Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.     

Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin

A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.     

Corynebacterium diphtheriae: a PTS view to the genome

We have surveyed the publicly available genome sequence of Corynebacterium diphtheriae (www.sanger.ac.uk) to identify components of the phosphotransferase system (PTS), which plays a central role in carbon metabolism in many bacteria. Three gene loci were found to contain putative pts genes. These comprise: (i) the genes of the general phosphotransferases enzyme I (ptsI) and HPr (ptsH), a fructose-specific enzyme IIABC permease (fruA), and a fructose 1-phosphate kinase (fruK); (ii) a gene that encodes an enzyme IIAB of the fructose/mannitol family, and a novel HPr-like gene, ptsF, that encodes an HPr domain fused to a domain of unknown function; (iii) and a gene for a glucose-specific enzyme IIBCA (ptsG). A search for genes that may be putative PTS-targets or that may operate in general carbon regulation revealed a possible regulatory gene encoding an antiterminator protein downstream from ptsG. Furthermore, genes were detected encoding glycerol kinase, glucose kinase, and a homologue of the global activator of carbon catabolite repression in Escherichia coli, CAP. The possible significance of these observations in carbon metabolism and the novel features of the detected genes are discussed.     

Preliminary investigation on the cytotoxicity of tellurite to cultured HeLa cells.

Cytotoxicity of tellurite to cultured HeLa cells was examined by cell viability, lactate dehydrogenase (LDH) assay, and tellurite uptake. The experimental results show that the toxicity of tellurite depends on its concentrations and exposure time. HeLa cells exposed to tellurite for 2 h at 9.1 x 10(-4) to 4.5 x 10(-3) mmol/L did not exhibit cytotoxic effects as measured by cell viability. Exposure to tellurite for 24 h at the same concentrations markedly reduced the cell viability to 57% of the control during the first 5 minutes. Additionally, HeLa cells incubated at 2.7 x 10(-2) to 0.27 mmol/L of tellurite for 2 h retained 53% to 67% of cell viability. Even after 24 h exposure, the HeLa cells incubated at 9.1 x 10(-4) to 4.5 x 10(-2) mmol/L of tellurite still retained 57% to 66% of cell viability. Furthermore, tellurite toxicity was also demonstrated in supernatant of the culture at 37 degrees C by LDH assay. It was found that exposure to tellurite for 90 minutes did not stimulate LDH activity. However, tellurite uptake seems to be more sensitive than the cell viability and LDH activity release tests, as it significantly increases with the increasing of exposure time.     

Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene

The effects of Geneticin (G418) selection on the growth and survival of cultured mammalian cells expressing the neomycin-resistance gene (neo) were studied by the analysis of cell clones from two retroviral neo vector-infected populations. We found a correlation between the neo expression level and growth rates in medium containing varying G418 concentrations. This relationship permits the use of differential selection schemes for the isolation of rare cells with increased expression. Comparison, by clone sampling, of vector-positive populations before and after selection with a G418 concentration in the range usually used for selection, showed different expression level and vector copy number distributions for the population infected with the vector of lower LTR activity, but not for the other. Such biasing effects of G418 selection may be important when selected cells are used for quantitative studies of gene expression.     

Clastogenicity of low pH to various cultured mammalian cells.

It has been reported that low pH itself can be clastogenic to Chinese hamster ovary cells or mouse lymphoma L5178Y cells. On the other hand, there was no indication that low pH is clastogenic to rat or human lymphocytes. Therefore, in order to evaluate the generality of clastogenicity of low pH conditions, chromosomal aberration tests were carried out on Chinese hamster cell line cells (CHO-K1, CHL, Don and V79 379A) and human cells (HeLa and peripheral lymphocytes used as whole-blood cultures). The cytotoxicity of low pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tris as an organic buffer to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCl. Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa cells had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectively. In V79 379A cells or human lymphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low (less than 20%). About 90% of aberrations induced in each cell line examined were chromatid-type gaps and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanges at pH 5.5 or pH 5.7, respectively. These results indicate that clastogenicity of low pH is a general finding, although the extent of it varies with cell type, and that the clastogenicity is associated with varying extents of cytotoxicity. The mechanisms of clastogenesis at low pH are not known, but might involve inhibition of DNA or protein synthesis or DNA-repair enzymes.     

Autofluorescence of viable cultured mammalian cells.

The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.     

Assessment of cytotoxicity and DNA damage exhibited by siloranes and oxiranes in cultured mammalian cells

The potential reactivity and structural properties of oxiranes (epoxides) are advantageous when considering polymers for medical devices. However, epoxy compounds are widely known to have genotoxic properties. The objective of the study was to evaluate the cytotoxicity and primary DNA damage effects induced by oxiranes and siloranes, silicon containing oxiranes. The siloranes, Ph-Sil, Tet-Sil, and Sil-Mix and the oxiranes Cyracure UVR-6105 and 1,3-bis[2-(2-oxiranylmethyl) phenoxy]pentane (OMP-5) were dissolved in organic solvents and dilutions containing less than 0.5% solvent were used in biological assays. The concentration that reduced the viability of 50% (TC(50)) of L929 cells was measured using the MTT assay and guided the selection of subtoxic doses for evaluation of DNA damage. Ph-Sil was more cytotoxic than OMP-5, Cyracure UVR-6105 and Sil-Mix. However, the TC(50) value of Tet-Sil could not be determined due to its poor solubility. DNA damage was evaluated in the sister chromatid exchange (SCE) assay with CHO cells, and the alkaline comet assay with L929 cells. In contrast to the siloranes, the oxiranes exhibited significant increases (p>0.05) in SCE frequencies and DNA migration relative to their solvent controls. Our findings support previous reports that siloranes have low genotoxic potential and can be suitable components for development of biomaterials.     

