Changes of the phagosomal elemental concentrations by Mycobacterium tuberculosis Mramp

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 323-332 ◽  
Author(s):  
Dirk Wagner ◽  
Jörg Maser ◽  
Ivana Moric ◽  
Neio Boechat ◽  
Stefan Vogt ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Divalent Cations ◽  
Peritoneal Macrophages ◽  
Bacterial Survival ◽  
Knockout Mutant ◽  
Elemental Concentrations ◽  
Restricted Environment ◽  
Mouse Peritoneal Macrophages ◽  
Strain H37rv

Pathogenic mycobacteria survive within phagosomes which are thought to represent a nutrient-restricted environment. Divalent cation transporters of the Nramp family in phagosomes and mycobacteria (Mramp) may compete for metals that are crucial for bacterial survival. The elemental concentrations in phagosomes of macrophages infected with wild-type Mycobacterium tuberculosis (M. tuberculosis strain H37Rv) and a M. tuberculosis Mramp knockout mutant (Mramp-KO), derived from a clinical isolate isogenic to the strain MT103, were compared. Time points of 1 and 24 h after infection of mouse peritoneal macrophages (bcg S) were compared in both cases. Increased concentrations of P, Ni and Zn and reduced Cl concentration in Mramp-KO after 1 h of infection were observed, compared to M. tuberculosis vacuoles. After 24 h of infection, significant differences in the P, Cl and Zn concentrations were still present. The Mramp-KO phagosome showed a significant increase of P, Ca, Mn, Fe and Zn concentrations between 1 and 24 h after infection, while the concentrations of K and Ni decreased. In the M. tuberculosis vacuole, the Fe concentration showed a similar increase, while the Cl concentration decreased. The fact that the concentration of several divalent cations increased in the Mramp-KO strain suggests that Mramp may have no impact on the import of these divalent cations into the mycobacterium, but may function as a cation efflux pump. The concordant increase of Fe concentrations within M. tuberculosis, as well as within the Mramp-KO vacuoles, implies that Mramp, in contrast to siderophores, might not be important for the attraction of Fe and its retention in phagosomes of unstimulated macrophages.

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

1975 ◽  
Vol 142 (1) ◽  
pp. 1-16 ◽  
Author(s):  
J A Armstrong ◽  
P D Hart
Keyword(s):  
Electron Microscopy ◽  
Rabbit Antiserum ◽  
Peritoneal Macrophages ◽  
Bacterial Survival ◽  
Virulent Challenge ◽  
Secondary Lysosomes ◽  
High Level ◽  
Mouse Peritoneal Macrophages ◽  
Specific Rabbit ◽  
Strain H37rv

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.

Phagocytosis by macrophages. II. The dissociation of the attachment and ingestion steps

1981 ◽  
Vol 51 (1) ◽  
pp. 189-201
Author(s):  
T. Ito ◽  
M.J. Ueda ◽  
T.S. Okada ◽  
S. Ohnishi
Keyword(s):  
Cell Surface ◽  
Divalent Cations ◽  
Peritoneal Macrophages ◽  
Metabolic Inhibitors ◽  
Assay Method ◽  
Coated Particles ◽  
Spin Resonance ◽  
Mouse Peritoneal Macrophages ◽  
Phagocytic Reaction ◽  
Number Of Particles

The phagocytic process of mouse peritoneal macrophages was dissociated, using bovine serum albumin (BSA)-coated particles containing spin-labelled cholestanone, into 2 steps: attachment of particles to the cell surface and ingestion of the particles into the cytoplasm. The number of particles was estimated from electron spin resonance (e.s.r.) measurements. The particles ingested into the cytoplasm were distinguished from those attached to the cell surface by treatment with a membrane-impermeable reducing agent, ascorbate. The validity of the assay method was tested under various conditions. The measurements provided accurate and reproducible data. The phagocytic reaction was followed as a function of time and the rate constants for the attachment and ingestion steps were obtained from the initial phase. Both steps were highly dependent on temperature. Divalent cations in the incubation medium were essential for the attachment step but apparently had no effect on the ingestion step. The metabolic inhibitors, KCN and 2-deoxyglucose, inhibited both steps. Cytochalasin B inhibited both steps, while colchicine inhibited only the attachment step but apparently had no effect on the ingestion step.

