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*
Departments of Molecular Microbiology and
Physiology and Cell Biology, Washington University, School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunoelectron microscopical studies by Clemens and Horwitz (4) on M. tuberculosis-infected MO revealed the presence of MHC class II and transferrin receptor in mycobacteria-containing phagosomes. Indeed, we demonstrated recently that MAC phagosomes, although restricted in acquisition of proton-ATPases, have access to cathepsins B, L, and D which enter phagosomes early in their maturation (10). In spite of this, the high phagosomal pH limits processing and activation of cathepsin D. The hypothesis that mycobacterial phagosomes represent early endosomes stabilized in this stage was given further credence in two studies showing their accessibility to transferrin, a marker for the recycling endosomal system (10, 11).
Several independent studies published during the past 10 years have
highlighted the role of certain cytokines in mycobacterial infections.
It is accepted that activation of MO by T cell-, NK cell-, or
macrophage-derived cytokines such as IFN-, IL-1,
granulocyte-macrophage-CSF, and TNF-
, alone or in concert, can
contribute to the antimycobacterial potential of these cells, resulting
in control of the infection in vitro (12, 13, 14, 15, 16, 17, 18, 19) and in vivo (20, 21).
Here, we have studied the influence of MO activation by IFN- and LPS
on the maturation of MAC phagosomes and correlated these changes with
mycobacterial survival. The data presented suggest that MAC phagosomes
are shifted from an early to a late endosomal stage of phagosome
maturation by MO activation, which is concomitant with a reduction in
mycobacterial growth and viability.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were used in this study: the mAb ID4B against
LAMP-1 was obtained from the Developmental Hybridoma Bank, Iowa City,
IA; mouse mAbs E11 and H9 against the vacuolar proton-ATPase E subunits
were generous gifts from Dr. S. Gluck (Washington University, St.
Louis, MO); rat mAb against transferrin receptor (R17/18, Tib217) were
obtained from American Type Culture Collection, mouse mAb
anti-digoxigenin was purchased from Boehringer Mannheim,
Indianapolis, IN. The rabbit polyclonal Ab to cathepsin D was a
generous gift from Dr. S. Kornfeld (Washington University). Secondary
species-specific Abs labeled with horseradish peroxidase were purchased
from Jackson Immunoresearch Laboratories, West Grove, PA. Iron-loaded
transferrin and human 2m were both purchased from
Calbiochem, San Diego, CA.
Bone marrow-derived MO and activation
MO were differentiated and maintained in culture as described
previously (3, 9). MO were grown in bacteriologic petri dishes and
split by placing them in cold PBS for 30 min followed by gentle
scraping. After splitting, MO were allowed to establish a monolayer for
at least 3 days in cell culture flasks before use. MO were activated
according to the following procedures: For early time points (2–4 h),
macrophages were incubated for 16 h with 400 U/ml recombinant
mouse IFN- and 200 to 500 ng/ml LPS for 2 h before infection.
For late time points (5 d), macrophages were incubated with 400 U/ml
recombinant mouse IFN- and 200 to 500 ng/ml LPS added on day 4
postinfection.
Bacteria, infection, colony-forming units
MAC 101 from frozen stocks derived from the first passage following isolation from a mouse was cultured in Middlebrook broth (Difco, Detroit, MI) and used within 3 days of thawing. Translucent colony appearance as an indication of virulence was tested before each experiment and routinely revealed <1% opaque (avirulent) colonies. MO cultures were infected with MAC 101 in a 10:1 ratio in DMEM without antibiotics supplemented with 5% L929 conditioned medium, 5% horse serum for 2 h, washed twice, and cultured for the time period indicated for each experiment.
MO cultures (1 x 104) were set up in 12-well tissue culture plates in duplicate and infected with MAC in a bacteria-MO ratio of 10:1. CFUs were determined as follows. The infected MO monolayers were lysed in 1 ml of PBS containing 0.5% Nonidet P-40, and lysates were passaged seven times through a 25-gauge tuberculin needle. The lysates were diluted in Middlebrook broth (1/100; 1/1000, 1/10,000), and 100-µl aliquots were plated in duplicate onto Middlebrook agar plates and incubated for 10 days.
Viability stain for mycobacteria
Following the protocol of McDonough and Kress (22), infected MO were incubated in DMEM/10% FCS containing 4 mg/ml carboxyfluorescein diacetate (Molecular Probes, Eugene, OR) for 10 min at 37°C, fixed in 4% formaldehyde, and counterstained with Evans blue (Sigma Chemical Co., St. Louis, MO). Mycobacteria-infected MO were examined under an epifluorescence microscope and scored blind for strong (metabolically active) or low/no fluorescence (metabolically inactive).
pH measurement
MAC labeled with NHS-carboxyfluorescein (Boehringer Mannheim) were used to infect MO on quartz glass. The fluorescence of the total cell population was measured at different time points fluorometrically and compared with a standard pH curve using NHS-carboxyfluorescein-labeled MAC in suspension and in nigericin-treated MO, as described previously (2, 23).
