Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor

Podbielski, Andreas; Blankenstein, Oliver; Lütticken, Rudolf

doi:10.1007/bf00198458

Cited by 62 publications

(48 citation statements)

References 46 publications

(31 reference statements)

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“…A BLAST (www.ncbi.nlm.nih.gov) search was performed. The coding sequence was found to deviate at two positions from the published sequence of CAMP factor of S. agalactiae (20) at both the nucleotide and amino acid levels. However, the coding sequence of the CAMP factor mature peptide exactly matched the amino acid sequence of protein B, the alias of CAMP factor of S. agalactiae (14).…”

Section: Cloning Recombinant Expression and Purification Of Campmentioning

confidence: 82%

Characterization of Streptococcus agalactiae CAMP Factor as a Pore-forming Toxin

Lang

Palmer

2003

Journal of Biological Chemistry

105

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A recombinant form of CAMP factor of Streptococcus agalactiae has been expressed as glutathione S-transferase-CAMP fusion protein in Escherichia coli. After thrombin cleavage of the fusion protein, the recombinant CAMP factor exhibited hemolytic activity comparable with that of the native form. Osmotic protection experiments with polyethylene glycols show that CAMP factor forms discrete transmembrane pores with a diameter upward of 1.6 nm on susceptible membranes; electron microscopy reveals circular membrane lesions of heterogeneous size, up to 12-15 nm in diameter. Liposome permeabilization studies show that pore formation is a highly cooperative process, which suggests that it involves the oligomerization of CAMP factor. Chemical cross-linking experiments also support an oligomeric mode of action.The CAMP reaction consists of a distinct zone of hemolysis on blood agar plates produced by Streptococcus agalactiae when grown near the colonies of Staphylococcus aureus (1). It has been used in diagnostic microbiology to identify the S. agalactiae strains ever since its discovery in 1944 by Christie et al. (1). The proteins responsible for the CAMP reaction are sphingomyelinase from S. aureus and CAMP factor, a protein secreted by S. agalactiae that has a molecular weight of 25 kDa and a pI of 8.9 (2). Sphingomyelinase initially hydrolyzes sphingomyelin to ceramide, which renders the erythrocytes susceptible to the lytic activity of CAMP factor. Erythrocytes from different mammalian species support the CAMP reaction to different extents depending on the sphingomyelin content in their cell membranes (3). Sheep red blood cells are the most susceptible, in keeping with their sphingomyelin content as high as 51% (by moles) (4). Human red blood cells and rabbit red blood cells, with 26 and 19% mol of sphingomyelin, respectively (4, 5), were reportedly not sensitive to CAMP factor after sphingomyelinase treatment (3). The latter, however, were rendered susceptible to the CAMP factor after phospholipase C treatment, which converted the glycerophospholipids to diacylglycerol (6), indicating that ceramide is not specifically required for CAMP activity. Previous work with liposomes also suggested that the fraction of cholesterol in the membrane influences the activity of CAMP factor (6, 7). CAMP factor has long been known as a pathogenic determinant that exerted lethal effects when administered to rabbits and mice (8). Apart from its membrane damaging activity, CAMP factor was found to bind to the F c fragments of immunoglobulins, and it was therefore designated as protein B in analogy to protein A of S. aureus (9).The CAMP factor genes of S. agalactiae (10), Streptococcus uberis (11), and Streptococcus pyogenes (12) have been cloned in Escherichia coli, and their sequences were found to be highly homologous with each other (12). Co-hemolytic phenomena have also been reported with various other bacterial genera, but the proteins responsible for those, although sometimes referred to by the name "CAMP factor" as well, are not...

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Section: Cloning Recombinant Expression and Purification Of Campmentioning

confidence: 82%

Characterization of Streptococcus agalactiae CAMP Factor as a Pore-forming Toxin

Lang

Palmer

2003

Journal of Biological Chemistry

105

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“…This test utilizes real-time PCR to detect a conserved target within the 3= DNA region adjacent to cfb, the gene encoding the S. agalactiae CAMP factor (12)(13)(14). Results were compared to LIM broth-enriched culture with plating on 5% sheep blood agar as a gold standard.…”

