Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7

Mycobacterium tuberculosis and the closely related organism Mycobacterium bovis can survive and replicate inside macrophages. Intracellular survival is at least in part attributed to the failure of mycobacterial phagosomes to undergo fusion with lysosomes. The transformation of phagosomes into phagolysosomes involves gradual acquisition of markers from the endosomal compartment. Members of the rab family of small GTPases which confer fusion competence in the endocytic pathway are exchanged sequentially onto the phagosomal membranes in the course of their maturation. To identify the step at which the fusion capability of phagosomes containing mycobacteria is compromised, we purified green fluorescent protein-labeled M. bovis BCG phagosomal compartments (MPC) and compared GTP-binding protein profiles of these vesicles with latex bead phagosomal compartments (LBC). We report that the MPC do not acquire rab7, specific for late endosomes, even 7 days postinfection, whereas this GTP-binding protein is present on the LBC within hours after phagocytosis. By contrast, rab5 is retained and enriched with time on the MPC, suggesting fusion competence with an early endosomal compartment. Prior infection of macrophages with M. bovis BCG also affected the dynamics of rab5 and rab7 acquisition by subsequently formed LBC. Selective exclusion of rab7, coupled with the retention of rab5 on the mycobacterial phagosome, may allow organisms from the M. tuberculosis complex to avert the usual physiological destination of phagocytosed material.     

Mycobacterial phagosome maturation, rab proteins, and intracellular trafficking

One of the most prominent features of pathogenic mycobacteria, which include the potent human pathogens Mycobacterium tuberculosis and Mycobacterium leprae and their opportunistic relatives Mycobacterium avium and Mycobacterium marinum, is their ability to survive and multiply in phagosomes of mononuclear phagocytic cells. The phagocytosed mycobacteria reside in a vacuolar compartment which is exempted from maturation into the phagolysosome. Recently, the arrest of the maturation of phagosomes containing M. tuberculosis complex organisms (Mycobacterium bovis BCG) has been linked to the accumulation on the phagosomal membrane of the small GTP binding protein rab5, specific for the control of fusion within the early endosomal compartment. Furthermore, M. bovis BCG phagosome is devoid of rab7, a rab protein associated with the late endosome. The selective accumulation of rab5 and exclusion of rab7 defines the check point that has been compromised in mycobacterial phagosome maturation. Here we summarize these observations and relates them to other phenomena in the area of membrane and protein trafficking with the emphasis on phagosomes containing intracellular pathogens.     

Fusion of azurophil granules with phagosomes and activation of the tyrosine kinase Hck are specifically inhibited during phagocytosis of mycobacteria by human neutrophils

Pathogenic mycobacteria parasitize macrophages and reside within phagosomes, which do not fuse with lysosomal granules. Mycobacteria are also internalized by neutrophils, which possess at least two types of granules, specific and azurophil granules, the latter being specialized lysosomes. Here, we investigated the ability of mycobacteria to inhibit the fusion of these granules with their phagosomes in human neutrophils. It was found that when pathogenic (Mycobacterium kansasii and Mycobacterium avium) or nonpathogenic (Mycobacterium smegmatis and Mycobacterium phlei) mycobacteria were internalized by neutrophils, they induced the inhibition of azurophil granule fusion with phagosomes even when they were serum opsonized. In contrast, secretion of specific granule content and production of O2-, both of which contribute to the neutrophil bactericidal response, were triggered. Hck is a Src family tyrosine kinase associated with azurophil granules. During internalization of zymosan, azurophil granules fused with phagosomes and Hck was activated and translocated to the phagosomal membrane, whereas in neutrophils engulfing mycobacteria, Hck did not translocate and remained unactivated. The activation of the tyrosine kinase Fgr was not affected. These results indicate that 1) pathogenic and nonpathogenic mycobacteria trigger similar bactericidal responses in neutrophils, 2) phagocytosis and fusion of azurophil granules can be uncoupled by mycobacteria, and 3) Hck could be one of the key elements of the azurophil secretory pathway that are altered during phagocytosis of mycobacteria.     

Further studies on the intracellular behavior of Torulopsis glabrata

The growth of Torulopsis glabrata was inhibited in glucose-peptone broth containing 10 to 20% normal human serum. Addition of iron to the medium diminshed the fungistatic effect. The intracellular growth of T. glabrata was remarkably restricted within mouse macrophages maintained in vitro, but this growth restriction was not caused by the limitation of iron imposed by the serum in the medium. The intracellular growth of T. glabrata within a very small percentage of the macrophages was not obviously related to the failure of lysosomal fusion to the phagosomes in those cells. The studies did not permit definite conclusions regarding the viability of the inhibited yeasts, but results suggested that a large portion of them survived. Potentially misleading artifacts of the technique for assessment of the intracellular behavior of the fungus were detected and are discussed.     

Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells

We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated beta-glucuronidase-conjugated paramagnetic beads (1-2 microm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPgammaS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Dietary cholesterol impairs memory and memory increases brain cholesterol and sulfatide levels.

Cholesterol and sulfatides play many important roles in learning and memory. To date, our observations about the effects of cholesterol on learning have been assessed during response acquisition; that is, the learning of a new memory. Here, we report for the first time to our knowledge, on the effect of a cholesterol diet on a previously formed memory. Rabbits were given trace conditioning of the nictitating membrane response for 10 days, then fed a 2% cholesterol diet for 8 weeks, and then assessed for memory recall of the initially learned task. We show that dietary cholesterol had an adverse effect on memory recall. Second, we investigated whether dietary cholesterol caused an increase in brain cholesterol and sulfatide levels in four major brain structures (hippocampus, frontal lobe, brainstem, and cerebellum) using a technique for analyzing myelin and myelin-free fractions separately. Although our data confirm previous findings that dietary cholesterol does not directly affect cholesterol and establish that it does not affect sulfatide levels in the brain, these levels did increase rather significantly in the hippocampus and frontal lobe as a function of learning and memory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.     

The occurrence of protein kinase C theta and lambda isoforms in retina of different species

Salmonella species are intracellular facultative pathogens which survive within phagocytic cells such as macrophages and proliferate inside vacuoles of epithelial cells. Early reports suggested that the capacity for surviving within macrophages was due to the inhibitory effect on the phagosome-lysosome fusion event induced by intracellular Salmonella. However, recent cell biology data, obtained both with phagocytic and epithelial cells, have shown that Salmonella-containing phagosomes have large amounts of lysosomal membrane glycoproteins (lgp), major components of the lysosomal membrane. This apparent discrepancy has partly been clarified at least in epithelial cells: the Salmonella-containing phagosome fuses with lgp-rich compartment different from the classical mature lysosome, as they do not contain certain lysosomal enzymes and are not connected with the endocytic route. Therefore, Salmonella seems to use an alternative strategy not merely based on the inhibition of phagosome-lysosome fusion event. This strategy essentially involves acquisition of only certain lysosomal components to form a specialized phagosomal compartment in which to survive or proliferate intracellularly. These observations have also exemplified the potential use of intracellular bacterial pathogens as biological probes to understand normal biological aspects of the eukaryotic cell. The intracellular lifestyle of Salmonella will undoubtedly provide new insights into the process of lysosome biogenesis.     

In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.     

Electrospray ionization tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets

The sphingolipid sulfatide is a component of myelin and some non-neuronal cells. Antibodies to sulfatide occur in some patients with autoimmune neuropathies and in patients with insulin-dependent diabetes mellitus (IDDM) caused by immunologic destruction of insulin-secreting pancreatic islet beta-cells. Distinct sulfatide molecular species may differ in immunogenicity, and facile means to identify sulfatide species in islets and other tissues obtainable in only small amounts could be useful. Electrospray ionization mass spectrometry (ESI/MS) permits structural determination of small quantities of phospholipids and is applied here to sulfatide analysis. We find that sulfatide standards are readily analyzed by negative ion ESI/MS, and tandem mass spectra of individual species exhibit some ions common to all species and other ions that reflect distinct fatty acid substituents in different sulfatide molecules. A signature ion cluster resulting from cleavage directed by the alpha-hydroxy group of sulfatide species with a hydroxylated fatty acid substituent identifies such species. Sulfatide profiles in tissue lipid extracts can be obtained by ESI/MS/MS scanning for common sulfatide ions and for ions reflecting fatty acid substituents. Islets are demonstrated to contain sulfatide and to exhibit a profile of species different from that of brain.     