Protein labeling
Transferrin was labeled covalently with NHS-digoxigenin
(Molecular Probes) at a 10:1 molar excess in PBS, pH 7.8, for 30 min on
ice and purified over Kwiksep exocellulose columns (Pierce Chemical
Co., Rockford, IL). Human 2m was labeled with
125I (Amersham, Arlington Heights, IL) by Iodobeads (Pierce
Chemical Co.) according to the manufacturer’s instructions.
Phagosome isolation
Phagosomes containing MAC were isolated according to a protocol described earlier (9, 10). Contamination of phagosomes with other cellular material or digoxigenin-transferrin was evaluated for each experimental preparation by a crossover method described (9, 10). In brief, bacilli were added to unlabeled macrophages that were scraped and combined with an equal number of macrophages that were either labeled metabolically with [35S]methionine or incubated in digoxigenin transferrin. The level of contamination was 3 to 10% for metabolically labeled macrophages and below the level of detection for digoxigenin-transferrin macrophages.
PAGE and Western blot
Isolated phagosomes were lysed in 3x SDS buffer, boiled, and separated by SDS-PAGE (12%) under reducing conditions. After blotting onto nitrocellulose, blots were blocked in PBS containing 0.05% Triton X-114, 0.05% Tween-20, 10% goat serum, and 5% milk powder; incubated in the respective Abs; and developed using the Lumiglo system (Pierce Chemical Co.).
Electron microscopy
Macrophages activated before or postinfection were fixed in 2% glutaraldehyde in PBS, osmicated, dehydrated through ethanol, and embedded in Spurr’s resin. Thin sections were cut, contrasted with uranyl acetate and Reynold’s lead, and examined in a Jeol 100CX electron microscope. The distribution of bacteria/vacuoles were scored by examining 200 vacuoles for each condition. No more than 6 vacuoles were scored per cell.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the effect of MO activation on MAC infection in vitro, we
performed infection experiments on both resting and activated murine
MO. Preliminary experiments confirmed previous reports that activation
with IFN- (400 U/ml) alone postinfection did not induce a strong
microbicidal response or the marked alteration in vacuole physiology
detailed below. Full mycobactericidal activity and iNOS expression
requires a second signal, TNF-, which can be induced by LPS (24, 25). We therefore added LPS at 200 ng/ml in concert with IFN- to
maximize MO stimulation. Cell monolayers of MO were infected in a ratio
of 10 bacilli/MO which infected >90% of the cells. MO were cultivated
in medium plus or minus IFN-, and LPS was added before infection or
4 days postinfection (Fig. 1
). In resting
MO cultures, the number of recoverable CFU showed a modest
decline over the first 4 days until the numbers could be seen to be
increasing from 5 days postinfection. In contrast, in activated MO,
this decline was more pronounced and sustained until the macrophage
monolayer started to disintegrate at 6 days postinfection. Despite the
ability of activated macrophages to regulate the bacterial population,
bacterial death was neither rapid nor efficient, and CFU analysis
indicated that many bacteria persisted within activated
macrophages.
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and LPS 4 days
postinfection when the bacteria were entering exponential growth phase
(Fig. 1
). In nonactivated MOs, CFUs increased from 4 days postinfection
and stayed at high levels until 10 days postinfection. In contrast, the
CFUs from MO cultures activated 4 days postinfection peaked 2 days
after activation and slowly decreased until 6 days after activation (10
days postinfection) (Fig. 1). During this same time, bacteria in
resting MO had entered into exponential growth. To further characterize
the effect of MO activation on MAC viability, we used a viability stain
method based on bacterial esterase activity (22). The proportion of
metabolically active (esterase-positive) mycobacteria increased during
the entire observation period in resting MO; whereas in MO activated 4
days postinfection, the number of metabolically active mycobacteria
stayed low and decreased until 6 days postactivation (10 days
postinfection) (Fig. 1b).
Although the trend is comparable, the viability stain method
"overestimated" the relative number of live bacilli in activated
macrophages relative to the CFU data. This discrepancy may reflect
bacterial aggregation on isolation from activated macrophages
before plating or persistence of esterase activity in
nonreplicative bacteria. Despite this variation, both data sets argue
that activation with IFN- and LPS renders murine bone marrow-derived
MO capable of controlling an established MAC infection.
