Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis

Intracellular parasites (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, and some Chlamydiae) may promote their survival within the host by acting from within phagosomes to prevent phagolysosome formation, thus avoiding exposure to the lysosomal hydrolases. The present studies demonstrate that when sulfatides of M. tuberculosis (anionic trehalose glycolipids largely responsible for the neutral red reactivity of virulent strains) are administered to cultured mouse peritoneal macrophages, they accumulate in the secondary lysosomes, which are rendered incompetent for fusion with phagosomes containing suitable target particles such as viable yeasts. This antifusion effect is also exhibited when small amounts of sulfatide are introduced directly into phagosomes by attachment to the target yeasts prior to their ingestion. The sulfatides evidently exert a selective inhibitory influence on membrane fusion, analogous to what occurs typically when macrophage cultures are infected with tubercle bacilli. This effect may be due to ionic interaction between the polyanionic micelles of bacterial sulfatide and organelle membranes, modifying the latter and inducing dysfunction.     

Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 +/- 0.06 versus pH 6.6 +/- 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton "leak", as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase-containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.     

The interaction between Mycobacterium and the macrophage analyzed by two-dimensional polyacrylamide gel electrophoresis

The intramacrophage pathogen Mycobacterium avium resides in a vacuole which displays unusual fusion characteristics, expressed as both a failure to mature into phagolysosomes and a continued access to the early recycling pathway. In contrast, compartments containing inert IgG-opsonized latex beads mature to phagolysosomes. Techniques were developed for the isolation of these particle-containing phagosomes from macrophages to facilitate analysis of phagosomal constituents by electrophoresis and autoradiography. Metabolic labeling of macrophages followed by phagosome isolation and two-dimensional polyacrylamide gel electrophoresis revealed only minor differences in the protein profiles between the M. avium and IgG-bead phagosomes despite the marked differences in the fusigenicity of the respective vacuoles. Pulse-chase labeling experiments revealed greater differences in the accessibility of Mycobacterium avium and IgG-bead phagosomes to newly synthesized proteins. These phagosome isolation techniques were extended to analyze the protein synthesis profile of intracellular M. avium for comparison with bacteria that were metabolically labeled in broth culture. Not surprisingly, the majority of polypeptides in the bacilli were common to both growth conditions. However, despite these similarities, intracellular M. avium express several unique proteins, most notably one abundant protein with a molecular weight of 51 kDa. In addition, the bacteria manifest a restricted set of proteins expressed while in stasis shortly after infection.     

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.     

Low-risk differentiated thyroid cancer: the need for selective treatment

By means of an electron microscopic study of the intestine in young rats infected with Cryptosporidium parvum we observed a mass migration of immunocompetent cells of the host (eosinophils, neutrophils and macrophages) into the lumen of intestine. Some lymphocytes were also observed. Immunocompetent cells (except lymphocytes) included inside phagosomes with different endogenic states of C. parvum. Macrophages with typical extracytoplasmic parasitophorous vacuoles formed by C. parvum were also observed in the intestine lumen. Almost all stages of C. parvum could be observed on a surface of such macrophages. However, we did not find in lamina propria of intestine villi any macrophages with parasites. The place of macrophages infection is unknown. We suggest that surviving of C. parvum in macrophages is principally possible.     

Identification of Legionella pneumophila mutants that have aberrant intracellular fates

After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum. A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy. To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI). Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 [correction of IgpI20] (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes. Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein. By these approaches, four phenotypic groups of mutants were classified. One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.     

Molecular aspects of the inflammatory process. Part II. Complement, lysosomal enzymes, lymphokinins, migration of the leucocytes (author''s transl)

Biochemical and electron microscope studies were conducted to determine the effects of traumatic shock on rabbit alveolar macrophages. Both resting and phagocytosing macrophages from the shocked animals, in comparison to comparable control macrophages, showed increased release of acid phosphatase from the cells into medium upon incubation in vitro, but decreases in the total content of acid phosphatase and beta-glucuronidase. Studies by electron microscopy showed ultrastructural alterations in macrophages from shocked animals consisting of a reduction in the number or a complete absence of lysosomes and, in some cases, increased amounts of rough endoplasmic reticulum and free ribosomes. In vitro incubation of macrophages from shocked animals with Pseudomonas aeruginosa showed that the process of bacterial ingestion was not impaired nor were the numbers of bacteria ingested decreased as compared to control macrophages. However, the ability of macrophages from shocked animals to destroy ingested bacteria appeared to be significantly altered. Extensive degradation of Pseudomonas was observed within phagocytic vacuoles of control macrophages after 15 minutes of incubation. In contrast, the majority of ingested organisms in macrophages from shocked animals showed no evidence of degradative changes.     

