cytochalasin-d and methylamine

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

Topics: Acanthamoeba; Animals; Artificial Gene Fusion; Bacterial Proteins; Base Sequence; Cycloheximide; Cytochalasin D; DNA Primers; Gene Expression; Genes, Bacterial; Genes, Reporter; Green Fluorescent Proteins; Immunophilins; Legionella pneumophila; Luminescent Proteins; Membrane Proteins; Methylamines; Peptidylprolyl Isomerase; Promoter Regions, Genetic; Superoxide Dismutase; Virulence

A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.

Topics: Acanthamoeba; Animals; Cell Division; Colony-Forming Units Assay; Culture Media; Cytochalasin D; Dose-Response Relationship, Immunologic; Host-Parasite Interactions; Legionella pneumophila; Methylamines; Temperature

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Topics: Analysis of Variance; Animals; Cell Line; Colony Count, Microbial; Cytochalasin D; Hartmannella; Humans; Legionella; Methylamines; Monocytes

The effect in vitro of some cytoplasmic structure and function inhibitors on the different stages of rabies virus infection was investigated. Treatment of fibroblasts (CER) and human neuroblastoma cells (IMR-32) with substances acting on low pH intracellular compartments (methylamine and monensin) prevented rabies virus genome delivery in the cytosol. An early inhibition of viral infection was also obtained in the presence of B and D cytochalasins and trifluoperazine which interact with microfilament structures. Treatment with colchicine and vinblastine did not affect rabies multiplication, suggesting that microtubules are not involved in this process. However, the multiplication of prebound virions did not take place in the presence of inhibitors of oxidative phosphorylation (sodium azide and CCCP) and of glycolysis (2-deoxy-D-glucose) indicating that rabies virus replication is largely energy-dependent in both host cells examined.

Topics: Animals; Azides; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Colchicine; Cytochalasin B; Cytochalasin D; Deoxyglucose; Dose-Response Relationship, Drug; Fibroblasts; Humans; Methylamines; Monensin; Neuroblastoma; Rabies virus; Sodium Azide; Trifluoperazine; Tumor Cells, Cultured; Vinblastine; Virus Replication

Human interferon alpha A, produced in Escherichia coli by recombinant DNA technology, was labeled with 125I to study its binding to receptors on human lymphoblastoid Daudi cells. This binding showed a marked temperature dependency, with maximum binding obtained at 30-37 degrees C. About 60% of the cell-bound radioactivity was released upon subsequent addition of unlabeled interferon, indicating that only part of the cell-bound interferon could be displaced by competitor. Moreover, about 30-50% of cell-bound interferon was not released by treating the cells with 0.2 N acetic acid, a procedure which removes polypeptide hormones on the cell surface, indicating that part of the interferon bound at 37 degrees C was internalized. This interferon was slowly degraded to acid-soluble products, which were released into the culture medium. Treatment of DAudi cells with the lysosomotropic amines chloroquine and methylamine inhibited the degradation of interferon. Methylamine, however, also inhibited the internalization of interferon. Daudi cells treated with interferon in the presence of chloroquine showed an increase in the interferon-induced enzyme 2',5'-oligo(A) polymerase comparable to that of cells treated with interferon alone. This enzyme increased to a similar extent in cells treated with interferon and cytochalasin, a drug which inhibited internalization of interferon by 50%. These results suggest that degradation and possibly internalization of interferon are not required for at least some of its biological activities.

Topics: Biological Transport; Burkitt Lymphoma; Cell Line; Chloroquine; Colchicine; Cytochalasin D; Cytochalasins; Humans; Interferon Type I; Kinetics; Lysosomes; Methylamines; Receptors, Cell Surface; Receptors, Interferon; Temperature