MAC are unable to block phagosome acidification in activated macrophages
In resting macrophages, phagosomes containing inert particles, or
other pathogens like Leishmania, rapidly acidify from the
extracellular pH to below pH 5.0 (2). In contrast, the pH within
phagosomes containing MAC shows a more restricted drop and equilibrates
to pH 6.2 (2). In this study, we compared the intraphagosomal pH of MAC
phagosomes in resting vs activated MO. Resting MO were compared with MO
activated for 16 h with IFN- and for 2 h with LPS before
infection. Carboxyfluorescein-labeled MAC were bound to the MO on ice,
unbound bacteria were washed off, and the MOs were placed at 37°C for
the time periods stipulated. As detailed previously (2), the pH of MAC
phagosomes in resting MO did not drop below pH 6.2 (Fig. 2). In contrast, in activated MO, the pH
in the MAC-containing phagosomes dropped to pH 5.2 within 180 min of
internalization (Fig. 2). There was an intriguing "rebound" in the
pH at 90 to 120 min postinfection which was observed in three
independent experiments.
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We had attributed the restricted acidification observed in
mycobacteria phagosomes to the paucity of vacuolar proton-ATPases in
this compartment (2). To test whether MO activation reverses this
phenotype, similar numbers of phagosomes were isolated from resting MO
or from MO activated with IFN- for 16 h and LPS for 2 h,
separated by SDS-PAGE, blotted, and probed for the E subunit of the
vacuolar proton-ATPase. At both 4 h (Fig. 3) and 5 days (Fig. 4) postinfection, minimal proton-ATPase
could be detected in phagosomes from resting MO. In contrast, MAC
phagosomes from activated MO infected for the same periods of time
contained significant amounts of proton-ATPase (Figs. 3 and 4).
Phagosome preparations were normalized for protein content (10);
however, to demonstrate that comparable amounts of protein were
loaded in the lanes, blots were subsequently probed for LAMP-1
(Figs. 3 and 4).
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Previous work from Clemens and Horwitz (4, 11), de Chastellier et
al. (5), and our laboratory (9, 10) had suggested that mycobacterial
phagosomes maintain communication with the early or recycling endosomal
system of their host cell. This was demonstrated by the
characterization of the flux of transferrin through MAC vacuoles even
in 9-day-old infections (10). If MAC vacuoles in activated MO undergo a
functional translocation to a later endosomal stage, this should be
mirrored by a loss of accessibility to transferrin. To test this, both
resting and activated MO infected with MAC 5 days previously were
incubated with digoxigenin-labeled transferrin for 45 min, washed
intensively, and lysed. Comparable numbers of phagosomes from resting
vs activated MO were separated by SDS-PAGE, blotted, and probed for
digoxigenin. MO lysates revealed that similar amounts of
digoxigenin-transferrin were taken up by both populations of MO (Fig. 4c). Also, as described previously, transferrin was
readily detected in MAC phagosomes from resting MO. In contrast, only
minimal amounts of digoxigenin-transferrin were present in MAC
phagosomes from activated MO (Fig. 4c). These were
the same phagosome preparations probed for proton-ATPase (E subunit)
and LAMP 1 as discussed. These data suggest that in activated MO,
MAC-containing phagosomes are shifted functionally toward a late stage
in endosomal maturation characterized by accumulation of proton-ATPases
and the loss of intersection with transferrin-carrying vesicles.
Additional experiments were performed using 125I-labeled
2m, which usually proceeds along the lysosomal pathway
following endosomal uptake. As expected, only small amounts of
2m could be detected in isolated MAC phagosomes from
resting MO, whereas up to four times more 2m was found
in phagosomes from activated MO (data not shown).
Alterations in phagosome physiology appears to precede the drop in MAC viability
It is still unclear whether the increased maturation of MAC phagosomes is symptomatic of or causal to the loss of bacterial viability. The complex responses of macrophages to cytokine activation render this question difficult to resolve. Furthermore, there are no data regarding the effects of macrophage activation on the regulation of phagosome/lysosome fusion independent of mycobacterial infections.
However, electron microscopic analysis of infected MO shortly after
activation indicates that one of the first phenotypic alterations in
activated macrophages is the coalescence of individual M.
avium-containing vacuoles into communal vacuoles with many bacilli
(Figs. 5 and 6). This is observed in
macrophages activated before infection and in macrophages activated 4
days postinfection and examined 5 days postinfection. Quantitation of
the distribution of bacilli in vacuoles (Fig. 6) demonstrates that at early time
points, 2 h postinfection, there is a marked decrease in the
numbers of bacilli in individual vacuoles in activated vs resting MO.
