Tracing iron and transferrin in the macrophage by visual means.

The purpose of the study was to investigate the movement of iron and transferrin in the macrophage using light and electron microscopy. First, depicted here are the phagocytosis of antibody sensitized murine red cells by the murine bone marrow derived macrophage and the formation of red cell phagosomes. Second, we show the fusion of the lysosomes with the red cell phagosome to form a lysophagosome and the lysis of the red cell using acid phosphatase as a lysosome marker. Third by autoradiography, the presence of 55Fe silver grains in the phagocytosed red cells and its delivery to the organelles of the macrophage are demonstrated. Fourth a transferrin species is shown in red cells of all ages, in the phagocytosed as well as the non-phagocytosed, and in the phagocytosed as well as the non-phagocytosed, and in the macrophage itself. Transferrin was detected using immunogold and fluorescence labelling. These studies suggest that iron, using vesicles as means of transport, moves from the effete red cells inside the macrophage to the outside possibly bound to transferrin.

HIV-1 infection of lung alveolar fibroblasts and macrophages in humans.

We have studied the infected cell populations in the lungs of four human immunodeficiency virus type 1 (HIV-1) seropositive patients suffering from lymphocytic alveolitis or lymphocytic interstitial pneumonitis. Adherent cells were obtained by bronchoalveolar lavage (BAL) and were analyzed by various technical approaches. The cells considered here were alveolar macrophages and fibroblasts, and could be clearly identified morphologically and by the expression of specific cell-surface markers using monoclonal antibodies. The presence of HIV-1 in both of these cell types was established by serological, virological, and molecular procedures. Our results show that alveolar macrophages and fibroblasts are naturally infected in the lungs of HIV+ patients. Both cell types express the CD4 receptor molecule, in contrast to skin fibroblasts which are negative. Alveolar macrophages and fibroblasts thus may act as eventual HIV-1 reservoirs in vivo, and are probably involved in the induction of inflammatory reactions because they are targets for CD8 cytotoxic T lymphocytes (CTL).

Modified lysosomal compartment as carrier of slowly and non-degradable tracers in macrophages.

A previous immunocytochemical study of macrophages infected with Bacillus subtilis showed that a cell wall antigen could be detected for several days in a population of small vesicles randomly distributed within the cells and apparently distinct from perinuclear lysosomes. These observations suggested the possibility that these vesicles might constitute a "storage" compartment for non-degradable compounds. In the present report we compared in pulse-chase experiments the location and fate of a series of degradable and non-degradable pinocytic tracers within the macrophages. The tracers, detected by fluorescent microscopy, were bovine serum albumin (BSA), hen egg ovalbumin (OVA), horseradish peroxidase (HRP). Lucifer Yellow, fluorescent dextran, and levan. BSA and OVA remained located in perinuclear lysosomes during the chase period until their disappearance occurring within 3 h. In contrast, the other tracers, although initially located in perinuclear lysosomes, were found after a 3 to 5-h chase in small vesicles homogeneously distributed in the macrophage cytoplasm where they remained visible for 2 to 3 days. The use of markers for different cell organelles indicated that these dispersed vesicles exhibited several of the lysosomal features. They were acidic, they contained the 100 kDa and the 120 kDa lysosomal proteins as well as some acid proteases albeit these markers were in lesser concentrations than in the perinuclear lysosomal compartment. The addition of bacteria to the macrophages previously loaded with fluorescent dextran showed that all dispersed vesicles have the same fusion property as lysosomes and that slowly degraded or non-degradable tracers turn over through the perinuclear lysosomal compartment by using the endocytic pathway. Measurement of the release of some of the tracers into the culture medium suggested that the "dispersed vesicles" were probably not implicated in exocytosis of the tracers.

Intracellular differentiation of Leishmania amazonensis promastigotes to amastigotes: presence of megasomes, cysteine proteinase activity and susceptibility to leucine-methyl ester.

Intracellular differentiation of Leishmania promastigotes to amastigotes is a critical step in the establishment of infection. In this report three related features of mexicana subspecies amastigotes were used to follow the differentiation of the parasites within macrophages. Early after infection, (a) parasites did not contain ultrastructurally recognizable megasomes, (b) cysteine proteinase activity of parasite lysates was not detected in gelatin-containing acrylamide gels, and (c) parasites were essentially resistant to L-leucine-methyl ester (Leu-OMe). Typical megasomes were first identified on the 5th day, were more prevalent on day 7, and underwent swelling in macrophages exposed to Leu-OMe. Cysteine proteinase activity was first detected on day 3 and increased thereafter. Susceptibility to Leu-OMe of parasites studied in situ or isolated from infected macrophages increased with time of intracellular residence and by 7 days approached that of amastigotes isolated from mouse lesions. In contrast, parasites derived from either promastigotes or amastigotes were equally susceptible to another leishmanicidal compound, tryptophanamide (Trp-NH2). The results provide additional support for the involvement of megasomes and their cysteine proteinases in parasite killing by Leu-OMe, and highlight the slow pace of the intracellular differentiation of L. amazonensis promastigotes to amastigotes.

