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

Abstract

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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“…4). This may suggest some flexibility in the pore architecture, as seen with several pore-forming toxins (35,40).…”

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“…CAMP factor is a protein initially discovered in S. agalactiae which produces the synergistic lysis of ovine erythrocytes in the presence of a ß-toxin S. aureus [69,70]. The deduced amino acid sequence of CAMP factor of S. uberis was found to be homologous to CFB amino acid sequence of S. agalactiae [46].…”

Carbon nanotubes inhibit the hemolytic activity of the pore-forming toxin pyolysin

“…The decrease in turbidity, i.e., optical density at 650 nm (OD 650 ), is directly correlated to lysis of human red blood cells (RBCs) [11]. The results were normalized and are shown in Fig.…”

“…A quantitative microtiter plate hemolysis assay [11] was used to characterize the hemolytic activity of PLO with and without SWNT-OX. The decrease in turbidity, i.e., optical density at 650 nm (OD 650 ), is directly correlated to lysis of human red blood cells (RBCs) [11].…”