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A UDP-glucose deficient mutant cell line as a model to study the cytotoxicity of Clostridium perfringens PLC

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posted on 2024-09-02, 22:01 authored by Marietta Flores Diaz

A Chinese Hamster fibroblast mutant cell line, deficient in UDP-glucose (UDP-Glc) and hypersensitive to Clostridium perfringens phospholipase C (PLC) was used in this study to determine some of the molecular consequences of a cellular UDP-Glc deficiency. Furthermore, using this cell as a model, structure/function studies were performed to identify residues critical for the cytotoxic activity of PLC.

It was found that the reason for the cellular UDP-Glc deficiency is a point mutation in the gene that encodes the UDP-Glc pyrophosphorylase (UDPG:PP), the enzyme that catalyzes UDP-Glc synthesis. The mutation changes the conserved glycine 115 to aspartic acid in the protein product. Protein analysis of cell lysates showed that the mutant cell overproduces seven stress proteins: one mitochondrial chaperone (GRP75) and six chaperones of the endoplasmic reticulum (GRP58, ERp72, GRP78, GRP94, GRP170 and calreticulin). These proteins are also upregulated in cells cultured under hypoxia or glucose starvation as well as in ischemic tissues.

To clarify whether there is a connection between the UDP-Glc deficiency and the overproduction of stress proteins and the hypersensitivity to the PLC, stable transfectant cells were prepared using a wild type UDPG:PP eDNA. Transfectant clones increased their UDP-Glc concentration and produce normal amounts of calreticulin and the GRPs, indicating that the UDP-Glc deficiency induces their overproduction. Exposure of the transfectant clones to PLC demonstrated that a cellular UDP-Glc deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase.

The UDP-Glc deficient cell was used to characterize the structural determinants responsible for the cytotoxic activity of PLC. Experiments with genetically engineered PLC variants showed that the sphingomyelinase activity and the C-terminal domain are required for its cytotoxic effect. In addition, in vivo experiments demonstrated that the sphingomyelinase activity and the C-terminal domain are also needed for myotoxicity. The toxic activities of PLC variants harboring single amino acid substitutions in aspartic acid residues, which bind calcium, and tyrosine residues of the putative membrane-interacting region at the C-terminal domain were studied. These residues were found to be critical for the hemolytic, cytotoxic and myotoxic activities of PLC.

Since UDP-Glc is required for the synthesis of membrane glycoconjugates, their role in the sensitivity to PLC was studied. It was found that inhibition of glycoproteins or proteoglycans synthesis/processing does not affect the sensitivity to PLC, whereas inhibition of glycosphingolipid synthesis sensitizes cells to this toxin. Furthermore it was demonstrated that complex gangliosides protect hypersensitive cells from the cytotoxic activity of PLC and prevent the membrane disrupting effect of this toxin in artificial membranes.

In conclusion, this work revealed that a cellular UDP-Glc deficiency induces the upregulation of a set of stress proteins important for cell survival under ischemic like conditions and furthermore provide new insights to understand the molecular mechanism of action of C. perfringens PLC.

List of scientific papers

I. Flores-Diaz M, Alape-Giron A, Persson B, Pollesello P, Moos M, von Eichel-Streiber C, Thelestam M, Florin I (1997). "Cellular UDP-glucose deficiency caused by a single point mutation in the UDP-glucose pyrophosphorylase gene. " J Biol Chem 272(38): 23784-91
https://pubmed.ncbi.nlm.nih.gov/9295324

II. Flores-Diaz M, Alape-Giron A, Florin I, Higuita JC, Pollesello P, Okada T, Moss M, Cordula C, Bergman T, Eichel-Streiber Cv, Mori K, Thelestam M (2001). "A cellular UDP-glucose deficiency causes overproduction of stress proteins in the mitochondria and the endoplasmic reticulum." (Manuscript)

III. Flores-Diaz M, Alape-Giron A, Titball RW, Moos M, Guillouard I, Cole S, Howells AM, von Eichel-Streiber C, Florin I, Thelestam M (1998). "UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of Clostridium perfringens phospholipase C. " J Biol Chem 273(38): 24433-8
https://pubmed.ncbi.nlm.nih.gov/9733734

IV. Alape-Giron A, Flores-Diaz M, Guillouard I, Naylor CE, Titball RW, Rucavado A, Lomonte B, Basak AK, Gutierrez JM, Cole ST, Thelestam M (2000). "Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene. " Eur J Biochem 267(16): 5191-7
https://pubmed.ncbi.nlm.nih.gov/10931204

V. Flores-Diaz M, Alape-Giron A, Clark G, Ichikawa S, Hirabayashi Y, Gutieerrez JM, Titball RW, Thelestam M (2001). "Cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C." J Biol Chem (Submitted)

History

Defence date

2001-11-30

Department

  • Department of Microbiology, Tumor and Cell Biology

Publication year

2001

Thesis type

  • Doctoral thesis

ISBN-10

91-7349-087-3

Number of supporting papers

5

Language

  • eng

Original publication date

2001-11-09

Author name in thesis

Flores Diaz, Marietta

Original department name

Microbiology and Tumor Biology Center (MTC)

Place of publication

Stockholm

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