Cultured Host Cells Research Articles

Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium.

Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid-like disease in mice, uses numerous virulence factors to infect its hosts. Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro. Many virulence genes are specifically induced at particular sites during infection or in cultured host cells. The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host. In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo. Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co-ordinate the appropriate expression of virulence factors at specific sites in vivo.

Assessment of immunity to mycobacterial infection with luciferase reporter constructs.

Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

An Edwardsiella tarda strain containing a mutation in a gene with homology to shlB and hpmB is defective for entry into epithelial cells in culture.

Edwardsiella tarda is an enteric pathogen that causes diarrhea, wound infections, and death due to septicemia. This species is capable of invading human epithelial cell lines, and we have now been able to follow the entry and replication of E. tarda within tissue culture host cells. E. tarda escapes from the endocytic vacuole within minutes of entry and then replicates within the cytoplasm. Unlike other well-studied bacteria that replicate and reside in the cytoplasm, we never observed this organism moving directly from cell to cell; instead the bacteria spread by lysing the plasma membrane after several rounds of replication. Efforts to study the interactions of E. tarda with tissue culture cells are complicated by the presence of a potent cytotoxin that the bacterium produces. Using transposon mutagenesis, we isolated a noncytotoxic strain of E. tarda. This mutant is also defective for hemolysin production. The dual phenotype of this strain is consistent with the hypothesis that cytotoxicity is due to the previously characterized E. tarda hemolysin activity. The nonhemolytic strain is also unable to enter HEp-2 cells. The disrupted gene has sequence similarity to members of a family of genes required for transport and activation of the hemolysin genes, shlA and hpmA. A cosmid bearing 40 kb of E. tarda DNA, including wild-type copies of the E. tarda homologs of the transporter-activator protein and the hemolysin itself, confers hemolytic, cytotoxic, and invasive abilities upon normally nonhemolytic, noncytotoxic, and noninvasive strains of Escherichia coli. Sequence data indicate that the genes required for hemolytic activity are linked to a transposable element, suggesting that they arose in the E. tarda genome by horizontal transfer.

Synthesis of DNA, rRNA, and protein by Rickettsia prowazekii growing in untreated or gamma interferon-treated mouse L929 cells

The syntheses of DNA, rRNA, and protein by Rickettsia prowazekii growing in mouse fibroblastic L929 cells were measured at various times after the addition of gamma interferon (IFN-gamma) to correlate the inhibition of a site of macromolecular synthesis with the established IFN-gamma-induced inhibition of rickettsial growth. A method was developed to measure the syntheses of DNA, rRNA, and protein by R. prowazekii during a 2-h pulse-labeling period while the rickettsiae were growing within cultured host cells that had intact macromolecular synthesis. This method involved incubation of the rickettsia-infected cells with a radioactive precursor (H3 32PO4 or Tran35S-label), purification of the rickettsiae, purification of rickettsial nucleic acids, and analysis of rickettsial nucleic acids and proteins by electrophoresis and autoradiography. A key feature of the method involved the use of calculated specific activities from a densitometric analysis of gels and autoradiograms, a procedure that made the data independent of rickettsial recovery. Rickettsial DNA and rRNA syntheses were both inhibited 12 h after the addition of IFN-gamma to infected cultures, whereas the synthesis of rickettsial proteins was not inhibited at this time. In contrast, at 20 h after the addition of IFN-gamma, rickettsial DNA, rRNA, and protein syntheses were all inhibited.

Interaction of chlamydiae and host cells in vitro

The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models.

Interaction of chlamydiae and host cells in vitro.

The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models.

