Suppressive Activity of Streptomycin on the Growth of Mycobacterium lepraemurium in Macrophage Cultures

The effect of streptomycin on the growth of an obligate intracellular bacterium was studied in a new host-parasite cell system. The system consisted of Mycobacterium lepraemurium grown in cultures of mouse peritoneal macrophages. Since these organisms do not grow in bacteriological media, the influence of extracellular bacterial growth can be ruled out. The suppressive activity of streptomycin was observed in a total of five experiments. At the end of 4 weeks, the average number of organisms per macrophage for the controls was 65.7; for cultures with streptomycin at concentrations of 0.5, 1, 5, 10, 50, and 100 mug/ml of medium, it was 45.4, 38.3, 28.7, 22.7, 13.4, and 8.2, respectively. A good dose-response relationship was evident. M. lepraemurium which had been treated in macrophage cultures with various concentrations of the antibiotic for 6 to 8 weeks was used to infect fresh macrophages. These cultures were in turn treated with streptomycin. Resistance of the organisms to streptomycin did not occur.     

Growth of Mycobacterium lepraemurium in Cultures of Mouse Peritoneal Macrophages

Successful growth of Mycobacterium lepraemurium was observed in cultures of mouse peritoneal macrophages. The optimal host cell maintenance medium was composed of 40% horse serum, 50% of the chemically defined medium NCTC 109, and 10% of a 1:5 dilution of beef embryo extract, supplemented with both liver extract and ferric nitrate. Multiplication of the bacilli was observed in 1 week and maximal growth in 6 to 7 weeks. All macrophages were filled with tens to hundreds of the organisms in cultures showing maximal growth. Glycerol caused an increase in the normal length of M. lepraemurium, without a corresponding increase in the number of the bacilli. Elongation of M. lepraemurium was observed 3 or 4 days after infection. Rapid and uniform growth of M. lepraemurium was achieved in serially transferred cultures (subcultures). The cumulative increase of the number of intracellular bacilli was 1.4 x 10(20)-fold in 14 transfers over a period of 68 weeks in one series, and 10(17)-fold in 12 transfers over a period of 56 weeks in another series. The generation time of M. lepraemurium was 7 days, a growth rate which approximates the fastest growth of the organisms in vivo. Organisms harvested from cultures at various stages of growth produced murine leprosy in mice, but showed no growth in bacteriological media. The present model offers an opportunity for studies on the host-parasite relationship without the complication of extracellular growth of the parasites.     

The Effect of Hyperglycaemia on In Vitro Cytokine Production and Macrophage Infection with Mycobacterium tuberculosis

Type 2 diabetes mellitus is an established risk factor for tuberculosis but the underlying mechanisms are largely unknown. We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis. Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22. Macrophage differentiation under hyperglycaemic conditions of 25 mmol/L glucose was also associated with increased cytokine production upon stimulation with M. tuberculosis lysate and LPS but in infection experiments no differences in M. tuberculosis killing or outgrowth was observed. The phagocytic capacity of these hyperglycaemic macrophages also remained unaltered. The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.     

In Vitro Growth of Rat Blastocyst under the Effect of Azathioprine

Rat blastocysts were isolated from the uterus on the 5th day after fertilization and set in culture. The effect of azathioprine (Imuran) on the blastocyst's development and on the early trophoblastic differentiation in vitro was investigated. Azathioprine, added to the medium of the blastocyst culture at various concentrations, dose dependently arrested development and had definite cytotoxic effect. In order to study the mechanism of action, a minimal dose of 5 μg/ml, which allowed the survival of about 60% of the blastocysts, was added to the medium after 48 hr of culturing. Under the effect of the substance the area of the spreading blastocyst cells was significantly restricted.
It was found, by autoradiographic methods, that the azathioprine affects the development by restraining DNA synthesis in the throphoblastic cells. Concomitantly RNA synthesis was inhibited and protein synthesis was reduced. The observations indicate, that the impairment of the in vitro differentiation of the blastocysts can be a result of the intracellular inhibitory action of the substance.     

The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.     

In Vitro Assay of Endotoxin by the Inhibition of Macrophage Migration

A quantitative in vitro technique was used to compare the ability of different endotoxins to inhibit the migration of macrophages from explants of rabbit spleen cultured in a coagulated plasma medium. The order of potency was different from that observed in chick embryo assays, and in assays with mice, of the same endotoxins. In general, however, the sensitivity of the macrophage inhibition test was comparable to that of other bioassay methods. A highly purified endotoxin from Salmonella enteritidis (Ribi) in a concentration of 0.004 mug/ml regularly inhibited macrophage migration. The in vitro method was used to detect a progressive loss of biological activity in fractions obtained during acid hydrolysis of the purified endotoxin. The selective toxicity of very low concentrations of endotoxin for mammalian macrophages was important in estimating the degree of specificity of the reaction. The pattern of cellular response in explant cultures made it possible to differentiate endotoxic damage from the specific cytotoxic action of antigen associated with delayed hypersensitivity.     

Cell Wall of Mycobacterium lepraemurium Strain Hawaii

The chemical properties of the cell wall of Mycobacterium lepraemurium strain Hawaii were investigated. Five subunits of the cell wall, arabinose mycolate, mycolic acids, tetrapeptide (Ala-Gln-diaminopimelic acid-Ala), disaccharide (N-acetylglucosaminyl-beta-1,4-N-glycolylmuramic acid), and arabinogalactan, were obtained, and their chemical structures were identified.     