Multiplication of Mycobacterium tuberculosis Within Normal and “Immune” Mouse Macrophages Cultivated With and Without Streptomycin

1970 ◽  
Vol 1 (1) ◽  
pp. 30-40
Author(s):  
Ronald J. Patterson ◽  
Guy P. Youmans
Keyword(s):  
Tissue Culture ◽  
Mycobacterium Tuberculosis ◽  
Culture Medium ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocytes ◽  
Immune Mouse ◽  
Mouse Macrophages ◽  
Intracellular Multiplication ◽  
Mycobacterial Growth ◽  
Mouse Peritoneal Macrophages

Acquired cellular immunity to infection with Mycobacterium tuberculosis is believed to reside in the capacity of mononuclear phagocytes of immunized animals to inhibit intracellular multiplication of the parasite. However, in macrophage tissue culture systems, it has been customary to employ streptomycin in the medium for the purpose of restricting extracellular, but not intracellular, growth of M. tuberculosis. In contrast, our data show that small amounts of streptomycin markedly inhibit intracellular as well as extracellular growth of M. tuberculosis in normal mouse peritoneal macrophages, and that the degree of this inhibition is directly proportional to the concentration of streptomycin used. In the absence of streptomycin, virulent tubercle bacilli grew as rapidly in “immune” macrophages as in normal macrophages. “Immune” macrophages, however, were slightly more resistant to destruction by the intracellularly multiplying mycobacteria. In the presence of streptomycin, however, intracellular mycobacterial growth was inhibited more in “immune” macrophages than in normal macrophages, and this effect also was directly proportional to the concentration of streptomycin used. Virulent mycobacteria grew somewhat more slowly within mouse peritoneal macrophages obtained after induction of a peritoneal exudate with glycogen than in noninduced cells. The rate of multiplication, though, was the same within normal and “immune” induced peritoneal cells except in the presence of streptomycin. As with noninduced macrophages, this drug inhibited the intracellular multiplication of virulent tubercle bacilli more effectively within “immune” induced than within normal induced cells. It would appear, therefore, that the greater inhibition of intracellular multiplication of virulent tubercle bacilli in “immune” macrophages in tissue culture noted by a number of investigators in the past may have been an artifact created by the use of streptomycin in the tissue culture medium.

scholarly journals RECEPTORS FOR COMPLEMENT ON LEUKOCYTES

1968 ◽  
Vol 128 (5) ◽  
pp. 991-1009 ◽  
Author(s):  
Waltraut H. Lay ◽  
Victor Nussenzweig
Keyword(s):  
Divalent Cations ◽  
Peritoneal Macrophages ◽  
Red Cells ◽  
Enzyme Treatment ◽  
Rabbit Serum ◽  
Mouse Serum ◽  
Polymorphonuclear Cells ◽  
Mouse Macrophages ◽  
Antibody Complex ◽  
Mouse Peritoneal Macrophages

