Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: Population genetics, human serologic response, and gene transcription

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase–PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

Ozonolysis for selectively depolymerizing polysaccharides containing β-d-aldosidic linkages

The depolymerization of polysaccharides, particularly those containing acid-sensitive components, into intact constituent repeating units can be very difficult. We describe a method using ozonolysis for depolymerizing polysaccharides containing β-d-aldosidic linkages into short-chain polysaccharides and oligosaccharides. This method is carried out on polysaccharides that have been fully acetylated whereby β-d-aldosidic linkages are selectively oxidized by ozone to form esters, from which the polysaccharides are subsequently cleaved with a nucleophile. Ozone oxidation of aldosidic linkages proceeds under strong stereoelectronic control, and reaction rates depend on the conformations of glycosidic linkages. Thus, β-d-aldosidic linkages with different conformations can have very different reaction rates even in the absence of substantial chemical differences. These rate differences allowed for very high selectivity in cleaving β-d-linkages of polysaccharides. Several polysaccharides from group B Streptococcus and other bacterial species were selectively depolymerized with this method. The repeating units of the group B Streptococcus polysaccharides all contain an acid-sensitive sialic acid residue in a terminal position on a side chain and several β-d-residues including galactose, glucose, and N-acetylglucosamine; however, with each polysaccharide, one type of linkage was more reactive than others. Selective cleavage of the most sensitive linkage occurs randomly throughout the polymer chain, yielding fragments of controllable and narrowly distributed sizes and the same repeating-unit structure. The average size of the molecules decreases exponentially, and desired sizes can be obtained by stopping the reaction at appropriate time points. With this method the labile sialic acid residue was not affected.

CM101-mediated recovery of walking ability in adult mice paralyzed by spinal cord injury

CM101, an antiangiogenic polysaccharide derived from group B streptococcus, was administered by i.v. injection 1 hr post-spinal-cord crush injury in an effort to prevent inflammatory angiogenesis and gliosis (scarring) in a mouse model. We postulated that gliosis would sterically prevent the reestablishment of neuronal connectivity; thus, treatment with CM101 was repeated every other day for five more infusions for the purpose of facilitating regeneration of neuronal function. Twenty-five of 26 mice treated with CM101 survived 28 days after surgery, and 24 of 26 recovered walking ability within 2–12 days. Only 6 of 14 mice in the control groups survived 24 hr after spinal cord injury, and none recovered function in paralyzed limbs. MRI analysis of injured untreated and treated animals showed that CM101 reduced the area of damage at the site of spinal cord compression, which was corroborated by histological analysis of spinal cord sections from treated and control animals. Electrophysiologic measurements on isolated central nervous system and neurons in culture showed that CM101 protected axons from Wallerian degeneration; reversed γ-aminobutyrate-mediated depolarization occurring in traumatized neurons; and improved recovery of neuronal conductivity of isolated central nervous system in culture.