The Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 replicates in mature phagolysosomes within J774 macrophages

Adherent-invasive Escherichia coli (AIEC) bacteria isolated from Crohn's disease patients are able to extensively replicate within macrophages in large vacuoles. The mechanism by which AIEC bacteria survive within phagocytic cells is unknown. This report describes the maturation of AIEC LF82-containing phagosomes within J774 macrophages. LF82-containing phagosomes traffic through the endocytic pathway as shown by the sequential acquisition and loss of EEA1 and Rab7 and by accumulation of Lamp-1, Lamp-2 and cathepsin D. We demonstrated that AIEC LF82-containing phagosomes mature into active phagolysosomes where bacteria are exposed to low pH and to the degradative activity of cathepsin D. Finally, we showed that an acidic environment is necessary for replication of AIEC LF82 bacteria within J774 macrophages. Thus, evidence is provided that AIEC LF82 bacteria do not escape from the endocytic pathway but undergo normal interaction with host endomembrane organelles and replicate within acidic and cathepsin D-positive vacuolar phagolysosomes.     

Baseline mechanical characterization of J774 macrophages

Macrophage cell lines like J774 cells are ideal model systems for establishing the biophysical foundations of autonomous deformation and motility of immune cells. To aid comparative studies on these and other types of motile cells, we report measurements of the cortical tension and cytoplasmic viscosity of J774 macrophages using micropipette aspiration. Passive J774 cells cultured in suspension exhibited a cortical resting tension of ∼0.14 mN/m and a viscosity (at room temperature) of 0.93 kPa·s. Both values are about one order of magnitude higher than the respective values obtained for human neutrophils, lending support to the hypothesis that a tight balance between cortical tension and cytoplasmic viscosity is a physical prerequisite for eukaryotic cell motility. The relatively large stiffness of passive J774 cells contrasts with their capacity for a more than fivefold increase in apparent surface area during active deformation in phagocytosis. Scanning electron micrographs show how microscopic membrane wrinkles are smoothed out and recruited into the apparent surface area during phagocytosis of large targets.     

Hyaluronan-binding proteins on cultured J 774 macrophages.

Cultivated macrophages of murine cell-line J 774 were found to bind high-molecular-weight (molecular weight average approx. 5.10(6) [3H]hyaluronan (HA) by a saturable mechanism at 4 degrees C. Half-maximal binding was observed at 7-8 microgram/ml (1.4-1.6 nM) and the maximal binding was reached at 30-40 microgram/ml. Scatchard plot analysis revealed that approx. 20,000 molecules could bind to each cell with a Kd of 1.5 nM. The binding could be effectively inhibited by unlabeled HA. Also chondroitin sulphate inhibited the binding, but only to about 50%. At 37 degrees C the J 774 cells took up and degraded the polysaccharide effectively. Affinity chromatography on HA coupled to agarose of solubilized surface-iodinated J 774 cells, revealed that a protein of approx. 60 kDa, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography, could be specifically eluted with HA-oligosaccharides. Our results suggest that J 774 macrophages can bind HA by a mechanism compatible with receptor-binding, and carry a 60 kDa HA-binding protein on their surface. This receptor-binding may mediate uptake and degradation of the polysaccharide and influence the levels and turnover of HA in interstitial fluid as well as the release of HA into the bloodstream.     

Electron microscopic analysis of DNA forks generated by Escherichia coli DNA helicase II

T7 phage DNA eroded with lambda exonuclease (to create 3'-protruding strands) or exonuclease III (to create 5'-protruding strands) was treated under unwinding assay conditions with DNA helicase II. Single-stranded DNA-binding protein (of Escherichia coli or phage T4) was added to disentangle the denatured DNA and the complexes were examined in the electron microscope. DNA helicase II complexes filtered through a gel column before assay retain the ability to generate forks suggesting that DNA helicase II unwinds in a preformed complex by translocating along the bound DNA strand. The enzyme initiates preferentially at the ends of the lambda-exonuclease-treated duplexes and is found at a fork on the initially protruding strand. It also initiates at the ends of the exonuclease-III-treated duplexes where, as with approximately 5% of the forks traceable back to a single-stranded gap, it is found on the initially recessed strand. The results are consistent with the view that DNA helicase II unwinds in the 3'-5' direction relative to the bound strand. They also confirm that the enzyme can initiate at the end of a fully base-paired strand. At a fork, DNA helicase II is bound as a tract of molecules of approximately 110 nm in length. Tracts of enzyme assemble from non-cooperatively bound molecules in the presence of ATP. During unwinding, DNA helicase II apparently can translocate to the displaced strand which conceivably can deplete the leading strand of the enzyme. Continued adsorption of enzyme to DNA might replenish forks arrested by strand switch of the unwinding enzyme.     

