material.- D. Metabolic Defect.- VI. Potentially Related Disorders.- A. Hematoside (GM3)-Gangliosidosis.- B. Animal Model Gangliosidoses.- C. In Vitro Model Studies.- VII. References.- 7 The Biological Role of Sialic Acid at the Surface of the Cell.- I. Introduction.- II. Occurrence, Forms, and Amounts of Sialic Acid Residues at the Surface of the Cell.- III. The Masking of Cell-Surface Antigens by Sialic Acid.- IV. Sialic Acid as a Receptor at Cell Surfaces.- A. Receptor for Lectins.- B. Receptor for Viruses.- C. Receptor for Mycoplasma.- D. Receptor for Hormones.- E. Receptor for Antibodies.- F. Receptor for Circulating Glycoproteins.- G. Receptor for Tetanus Toxin.- V. Sialic Acid in Normal and Malignant or Transformed Cells.- VI. Role of Sialic Acid in Cell-to-Cell Interaction.- A. Cellular Adhesion.- B. Intercellular Aggregation.- C. Agglutination.- VII. Physiological Role of Sialic Acid Residues.- A. Transport of Ions, Amino Acids, and Proteins.- B. Phagocytosis.- C. Anaphylactic Shock, Hypercapnia, and Brain Excitability.- D. Lymphocyte Stimulation.- E. Sperm Capacitation.- VIII. Conclusion.- IX. References.- 8 The Altered Metabolism of Sialic-Acid-Containing Compounds in Tumorigenic-Virus-Transformed Cells.- I. Introduction.- II. Experimental Procedures.- A. Cells and Cell Culture.- B. Isolation, Identification, and Quantification of Gangliosides.- C. Assay of Enzymes Involved in Glycolipid Metabolism.- III. Ganglioside Metabolism in Cultured Mouse Cell Lines.- A. Distribution of Gangliosides in Normal and Virally Transformed Cells.- B. Enzymatic Studies.- C. Effect of Growth and Culture Conditions on Ganglioside Metabolism.- D. Sialic-Acid-Containing Glycolipids in Transformed Cells Obtained from Other Species.- VI. Sialic Acid and Glycoproteins in Transformed Cells.- A. Sialic Acid and Sialyltransferase Activity in Transformed Cells.- B. Membrane Glycoproteins.- C. Glycopeptides of Transformed Cells.- D. Role of Sialic Acid and Sialyltransferase.- E. Comments.- V. Relationship between Viral Transformation and Altered Ganglioside Metabolism.- A. Productive Infection of Mouse Cells.- B. Ganglioside Metabolism in Flat Revertant Cell Lines.- C. Specificity of the Altered Ganglioside Metabolism.- D. Generality of the Phenomenon.- E. Transformation of Mouse Cells by RNA Tumor Viruses and Other Agents.- VI. Discussion.- A. Molecular Basis of Altered Ganglioside Metabolism.- B. Significance.- VII. Concluding Remarks.- VIII. References.- 9 Circulating Sialyl Compounds.- I. Introduction.- II. Normal Plasma Constituents.- A. Circulating Sialoenzymes.- B. Serum Sialoglobulins.- C. Sialoglycoprotein Hormones.- III. Circulating Sialoglycoproteins in Abnormal Physiological States.- A. Diabetes.- B. Inflammatory Reactions.- C. Infectious Psychoses.- D. The Effect of Steroid Hormones.- E. Liver Disease.- F. Virus Inhibition of Hemagglutination.- G. Cancer.- H. Diet.- IV. Role of Sialic Acid in Circulating Sialoglycocompounds.- V. References.- 10 Sialidases.- I. Background and Nomenclature.- II. Bacterial Sialidases.- A. Occurrence of Microbial Sialidases.- B. Organismic Characterization and Induction of Bacterial Sialidases.- C. Purification of Bacterial Sialidases.- D. Size and Properties of Bacterial Sialidases.- E. Mode of Action of Bacterial Sialidases.- F. Biological Roles for Bacterial Sialidases.- III. Viral Sialidases.- A. Morphology and Genetics of Viral Sialidases.- B. Purification of Viral Sialidases.- C. Size of Viral Sialidases.- D. Properties of Viral Sialidases.- E. Possible Biological Roles for Viral Sialidases.- IV. Experimental Use of Microbial Sialidases.- V. Mammalian Sialidases.- A. Organ Distribution of Mammalian Sialidases.- B. Subcellular Distribution of Mammalian Sialidases.- C. Purification of Mammalian Sialidases.- D. Assay of Mammalian Sialidases.- E. Physical Properties of Mammalian Sialidases.- F. Developmental Studies of Mammalian Sialidases.- G. Possible Biological Roles of Mammalian Sialidases.- H. Sialidase Activity in Cells in Tissue Culture.- VI. References.

Biological roles of sialic acid

1. Tara M. Randis, MD, MS*
2. Jacqueline A. Baker, MD†
3. Adam J. Ratner, MD, MPH*,‡


 

1. Departments of *Pediatrics and

2. ‡Microbiology, New York University School of Medicine, New York, NY

3. †Department of Pediatrics, Columbia University College of Physicians & Surgeons, New York, NY

* Abbreviations:
 CDC: : Centers for Disease Control and Prevention
 CFR: : case fatality rate
 CSF: : cerebrospinal fluid
 EO: : early-onset
 GBS: : group B Streptococcus HIV: : human immunodeficiency virus
 IAP: : intrapartum antibiotic prophylaxis
 LO: : late-onset
 PCR: : polymerase chain reaction

Despite recommendations for universal screening and intrapartum antibiotic prophylaxis strategies for pregnant women, group B Streptococcus remains a major cause of neonatal disease.

