Tyrosine 331 and phenylalanine 334 in Clostridium perfringens α-toxin are essential for cytotoxic activity

AbstractDifferences in the biological properties of the Clostridium perfringens phospholipase C (α-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of α-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of α-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp

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

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

Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing

Background: A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. Results: The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani. Conclusions: Our comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.Universidad de Costa Rica, Instituto Clodomiro PicadoUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP