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Escherichia coli K1 isolates synthesize a polysialic acid (polySia) capsule, are components of the adult gastrointestinal microbiota and may cause lethal bacteraemia and meningitis if acquired maternally by newborn infants. We used a neonatal rat pup K1 infection model to establish that prompt administration of a selective capsule depolymerase reverses the bacteraemic state and prevents death of almost all pups. In untreated animals, bacteria colonize the gastrointestinal tract and gain entry to the blood compartment, where they express the non-O-acetylated form of polySia. The bacteria invade the major organs of the host; histological and histochemical analysis of brain sections revealed that at least some bacteria enter the central nervous system through the blood-cerebrospinal fluid barrier at the choroid plexus prior to colonization of the meninges. Once in this location, they cease expression of polySia. The unexpected abrogation of polySia, a factor associated with the pathogenesis of meningitis and essential for transit through the blood, suggests that the neuropathogen dispenses with its protective capsule once it has colonized protected niches. Thus, systemic infections due to encapsulated pathogens may be resolved by capsule depolymerization only if the enzyme modifies the bacteria whilst they are in the blood compartment.

Streptococcus pyogenes (group A streptococcus [GAS]), a catalase-negative gram-positive bacterium, is aerotolerant and survives [H.sub.2][O.sub.2] exposures that kill many catalase-positive bacteria. The molecular basis of the [H.sub.2][O.sub.2] resistance is poorly known. Here, we demonstrate that serotype M49 GAS lacking the Rgg regulator is more resistant to [H.sub.2][O.sub.2] and also decomposes more [H.sub.2][O.sub.2] than the parental strain. Subgenomic transcriptional profiling and genome-integrated green fluorescent protein reporters showed that a bicistronic operon, a homolog of the Streptococcus mutans ahpCF operon, is transcriptionally up-regulated in the absence of Rgg. Phenotypic assays with ahpCF operon knockouts demonstrated that the gene products decompose [H.sub.2][O.sub.2] and protect GAS against peroxide stress. In a murine intraperitoneal-infection model, Rgg deficiency increased the virulence of GAS, although in an ahpCF-independent manner. Rgg-mediated repression of [H.sub.2][O.sub.2] resistance is divergent from the previously characterized peroxide resistance repressor PerR. Moreover, Rgg-mediated repression of [H.sub.2][O.sub.2] resistance is inducible by cellular stresses of diverse natures--ethanol, organic hydroperoxide, and [H.sub.2][O.sub.2]. Rgg is thus identified as a novel sensoregulator of streptococcal [H.sub.2][O.sub.2] resistance with potential implications for the virulence of the catalase-negative GAS.

Transposon mutagenesis is a powerful technique for generating collections of insertion mutants for genetic studies. This paper describes how phage Mu DNA transposition complexes, transpososomes, can be exploited for gene delivery to efficiently introduce selectable markers to genomes of Gram-positive bacteria. Mu transpososomes were assembled in vitro with custom-designed mini-Mu transposons, concentrated, and electroporated into cells of three Gram-positive bacterial species: Staphylococcus aureus, Streptococcus pyogenes and Streptococcus suis. Within cells, the complexes reproduced an authentic DNA transposition reaction and integrated the delivered transposons into the bacterial genomes, yielding single-copy insertions. The integration efficiency among different species and strains of Gram-positive bacteria ranged from 1 x [10.sup.1] to 2 x [10.sup.4] c.f.u. [([micro]g introduced transposon DNA). sup.-1]. The strategy should be applicable to a variety of other Gram-positive species after initial optimization of certain key factors affecting transposon delivery, such as the preparation method of competent cells and physical parameters of electroporation. This study extends the scope of the Mu transpososome delivery-based genomic DNA integration strategy to Gram-positive bacteria. Thus, a straightforward generation of sizeable mutant banks is feasible for these bacteria, potentiating several types of genomic-level approaches for studies of a variety of important bacterial processes, such as pathogenicity.

Results reveal that in the majority of Streptococcus pyognes strains a glycoprotein-binding activity is detected in thyroglobulin, fetuin, asialofetuin and mucin glycoproteins. Data further indicate that the binding activity is regulated by the bacterial virulence factor regulating gene Mga.

Inhibition of bacterial adhesion was demonstrated in a study conducted on di-, tri-, and tetravalent dendritic galabiosides that inhibit hemagglutination by Streptococcus suis at nanomolar concentration. To form such compounds, coupling of galabiose derivatives to carrier molecules was initiated. Findings revealed the relationship between the number of galabiose units found in an inhibitor and inhibitory efficacy.

The role of the phage depolymerase in binding and degradation was investigated. Phage mutants that are specific for Escherichia coli bacteria that have a low level of K1 polysialic acid capsule were isolated. It was shown that these mutants have lost the cleaving activity of the phage endosialidase while retaining the binding activity. The results suggest that the polysaccharide-binding activity of the enzyme is necessary for phage infection, whereas the cleavage of the polysaccharide is required for penetration of the dense polysaccharide of wild-type bacteria but is inhibitory in the infection of bacteria with a sparsecapsule.