Contribution of electron microscopy in the study of the interactions between pathogenic bacteria and their host cells.

Our knowledge on the functional anatomy of bacteria is based on the electron microscopic (EM) studies performed during the last forty years. Most pathogenic properties however cannot be visualized in EM because they are not related to defined structures. In contrast, EM studies have provided important data on the behaviour of pathogenic bacteria in their host cells. They have shown that many bacterial species have developed different stratagems to survive and multiply in their host cell. Some are even able to use the host cell machinery to move and invade adjacent cells.

Immunolabelling of Shiga toxin in macrophages infected with Shigella dysenteriae 1.

Immunolabelling of Shiga toxin in macrophages infected with a non-invasive Shigella dysenteriae 1 isolate showed that bacteria remained alive for 3 h after ingestion within the phagocytic vacuole and synthesized Shiga toxin. The normal process of toxin secretion was, however, impaired by the phagosomal environment and toxin molecules accumulated within the bacterial cytoplasm.

Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2.

Listeria monocytogenes penetrates and multiplies within professional phagocytes and other cells such as the Caco-2 human enterocytelike cell line. Listeriolysin O, a membrane-damaging cytotoxin accounts for intracellular multiplication through lysis of the membrane-bound phagocytic vacuole. This work demonstrates that once released within the cytosol, L. monocytogenes acquires the capacity to spread intracellularly and infect adjacent cells by interacting with host cell microfilaments. Such evidence was obtained by using drugs which disrupt the cell cytoskeleton. Nocodazole, which blocks polymerization of microtubules, did not affect intracellular spread, whereas cytochalasin D, which blocks polymerization of G-actin, inhibited the intracellular motility of the bacteria. By using fluorescence staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), transmission electron microscopy, and immunogold labeling, direct evidence was obtained that intracellular bacteria were enveloped with a thick layer of F-actin. Within 2 h after entry, it was demonstrated by confocal microscopy that bacteria were following highly organized routes corresponding to stress fibers. Four hours after entry, some bacteria presented random movements which could be seen by the presence of a large trail of F-actin. Such movements also caused protrusions which deeply penetrated adjacent cells and resulted in the formation of vacuoles limited by a double membrane. After subsequent lysis of these membranes, bacteria released within the cytoplasm were able to multiply and invade new cells. In contrast, an hly::Tn1545 mutant of the wild-type microorganism demonstrated almost no intracellular spread. Only a few bacteria displaying delayed lysis of the phagocytic vacuole behaved like the wild-type strain. Hemolysin-mediated lysis of the phagocytic vacuole and subsequent interaction with host cell microfilaments may represent a major virulence factor allowing tissue colonization during listeriosis.

Improved sectioning and ultrastructure of bacteria and animal cells embedded in Lowicryl.

Lowicryl K4M-embedded Gram-positive and Gram-negative bacteria have a tendency to separate between the cell surface and the resin. This often leads to distortion of bacteria and more especially of mycobacteria. We describe attempts made to overcome this technical problem. Different assays were made on Bacillus subtilis, Escherichia coli, and Mycobacterium avium: 1) Modification of the bacterial surface by coating of bacteria with proteinic compounds; 2) treatment of bacteria with metallic salts known to modify cell wall polysaccharides; and 3) comparison between Lowicryl K4M and HM20. Conditions have been found in which the separation of all bacterial species from the resin is abolished. The most important factor appeared to be the treatment of bacteria before dehydration, with 0.5% uranyl acetate for 30 min. The second most important factor, especially for M. avium and to a lower extent for Gram-negative bacteria, was the use of Lowicryl HM20. No differences were observed with Gram-positive bacteria between K4M and HM20. Pre-embedding in gelatin instead of agar improved sectioning of M. avium, but had no effects on the other bacterial species. These conditions applied to macrophages infected with Shigella dysenteriae or M. avium also gave excellent results. In addition to sectioning improvement of bacteria, uranyl acetate improved the ultrastructure of bacteria and macrophages. All organelles were more clearly delineated and, hence, more easily identified. Finally, it was shown that UA treatment did not affect immunogold labeling of a variety of antigens.

Immunodetection of lipopolysaccharide in macrophages during the processing of non invasive Shigella dysenteriae.

