PHASE VARIATION IN THE PATHOGENESIS OF GBS INFECTION

B群链球菌（GBS）是目前最常见的严重病因

Group B streptococci (GBS) are presently the most frequent cause of serious bacterial infections of neonates in the United States. The long-term goal of our studies is to find out why GBS, a commensal organism in about a third of the normal population of pregnant women, should cause serious, often fatal, infection in about 4 of every 1000 live-born infants. Our hypothesis is that GBS are not normally pathogenic but have pathogenic potential when as yet unknown environmental factors trigger a major phenotypic change that includes the production of increased amounts of capsular polysaccharide. The biochemical basis for capsule size variation in GBS will be studied. This will include determining if the type-specific polysaccharide chains are longer in large capsule strains, if there are more chains, or if there are other structural changes. Possible coordinate regulation of capsule size variation will be studied, looking specifically for concurrent changes in teichoic acid, neuraminidase, and other bacterial components. The molecular genetic basis of capsule size variation will be studied using transposon insertional mutagenesis to produce mutations in genes that affect capsule size. A new electroporation technique, along with a recently constructed plasmid suitable for use with tetracycline-resistant GBS strains, will be employed to yield mutants containing single transposon inserts. The DNA flanking the transposon inserts in mutants of interest will be cloned and the gene products involved in capsule size phase variation will be identified. Defined mutations will be made in some of the cloned genes which will then be used for the allelic replacement of the corresponding chromosomal genes by homologous recombination. The role of capsular size variation in GBS infection and in asymptomatic colonization will be studied. The hypothesis that disease causing strains possess large capsules that revert to small capsule forms upon culture will be tested by assessing capsule size by buoyant density centrifugation and electron microscopy Of organisms prior to culture on artificial media. Capsule size in asymptomatic vaginal colonization will be studied using two new complementary procedures. The first involves dissociation of GBS bound to vaginal epithelial cells using Triton X-100 prior to density gradient centrifugation. The second method involves electron microscopy of GBS in vaginal secretions selectively enriched for GBS on nickel grids coated with specific antibody. Capsule size will be correlated with levels of type-specific antibody in the vaginal secretions to determine if antibody plays a role in suppressing large-capsule forms of GBS. The effect of capsule size as a virulence factor will be studied in mouse protection tests, and the amount of antibody required for opsonization of large and small capsule strains will be studied in opsonophagocidal assays. Mucin enhances the virulence of certain GBS strains and appears to induce a phase shift. We plan to determine if the presence of sialic acid in mucin is essential for its ability to enhance virulence. The role of GBS neuraminidase in this process will be assessed using neuraminidase-negative mutants.