Mycobacterium avium complex infection in mice: lack of exacerbation after LP-BM5 murine leukemia virus infection

The murine leukemia virus LP-BM5 has been used to reproduce the model of murine AIDS in order to evaluate the course of infection with the MO-1 strain of Mycobacterium avium complex (MAC). LP-BM5 was inoculated in C57BL/6 mice by intravenous (i.v.) injection either 8 weeks before an i.v. challenge with 10(3) or 10(6) CFU of MAC (coinfection 1) or 10 days after an i.v. challenge with 10(3) CFU of MAC (coinfection 2). During coinfection 2 experiments, the phenotypic alterations in blood lymphocyte subsets were analyzed. During coinfection 1, LP-BM5 infection tended to decrease the mycobacterial growth, with the difference reaching statistical significance for the lower inoculum (10(3) CFU of MAC) (P<0.001). During coinfection 2, LP-BM5 did not exacerbate MAC infection except in the spleen, at day 90 after LP-BM5 challenge (P<0.001). LP-BM5 infection and the LP-BM5-MAC coinfection increased the numbers of activated CD4+ lymphocytes (CD4+ Ly6AE+) (P<0.001), activated CD8+ lymphocytes (CD8+ Ly6AE+) (P<0.001), and activated B lymphocytes (Ly5+ Ly6AE+) (P<0.001). This activation of T lymphocytes could explain the lack of exacerbation of MAC infection and even the trend to a lower level of MAC infection. Thus, this model of retroviral infection of mice does not seem to be a reliable model of immunodepression for the study of MAC infection and its treatments.     

Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.     

Attempts to elucidate reasons why mycobacterial infections are intractable, by using an experimental mouse infection model

This paper reviews some recent studies which have been performed by us and other investigators, in order to clarify the reason why most mycobacterial infections such as due to Mycobacterium tuberculosis and M. avium complex infections are intractable, that is, why these organisms can escape from attack by microbicidal mechanisms of host macrophages and consequently persist for long time at sites of infection. This paper mainly dealt with the two major subjects, which were studied by using an experimental model for murine M. avium infection. The first subject is on the modes and mechanisms of mycobacterial killing in host macrophages and the mechanisms of bacterial escape from an onslaught by macrophages. The second is on the characteristics of immunosuppressive macrophages induced in M. avium complex infection and the role of the suppressor macrophages in the establishment of immune unresponsiveness of host mice in the progressed stage of infection.     

Lethal tuberculosis in interleukin-6-deficient mutant mice

Tuberculosis is a chronic infectious disease which causes major health problems globally. Acquired resistance is mediated by T lymphocytes and executed by activated macrophages. In vitro studies have emphasized the importance of macrophage activation for mycobacterial growth inhibition. In vivo, the protective host response is focused on granulomatous lesions in which Mycobacterium tuberculosis is contained. A cellular immune response of the T helper 1 (Th1) type is considered central for control of tuberculosis. Using interleukin-6 (IL-6)-deficient mice, we here demonstrate a crucial role of this pluripotent cytokine in protection against M. tuberculosis but not against Mycobacterium bovis BCG. Infection with M. tuberculosis was lethal for the IL-6-deficient mice at inocula that were still controlled by IL-6-competent mice. Spleen cells from M. tuberculosis-infected IL-6-/- mouse mutants produced elevated levels of IL-4 and reduced levels of gamma interferon compared to the control levels. Cytofluorometric analyses of spleen cells from M. tuberculosis-infected mice revealed more-profound alterations in T-cell ratios in IL-6-/- mice than in control mice. We assume that IL-6 contributes to host resistance by its proinflammatory activity and by its influence on cytokine secretion.     

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.     

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.     

Altered cytokine production and impaired antimycobacterial immunity in protein-malnourished guinea pigs

Protein malnutrition leads to multiple detrimental alterations of host immune responses to mycobacterial infection. In this study, we demonstrated that splenocytes from low-protein (LP) guinea pigs vaccinated 6 weeks previously with attenuated Mycobacterium tuberculosis H37Ra failed to control the accumulation of virulent M. tuberculosis H37Rv in cocultured autologous peritoneal macrophages, despite the fact that they were able to control the accumulation of virulent tubercle bacilli in cocultured syngeneic peritoneal macrophages from normally nourished guinea pigs as successfully as did those from high-protein (HP) counterparts. Vaccine-induced growth control of virulent M. tuberculosis H37Rv in these cocultures appeared to be mediated by CD4 lymphocytes but not CD8 cells. Tuberculin (purified protein derivative [PPD])-induced lymphoproliferation was markedly impaired in vaccinated LP guinea pigs, and the depletion of CD4 lymphocytes significantly decreased lymphocyte proliferation whereas CD8 cell depletion did not. Protein malnutrition also impaired the abilities of cells from vaccinated LP guinea pigs to produce cytokines, including interferon, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), in response to PPD, despite the demonstration of higher serum levels of TNF-alpha and TGF-beta after an intravenous injection of PPD into LP guinea pigs. In contrast, peritoneal macrophages from protein-malnourished guinea pigs produced a higher level of TGF-beta 4 days after infection in vitro with M. tuberculosis H37Rv than did those from protein adequate controls. These results suggest that dietary protein malnutrition impairs vaccine-induced resistance to M. tuberculosis, in part, by altering the cytokine profile to favor macrophage deactivation.     

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.     

Change in colony morphology influences the virulence as well as the biochemical properties of the Mycobacterium avium complex

Factors that influence colony morphology are of crucial importance for drug development as well as for understanding the virulence of Mycobacterium avium complex (MAC) strains. The MAC 101 strain used in the present study grows as smooth transparent (SmT) colonies that tend to become opaque and pigmented when incubated for long periods of time. However, when MAC was passaged in animals, two types of colonies were recovered. The new rough transparent (RgT) colony morphology appeared more flat and transparent, having a central spot, irregular edges at times, and a dry, granular appearance like that of the rough mutants. In animal studies, the RgT bacilli multiplied at a much faster rate than that of the SmT bacilli, causing 60-80% mortality compared with the 10% mortality observed in mice infected with SmT. In vitro studies indicated that the SmT MAC did not grow and multiply as well in resident peritoneal macrophages as the RgT MAC did. The two morphotypes did not differ in their growth ratesin vitro but the RgT MAC failed to reduce dimethylthiazol-diphenyltetrazolium bromide (MTT), alamar blue and neutral red, suggesting that there might be significant changes in the cell wall or elsewhere causing changes in cellular permeability. These two morphotypes could serve as models for studying the biochemical markers or the identification of factors responsible for the virulence of the MAC.     

High sensitivity of transgenic mice expressing soluble TNFR1 fusion protein to mycobacterial infections: synergistic action of TNF and IFN-gamma in the differentiation of protective granulomas

To investigate the role of tumor necrosis factor (TNF) in protective immune responses to Mycobacterium tuberculosis and M. bovis Bacillus Calmette Guérin (BCG), we have used transgenic mice unable to use TNF because of the expression of high amounts of a soluble TNF receptor (R) type I (sTNFR1) fusion protein, and studied resistance of these mice to infection by lethality assays, evaluation of bacterial recovery and histologic examination. These mice showed a strongly increased sensitivity to M. tuberculosis and BCG infections, with bacterial overgrowth and marked inhibition of macrophage differentiation within granulomas; after M. tuberculosis infection, this resulted in extensive lesions of caseous necrosis in the lung. To explore the respective roles of TNF and interferon (IFN)-gamma in resistance to BCG and granuloma differentiation, controls and sTNFR1-transgenic mice were compared to IFN-gammaR mutant mice and mice double defective in TNF and IFN-gamma activity (obtained by crossing transgenic and mutant mice). The three groups of deficient mice showed a strongly enhanced susceptibility to BCG infection, with the following decreasing order of sensitivity between groups: TNF + IFN-gamma --> TNF --> IFN-gamma-deficient mice. The hepatic granulomas of IFN-gammaR mutant mice were small and contained eosinophils but few differentiated macrophages; compared to those of sTNFR1-transgenic mice, acid-fast bacilli were less numerous within the macrophages. Granulomas of double-deficient mice were strikingly different by their very large size and cellular content, made up large numbers of polymorphonuclears, eosinophils, and cells undergoing apoptosis, but without detectable differentiated macrophages; acid-fast bacilli were spread in the lesions. These studies show the essential role of both TNF and IFN-gamma in the development, during mycobacterial infections, of protective granulomas containing highly differentiated macrophages capable of destroying ingested bacteria, and emphasize that these two cytokines act synergistically in granuloma formation.     

Isolation and characterization of a 70 kDa protein from Mycobacterium avium

Mycobacterium avium complex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein of M. avium (Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) of M. leprae (Ml) and M. tuberculosis (Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in an in vitro attachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-alpha by MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-alpha by these cells.