Assessment of cytotoxicity and DNA damage exhibited by siloranes and oxiranes in cultured mammalian cells

The potential reactivity and structural properties of oxiranes (epoxides) are advantageous when considering polymers for medical devices. However, epoxy compounds are widely known to have genotoxic properties. The objective of the study was to evaluate the cytotoxicity and primary DNA damage effects induced by oxiranes and siloranes, silicon containing oxiranes. The siloranes, Ph-Sil, Tet-Sil, and Sil-Mix and the oxiranes Cyracure™ UVR-6105 and 1,3-bis[2-(2-oxiranylmethyl) phenoxy]pentane (OMP-5) were dissolved in organic solvents and dilutions containing less than 0.5% solvent were used in biological assays. The concentration that reduced the viability of 50% (TC₅₀) of L929 cells was measured using the MTT assay and guided the selection of subtoxic doses for evaluation of DNA damage. Ph-Sil was more cytotoxic than OMP-5, Cyracure™ UVR-6105 and Sil-Mix. However, the TC₅₀ value of Tet-Sil could not be determined due to its poor solubility. DNA damage was evaluated in the sister chromatid exchange (SCE) assay with CHO cells, and the alkaline comet assay with L929 cells. In contrast to the siloranes, the oxiranes exhibited significant increases (p > 0.05) in SCE frequencies and DNA migration relative to their solvent controls. Our findings support previous reports that siloranes have low genotoxic potential and can be suitable components for development of biomaterials.     

Implications of oxidative stress in the cytotoxicity of Pseudomonas aeruginosa ExoU

ExoU PLA2-like activity has been shown to account for membrane lysis and acute death of infected cells. Translocation of effector proteins by the type III secretion systems depends on close contact between microbial and host cells. Our finding that both the ExoU-producing PA103 Pseudomonas aeruginosa and its mutant obtained by deletion of exoU adhered poorly to endothelial cells (EC) led to the hypothesis that, in some cells, the amount of injected toxin may not be enough to induce cell lysis but cells would suffer from a long-term effect of ExoU intoxication. To address this question, cells were exposed to both bacteria for 1 h and then treated with gentamicin-containing medium, to eliminate infecting microorganisms. After 24 h, the percentage of viable EC in PA103-infected cultures was significantly lower than in cultures exposed to the mutant, as determined by the MTT assay. Cell death was not likely to depend on the ExoU lytic activity since cell labeling with propidium iodide was similar in cultures infected with both bacterial strains. Bacterial cytotoxicity was significantly reduced by MAFP, a specific inhibitor of cPLA2 and iPLA2. Since the PLA2 activity on membrane phospholipids generates free fatty acid, including arachidonic acid (AA), we next compared the bacterial ability to release AA from infected EC. PA103 was shown to induce a potent AA release that was inhibited by MAFP. AA oxidation by oxygenases generates eicosanoids, known to induce both cell death and proliferation. However neither inhibitors of cyclooxygenases (ibuprofen) nor lipoxygenases (NDGA) reduced the ExoU toxicity. Since non-enzymatic oxidation of AA generates reactive radicals, we next investigated the PA103 ability to induce oxidative stress in infected cells. FACS analysis of cell labeling with the C-11 fluor probe and with anti-4-hydroxynonel antibody revealed a significant peroxidation of cell membrane lipids. These results, together with our finding that PA103-infected EC death was significantly attenuated by alpha-tocopherol, led to the conclusion that AA-induced oxidative stress may be another mechanism of cell damage in the course of infection by ExoU-producing P. aeruginosa.     

Improvement of the preliminary classification of Corynebacterium diphtheriae v. gravis

In this study the previously published preliminary scheme for the subdivision of toxigenic and nontoxigenic Corynebacterium diphtheriae, classified with cultivar gravis, is made more precise. 3 groups remain in this scheme: I, II and III; each of them contains toxigenic C. diphtheriae (subgroup a) and nontoxigenic precursors of C. diphtheriae (subgroup b). For the first time nontoxigenic analogs of C. diphtheriae, phagovar OPQSTg, have been introduced into group I and newly discovered toxigenic C. diphtheriae, phagovar K, with their nontoxigenic precursors converted by phages 5 tox+, 6 tox+ and W tox+ have been introduced into group III. Group IV has been provisionally excluded from the scheme because this group comprises a small number of strains (3 strains). This classification can already be used in research practice for a finer differentiation of strains classified with cultivar gravis and for correct epidemic orientation.