Leishmania donovani lipophosphoglycan disrupts phagosome microdomains in J774 macrophages

Clearance of pathogens by phagocytosis and their killing in phagolysosomes is a key aspect of our innate ability to fight infectious agents. Leishmania parasites have evolved ways to survive and replicate in macrophages by inhibiting phagosome maturation and avoiding the harsh environment of phagolysosomes. We describe here that during this process Leishmania donovani uses a novel strategy involving its surface lipophosphoglycan (LPG), a virulence factor impeding many host functions, to prevent the formation or disrupt lipid microdomains on the phagosome membrane. LPG acts locally on the membrane and requires its repetitive carbohydrate moieties to alter the organization of microdomains. Targeting and disruption of functional foci, where proteins involved in key aspects of phagolysosome biogenesis assemble, is likely to confer a survival advantage to the parasite.     

mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages

Previous studies demonstrated that macrophages are difficult to transfect. In the present study, we investigated whether J774A.1 macrophages can be efficiently transfected using nucleofector technology. Nucleofection of J774A.1 macrophages with mRNA resulted in transfection efficiencies up to 75% without cell death as compared to control pulsed macrophages. In contrast, introduction of DNA into J774A.1 cells caused apoptosis without expression of the gene of interest. Our results show that mRNA nucleofection is a new high-speed transfection method for macrophages.     

An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli

Representative strains of the different diarrheagenic Escherichia coli virotypes were tested for their potential cytotoxicity in the J774 macrophage cell line. All the seven virotypes of E. coli were cytotoxic to J774 macrophages, and in most cases the bacteria induced an apoptotic response. With the exception of the enterotoxigenic E. coli (ETEC) strain, all the other six virotypes caused induction of apoptosis as evidenced by quantitative analysis of the characteristic DNA fragmentation at the individual cell level. These results suggest that apoptosis could be one of the mechanisms contributing to the diarrheal disease development.     

Defective catabolism of oxidized LDL by J774 murine macrophages.

In J774 murine macrophages, chemically oxidized LDL (OxLDL) and biologically oxidized LDL (BioOxLDL) have similar metabolic fates, characterized by a relatively poor degradation when compared with acetylated LDL (AcLDL), and a modest ability to activate acyl-CoA:cholesterol acyltransferase (ACAT) (850 and 754 pmol [14C]oleate/mg cell protein in OxLDL- and BioOxLDL-incubated cells, versus 425 and 7070 pmol [14C]cholesteryl oleate/mg cell protein in control and AcLDL-incubated cells) with a massive increase of cellular free cholesterol. Therefore, OxLDL were used to investigate the cellular processing of oxidatively modified LDL. Binding and fluorescence microscopy studies demonstrated that OxLDL are effectively bound and internalized by macrophages and accumulate in organelles with density properties similar to those of endo/lysosomes. Although the overall metabolism of OxLDL is modestly affected by 100 microM chloroquine, owing to the poor cellular degradation of the substrate, the drug can further depress OxLDL degradation, indicating that this process takes place in an acidic compartment. Failure to detect products of extensive degradation of OxLDL in the medium is due to their relative resistance to enzymatic hydrolysis, as demonstrated also by in vitro experiments with partially purified lysosomal enzymes, rather than to the intracellular accumulation of degradation products (degraded intracellular protein is, at most, 8.5% of total). This sluggish degradation process is not due to a cytotoxic effect since OxLDL do not affect the intracellular processing of other ligands like AcLDL or IgG. The accumulation of OxLDL-derived products within macrophages may elicit cellular responses, the relevance of which in the atherosclerotic process remains to be addressed.     

Repair of ozone-induced DNA lesions in Escherichia coli B cells

Alkaline sucrose gradient sedimentation indicates that ozone can produce DNA single- and double-strand breaks in wild-type E. coli and ozone-sensitive mutant MQ1844(ozrB). Another type of DNA damage repaired only by the ozrB gene product may also be responsible for the killing effect of ozone on E. coli cells.     

Specific modulation of surface receptors in J.774 macrophages by anchorage

The J.774 murine macrophage cells were cultured in suspension in Teflon flasks. When allowed to attach on culture plastic dishes, a 2-3-fold increase in transferrin binding was observed. This occurred in 10 min, reached a steady state at 60 min, remained stable for several hours and was reversible after resuspension of the cells at 37 degrees C. The phenomenon was not dependent on the synthesis of new protein. An opposite change of acetyl LDL receptors was observed, with a threefold decrease of the binding 1 h after the attachment of the cells. The increase of transferrin binding affected almost equally the cell surface and the intracellular sites; therefore it could not be related to a simple shift between these two compartments. It is suggested that the attachment of the cells induced a recruitment of binding sites from a 'silent' pool of receptors. Serum factors, as well as phorbol esters and db-cAMP potentiated the effect of anchorage.     

