Intracellular Growth Rate Research Articles

Overexpression of cloned genes using recombinant escherichia coli regulated by a T7 promoter: II. Two‐stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures.

Analysis of two-stage recombinant bacterial fermentations using a structured kinetic model

A structured kinetic model has been employed to analyze the performance of a two-stage continuous fermentation of a recombinant Escherichia coli. Separating the cell growth phase from the gene expression phase in two fermentors minimizes the growth rate difference between the recombinant cells and the plasmid-free cells in the first fermentor, thereby increasing the plasmid stability. The plasmid-harboring cells from the first fermentor are continuously fed into the second fermentor, in which the foreign protein synthesis is turned on by the addition of the inducer. Consequently, the recombinant cells experience an immediate reduction in growth rates as soon as they enter the second stage and then recover to synthesize the foreign protein. To analyze the fermentation performance contributed by these cells with different intracellular foreign protein levels and growth rates, a novel method for determining the residence time distribution of the growing cells in the second stage has been formulated. Combined with this method, the structured kinetic model for recombinant bacterial cells is used to predict the plasmid stability and foreign productivity at various operation conditions, such as induction strength and dilution rates. This model can provide us with thorough understanding of the characteristics of the two-stage fermentations, and is useful for the development of large scale continuous cultures of recombinant bacteria.

A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection

To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.

Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox- mutant of Shigella dysenteriae 1.

A Tox- mutant of Shigella dysenteriae 1, SC501, was genetically engineered by cloning the Shiga toxin operon, inserting a cassette into the A subunit gene, and exchanging this in vitro-mutagenized sequence with the wild-type gene. SC501 produced a low amount of residual cytotoxicity which was not neutralized by a rabbit immune serum directed against Shiga toxin. Invasion of cultured cells demonstrated that Shiga toxin had no effect on the rate of intracellular growth of bacteria or on the rapid killing of invaded host cells. On the other hand, several significant differences were observed in macaque monkeys infected intragastrically with either the wild-type strain or its mutant. The production of Shiga toxin by the invading strain was correlated with the presence of blood within stools, a sharp drop in blood polymorphonuclear cells, and histopathological alterations, such as the destruction of capillary vessels within the connective tissue of the colonic mucosa, severe inflammatory vasculitis of the peritoneal mesothelium, and major efflux of inflammatory cells to the intestinal lumen. It is proposed that Shiga toxin influences the severity of bacillary dysentery by inducing colonic vascular damage, which accounts for bloody stools, intestinal ischemia, and inflation of a polymorphonuclear intestinal compartment during the infectious process.

Growth of Mycobacterium avium in activated macrophages harvested from inbred mice with differing innate susceptibilities to mycobacterial infection.

The growth of Mycobacterium avium in macrophages obtained from Mycobacterium bovis BCG-infected mice was compared with that in macrophages from uninfected mice. BCG vaccination resulted in substantial macrophage activation, measured as increased acid phosphatase and superoxide anion production, as well as enhanced leishmanicidal activity. However, the activated macrophages were only able to reduce the rate of intracellular growth by Listeria monocytogenes and M. avium in vivo and did not express detectable levels of mycobactericidal activity in vitro. Exposure of the macrophage monolayers to concanavalin A-stimulated spleen cell supernatant fluid and lipopolysaccharide did not further enhance the ability of the BCG-activated macrophages to control the intracellular replication of the M. avium. Macrophages from BCG-infected C57BL/6 (BCGs) mice were quantitatively better able to control the intracellular replication of the M. avium challenge than were similar phagocytes obtained from BCGr (A/J) mice. These findings have important implications with respect to the expression of acquired resistance to these atypical mycobacterial infections.

On periodic algal cyclostat populations

A number of useful relations for population growth rate, uptake of limiting nutrient, amount of intracellular growth limiting nutrient and extracellular limiting nutrient concentration are presented in terms of their period averaged functions. These period averages may provide “species specific” information. The period length is shown to be unique for a given dilution rate and amount of intracellular growth limiting nutrient. A plot of residual limiting nutrient concentration versus cell density describes a closed trajectory (or “phase” loop). The area of this loop may be evaluated in terms of certain period averages of the parameters of the cyclostat population. This area is characteristic for a species at any given dilution rate and growth limiting nutrient concentration entering the cyclostat vessel. Equations for describing multispecies cyclostats are presented. Also, some necessary conditions for stable coexistence in such a system are described.

Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival.

