Molecular epidemiology of group B streptococcal infections

Streptococcus agalactiae (GBS) is a causative agent of sepsis and meningitis in newborns and diseases in pregnant women and nonpregnant adults. Various approaches, including both nongenetic and genetic techniques, are currently used for the study of epidemiology of GBS infections. In the present paper the different methods of molecular epidemiology of GBS infections are reviewed, and several novel approaches are introduced. The advantages and disadvantages of molecular methods are discussed and compared with traditional serotyping technique. The possible use of the molecular approaches for identification of different genetic lineages in GBS as well as for identification and control of the epidemiologically actual clones is discussed.     

Pathogenesis of neonatal Streptococcus agalactiae infections

Streptococcus agalactiae is an important human pathogen causing severe neonatal infections. During the course of infection, S. agalactiae colonizes and invades a number of different host compartments. Bacterial molecules including the polysaccharide capsule, the hemolysin, the C5a peptidase, the C-proteins, the hyaluronate lyase and a number of unknown bacterial components determine the interaction with host tissues. This review summarizes our current knowledge about these interactions.     

Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants

Neonatal mice delivered from mothers preimmunized with heated or formalinized whole cell vaccines of type Ia, Ia/c and III/c group B streptococci were infected with each type of bacteria, and then serum antibodies of mothers and neonates who survived the experiments were measured by enzyme-linked immunosorbent assay. The relationship between the protectivity in neonate mice and the antibody titers to the type specific polysaccharide antigens and the protein c antigen of their sera were examined. In the Ia-immunized group which showed high protection against the type Ia infection, anti-Ia IgG antibody titers were low, and anti-protein c IgG antibody was not detected. Type Ia/c and III/c vaccines were highly effective against both type Ia/c and III/c infection, but less effective in type Ia infection. The protein c antigen was identified in both type strains by the double diffusion assay, and the IgG antibodies to the protein c were significantly high in sera of both maternal mice immunized with types Ia/c or III/c organisms and their newborn infants. High titers of the protein c IgG antibody retained 3 to 4 weeks after the last injection of vaccines which corresponded to the period of pregnancy and lactation. Small amounts of IgM antibody to all antigens were detected only in maternal sera. These results suggest that IgG antibodies to the protein c antigen and to the type-specific polysaccharide antigens are equally important protective factors which are transferable from preimmunized mothers to their newborn infants through placenta and/or lactation.     

Characterization of the active site of group B streptococcal hyaluronan lyase

Xin Li was inadvertently dropped from the list of authors for this article. The authors regret this error.     

Characterization of the active site of group B streptococcal hyaluronan lyase

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.     

New DNA-based PCR approaches for rapid real-time detection and prevention of group B streptococcal infections in newborns and pregnant women

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis, and maternal infection. Although the pathogenesis of GBS infection is not well understood, several virulence factors have been identified. Two prevention strategies have been proposed: chemoprophylaxis and immunoprophylaxis. Implementation of selective intrapartum chemoprophylaxis on the basis of either screening or risk assessment has led to a substantial decrease in the morbidity and mortality of GBS disease in both mothers and infants. Penicillin remains the antibiotic of choice with no reported resistant GBS so far, whereas resistance of 10-20% of GBS to erythromycin and clindamycin has been reported in North America. Chemoprophylaxis based on screening requires optimal detection methods for GBS, which involve selective broth culture of combined vaginal and anal samples. Other conventional methods are useful for rapid identification of heavily colonised women, but are unreliable for the detection of light GBS colonisation because of poor sensitivity. GBS-specific polymerase chain reaction (PCR) assays using real-time PCR coupled with fluorescence-labelling technology offer powerful tools for sensitive and specific, yet rapid (less than 1 h), detection of GBS directly from clinical specimens at the time of delivery. The application of these assays to the current prevention strategies will simplify the prevention practice and rationalise the use of antibiotics. Immunoprophylaxis relies on the development of new vaccines against GBS, and active research is being conducted in this area.     

Immunochemical analysis of the types Ia and Ib group B streptococcal polysaccharides

The types Ia and Ib group B streptococcal type-specific polysaccharides have remarkable immunologic differences despite a great deal of structural similarity. Although these two complex polysaccharides differ only by a single glycosidic linkage, they are antigenically distinct. Furthermore, terminal sialic acid residues appear to be critical to the immunodeterminant on the type Ia polysaccharide, whereas the antigenicity of the type Ib polysaccharide does not show this dependence on sialic acid. In the current investigation we defined better the immunodeterminant of these polysaccharides. With homologous rabbit antiserum, the type Ia native and core polysaccharides demonstrated partial serologic identity, whereas the type Ib native and core polysaccharides demonstrated complete serologic identity. Surprisingly, the type I degalactosylated polysaccharide, degraded structure, was capable of reacting with a population of antibodies present in type Ia antiserum similar to the complete type Ia native polysaccharide, although demonstrating a reduced level of immunodeterminant expression. Unlike the reactions of the type Ia polysaccharides with homologous rabbit antiserum, the Ib native and core polysaccharides were able to react with identical populations of antibodies in type Ib-specific antiserum. A minor population of antibodies was demonstrated in the type Ib antiserum, which was reactive with the degalactosylated polysaccharide. That a population of antibodies reactive toward the degalactosylated polysaccharide is present in both type Ia and type Ib antisera suggests that the Iabc cross-reacting determinant is due to the presence of serum antibodies reactive with this trisaccharide repeating unit, which is shared by both the type Ia and the type Ib native and core polysaccharides.     