Section: S Treptococcus Agalactiae (Group B Streptococcus [Gbs]) Is Amentioning

confidence: 99%

Multicenter Clinical Evaluation of the Xpert GBS LB Assay for Detection of Group B Streptococcus in Prenatal Screening Specimens

et al. 2015

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dNeonatal infection with Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis and meningitis in newborns. Recent guidelines have recommended universal screening of all pregnant women to identify those colonized with GBS and administration of peripartum prophylaxis to those identified as carriers to reduce the risk of early-onset GBS disease in neonates. Enriched culture methods are the current standard for prenatal GBS screening; however, the implementation of more sensitive molecular diagnostic tests may be able to further reduce the risk of early-onset GBS infection. We report a clinical evaluation of the Xpert GBS LB assay, a molecular diagnostic test for the identification of GBS from broth-enriched vaginal/rectal specimens obtained during routine prenatal screening. A total of 826 specimens were collected from women undergoing prenatal screening (35 to 37 weeks' gestation) and tested at one of three clinical centers. Each swab specimen was tested directly prior to enrichment using the Xpert GBS assay. Following 18 to 24 h of broth enrichment, each specimen was tested using the Xpert GBS LB assay and the FDA-cleared Smart GBS assay as a molecular diagnostic comparator. Results obtained using all three molecular tests were compared to those for broth-enriched culture as the gold standard. The sensitivity and specificity of the Xpert GBS LB assay were 99.0% and 92.4%, respectively, compared to those for the gold standard culture. The Smart GBS molecular test demonstrated sensitivity and specificity of 96.8% and 95.5%, respectively. The sensitivities of the two broth-enriched molecular methods were superior to those for direct testing of specimens using the Xpert GBS assay, which demonstrated sensitivity and specificity of 85.7% and 96.2%, respectively. S treptococcus agalactiae (group B Streptococcus [GBS]) is aGram-positive bacterium associated with transient colonization of mucosal membranes throughout the body, including the vagina, gastrointestinal tract, and urethra (1). GBS rarely causes disease in healthy individuals but can cause serious illness in immunocompromised patients, elderly individuals, and newborn infants (2). Of particular concern is neonatal infection caused by vertical transmission during labor and birthing. Transmission from an asymptomatically colonized mother to the neonate can result in early-onset invasive GBS disease, which is a leading cause of sepsis and meningitis in newborns in the United States (3). Early-onset GBS disease in newborns can result in death or longterm disabilities such as mental retardation and hearing or vision loss (4). Because these infections are acquired through direct contact of the neonate with GBS in the mother's urogenital tract during delivery, it has become a universal practice to screen pregnant women for vaginal/rectal colonization with GBS at 35 to 37 weeks' gestation (5). The identification of GBS during routine screening results in administration of intrapartum prophylaxis to mitigate transmission of bacteria and red...

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“…Binding of this protein to the Fc region of immunoglobulins has been demonstrated (12,19), and the protein seems to be lethal to rabbits and mice (30). Protein B seems to be essential for S. agalactiae, as the cfb gene encoding this protein (24) was found to be universally present in the genomes of 162 GBS strains from different geographic areas (20). Furthermore, 96 to 99% of all human GBS isolates have been found to be positive by the CAMP reaction (11,14,23,25).…”

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confidence: 99%

Method for Quantitative Detection and Presumptive Identification of Group B Streptococci on Primary Plating

Hansen

Sørensen

2003

J Clin Microbiol

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Maternal prenatal screening for group B streptococci (GBS) followed by offering of intrapartum chemoprophylaxis to carriers is one of the strategies used to reduce the incidence of neonatal early-onset GBS infections. Culturing of vaginal and anorectal swab specimens in selective broth is the screening procedure recommended by the Centers for Disease Control and Prevention. This technique is sensitive; it does not, however, allow either evaluation of the degree of colonization or detection of cocolonization with several GBS clones. We have examined the carriage rate and population dynamics of GBS in a group of Danish women during pregnancy and 1 year after delivery using a new detection method. In the present paper we describe a mixed blood agar medium (MB agar) that identifies GBS in the primary cultures by detection of a double hemolysis pattern consisting of characteristic, large zones of partial hemolysis ("CAMP zones") and of narrow zones of complete hemolysis. The MB agar was at least as sensitive as culturing in selective broth for detection of GBS in vaginal and anorectal swab specimens, and GBS strains could be identified directly on the primary plate due to the CAMP zones without the need for subculturing. The carriage rate of GBS in a group of Danish women was found to be more than 30%, a figure considerably higher than the rate that was reported previously.

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Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor

Cited by 62 publications

References 46 publications

Characterization of Streptococcus agalactiae CAMP Factor as a Pore-forming Toxin

Characterization of Streptococcus agalactiae CAMP Factor as a Pore-forming Toxin

Multicenter Clinical Evaluation of the Xpert GBS LB Assay for Detection of Group B Streptococcus in Prenatal Screening Specimens

Method for Quantitative Detection and Presumptive Identification of Group B Streptococci on Primary Plating

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