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.     

Restricted diffusion of DNA segments within the isolated Escherichia coli nucleoid

To study the dynamics and organization of the DNA within isolated Escherichia coli nucleoids, we track the movement of a specific DNA region. Labeling of such a region is achieved using the Lac-O/Lac-I system. The Lac repressor-GFP fusion protein binds to the DNA section where tandem repeats of the Lac operator are inserted, which allows us to monitor the motion of the DNA. The movement of such a GFP spot is followed at 48 ms temporal resolution during 12s. The spots are found to diffuse within a confined space, so that the nucleoid appears to behave like a viscoelastic network. The distribution of the "particle" position in time can be fitted to a Gaussian function indicating that the motion of the particle is Brownian. An average self-diffusion constant Ds=0.12 microm(2) s-1 is derived via the time auto-correlation functions of the displacement and is compatible with the collective diffusion coefficient measured previously by dynamic light scattering. Restriction of a DNA sequence to a small region of the nucleoid is tentatively related to the existence of so-called supercoiling domains.     

Characterization of Abcc4 gene amplification in stepwise-selected mouse J774 macrophages resistant to the topoisomerase II inhibitor ciprofloxacin

Exposure of J774 mouse macrophages to stepwise increasing concentrations of ciprofloxacin, an antibiotic inhibiting bacterial topoisomerases, selects for resistant cells that overexpress the efflux transporter Abcc4 (Marquez et al. [2009] Antimicrob. Agents Chemother. 53: 2410-2416), encoded by the Abcc4 gene located on Chromosome 14qE4. In this study, we report the genomic alterations occurring along the selection process. Abcc4 expression progressively increased upon selection rounds, with exponential changes observed between cells exposed to 150 and 200 μM of ciprofloxacin, accompanied by a commensurate decrease in ciprofloxacin accumulation. Molecular cytogenetics experiments showed that this overexpression is linked to Abcc4 gene overrepresentation, grading from a partial trisomy of Chr 14 at the first step of selection (cells exposed to 100 μM ciprofloxacin), to low-level amplifications (around three copies) of Abcc4 locus on 1 or 2 Chr 14 (cells exposed to 150 μM ciprofloxacin), followed by high-level amplification of Abcc4 as homogeneous staining region (hsr), inserted on 3 different derivative Chromosomes (cells exposed to 200 μM ciprofloxacin). In revertant cells obtained after more than 60 passages of culture without drug, the Abcc4 hsr amplification was lost in approx. 70% of the population. These data suggest that exposing cells to sufficient concentrations of an antibiotic with low affinity for eukaryotic topoisomerases can cause major genomic alterations that may lead to the overexpression of the transporter responsible for its efflux. Gene amplification appears therefore as a mechanism of resistance that can be triggered by non-anticancer agents but contribute to cross-resistance, and is partially and slowly reversible.     

Characterization of the interactions within the mazEF addiction module of Escherichia coli

In bacteria, programmed cell death is mediated through the unique genetic system called "addiction module," which consists of a pair of genes encoding a stable toxin and an unstable antitoxin. The mazEF system is known as an addiction module located on the Escherichia coli chromosome. MazF is a stable toxin, and MazE is a labile antitoxin interacting with MazF to form a complex. MazE and the MazE-MazF complex can bind to the mazEF promoter region to regulate the mazEF expression. Here we show that the binding of purified (His)6MazE to the mazEF promoter DNA was enhanced by MazF. The site-directed mutations at the conserved amino acid residues in MazE N-terminal region (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)6MazE and the MazE-MazF-(His)6 complex, suggesting that MazE binds to the mazEF promoter DNA through the N-terminal domain. The ratio of MazE to MazF(His)6 in the MazE-MazF(His)6 complex is about 1:2. Because both MazE and MazF-(His)6 exist as dimers by themselves, the MazE-MazF-(His)6 complex (76.9 kDa) is predicted to consist of one MazE dimer and two MazF(His)6 dimers. The interaction between MazE and MazF was also characterized with the yeast two-hybrid system. It was found that the region from residues 38 to 75 of MazE was required for its binding to MazF. Site-directed mutagenesis at this region revealed that Leu55 and Leu58 play an important role in the MazE-MazF complex formation but not in MazE binding to the mazEF promoter DNA. The present results demonstrate that MazE is composed of two domains, the N-terminal DNA-binding domain and the C-terminal domain interacting with MazF.     

Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages

After internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago-lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic'Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.     

Effects of PM10 in human peripheral blood monocytes and J774 macrophages

Background

Asthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma.

Methods

To test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response.

Results

We found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines.

Conclusion

We conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.