Lipoproteins are critical TLR2 activating toxins in group B streptococcal sepsis

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.     

Analysis of recombinant group B streptococcal protein ScaAB and evaluation of its immunogenicity

Group B streptococcal (GBS) gene encoding the putative lipoprotein and adherence factor ScaAB was cloned and expressed in E. coli. Recombinant ScaAB protein was isolated. Signal sequence of ScaAB was found to be cleaved in the E. coli host. ScaAB recombinant protein was immunogenic in mice and antibodies against this protein were discovered in mice sera after GBS infection. The perspectives of the use of ScaAB protein in GBS vaccine are discussed.     

Comparative genetic study of group B streptococcal strains of human and bovine origin

The presence and restriction fragment length polymorphism (RFLP) of DNA fragments hybridizing with virulence and "house keeping" gene probes were analyzed for 87 group B streptococcal (GBS) strains of human and bovine origin. Most characteristics obtained for bovine strains were similar when compared with those for human strains. The most significant degree of RFLP was discovered for the sizes of HindIII fragments containing bca gene. Human GBS strains with bac gene, encoding beta antigen with IgA binding capacity, were characterized by almost identical complex hybridization patterns with multiple gene probes. At the same time bac gene was not found among bovine GBS strains. Gene scpB that encodes C5a peptidase in all human GBS strains was detected only in 9 of 39 strains of bovine origin. These two characteristics effectively distinguished bovine GBS strains from GBS strains of human origin.     

An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections

The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.     

Characteristics of group A streptococcal bacteriophages

Friend, Patric L. (Northwestern University Medical School, Chicago, Ill.), and Hutton D. Slade. Characteristics of group A streptococcal bacteriophages. J. Bacteriol. 92:148-154. 1966.-A medium for the growth of group A streptococcal phages is described, consisting of Brain Heart Infusion broth supplemented with 0.2% yeast extract, 10(-4)m CaCl(2), and 10 mug/ml of dl-tryptophan. Cell and phage growth in this medium was excellent, and did not require the addition of serum or other proteins as indicated by other workers. Growth of one phage has also been achieved in a completely synthetic medium. The adsorption characteristics of two group A phages in protein broth and synthetic broth were studied, and the initial adsorption of phage was found to be more extensive in synthetic broth. However, the final amounts of adsorption in both were similar. The addition of purified group A carbohydrate antigen to the adsorption mixture in synthetic broth had no effect on the adsorption, and cells containing type-specific M protein adsorbed phage at the same rate as those lacking M protein. It was concluded that neither the group antigen nor the type antigen was the primary site of phage adsorption. One-step growth curves of the two phages showed a second step or burst occurring. Sonic oscillation of the bacterial cultures, which broke up the chains to single cells, abolished the second step of the growth curve. It appears that the second step is a function of the chain formation of streptococcal cells.     

Identification of the actin and plasminogen binding regions of group B streptococcal phosphoglycerate kinase

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in sign ificantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.     

Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide

Immunization of rabbits with group B type III streptococcus organisms induces two distinct populations of antibodies with a specificity for determinants on the native capsular polysaccharide antigen of these organisms. Some of the structural and conformational features of the two determinants responsible for the formation of these antibodies were elucidated by (13)C NMR and serological studies on the native type III polysaccharide and some of its structurally modified analogues. The specificity of the determinant corresponding to the major population of antibodies is dependent of the presence of sialic acid residues on the native type III antigen, and although these residues are not an integral part of the determinant, they exert conformational control over it. The carboxylate groups of the sialic acid residues are an important factor in this control mechanism which could possibly involve intramolecular hydrogen bonding. The terminal sialic acid residues control the orientation of the penultimate beta-d-galactopyranose residues with respect to the backbone of the native antigen. The orientation of these residues is critical to the determinant because the determinant is probably small and is located precisely at the junction of the same beta-d-galactopyranose residues with the backbone of the native type III antigen. The determinant corresponding to the other population of antibodies is not sialic acid dependent. This determinant is located on the backbone of the native antigen in the vicinity of the other determinant but on the opposite side to the oligosaccharide branches. In this position, its conformation is unaffected even by the removal of the oligosaccharide branches from the native antigen.