We developed a group B streptococcus multiplex PCR assay which allows, by direct analysis of the amplicon size, determination of the surface protein antigen genes of alpha-C protein, epsilon protein, Rib, Alp2, Alp3, and Alp4. The multiplex PCR assay offers a rapid and simple method of subtyping Streptococcus agalactiae based on surface protein genes.
SUMMARYGroup B streptococci (GBS ) are an important cause of neonatal sepsis, pneumonia and meningitis. In the early phase of infection, macrophages and polymorphonuclear cells (PMN ) are the first immune cells that interact with GBS. In this in vitro study, to gain insight into GBS-macrophage interaction in the absence of type-specific antibodies, we examined the features of GBS survival in thioglycollate-elicited murine peritoneal macrophages and the effect of GBS on the protein kinase C (PKC )-dependent transduction pathway. Our results demonstrate that type Ia GBS, strain 090 (GBS-Ia) and type III GBS strain COH 31r/s (GBS-III ), after in vitro phagocytosis survive and persist intracellularly in macrophages for up to 24 and 48 hr, respectively. However, macrophage activation by interferon-c (IFN-c) and lipopolysaccharide from Escherichia coli (LPS ) caused a significant reduction in the time of intracellular persistence. Macrophage activation by IFN-c and LPS seems to be a multifactorial event involving multiple intracellular signal pathways also including PKC. Since PKC is one of the components in the signal network leading to macrophage activation and an important target for several intracellular micro-organisms, we wondered whether PKC could have a role in intracellular GBS survival. Both PKC depletion by treatment with phorbol 12-myristate 13-acetate (PMA) for 18 hr and PKC inhibition by Calphostin C rendered macrophages more permissive for the intracellular GBS survival. Furthermore, GBSinfected macrophages were unable to respond to PMA and LPS, activators of PKC, by inducing antimicrobial activity. The ability of GBS to impair PKC-dependent cell signalling was also demonstrated by the reduced c-fos gene expression in GBS-infected macrophages with respect to control macrophages, after LPS stimulation. In conclusion, our results indicate that GBS survive in macrophages and impairment of PKC signal transduction contributes to their intracellular survival. INTRODUCTIONcorrelates with the susceptibility or resistance of neonates to GBS infection.15,16 Group B streptococci (GBS ) are the major cause of pneuThe discovery that macrophages can phagocytose GBS in monia, sepsis and meningitis in neonates and a serious cause the absence of immune serum by C3-dependent binding17 and of mortality or morbidity in immunocompromised adults.1,2 C3-independent binding using complement receptor type three The main virulence factor of GBS is thought to be the capsular (CR3)18 suggests that there is also a potential role for antibodypolysaccharide because of its antiphagocytic properties.3,4 In independent mechanisms in resistance to GBS infection. resistance to GBS infection, a central role is played by antiHowever, the recent demonstration that type III GBS phagobodies to the type-specific capsular polysaccharide and complecytosed by a macrophage-like line J774 in the absence of typement which potentiate in vitro phagocytosis and GBS killing specific antibodies survived within its host cell,19 seems to by phagocytic cells...
BackgroundNon-toxigenic Corynebacterium diphtheriae strains are emerging as a major cause of severe pharyngitis and tonsillitis as well as invasive diseases such as endocarditis, septic arthritis, splenic abscesses and osteomyelitis. C. diphtheriae strains have been reported to vary in their ability to adhere and invade different cell lines. To identify the genetic basis of variation in the degrees of pathogenicity, we sequenced the genomes of four strains of C. diphtheriae (ISS 3319, ISS 4060, ISS 4746 and ISS 4749) that are well characterised in terms of their ability to adhere and invade mammalian cells.ResultsComparative analyses of 20 C. diphtheriae genome sequences, including 16 publicly available genomes, revealed a pan-genome comprising 3,989 protein coding sequences that include 1,625 core genes and 2,364 accessory genes. Most of the genomic variation between these strains relates to uncharacterised genes encoding hypothetical proteins or transposases. Further analyses of protein sequences using an array of bioinformatic tools predicted most of the accessory proteome to be located in the cytoplasm. The membrane-associated and secreted proteins are generally involved in adhesion and virulence characteristics. The genes encoding membrane-associated proteins, especially the number and organisation of the pilus gene clusters (spa) including the number of genes encoding surface proteins with LPXTG motifs differed between different strains. Other variations were among the genes encoding extracellular proteins, especially substrate binding proteins of different functional classes of ABC transport systems and ‘non-classical’ secreted proteins.ConclusionsThe structure and organisation of the spa gene clusters correlates with differences in the ability of C. diphtheriae strains to adhere and invade the host cells. Furthermore, differences in the number of genes encoding membrane-associated proteins, e.g., additional proteins with LPXTG motifs could also result in variation in the adhesive properties between different strains. The variation in the secreted proteome may be associated with the degree of pathogenesis. While the role of the ‘non-classical’ secretome in virulence remains unclear, differences in the substrate binding proteins of various ABC transport systems and cytoplasmic proteins potentially suggest strain variation in nutritional requirements or a differential ability to utilize various carbon sources.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1980-8) contains supplementary material, which is available to authorised users.
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