Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice.

The ability of the host to resist infection to a variety of intracellular pathogens, including mycobacteria, is strongly dependent upon the expression of the Bcg gene. Mouse strains which express the resistance phenotype (Bcgr) restrict bacterial growth, whereas susceptible strains (Bcgs) allow bacterial growth. Expression of the Bcg allele is known to influence the priming of host macrophages (M phi s) for bactericidal function. In the present work, bone marrow-derived M phi s from congenic BALB/c (Bcgs) and C.D2 (BALB/c.Bcgr) mice were infected with the virulent strain Mycobacterium avium TMC 724 to define the mechanism involved in growth restriction of M. avium. By combining CFU measurements and ultrastructural analyses, we show that growth of this bacterium is restricted in marrow M phi s from resistant mice. Using acid phosphatase as a lysosomal marker, we provide evidence that the hydrolytic activity of M phi s, as measured by the capacity of lysosomes to fuse with and transfer active hydrolytic enzymes to phagosomes in which M. avium resides, is an expression of the Bcg gene and that this phenomenon is a key antibacterial activity responsible for growth restriction of M. avium: (i) the percentage of phagosome-lysosome fusions was twice as high in Bcgr M phi s as in Bcgs M phi s, and (ii) the percentage of intact viable bacteria residing in acid phosphatase-negative phagosomes was twice as low in Bcgr M phi s as in the Bcgs counterparts. These differences are not due to a lower activity of the enzyme in Bcgr M phi s. The mechanism by which the Bcg gene exerts control over the phagolysosomal fusion is discussed.     

Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction.

Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium.     

Growth of Mycobacterium tuberculosis in a defined medium is very restricted by acid pH and Mg(2+) levels

Mycobacterium tuberculosis grows within the phagocytic vacuoles of macrophages, where it encounters a moderately acidic and possibly nutrient-restricted environment. Other mycobacterial species encounter acidic conditions in soil and aquatic environments. We have evaluated the influence of pH and divalent cation levels on the growth of M. tuberculosis and seven other mycobacterial species. In a defined medium, the growth of M. tuberculosis was very restricted by acidic pH. Higher levels of Mg(2+) were required for growth of M. tuberculosis in mildly acidic media (pH 6.0 to 6.5) compared to pH 7. 0 medium. The divalent cations Ca(2+), Zn(2+), or Mn(2+) could not replace Mg(2+) during growth at pH 6.25, but Ca(2+) could at least partially substitute for Mg(2+) during growth at pH 7.0. Among eight species of mycobacteria tested, there was a diversity of growth rates in media with acidic pH and low Mg(2+) levels. M. tuberculosis was the most restricted in growth at pH 6.0, and all of this growth required elevated levels of Mg(2+). M. kansasii and M. smegmatis also grew very poorly in acidic media with limiting Mg(2+). M. fortuitum, M. marinum, M. scrofulaceum, M. avium, and M. chelonae grew at pH 6.0 in an unrestricted manner. These results demonstrate that M. tuberculosis is unique among the mycobacteria in its extreme sensitivity to acid and indicate that M. tuberculosis must acquire sufficient Mg(2+) in order to grow in a mildly acidic environment such as within the phagosome of macrophages.     

Interaction of Mycobacterium avium with environmental amoebae enhances virulence.

Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium.     

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.     

Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.     

Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.     

Identification of Mycobacterium tuberculosis and Mycobacterium avium by non-radioactive probes: evaluation of the Snap Syngene system]

We evaluated an alkaline phosphatase-labeled oligonucleotide probe for the rapid identification of Mycobacterium tuberculosis and mycobacteria belonging to the M avium and M intracellulare complex (MAIS). Sixty-two strains of mycobacteria and eight strains belonging to related genera were studied. All M tuberculosis strains hybridized with the tuberculosis probe. All M avium and M intracellulare gave a strong signal with their probes. However the 3 M xenopi strains tested hybridized with all probes for MAIS complex.     