Tubercle bacilli of the pathogenic human strain H37Rv had previously been shown to multiply, after ingestion by cultured mouse peritoneal macrophages, within phagosomes that tended to remain unfused with secondary lysosomes. Means were sought therefore for promoting experimentally a modification of the host response so as to attain a high level of phagolysosome formation, enabling tests to be made of any effects on the course and outcome of the intracellular infection. This was achieved by exposing viable bacilli to specific rabbit antiserum before their ingestion. Quantitative assessments, using electron microscopy, now showed that a majority of the phagosomes containing intact bacilli had fused with ferritin-labeled lysosomes, and frequently the fusion was massive. Bacterial viability studies established that the serum pretreatment was not itsel bactericidal. In the course of progressive infections with strain H37Rv, monitored by counts both of viable bacterial units and of intracellular acid-fast organisms, no appreciable difference was found between the intracellular growth rates of control and antiserum-treated bacilli. Concurrent electron microscopy showed that bacilli could remain intact and multiply both in phaagolysosomes and in unfused phagosomes, ruling out the possibility of selective growth of antiserum-pretreated bacilli within the minority of phagosomes that remained unfused. It was concluded that "turning on" phagosome-lysosome fusion in normal macrophages did not influence the outcome of infection with virulent M. tuberculosis; lysosome contents manifestly failed to exercise an antibacterial effect on this organism. Nevertheless, the possibility remains that the lysosomes of specific immune macrophages have antituberculous potentiality. In that case the experimental "turning on or off" of fusion could be a decisive factor in the outcome of a virulent challenge. Should it not be, the antibacterial capabilities of immune cells would need to be ascribed to factors other than lysosomal attack, the latter being essentially for disposal of the dead organisms.

Adaptation of potassium metabolism and restoration of mitosis during prolonged treatment of mouse lymphoblasts with ouabain.

The effects of ouabain on the growth of murine lymphoblasts in vitro have been studied. Exposure of cells to ouabain (0.1 mM) initially inhibited 86Rb+ uptake rate, reduced the intracellular potassium concentration, and decreased population growth rates. Continued exposure to the same ouabain concentration resulted in an increase of 86Rb+ uptake rate, intracellular potassium content and population growth rates to control values (adaptation). When treated cells were resuspended in medium free of ouabain after 12 to 15 hours of ouabain treatment, 86Rb+ uptake rates and intracellular potassium levels exceeded those of untreated cells. Adaptation was inhibited by cycloheximide (3 mug/ml) and by actinomycin D (0.05 mug/ml). Kinetic analysis of transport suggested that while the total capacity of the Na/, K+ transport system increased, the affinity for both the cation (86Rb+) and ouabain decreased.

INTRACELLULAR GROWTH OF HISTOPLASMA CAPSULATUM.

Howard, Dexter H. (University of California, Los Angeles). Intracellular growth of Histoplasma capsulatum. J. Bacteriol. 89:518-523. 1965.-Under a variety of experimental conditions, the generation time of Histoplasma capsulatum in mammalian histiocytes was remarkably constant. The rate of intracellular growth of five different isolates of H. capsulatum was very similar. The generation time of the fungus within guinea pig cells was nearly the same as it was within mouse cells under comparable conditions. Preliminary exposure of yeast cells to specific antibodies and complement did not affect intracellular proliferation of the fungus during the first 24 hr after phagocytosis by mouse histiocytes. The rate of growth of the fungus within cells from immunized mice and guinea pigs was comparable to that in cells from normal animals. This latter result was not altered by preliminary exposure of the fungus to hyperimmune serum and complement.

Phagocytosis of Microorganisms by Hela Cells

Summary Bovine fetal serum promotes the uptake of mycobacteria by HeLa cells and has been found superior to horse serum and chicken serum in infection medium. There has been little variation between lots. When bovine fetal serum has been used in the infection medium the subsequent intracellular growth rate of tubercle bacilli is faster than it is when horse or chicken serum is used in the infection medium. The time required to “fill” the cells with tubercle bacilli is shortened by about 20%. The phagocytosis of staphylococci and streptococci is also promoted by bovine fetal serum and by horse serum. Absorption with Zymosan of the bovine fetal serum before its inclusion in infection medium leads to more rapid intracellular growth of these organisms. Dilution of these organisms in broth, rather than salt solution, also increases the rate of their subsequent intracellular growth. A combination of two purified proteins, bovine fetuin and human albumin, also promotes the uptake of mycobacteria, staphylococci, and group A streptococci. A medium containing these two proteins appears to be the preferable infection medium for the growth of these organisms in HeLa cells.