Histoplasma capsulatum modulates the acidification of phagolysosomes

The phagolysosome is perhaps the most effective antimicrobial site within macrophages due both to its acidity and to its variety of hydrolytic enzymes. Few species of pathogens survive and multiply in these vesicles. However, one strategy for microbial survival would be to induce a higher pH within these organelles, thus interfering with the activity of many lysosomal enzymes. Altering the intravesicular milieu might also profoundly influence antigen processing, antimicrobial drug delivery, and drug activity. Here we report the first example of an organism proliferating within phagolysosomes that maintain a relatively neutral pH for a sustained period of time. We inoculated P388D1 macrophages with fluorescein isothiocyanate (FITC)-labeled Histoplasma capsulatum or zymosan. Using the ratio of fluorescence excitations at 495 and 450 nm, we determined that vesicles containing either virulent or avirulent FITC-labeled H. capsulatum yeasts had a pH one to two units higher than vesicles containing either zymosan or methanol-killed H. capsulatum. The difference in pH remained stable for at least 5.5 h postinoculation. Longer-term studies using cells preincubated with acridine orange indicated that phagolysosomes containing live Histoplasma continued to maintain a relatively neutral pH for at least 30 h. Many agents raise the pH of multiple vesicles within the same cell. In contrast, H. capsulatum affects only the phagolysosome in which it is located; during coinoculation of cells with unlabeled Histoplasma and labeled zymosan, organelles containing zymosan still acidified normally. Similarly, unlabeled zymosan had no influence on the elevated pH of vesicles housing labeled Histoplasma. Thus, zymosan and Histoplasma were segregated into separate phagolysosomes that responded independently to their phagocytized contents. This localized effect might reflect an intrinsic difference between phagosomes housing the two particle types, active buffering by the microbe, or altered ion transport across the phagolysosomal membrane such that acidification is inhibited.     

Cell-to-cell interaction is required to induce proteinuria in in situ immune complex glomerulonephritis

This experiment was performed to study the roles of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), and another adhesion molecule, selectin, in the development of cationized antigen-induced in situ immune complex glomerulonephritis (CAICGN). CAICGN was induced in preimmunized rats by perfusing cationized human immunoglobulin G (CaIgG) through the left kidney. Albuminuria developed within 2 days of CaIgG perfusion and peaked around day 7. Marked polymorphonuclear leukocyte (PMN) infiltration was observed in the glomeruli 1 hour after CaIgG perfusion, but the infiltrate resolved by day 7. Immunofluorescent studies disclosed linear deposition of rat IgG and C3 along glomerular capillary walls 1 hour after CaIgG perfusion. Treatment with monoclonal antibodies (mAbs) to both ICAM-1 and LFA-1, as well as with a sulfatide, a ligand of L- and P-selectin, started within 2 days after CaIgG perfusion completely suppressed the development of proteinuria without affecting the glomerular deposition of immunoreactants. Although sulfatide attenuated the PMN response 1 hour after CaIgG perfusion, ICAM-1 and LFA-1 mAb treatment did not alter PMN infiltration. Treatment with ICAM-1 and LFA-1 mAbs started on day 5, or treatment with sulfatide started on day 4, after CaIgG perfusion did not affect albuminuria. These findings suggest that adhesion molecules play an important role in the development of proteinuria in CAICGN. The contribution of these molecules was evident for only a short interval after the induction of nephritis, when a significant infiltration of PMNs was observed.     

The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins

The human cytomegalovirus (CMV) US28 gene encodes a functional CC chemokine receptor. However, this activity was observed in cells transfected to express US28 and might not correspond to the actual role of the protein in the CMV life cycle. Expression of US28 allows human immunodeficiency virus type 1 (HIV-1) entry into certain CD4(+) cells and their fusion with cells expressing HIV-1 envelope (Env) proteins. Such properties were initially reported for the cellular chemokine receptors CCR5 and CXCR4, which behave as CD4-associated HIV-1 coreceptors. We found that coexpression of US28 and either CXCR4 or CCR5 in CD4(+) cells resulted in enhanced synctium formation with HIV-1 Env+ cells. This positive effect of US28 on cell fusion seems to be distinct from its HIV-1 coreceptor activity. Indeed, enhancement of cell fusion was also observed when US28 was expressed on the HIV-1 Env+ cells instead of an CD4(+) target cells. Furthermore, US28 could enhance cell fusion mediated by other viral proteins, in particular, the G protein of vesicular stomatitis virus (VSV-G). The HIV-1 coreceptor and fusion-enhancing activities could be affected by mutations in different domains of US28. The fusion-enhancing activity of US28 seems to be cell type dependent. Indeed, cells coexpressing VSV-G and US28 fused more efficiently with human, simian, or feline target cells, while US28 had no apparent effect on fusion with the three mouse or rat cell lines tested. The positive effect of US28 on cell fusion might therefore require its interaction with a cell-specific factor. We discuss a possible role for US28 in the fusion of the CMV envelope with target cells and CMV entry.     