Mycobacterium tuberculosis and Mycobacterium avium inhibit IFN- gamma -induced gene expression by TLR2-dependent and independent pathways.

Mycobacteria-infected macrophages are poor responders to interferon-gamma (IFN-gamma), resulting in decreased expression of IFN-gamma-induced genes. In the present study, we examined the inhibition of IFN-gamma-induced gene expression by Mycobacterium tuberculosis and four different Mycobacterium avium strains in mouse RAW264.7 macrophages. Gamma-irradiated M. tuberculosis inhibited mRNA expression of a panel of six different IFN- gamma-induced genes. All four of the M. avium strains completely inhibited IFN-gamma-induced expression of MHC class II Aalpha and Ebeta mRNA. However, the Mac101 strain, which is serovar 1, inhibited IFN-gamma induction of IFN regulatory factor-1 (IRF-1) and guanylate-binding protein-1 (GBP-1) mRNA to a greater extent than the other M. avium strains, which are serovar 2. In this study, we also show that mycobacteria inhibit gene expression by both toll-like receptor 2 (TLR2)-dependent and independent pathways. The inhibition of IFN-gamma-induced gene expression by M. avium was reduced but not completely blocked in macrophages from TLR2(/) mice. IFN-gamma-induced gene expression was also inhibited by mycobacteria in RAW264.7 cells expressing dominantnegative TLR2 or myeloid differentiation factor 88 (MyD88), further indicating the existence of a pathway independent of TLR2 and MyD88. These data suggest that mycobacteria inhibit IFN-gamma-induced gene expression by multiple pathways involving both TLR2 and non-TLR receptors.     

Rapid identification of Mycobacterium species

Identification of Mycobacterium species is currently a long and fastidious procedure. We have developed a rapid (5-h) standard method using the API-ZYM system and rapid nitratase, urease and catalase tests. Pigmentation and growth rate were noted (but were only necessary for complete identification of 6% of strains). The tests were assigned numerical values from which a profile number was derived. A total of 716 strains were studied: 434 belonging to the M. tuberculosis complex and 282 other mycobacteria including 21 from M. avium complex. All M. tuberculosis complex strains were differentiated from all other mycobacteria and M. bovis was clearly separated from M. tuberculosis. All M. avium complex strains were differentiated from other mycobacteria. Among mycobacteria other than tubercle bacilli, 97% of the strains studied were identified. The method has proven to be simple, rapid and standardizable. It is suggested that the use of a code list could permit identification of most mycobacteria.     

Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic.

Mycobacterium tuberculosis and Mycobacterium avium multiply in cultured human macrophages (MP) within membrane-enclosed vesicles. These vesicles are generally assumed to be acidic. The evidence most frequently cited for this assumption is that pyrazinamide, which requires an acid pH to be effective, is effective and streptomycin, which loses most of its activity at a low pH, is poorly effective against tubercle bacilli. This assumption was tested by using the two weak bases chloroquine and NH4Cl to raise the pH of acidic vesicles in MP experimentally infected with M. tuberculosis or M. avium. An immunocytochemical locator of acidic regions in the MP was used to monitor the association of intracellular bacilli with acidity. MP were infected with M. tuberculosis or M. avium and incubated with various combinations of the drugs and the weak bases. Replication of the bacteria in the MP was measured by culture counts. Intracellular associations of the mycobacteria with acidity were assessed by electron micrographs and by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, which was detected with colloidal gold-labeled antibodies. It was confirmed by immunocytochemistry that both chloroquine and NH4Cl raise the pH of acidic vesicles in the infected MP. However, neither caused any pH-related change in the antimycobacterial activities of pyrazinamide or streptomycin or of the pH-independent drug isoniazid. Immunochemical analyses showed acidity to be associated with killed but not living mycobacteria in the MP. These findings suggest that living M. tuberculosis and M. avium are located in human MP in vesicles which are not acidic.     