Spread and Persistence of Infection with a Trachoma Biovar Strain of Chlamydia trachomatis in Multiplying and Nonmultiplying McCoy Cells

The trachoma biovar of Chlamydia trachomatis enters host cells in culture with difficulty, and cell-to-cell spread resulting in amplification of the initial growth usually does not occur. An experimental model was devised to demonstrate that, by using conditions that more closely approximate those encountered in man, the trachoma biovar of C. trachomatis can readily achieve cell-to-cell passage. Fresh McCoy cells were sequentially added to monolayers that had been inoculated with a trachoma biovar strain of C. trachomatis 3, 6, and 9 days earlier. Subsequent incubation resulted in intercellular propagation, with an increase in the number of inclusions from 500 to 80,000 per coverslip. A second experiment demonstrated the reproducibility of this phenomenon and showed that cell-to-cell spread can occur at a low rate in overcrowded, not overlaid, cell layers; it also showed that, in multiplying cell layers, the infection tends to become persistent.

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites.

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.

New Soluble-Formazan Assay for HIV-1 Cytopathic Effects: Application to High-Flux Screening of Synthetic and Natural Products for AIDS-Antiviral Activity

We have developed an effective and optimally safe microculture method for rapid and convenient assay of the in vitro cytopathic effects of human immunodeficiency virus (HIV-1) on human lymphoblastoid or other suitable host cells. The assay procedure is applicable to the evaluation of drug effects on in vitro infections induced directly in cultured host cells by cell-free HIV-1 or by coculture with H9 cells chronically infected with HIV-1. The assay uses a newly developed tetrazolium reagent that is metabolically reduced by viable cells to yield a soluble, colored formazan product measurable by conventional colorimetric techniques. This simple microassay minimizes the number of plate manipulations typically required with other assay methods and, coupled with computerized data collection and analysis, facilitates large-scale screening of agents for potential antiviral activity. To support and enhance the discovery of new anti-HIV-1 agents, the National Cancer Institute is offering investigators worldwide the opportunity to submit new candidate agents for anti-HIV-1 screening with this method.

Structural bases of the cytolytic mechanisms of Entamoeba histolytica.

The cellular bases of the powerful cytolytic activity of the human protozoan parasite Entamoeba histolytica were explored by studying the effect of the virulent strain HM1:IMSS on epithelial monolayers of MDCK cells using a combination of time-lapse microcinematography and transmission and scanning electron microscopy. Early alterations of the epithelial cell membranes were detected by measuring changes in the transepithelial electrical resistance of MDCK monolayers mounted in Ussing chambers. The aggressive mechanism of E. histolytica trophozoites was found to be a complex, multifactorial phenomenon that included hit-and-run damage to the plasma membrane of effector cells mediated through contact, phagocytosis of lysed or apparently intact, but detached, MDCK cells, and intracellular degradation of ingested cells. Following contact with amebas, the epithelial monolayers showed a pronounced lowering of transepithelial resistance, opening of tight junctions, distortion of microvilli, surface blebbing, and the presence of minute focal discontinuities in the plasma membrane. There was no evidence of amebic exocytosis, membrane fusion, or junction formation between the parasite and host plasma membranes. Although modifications in the epithelial cell membranes usually preceded lysis, the cytolytic activity of the parasite did not exclusively involve damage to the plasma membrane of the cultured host cells but also was mediated by avid phagocytosis, the displacement and separation of neighboring cells by means of pseudopodial activity, and the "pinching-off" of the peripheral cytoplasm of epithelial cells.

Monoclonal antibody with hemagglutination, immobilization, and neutralization activities defines an immunodominant, 47,000 mol wt, surface-exposed immunogen of Treponema pallidum (Nichols).