Sheep red blood cells sensitized by 7S, but not by 19S rabbit anti-Forssman antibodies, adhere and form rosettes on mouse macrophages and on a few monocytes and polymorphonuclear cells (PMN). When, however, C' factors from mouse serum are added to the antigen-19S antibody complex (EAC'), rosettes are formed on most mouse peritoneal macrophages and PMN and on a few monocytes. In addition EAC' also adheres to 10–25% of lymph node lymphocytes but not to thymus lymphocytes. EAC' prepared with 7S anti-Forssman antibodies has identical properties. The adherence of red cells induces an increase in the membrane activity of the leukocytes and causes injury to the red cells which rapidly become deformed and fragmented. Adherence of EAC' occurs at 37°C and is minimal at 4°C. Probably only the first four C' components are involved in this phenomenon as mouse serum deficient in C'5 or rabbit serum, deficient in C'6 can be used as a source of C' components. Treatment of EAC' with EDTA does not modify its leukocyte-adherence properties. The adherence of EAC' to the leukocytes is not inhibited in the presence of serum. The receptors for C' on macrophages, PMN, and monocytes differ from those found on lymphocytes. Rosette formation by EAC' on macrophages, PMN, and monocytes depends on divalent cations (Mg++) and can be reversed by Na3H EDTA, while adherence to lymphocytes is independent of these ions and occurs in the presence of 0.01 M Na3H EDTA. Both types of receptors for C' components are destroyed by trypsin treatment of the leukocytes, in contrast with the receptors for 7S antibodies on the same cells which persist after enzyme treatment.

scholarly journals Growth of Mycobacterium tuberculosis in a Defined Medium Is Very Restricted by Acid pH and Mg2+ Levels

2000 ◽  
Vol 68 (8) ◽  
pp. 4518-4522 ◽  
Author(s):  
Debra L. Piddington ◽  
Ali Kashkouli ◽  
Nancy A. Buchmeier
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Divalent Cations ◽  
Defined Medium ◽  
Acidic Ph ◽  
Mycobacterial Species ◽  
Acidic Media ◽  
Restricted Environment ◽  
Extreme Sensitivity ◽  
Acidic Conditions ◽  
Influence Of Ph

ABSTRACT Mycobacterium tuberculosis grows within the phagocytic vacuoles of macrophages, where it encounters a moderately acidic and possibly nutrient-restricted environment. Other mycobacterial species encounter acidic conditions in soil and aquatic environments. We have evaluated the influence of pH and divalent cation levels on the growth of M. tuberculosis and seven other mycobacterial species. In a defined medium, the growth of M. tuberculosis was very restricted by acidic pH. Higher levels of Mg2+ were required for growth of M. tuberculosis in mildly acidic media (pH 6.0 to 6.5) compared to pH 7.0 medium. The divalent cations Ca2+, Zn2+, or Mn2+ could not replace Mg2+ during growth at pH 6.25, but Ca2+could at least partially substitute for Mg2+ during growth at pH 7.0. Among eight species of mycobacteria tested, there was a diversity of growth rates in media with acidic pH and low Mg2+ levels. M. tuberculosis was the most restricted in growth at pH 6.0, and all of this growth required elevated levels of Mg2+. M. kansasii andM. smegmatis also grew very poorly in acidic media with limiting Mg2+. M. fortuitum, M. marinum, M. scrofulaceum, M. avium, andM. chelonae grew at pH 6.0 in an unrestricted manner. These results demonstrate that M. tuberculosis is unique among the mycobacteria in its extreme sensitivity to acid and indicate thatM. tuberculosis must acquire sufficient Mg2+ in order to grow in a mildly acidic environment such as within the phagosome of macrophages.

Immunostimulatory Effects of Purple Bamboo Salts Composed with Rubus coreanus in Raw264.7 Cells and Mouse Peritoneal Macrophages

2017 ◽  
Vol 46 (3) ◽  
pp. 306-313
Author(s):  
Heejeon Park ◽  
Sokho Kim ◽  
Sohee Jeong ◽  
Heeran Park ◽  
Jin-Hyung Kim ◽  
...  
Keyword(s):  
Peritoneal Macrophages ◽  
Raw264.7 Cells ◽  
Rubus Coreanus ◽  
Mouse Peritoneal Macrophages

Harvest and Culture of Mouse Peritoneal Macrophages

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (22) ◽  
Author(s):  
Mingfang Lu ◽  
Alan Varley
Keyword(s):  
Peritoneal Macrophages ◽  
Mouse Peritoneal Macrophages
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