SPC3, a V3 loop-derived synthetic peptide inhibitor of HIV-1 infection, binds to cell surface glycosphingolipids

Synthetic multibranched peptides derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120 inhibit HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms: competitive inhibition of HIV-1 binding to CD4-/GalCer+ colon cells and postbinding inhibition of HIV-1 fusion with CD4+ lymphocytes. In the present study, we have characterized the cellular binding sites for the V3 peptide SPC3, which possesses eight V3 consensus motifs GPGRAF radially branched on a neutral polyLys core matrix. These binding sites are glycosphingolipids that share a common structural determinant, i.e., a terminal galactose residue with a free hydroxyl group in position 4: GalCer/sulfatide on CD4-/GalCer+ colon cells; LacCer and its sialosyl derivatives GM3 and GD3 on CD4+ human lymphocytes. These data suggest that the V3 peptide binds to the GalCer/sulfatide receptor for HIV-1 gp120 on HT-29 cells and thus acts as a competitive inhibitor of virus binding to these CD4- cells, in full agreement with previously published virological data. In contrast, SPC3 does not bind to the CD4 receptor, in agreement with the data showing that the peptide inhibits HIV-1 infection of CD4+ cells by acting at a postattachment step. The binding of SPC3 to LacCer, GM3, and GD3, expressed by CD4+ lymphocytes, suggests a role for these glycosphingolipids in the fusion process between the viral envelope and the plasma membrane of CD4+ cells. Since the multivalent peptide can theoretically bind to several of these glycosphingolipids, we hypothesize that the resulting cross-linking of membrane components may affect the fluidity of the plasma membrane and/or membrane curvature, altering the virus-cell fusion mechanism.     

Potentiating interactions between morphogenetic protein and neurotrophic factors in developing neurons

A highly specific antibody against sulfatide, a myelin-associated glycolipid, has been investigated using indirect double immunocytochemistry in rat primary astroglial cultures from cerebral cortex. Sulfatide was expressed in a selected subpopulation of astrocytes (2-3%) and was found to be exclusively located intracellularly. The sulfatide-positive cells appeared in two different morphologies: flat and stellate. Immunolabeling of the astroglial cultures showed that sulfatide always co-existed with GFAP or S-100, and in some cells with GD3 (flat 90% and stellate 50%) or A2B5 (1%) antibody. The sulfatide-positive cells did not bind the O1 antibody, which is used as a marker for oligodendrocytes. Glial cultures from other regions and mixed cultures, with both neurons and glial cells, were examined and showed similar results. Biochemical analysis by TLC-ELISA verified the presence of sulfatide in the astroglial culture and showed decreasing amounts of sulfatide with days in vitro; 0.05 nmol/mg protein at day 10 and 0.01 nmol/mg protein at day 17. This analysis also showed that neither sulpholactosylceramide nor seminolipid was present, each of which also has affinity for the sulfatide antibody. This selective and intracellular expression encourages further identification of the astrocytes expressing sulfatide and the biological role of sulfatide in these cells.     

Hydration and stability of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles

The effect of sulfatide on membrane hydration of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations >/=30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.     

Membrane-induced step in the activation of Sendai virus fusion protein

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low alpha-helical structure. However, when the two peptides are mixed together, their alpha-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into alpha-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion.     

The mannose receptor mediates uptake of pathogenic and nonpathogenic mycobacteria and bypasses bactericidal responses in human macrophages

The mannose receptor (MR) is involved in the phagocytosis of pathogenic microorganisms. Here we investigated its role in the bactericidal functions of human monocyte-derived macrophages (MDMs), using (i) trimannoside-bovine serum albumin (BSA)-coated latex beads and zymosan as particulate ligands of the MR, and (ii) mannan and mannose-BSA as soluble ligands. We show that phagocytosis of mannosylated latex beads did not elicit the production of O2-. Zymosan, which is composed of alpha-mannan and beta-glucan, was internalized by the MR and a beta-glucan receptor, but the production of O2- was triggered only by phagocytosis through the beta-glucan receptor. Activation and translocation of Hck, a Src family tyrosine kinase located on lysosomes, has previously been used as a marker of fusion between lysosomes and phagosomes in human neutrophils. In MDMs, Hck was activated and recruited to phagosomes containing zymosan later than LAMP-1 and CD63. Phagosomes containing mannosylated latex beads fused with LAMP-1 and CD63 vesicles but not with the Hck compartment, and the kinase was not activated. We also demonstrate that the MR was unable to distinguish between nonpathogenic and pathogenic mycobacteria, as they were internalized at similar rates by this receptor, indicating that this route of entry cannot be considered as a differential determinant of the intracellular fate of mycobacteria. In conclusion, MR-dependent phagocytosis is coupled neither to the activation of NADPH oxidase nor to the maturation of phagosomes until fusion with the Hck compartment and therefore constitutes a safe portal of entry for microorganisms.