Radioimmunoprecipitation (RIP) analyses performed on 125I-surface-labeled Treponema pallidum cells using various immune sera revealed the presence of six major surface antigens (immunogens) with apparent molecular weights of 47 K, 36 K, 34 K, 32 K, 29 K, and 13 K. Among these, the 47 K surface antigen was most abundant. Radioimmunoprecipitation assays using 125I-labeled T. phagedenis biotype Reiter or immunoblot analyses using the same strain, failed to reveal the presence of the 47 K mol wt antigen in the representative nonpathogenic treponeme. Preabsorption of anti-T. pallidum immune rabbit serum (IRS) with the Reiter organism did not remove anti-T. pallidum antibodies from immune serum that reacted with the 47 K mol wt immunogen or other immunogens of T. pallidum present in the characteristic antigenic profile. Monoclonal antibodies (mAb) directed specifically against the 47 K mol wt immunogen of T. pallidum also failed to react with an analogous 47 K mol wt component in Treponema phagedenis biotype Reiter, further suggesting the unique presence of this antigen in pathogenic treponemes. The presence of the 47 K mol wt surface immunogen in pathogenic treponemes other than T. pallidum subspecies pallidum was also observed (43). Anti-47 K immunogen mAb was nonreactive against rabbit IgG or IgM. mAb directed specifically against the 47 K mol wt immunogen of T. pallidum was examined for strategic functional activities. It was found to be reactive in the microhemagglutination assay for T. pallidum antibodies, the T. pallidum immobilization test, and was found to be capable of significant blockage of attachment of virulent T. pallidum to host cells in tissue culture. Additional significant biological activity for the anti-47 K mol wt immunogen mAb was revealed through results of the in vitro-in vivo neutralization test of Bishop and Miller, in which a 99% or 100% neutralizing activity was demonstrated. The combined data of this study suggest that the 47 K mol wt immunogen of T. pallidum represents an abundant, immunodominant, surface-exposed immunogen possessing potential biological importance in the pathogenesis and immunology of T. pallidum infection. These studies serve to establish the first functionally defined immunogen for T. pallidum, which may represent the major immunogen of the organism.

CLINICAL AND LABORATORY EVALUATION OF IDOXURIDINE (IDU) THERAPY IN HERPES SIMPLEX KERATITIS.

Introduction In 1960 Tamm 1 reported that inhibitors of DNA synthesis could suppress the multiplication of herpes simplex virus in tissue culture. The treatment of herpes keratitis with idoxuridine (5-iodo-2-desoxyuridine, IDU) however, was first suggested by Kaufman. 2 He and other authors have reported their generally good results in subsequent communications 3-16 in experimentally induced disease in rabbits as well as in the naturally occurring infection in man. Attempts to repeat this work yielded somewhat less encouraging results in our hands and, therefore, a more extensive investigation was initiated. Our studies reported in this paper include: (1) the effect of idoxuridine on herpes keratitis in rabbits and patients, correlated with the presence or absence of demonstrable virus; (2) the influence of the immune status of rabbits on the clinical and virological course of the disease; and (3) the influence of idoxuridine on virus and host cells in a tissue culture

A Biologic Comparison of Two Strains of Herpesvirus Hominis

Summary Two strains of Herpesvirus hominis which differ primarily in the cytopathic effects they induce in various host cells in tissue culture have been described. One strain (GC) causes the formation of syncytial giant cells, whereas the other strain (P) produces rounding and piling up of the cells with a few small giant cells but no syncytia. A number of other characteristics of these two strains were studied. Each was able to grow to the same titer in several lines of cultured cells. Their rates of multiplication were similar and new virus appeared in the medium between 5 and 7 hr after inoculation. Each was similarly inactivated by heat, the half life being 1 hr and 30 min at 37°C and 3 hr and 48 min at 30–31°C. Each was inactivated by u.v. light at the same rate with a half life of 7 sec. There was no difference in the rate of adsorption or penetration, 87% and 91% being adsorbed and 100% and 88% of the adsorbed P and GC strains, respectively, having penetrated within 2 hr at 37°C. Both strains were equally infective for mice and rabbits. They did not differ in antigenicity. The electronmicroscope showed particles of identical size (medians 154 mμ and 157 mμ respectively). G6P dehydrogenase and hexokinase activities in cells infected with either strain were increased over controls at 12 hr after inoculation, indicating increased permeability of cell membranes. At 3 and 6 hr after inoculation the hexokinase activity in GC infected cells differed from that in P infected cells. Some observations have been made on the toxic effect of neutral red on RK cells.