Studies of Mycobacterium lepraemurium in Cell Culture

A serially diluted bacterial suspension of the Kurume-42 strain of Mycobacterium lepraemurium maintained for 1255 days in a mouse foot pad (MFP) cell culture was inoculated in mice subcutaneously. The ID₅₀ value was estimated at more than 10.7 and less than 85 organisms, indicating that pathogenicity of the organism had been maintained well in a long-term cell culture. The cells infected and maintained for a long period in the cell culture showed all the stages of cell mitosis. This suggests that the bacterial increase in cell cultures of M. lepraemurium is not only due to rephagocytosis of the bacilli released from the infected cells but also to a constant intracellular growth cycle of the bacilli accompanied by mitosis of the infected cells. In acid phosphatase activity, no appreciable differences were noted between the infected and uninfected cells as far as the present cell culture system was concerned. Most of the bacilli within the cells were ultrastructurally normal. Solid bacilli in phagosomes were surrounded by less electron-dense clear zones.     

Studies of Mycobacterium lepraemurium in Cell Culture

A serial increase in the number of Mycobacterium lepraemurium with successful subcultures has been obtained in the mouse foot pad (MFP) cell culture. Special attention has been given to maintaining the infected cells for longer periods; 1) the infected cells were incubated at 30 C rather than at 37 C, and 2) the concentration of serum in the culture medium was reduced from 10 to 2% as soon as a monolayer growth of the transferred cells was obtained. There have been cumulative bacterial increases of 1.47 × 10¹⁷ and 1.84 × 10¹⁵ fold for the Kurume-42 strain during a period of 1255 days, and 2.23 × 10⁹ and 3.89 × 10⁵ fold for the Hawaiian strain during periods of 831 and 572 days. The overall generation times were estimated at 22.0, 24.8, 26.8, and 30.8 days, respectively. All attempts to grow the acid-fast bacilli obtained in cell cultures on artificial culture media have failed. The ability of the organisms to produce typical lesions in mice has been well preserved.     

Inhibition of Macrophage Phagocytosis by Pseudomonas aeruginosa Rhamnolipids In Vitro and In Vivo

Patients with cystic fibrosis often have chronic and ultimately lethal pulmonary infections with Pseudomonas aeruginosa. In order to understand why these bacteria resist pulmonary clearance, we have investigated the interaction of P. aeruginosa and phagocytic cells. In an earlier study we reported that sub-lytic concentrations of two glycolipids produced by P. aeruginosa (the mono- and dirhamnolipids) caused structural changes in human monocyte-derived macrophages, and at lower concentrations inhibited the phagocytosis of Staphylococcus epidermidis by these cells. In the present study we demonstrate that rhamnolipids also inhibit the in vitro phagocytosis of both P. aeruginosa and Saccharomyces cerevisiae by thioglycollate-elicited mouse peritoneal macrophages. Using lucifer yellow to label the lysosomal compartments of macrophages, we determined that rhamnolipids interfere with the internalization of attached particles and reduce the level of phagosome-lysosome fusion of internalized targets within macrophages. We also demonstrate that physiologically relevant concentrations of rhamnolipids injected intratracheally into rat lungs inhibited the response of alveolar macrophages to a challenge of zymosan particles in vivo. These studies further demonstrate the profound inhibitory effects of P. aeruginosa rhamnolipids on macrophage function and are consistent with our hypothesis that the in situ production of these rhamnolipids directly contributes to the persistence of this pathogen in cystic fibrosis patient lungs. Received: 15 December 1995 / Accepted: 22 January 1996     

Impact of Zinc Oxide Nanoparticles on Pomegranate Growth under In Vitro Conditions

Russian Journal of Plant Physiology - Nanotechnology is a promising tool to achieve great advancements in the global agricultural systems and food production. The widespread application of...     

Radiometric Measurement of Metabolic Activity of Mycobacterium lepraemurium

A sensitive and nondestructive radiometric method has been applied to the detection of metabolism of Mycobacterium lepraemurium, as a model for the study of the metabolism and substrate requirements of M. leprae. The method is based on the measurement of the (14)CO(2) produced through the bacterial conversion of [U-(14)C]acetate or [U-(14)C]glycerol by 7 x 10(9) bacteria suspended in 10 ml of either a simple buffer system (K-36) or a complex medium (NC-5). Metabolism of the bacilli was easily detected within 3 days after inoculation and was measured daily. NC-5 medium supported metabolism of M. lepraemurium for several weeks longer than the simple K-36 buffer. The radiometric technique shows promise as a rapid and efficient system for evaluating the metabolism of mycobacteria without introducing any changes in the physiologic state of the organisms, studying their metabolic pathways, determining conditions potentially favorable for multiplication of these organisms in vitro, and studying their susceptibility to inhibition by drugs.     

Comparative ultrastructure of Mycobacterium leprae and Mycobacterium lepraemurium cell envelopes.

The structural properties of the cell envelopes of Mycobacterium leprae and Mycobacterium lepraemurium were investigated by freeze-fracture, freeze-etching, and negative-staining techniques. Freeze-fracture split the cell wall and exposed the internal features of the peptidoglycolipid mycosidic filamentous network. The cell membrane was also split into two asymmetric faces. The external fracture face was characterized by linear arrays of intramembranous particles, whereas the protoplasmic fracture face showed randomly distributed clusters of particulate entities. Comparative analysis of the ultrastructural features observed in M. leprae and M. lepraemurium indicated that the organization of the cell envelope in these two species differed particularly with respect to the amount and complexity of the superficial peptidoglycolipid and mycosidic integument, which is poorly developed in the mycobacterium responsible for human disease.     

In Vitro Cultivation of Borrelia duttonii on Cultures of SflEp Cells

Borrelia duttonii strain 406 K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0 × 10⁵ organisms per ml from infected mice, were 23-fold and 150-fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.