The location of lipopolysaccharide (LPS) was studied by immunofluorescence and immunoelectron microscopy in macrophages infected with a non-invasive Shigella dysenteriae 1 strain. Bacterial degradation began only 3 h after the end of infection. The first visible sign of degradation was detected by immunogold labelling at the level of LPS which detached from the bacterial surface and was transferred to the perinuclear lysosomes. After a few hours, it was found in small vesicles spread over the whole macrophage cytoplasm in which it remained visible for 72 h. These vesicles seemed to belong to a compartment in which slowly or non-degradable compounds are stored. LPS separation from the bacterial surface was immediately followed by the degradation of the intrabacterial constituents. The long lag period observed before initiation of bacterial degradation was not due to a lack of phagosome acidification, since DAMP, a lysosomotropic drug was found in all phagosomes at the end of the ingestion period. The frequency of phagosome-lysosome fusion was 30% for S dysenteriae and 72% for B subtilis used as a reference of high fusion frequency. The low frequency of fusion of S dysenteriae may play an important role in the survival of the virulent strains in macrophage by providing bacteria enough time to lyse the phagosome membrane before lysosome fusion occurs.

Analysis of the subcellular location of pullulanase produced by Escherichia coli carrying the pulA gene from Klebsiella pneumoniae strain UNF5023.

Three different techniques, protease accessibility, cell fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion-competent and secretion-incompetent, respectively). Pullulanase produced by secretion-competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N-terminal fatty-acylated cysteine residue (and probably also a short N-terminal segment of the pullulanase polypeptide), confirming that the N-terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion-incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electronmicroscopy of vesicles labelled with antipullulanase serum and protein A-gold confirmed that pullulanase was associated with both types of vesicles. When thin-sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion-competent cells and mainly in the proximity of the inner membrane in the secretion-incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion-competent cells, this could not be confirmed by cell-fractionation techniques. Possible explanations for this discrepancy are discussed.

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1 beta.

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.

Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12.

LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages. A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm. This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein. In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences. We chose four regions. Three of them [aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)] are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)] is predicted to be periplasmic. By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions. In some cases, the recognition sites for antibodies were narrowed down to parts of a region. In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4. Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)

Megasomes as the targets of leucine methyl ester in Leishmania amazonensis amastigotes.

Certain L-amino acid esters, such as L-leucine methyl ester (Leu-OMe), can kill intracellular and isolated Leishmania amazonensis amastigotes. Killing appears to involve ester trapping and hydrolysis within an acidified parasite compartment (M. Rabinovitch and S. C. Alfieri, 1987, Brazilian Journal of Medical and Biological Research 20, 665-74). We show here by acid phosphatase light microscopic cytochemistry and by ultrastructural morphometry that megasomes, lysosome-like amastigote organelles, are the putative parasite targets of Leu-OMe. This conclusion is supported by the following observations. (a) Control amastigotes displayed a string of electron-dense, acid phosphatase-positive megasomes mostly located in the cellular poles opposite the flagellar pockets. Incubation of the amastigotes with Leu-OMe resulted in concentration-dependent swelling and fusion of the organelles as well as decreased electron density of the internal contents. These changes, which preceded parasite disruption, were followed by the progressive loss of parasite viability and the release of acid phosphatase activity into the medium. (b) Incubation of the amastigotes with L-isoleucine methyl ester, a non-leishmanicidal compound, induced only moderate fusion of the megasomes. (c) Pre-incubation of the parasites with the proteinase inhibitors antipain and chymostatin, previously shown to confer protection from Leu-OMe toxicity, nearly completely prevented the morphological changes of megasomes. (d) Exposure of amastigotes to tryptophanamide (Trp-NH2), the leishmanicidal activity of which is not reduced by antipain and chymostatin, did not result in swelling and fusion of the megasomes. This last finding suggests that different mechanisms underlie the destruction of amastigotes by Trp-NH2 and Leu-OMe.(ABSTRACT TRUNCATED AT 250 WORDS)

Leishmania amazonensis: acidic organelles in amastigotes.