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### Organism

Streptococcus agalactiae , or group B Streptococcus (GBS), was first recognized as a distinct entity in the 1930s by Rebecca Lancefield, who used immunologic typing of carbohydrate antigens as a means to classify streptococci. (1) Early studies by her group and others indicated that GBS was an uncommon cause of human disease and was more frequently isolated as an etiologic agent of bovine mastitis. Sporadic case reports from that time demonstrated the potential for GBS to cause invasive infections, especially in peripartum women, although such infections were believed to be infrequent. However, over the period of the 1950s to the early 1970s, GBS became the major cause of neonatal sepsis in the United States and worldwide.

### Colonization and Pathogenesis

GBS asymptomatically colonizes the lower gastrointestinal and female genital tracts. Colonization rates …

Group B Streptococcal Infections

In the last few years an increasing number of enzymes were found to be associated with virus particles. For a long time neuraminidase was the only virus specific enzyme known to be an integral part of myxoviruses. Therefore, it is not surprising that it is the best known among the enzymes associated with animal virions.

Viral and bacterial neuraminidases.

A group of enzymes were prepared from the culture fluids of streptococci belonging to groups A, B, C, G, and L, and from a strain of Streptococcus sanguis. These streptococcal enzymes (designated St-sialidases) released a substance shown to belong to the sialic acid group from the specific substrate BSM-St, a sialomucoid prepared from bovine submaxillary gland. They were inactive on N-acetylneuramin lactose prepared from bovine colustrum and on a sialomucoid prepared from bovine submaxillary mucin, whereas these substances are susceptible to sialidases produced by group K streptococci and by Vibrio cholerae. Some of the St-sialidases were markedly activated by divalent cations, but others showed little response. The heat stability of the enzymes produced by the different strains varied. The optimal pH was between 5.5 and 6.5 with acetate buffer and was about 7 with phosphate buffer. K(m) values were determined for the St-sialidases with BSM-St as substrate.

Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

A survey has been made of the activity of a wide variety of standard strains of streptococci against bovine submaxillary mucin. Strain 6646 (group K) and strain D 168A “X” (group M) completely broke down and strain H 60R (group F) incompletely broke down bound sialic acid of bovine submaxillary mucin added to the growth medium. Among these strains, strain 6646 (group K) produced sialidase in the cell and in the culture fluid. An appropriate amount of glucose in the culture medium stimulated growth and the production of enzyme, but an excess of glucose in the culture medium caused abundant growth without production of the enzyme. The streptococcal sialidase was precipitated from the culture fluid by ammonium sulfate at 50% saturation, and further purification was achieved by diethylaminoethyl cellulose chromatography. Ca++ and Co++ stimulated the sialidase activity, and Mn++, Zn++, and ethylenediaminetetraacetate inhibited it. With acetate buffer, the optimal pH lay between 5 and 6. Sialic acid was detected in the reaction product of the streptococcal sialidase and bovine submaxillary mucin.

Streptococcal Sialidase I. Isolation and Properties of Sialidase Produced by Group K Streptococcus

The occurrence of a streptococcal sialidase (designated St-sialidase) in culture fluids of various streptococci was investigated. St-sialidase was found to occur in strains belonging to groups A, B, C, E, G, H, and L, and the unclassified strains, Streptococcus sanguis and Streptococcus uberis. St-sialidase of group A was confined predominantly to types 4 and 22. St-sialidases, extracted from the culture fluids of some selected strains, were antigenic, eliciting the formation of antibody which effectively neutralized the enzymatic activity of the enzyme. Antisera to the St-sialidases of groups A, B, C, E, G, and L, and Streptococcus sanguis were produced in rabbits. The St-sialidases of groups A, B, and E streptococci were serologically distinct and group-specific. The St-sialidases from groups C, G, and L were serologically homologous, but distinct from St-sialidases of the other groups. Antiserum to the enzyme of strain 10557 (S. sanguis) cross-reacted with the St-sialidase of strain 9927 (S. uberis).

Distribution and serological specificity of sialidase produced by various groups of streptococci.

Kinetic and immunological studies were carried out on the sialidase produced by strain 6646, group K streptococcus (K-sialidase). The Km values of K-sialidase were 0.9 mm for sialyllactose and 0.17 mm for bovine submaxillary mucin. The antibody against K-sialidase was produced in rabbits immunized with this enzyme. An assay procedure for determination of the anti-K-sialidase activity in terms of reciprocal of the serum dilution corresponding to the 50% inhibition point is described. Anti-K-sialidase activity is widely distributed in human sera, but this has not yet been found to be correlated with streptococcal diseases, and no definite relationship was proved between the anti-K-sialidase titer and the anti-streptolysin O titer through this study. Anti-K-sialidase serum had no effect on Vibrio cholerae sialidase.

Seiki Hayano

Papers

Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

Streptococcal Sialidase I. Isolation and Properties of Sialidase Produced by Group K Streptococcus

Distribution and serological specificity of sialidase produced by various groups of streptococci.

Streptococcal sialidase. II. Kinetic and immunological studies of sialidase produced by group K streptococcus.