Use of Gen-Probe AccuProbes to identify Mycobacterium avium complex, Mycobacterium tuberculosis complex, Mycobacterium kansasii, and Mycobacterium gordonae directly from BACTEC TB broth cultures.

To evaluate the utility of Gen-Probe AccuProbes for the identification of mycobacteria directly from BACTEC TB 12B vials containing acid-fast bacilli, culture results for 11,375 clinical specimens other than blood received from 1 January 1992 to 30 September 1993 were reviewed retrospectively. During this period, a total of 359 of 11,375 BACTEC vials were positive for acid-fast bacilli and were evaluated for mycobacteria with one or more probes: 224 were probed for Mycobacterium tuberculosis complex, 253 were probed for Mycobacterium avium complex, 64 were probed for Mycobacterium kansasii, and 77 were probed for Mycobacterium gordonae. After initial testing with the probes, 75 vials were positive for M. tuberculosis complex, 99 were positive for M. avium complex, 11 were positive for M. kansasii, and 55 were positive for M. gordonae. Repeat testing of vials that were initially probe negative or testing of colonies from subcultures of these vials identified an additional 11 M. tuberculosis, 27 M. avium complex, 1 M. kansasii, and 9 M. gordonae that were not detected on initial screening. On the basis of these data, the percentage of organisms identified directly from the BACTEC TB 12B vials upon initial screening with each of the four AccuProbes was 87.2% for M. tuberculosis complex, 78.6% for M. avium complex, 91.7% for M. kansasii, and 85.9% for M. gordonae.     

The recovery of Mycobacterium avium complex and Mycobacterium tuberculosis from blood specimens of AIDS patients using the nonradiometric BACTEC NR 660 medium

The ability of the nonradiometric BACTEC NR 660 aerobic 6A blood culture medium to support mycobacterial growth was investigated. During a 19-month period blood cultures from 140 AIDS patients were sent to the microbiology laboratory. After the cultures were incubated for seven days, aliquots of medium from the vials were centrifuged, sediments examined microscopically for mycobacteria, and cultured to mycobacterial media. Seventy-one AIDS patients (51%) had at least one blood culture positive for mycobacteria. There was a significant difference in the percent of female AIDS patients positive for mycobacteria compared to male patients (72% vs. 44%, P less than 0.01). Forty-four percent of all subsequently positive cultures were detected by an acid fast stain of the specimen sediment. Subcultures from the BACTEC 6A suspensions were positive on mycobacterial media at one-seven weeks (mean three weeks) after planting. Sixty-nine of the isolates were Mycobacterium avium complex, while two were Mycobacterium tuberculosis. Some bacteremias with M. tuberculosis may have been undetected because growth experiments with a reference strain showed that, in contrast to M. avium complex, M. tuberculosis did not increase in concentration in 6A medium.     

The Mycobacterium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.     

Localization of CORO1A in the macrophages containing Mycobacterium leprae

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A.     

Urease activity represents an alternative pathway for Mycobacterium tuberculosis nitrogen metabolism

Urease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficient Mycobacterium tuberculosis strain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage on M. tuberculosis in macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impaired in vitro growth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity in M. tuberculosis nitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion that M. tuberculosis virulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment.     

In vivo efficacy of phage therapy for Mycobacterium avium infection as delivered by a nonvirulent mycobacterium

The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly.     

A PE protein expressed by Mycobacterium avium is an effective T-cell immunogen

Infection of mice with Mycobacterium avium or immunization with a novel PE gene expressed by M. avium (MaPE) showed that a dominant T-cell immune response was elicited. Immunization with an MaPE DNA vaccine protected mice against an aerosol challenge with Mycobacterium tuberculosis, suggesting that mycobacteria express PE antigens with cross-protective T-cell epitopes.     

Surfactant protein D increases fusion of Mycobacterium tuberculosis-containing phagosomes with lysosomes in human macrophages

Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst.