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.     

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.     

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.     

Influence of mouse strain and vaccine viability on T-cell responses induced by Mycobacterium bovis bacillus Calmette-Guérin

C57BL/6 and BALB/c mice were vaccinated with either live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin (BCG) organisms, and splenic T cells were used to screen the stimulatory potential of fractionated somatic and secreted mycobacterial proteins by production of gamma interferon (IFN-gamma). Maximum responses were obtained with fractionated secreted proteins of Mycobacterium tuberculosis. There was no single dominant antigen, but five regions of mycobacterial proteins induced high concentrations of IFN-gamma. However, only two of the five regions stimulated T cells from both mouse strains: two were exclusively recognized by T cells from BALB/c mice, and one was exclusively recognized by T cells from C57BL/6 mice. T cells from mice vaccinated with heat-killed M. bovis BCG organisms failed to respond to fractionated secreted proteins but recognized several somatic antigen fractions. As late as 1 year after primary vaccination, memory T cells responded to similar protein regions, and IFN-gamma production was intensified by secondary infection. Our data confirm a central role for secreted proteins in immunity to mycobacteria. Moreover, we demonstrate that a major set of mycobacterium-reactive T cells is stimulated only by vaccination with live but not with heat-killed M. bovis BCG organisms. Because a major impact of genetic host factors on antigen recognition was observed, we favor the use of live carrier organisms which secrete mycobacterial proteins over subunit vaccines as an improved antituberculosis vaccine.     

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.     

Structure of a hydroxymycolic acid potentially involved in the synthesis of oxygenated mycolic acids of the Mycobacterium tuberculosis complex

Mycolic acids are believed to play a crucial role in the architecture of the mycobacterial envelope. However, very few steps of their biosynthetic pathway have yet been elucidated. We previously isolated [Dubnau, E., Lanéelle, M. A., Soares, S., Bénichou, A., Vaz, T., Promé, D., Promé, J. C., Daffé, M. & Quémard, A. (1997) Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto- and hydroxy-mycolic acids, Mol. Microbiol. 23, 313-322] a gene cluster from Mycobacterium bovis BCG, cmaA-D, which confers upon M. smegmatis the ability to synthesize cyclopropanated ketomycolic acid, and a new type of mycolic acid which is hydroxylated. A meticulous analysis of all the mycolic-like fatty acids of M. bovis BCG and M. tuberculosis showed that these organisms produce small amounts of the hydroxymycolic acid. The structure of this molecule, determined by NMR spectroscopy, mass spectrometry and stereochemical studies, strongly suggests that there is a direct biosynthetic relationship between the keto- and the hydroxy-mycolic acids.     

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.     

Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex

A PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. The senX3-regX3 IR contains a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs within the senX3-regX3 IR, whereas all non-BCG M. tuberculosis complex strains contained a 53-bp MIRU, in addition to the 77-bp MIRUs. All 148 strains analyzed so far could be divided into eight different groups according to the copy numbers of the 77-bp MIRU and to the presence or absence of the 53-bp MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The other strains contained one to five 77-bp MIRUs invariably followed by a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains and its presence in virulent strains allowed us to develop an enzyme-linked immunosorbent assay using specific capture oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.     

Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis

Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.     

Abnormal prostate development in C3(1)-bcl-2 transgenic mice

Two bacillus Calmette-Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum. Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-gamma production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum-infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis.     

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.     

Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages

Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial infections, IFN-gamma-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH 5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases. Furthermore, the lower pH is reflected in the transition of MAC phagosomes to a point no longer accessible to transferrin, a marker of the recycling endosomal system. These alterations parallel the coalescence of bacterial vacuoles from individual bacilli in single vacuoles to communal vacuoles with multiple bacilli. These data demonstrate that bacteriostatic and bactericidal activities of activated macrophages are concomitant with alterations in the physiology of the mycobacterial phagosome.     

Activation of CD8 T cells with specificity for mycobacterial heat shock protein 60 in Mycobacterium bovis bacillus Calmette-Guérin-vaccinated mice

Heat shock protein 60 (hsp60)-specific CD8 T cells lysed Mycobacterium bovis BCG-infected macrophages in vitro and adoptively transferred protection against mycobacterial infection. Moreover, CD8 T cells with this hsp60 specificity were activated in vivo by BCG vaccination. Our studies suggest there is participation of hsp60-specific CD8 T cells in BCG-induced immunity.     

Identification and strain differentiation of Mycobacterium species on the basis of DNA 16S-23S spacer region polymorphism

Amplification of the region separating the genes coding for the two rRNA species 16S and 23S was performed to identify 56 mycobacterial strains, belonging to eleven species: Mycobacterium tuberculosis, M. avium, M. kansasii, M. gordonae, M. abscessus, M. fortuitum, M. xenopi, M. bovis, M. bovis/BCG, M. africanum and M. intracellulare. Reproducible amplification patterns were obtained with most species with the exception of M. kansasii which showed heterogeneity, confirming the existence of a genetically distinct subspecies within this species. In addition, we used the amplified products as target DNA for restriction endonuclease digestion and RAPD (randomly amplified polymorphic DNA) analysis to compare strains of M. abscessus, M. tuberculosis and M. avium. The discriminatory power of these two typing methods was higher than when whole genomic DNA is used as target. Our results demonstrate that the two-step approach to identification and typing on the basis of the hypervariability of 16S-23S spacer region is reliable, rapid and simple, and consequently could be an epidemiological tool in clinical laboratories.     

Haemolytic activity of Mycobacterium spp

Haemolytic activity of clinical isolates of Mycobacterium bacilli (98) was determined by the method of King et al., 1993. During 3-h incubation, all M. tuberculosis (MTB) isolates (28) and one out of 38 M. avium-intracellulare (MAI) strains, produced a strong contact-dependent haemolysin (CDH). Six MAI strains expressed a weak CDH. One MAI isolate produced a strong and five other MAI strains a weak contact-independent haemolysin (CIH). Two M. bovis BCG strains and 7 M. vaccae strains did not demonstrate haemolytic activity. The persistence of chosen Mycobacterium strains differing by haemolytic activity, in the spleens of infected C57BL/6 mice was examined. Mycobacteria producing a strong CDH (MTB H37Rv, MTB 101/92, MAI 83/93) or CIH (MAI 475/93) survived in the spleens of nonimmunized or M. bovis BCG-immunized mice for longer time than MAI strains expressing weak haemolytic activity or M. bovis BCG vaccine strain.     

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.