Role of iron in Nramp1-mediated inhibition of mycobacterial growth.
(1/1859)
Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth. (+info)
Killing kinetics of intracellular Afipia felis treated with amikacin.
(2/1859)
Afipia felis is a facultative intracellular bacterium which multiplies in macrophages following inhibition of phagosome-lysosome (P-L) fusion. When A. felis-infected cells are incubated for 72 h with various antibiotics, only aminoglycosides are found to be bactericidal. We therefore studied the killing of intracellular A. felis by amikacin, and its relationship with the restoration of P-L fusion. Amikacin reduced the number of A. felis from 8.5 x 10(5) to 3.5 x 102 cfu/mL within 94 h. P-L fusion was restored after 30-40 h of incubation with amikacin. Both mechanisms may participate in the intracellular killing of bacteria. (+info)
Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan.
(3/1859)
Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = 0 became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms. (+info)
Phagosomes are fully competent antigen-processing organelles that mediate the formation of peptide:class II MHC complexes.
(4/1859)
During the processing of particulate Ags, it is unclear whether peptide:class II MHC (MHC-II) complexes are formed within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes. Murine macrophages were pulsed with latex beads conjugated with OVA. Flow or Western blot analysis of isolated phagosomes showed extensive acquisition of MHC-II, H-2M, and invariant chain within 30 min, with concurrent degradation of OVA. T hybridoma responses to isolated subcellular fractions demonstrated OVA (323-339):I-Ad complexes in phagosomes and plasma membrane but not within dense late endocytic compartments. Furthermore, when two physically separable sets of phagosomes were present within the same cells, OVA(323-339):I-Ad complexes were demonstrated in latex-OVA phagosomes but not in phagosomes containing latex beads conjugated with another protein. This implies that these complexes were formed specifically within phagosomes and were not formed elsewhere and subsequently transported to phagosomes. In addition, peptide:MHC-II complexes were shown to traffic from phagosomes to the cell surface. In conclusion, phagosomes are fully competent to process Ags and generate peptide:MHC-II complexes that are transported to the cell surface and presented to T cells. (+info)
Modulation of endocytosis in nuclear factor IL-6(-/-) macrophages is responsible for a high susceptibility to intracellular bacterial infection.
(5/1859)
Activated macrophages kill bacteria, a function known to depend on the expression of NF-IL-6. Here, it is demonstrated that the attenuated Brucella abortus vaccine strain 19 replicates much better in NF-IL-6-/- than in NF-IL-6(+/+) and NF-IL-6(+/+)-activated murine macrophages and at levels comparable to those observed in normal macrophages infected with the pathogenic strain 2308. The role of NF-IL-6 in the inhibition of intracellular bacterial replication is related to its control of endocytosis and membrane fusion between endosomes and Brucella-containing phagosomes. Addition of the granulocyte-CSF (G-CSF), whose induction is impaired in NF-IL-6(-/-) macrophages, restores both endocytosis and the morphology of endosomes, together with bactericidal activity. Regulation of membrane traffic in endocytosis by G-CSF whose expression is controlled by NF-IL-6 may explain how a host cell can control intracellular bacterial replication. (+info)
Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.
(6/1859)
An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation. (+info)
RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes.
(7/1859)
Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64-69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP-RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP-RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes. (+info)
Increased expression of Rab5a correlates directly with accelerated maturation of Listeria monocytogenes phagosomes.
(8/1859)
Previous studies have shown that Listeria monocytogenes (LM) modulates phagocytic membrane traffic. Here we explore whether Rab5a, a GTPase associated with phagosome-endosome fusion, is related to phagosome maturation and to the intracellular survival of LM. Stable transfection of Rab5a cDNA into macrophages accelerates intracellular degradation of LM. Morphological studies confirmed that phagosome maturation and phagosome-lysosome fusion is enhanced by overexpression of Rab5a. Down-regulation experiments using antisense oligonucleotides targeted to the Rab5a mRNA efficiently reduced Rab5a synthesis, reduced phagosome-endosome traffic, blocked phagosome-lysosome fusion, and extended intraphagosomal survival of LM. Down-regulation of Rab5a had no effect on LM internalization. Down-regulation of Rab5c had no effect on phagosome maturation and phagosome-lysosome fusion. The results indicate that Rab5a controls early phagosome-endosome interactions and governs the maturation of the early phagosome leading to phagosome-lysosome fusion. (+info)