Leishmania amastigotes are intracellular protozoan parasites which exclusively invade cells of the macrophage series and multiply within phagolysosomes. Recent studies showed that intracellular and isolated amastigotes of L. amazonesis are killed by amino acid esters which appear to be trapped within as yet unidentified, possibly acidified, "lysosome-like" parasite compartments and cleaved by hydrolytic enzyme(s) (M. Rabinovitch, V. Zilberfarb, and C. Ramazeilles, 1986, Journal of Experimental Medicine 163, 520-535). In the present study, we have localized acidic compartments of Leishmania amastigotes using as a probe the weak base 3-(2,4 dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP). This indicator, which can be detected within cells by light and electron microscopy using immunocytochemical immunocytochemical methods, mainly accumulates within megasomes and in dense inclusion vacuoles. With the help of quantitative assays to titrate cell-associated DAMP, it was found that (a) its uptake is temperature dependent and thus probably requires an energy supply, (b) the proton ionophore monensin partially inhibits the trapping of DAMP, and (c) monensin greatly increases its efflux from cells. These results, as well as those obtained by quantitative ultrastructural immunocytochemistry of cells incubated with DAMP in the absence or presence of monensin, show that megasomes and inclusion vacuoles have a low pH probably maintained by an active process. Furthermore, confirming the report of H. F. Hassan and G. H. Coombs (1987, Molecular and Biochemical Parasitology 23, 285-296) megasomes were found to display acid phosphatase activity at both light and electron microscope levels. This, together with the demonstration that megasomes are acidified, suggests that these organelles may be targets for amino acid derivatives.

Endocytic membrane traffic with respect to phagosomes in macrophages infected with non-pathogenic bacteria: phagosomal membrane acquires the same composition as lysosomal membrane.

A morphometric analysis was made to study membrane traffic in bone marrow-derived macrophages, containing phagosomes with partially degraded Bacillus subtilis. Cell surface glycoproteins, labeled with radioactive galactose by terminal glycosylation, provided a covalent autoradiographic membrane marker. Membrane compartments were characterized in terms of cytochemical staining for horseradish peroxidase taken up by receptor-mediated endocytosis. The area, composition, and exchange rates of endocytic membrane compartments were measured as in a previous analysis for non-infected macrophages, devoid of phagosomes. In direct comparison with this earlier study, the present data allowed an assessment of the involvement of phagosomes in the interactions between endocytic membrane compartments. The presence of phagosomes led to a 30% reduction of lysosomal membrane area. The rate at which cell surface-derived label flowed into the lysosomal membrane pool was reduced by the same fractional amount. This suggested a linear relationship between flow rate and membrane area. The initial flow rate of label into phagosomes was higher than expected, based on their membrane area being only about 60% that of lysosomes. This rate could only be measured during the early phase of the experiments when phagosomes were younger, therefore displaying a fast exchange rate, reminiscent of the endosome compartment. However, steady-state conditions, at late times, strongly suggested that phagosomes with degraded contents finally acquire membrane of lysosomal origin. First, the composition of phagosome membrane became the same as that of lysosomes, remaining unchanged as compared to non-infected cells. Second, the membrane area of phagosomes amounted to the loss of lysosomal membrane area in infected cells.

Induction of a stable morphological change in Propionibacterium freudenreichii.

When cells of Propionibacterium freudenreichii were incubated under fasting conditions and then plated in the presence of an inhibitor of protein synthesis, a variable but significant (greater than 10(-2) fraction of the population changed their morphology from rod to sphere, with a considerable thickening of the cell wall. This change was accompanied by metabolic and antibiotic-resistance modifications, including the synthesis of at least one new enzyme (alpha-glucosidase), and by the simultaneous appearance of several new species of DNA, presumably plasmids. The round cells grew faster than the parent strain and maintained their morphology indefinitely when propagated on complex medium containing glucose as the main carbon source. However, when glucose was omitted, cells returned to the rod form and regained their previous characteristics, including the absence of detectable plasmids.

Bacterial antigen immunolabeling in macrophages after phagocytosis and degradation of Bacillus subtilis.

After phagocytosis of Bacillus subtilis 168 by bone marrow-derived macrophages, the intracellular pathway followed by different antigens was studied by immunofluorescence and immunoelectron microscopy. Three different rabbit antisera were used: (i) an antiserum to B. subtilis whole cells mainly recognizing the cell wall constituents, (ii) an antiserum to teichoic acid, and (iii) an antiserum to peptidoglycan recognizing the disaccharide tetrapeptide molecules resulting from peptidoglycan degradation. During the first 3 h after phagocytosis of B. subtilis, the three antisera were confined to the same vacuolar compartments, as follows. They were first found in phagosomes gathered in the perinuclear region. Upon bacterial degradation, the three antisera colocalized in an increasing number of small dense vesicles, located in the perinuclear region, that seemed to result from the fragmentation of phagolysosomes. These vesicles correspond to an acidic compartment since they also stained for 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, a drug known to accumulate in the acidic compartments of cells. At later time points, the antigens recognized by the three antisera followed different pathways. After 18 h, teichoic acid and peptidoglycan were no longer detectable in macrophages whereas an antigen(s) labeled with antiserum to B. subtilis whole cells remained stocked for several days in small acidic vesicles randomly distributed throughout the macrophage. This compartment appeared to be different from the one labeled during the first 3 h after ingestion of bacteria. These results suggest that the transport rate and the compartments implicated in antigen processing differ according to the antigen.