An Arcanobacterium (Actinomyces) pyogenes mutant deficient in production of the pore-forming cytolysin pyolysin has reduced virulence.
(1/1527)
Pyolysin (PLO), the hemolytic exotoxin expressed by Arcanobacterium (Actinomyces) pyogenes, is a member of the thiol-activated cytolysin family of bacterial toxins. Insertional inactivation of the plo gene results in loss of expression of PLO with a concomitant loss in hemolytic activity. The plo mutant, PLO-1, has an approximately 1. 8-log10 reduction in the 50% infectious dose compared to that for wild-type A. pyogenes in a mouse intraperitoneal infection model. Studies involving cochallenge of wild-type and PLO-1 bacteria resulted in recovery of similar numbers of both strains, suggesting that PLO production is required for survival in vivo. Recombinant, His-tagged PLO (His-PLO) is cytotoxic for mouse peritoneal macrophages and J774 cells in a dose-dependent manner. Protection against challenge with A. pyogenes could be afforded by vaccination with formalin-inactivated His-PLO, suggesting that PLO is a host-protective antigen, as well as a virulence determinant. (+info)
Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin.
(2/1527)
Helicobacter pylori secretes a cytotoxin (VacA) that induces the formation of large vacuoles originating from late endocytic vesicles in sensitive mammalian cells. Although evidence is accumulating that VacA is an A-B toxin, distinct A and B fragments have not been identified. To localize the putative catalytic A-fragment, we transfected HeLa cells with plasmids encoding truncated forms of VacA fused to green fluorescence protein. By analyzing truncated VacA fragments for intracellular vacuolating activity, we reduced the minimal functional domain to the amino-terminal 422 residues of VacA, which is less than one-half of the full-length protein (953 amino acids). VacA is frequently isolated as a proteolytically nicked protein of two fragments that remain noncovalently associated and retain vacuolating activity. Neither the amino-terminal 311 residue fragment (p33) nor the carboxyl-terminal 642 residue fragment (p70) of proteolytically nicked VacA are able to induce cellular vacuolation by themselves. However, co-transfection of HeLa cells with separate plasmids expressing both p33 and p70 resulted in vacuolated cells. Further analysis revealed that a minimal fragment comprising just residues 312-478 functionally complemented p33. Collectively, our results suggest a novel molecular architecture for VacA, with cytosolic localization of both fragments of nicked toxin required to mediate intracellular vacuolating activity. (+info)
Design of highly specific cytotoxins by using trans-splicing ribozymes.
(3/1527)
We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins. (+info)
Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells.
(4/1527)
The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III. (+info)
Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA.
(5/1527)
The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer. (+info)
A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins.
(6/1527)
The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for TcsL-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracellular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and beta3-integrin remaining at the tips of cellular protrusions. These components were absent from cells treated with the R-Ras-inactivating toxins. The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras. (+info)
Bacterial toxins and the Rho GTP-binding protein: what microbes teach us about cell regulation.
(7/1527)
In the present review activities of two bacterial toxins, Clostridium botulinum exoenzyme C3 and Escherichia coli CNF1, both acting on the GTP-binding protein Rho are analyzed. Proteins belonging to the Rho family regulate the actin cytoskeleton and act as molecular switches in a number of signal transduction pathways. C3 and CNF1 have opposite effects on Rho thus representing useful tools for studies on cell division, cell differentiation and apoptosis. (+info)
Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells: mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.
(8/1527)
Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs. (+info)