A cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C.
ABSTRACT Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.
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ABSTRACT: Myonecrotic isolates of Clostridium perfringens secrete multimodular sialidases, often termed "large sialidases", that contribute to the virulence of this bacterium. NanJ is the largest of the two secreted sialidases at 1173 amino acids and comprises 6 different modules which are, from the N-terminus, a family 32 carbohydrate binding module (CBM), a family 40 CBM, a family 33 glycoside hydrolase, a module of unknown function, a family 82 "X-module" of unknown function, and a module with amino acid similarity to fibronectin type III domains. The hydrolase activity of clostridial sialidases is quite well documented; however, the functions of their accessory domains are entirely uninvestigated. Here we describe the carbohydrate binding activity of the isolated family 32 CBM (CBM32) and the isolated family 40 CBM (CBM40). CBM32 is shown to bind galactose or N-acetylgalactosamine, while CBM40 is sialic acid specific, though both CBMs appear to bind with very low affinities. The crystal structure of CBM32 was determined at 2.25 A in complex with galactose. This revealed what appears to be a very simple galactose binding site. The crystal structure of CBM40 was determined at 2.20 A in complex with a sialic acid containing molecule that it fortuitously crystallized with, revealing the molecular details of the CBM40-sialic acid interaction. Overall, the results indicate that NanJ contains carbohydrate specific binding modules that likely function to target the enzyme to molecules or cells bearing mixed populations of glycans that terminate in either galactose/N-acetylgalactosamine or sialic acid.Biochemistry 11/2007; 46(40):11352-60. · 3.42 Impact Factor
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ABSTRACT: We identified 1113 articles (103 reviews, 1010 primary research articles) published in 2005 that describe experiments performed using commercially available optical biosensors. While this number of publications is impressive, we find that the quality of the biosensor work in these articles is often pretty poor. It is a little disappointing that there appears to be only a small set of researchers who know how to properly perform, analyze, and present biosensor data. To help focus the field, we spotlight work published by 10 research groups that exemplify the quality of data one should expect to see from a biosensor experiment. Also, in an effort to raise awareness of the common problems in the biosensor field, we provide side-by-side examples of good and bad data sets from the 2005 literature.Journal of Molecular Recognition 19(6):478-534. · 3.31 Impact Factor