Furthermore, the more established the infection, such as 5-day
infections in resting MO, the higher is the proportion of single
bacteria in individual vacuoles. However, at both the 2-h and 5-day
time points, the majority of bacilli show few signs of damage or
degradation, even in the communal vacuoles of activated MO. These data
provide a preliminary indication that the merging of vacuoles precedes
any marked drop in bacterial viability, as assessed by CFUs shown in
Figure 1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of cytokine-activated MO in the modulation of mycobacterial
infections shows qualitative variation with mouse strain and bacterial
species but has been shown to be critical in protection through
experiments conducted on IFN- and TNF- receptor knockout mice
(26, 27). In vitro, IFN-- and/or TNF--activated murine MO are
able to inhibit growth of Mycobacterium bovis and M.
tuberculosis (12, 15, 16, 17, 26). Similarly, Appelberg and Orme (18)
showed bacteriostasis by IFN--activated murine MO toward some but
not all MAC isolates tested. The levels of MO-derived TNF- produced
by infected MO varied with bacterial isolate and appeared to be
crucially involved in the protective response (18, 19, 27). In systems
where mycobactericidal behavior has been induced, there is some debate
as to the mechanism(s) involved. Growth inhibition of M.
tuberculosis and M. bovis by activated MO has been
attributed to the release of NO but not to reactivated oxygen
metabolites (16). In contrast, growth inhibition of MAC by activated MO
has been suggested to be independent of NO (18) and mediated by
superoxide production (19), possibly in the context of enhanced
phagosome/lysosome fusion (18).
Despite the body of data demonstrating the central role of iNOS in the regulation of murine mycobacterial infections and other intracellular pathogens, the evidence is all of the same type, either protection through the use of inhibitors such as nonhydrolyzable arginine analogues or the use of iNOS knockout mice (28, 29, 30). Although these experiments all indicate that iNOS is necessary for protection, they do not shed light on whether it is sufficient. Obviously, iNOS fulfills a necessary function in the regulation of many infections, but a full appreciation of its mode of action must take into account the cascade of other physiologic changes that occur during macrophage activation.
The maintenance of mycobacterial vacuoles within the early endosomal machinery appears to require metabolic activity because dead bacilli are internalized into vacuoles that acidify and fuse with lysosomes (11) (S. Sturgill-Koszycki et al., unpublished observations). It is therefore important to appreciate which event comes first: the death, or compromise of the infecting microbe; or the differentiation of their compartment into an acidic, hydrolytically competent lysosome. If the latter is true, this translocation could drastically alter both the environment and cofactors that would potentiate the efficacy of NO. Our data on murine macrophages infected with Mycobacterium avium indicate that activation facilitates acidification of mycobacterial vacuoles in both de novo and established infections. The functional translocation toward more lysosomal compartments appears to precede any marked drop in microbial viability, suggesting that it is the product of an alteration in macrophage physiology, rather than a consequence of microbial death.
The lysosomal environment of activated macrophages could potentiate NO toxicity in several ways (24, 31). Oxidation of NO to nitrite and nitrate will be retarded at acid pH. NO can combine with H2O2, the production of which is up-regulated in activated macrophages, to make peroxynitrite (ONOO-). NO can release metal ions, such as Fe2+, from metalloproteins which can combine with H2O2 to produce ·OH and hypervalent iron. Furthermore, the activity of lysosomal hydrolases on the microbial cell wall likely exposes more targets to oxidative attack. The microbicidal responses of activated macrophages are probably based on the complex interactions of several antimicrobial phenomena, and more work is required on the effects of activation on the regulation of intracellular fusion within the endosomal/lysosomal continuum before these interactions can be appreciated.
Macrophage activation may also influence the availability or accessibility of nutrients; e.g., retention within the recycling endosomal machinery facilitates access to nutrients such as iron. M. tuberculosis possess high affinity iron-binding proteins, and it has been suggested that these are necessary because iron is a vital factor for mycobacterial growth (32). This hypothesis is given further credence by the report that the anti-MAC activity in human serum is due to transferrin-mediated iron depletion and that apotransferrin can reduce intracellular growth of MAC (33, 34).
Results detailed in this study indicate that the loss of mycobacterial viability is a gradual process which appears subsequent to the functional translocation of mycobacterial vacuoles to later endosomal and lysosomal compartments. This transition transfers mycobacteria from a relatively nonhostile environment and renders them accessible to low pH, reducing conditions, acid hydrolases, toxic peptides, and the potentiated effects of O and NO radicals at low pH (33, 35). Under these conditions, many of the bacilli appear static but viable. How long this condition could persist is unclear but it may mirror dormant M. tuberculosis infections in vivo before reactivation (36). A fuller appreciation of the complex interrelationships active during killing of Mycobacterium awaits elucidation of the mechanism(s) whereby Mycobacterium spp. arrest endosomal maturation and an understanding of the regulation of endosomal/lysosomal fusion in activated macrophages.
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Footnotes |
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2 Current address: MPI fur Infektionsbiologie, Monbijoustrasse 2, D-10117 Berlin, Germany.
3 Address correspondence and reprint requests to Dr. David G. Russell, Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail address:
4 Abbreviations used in this paper: MAC, Mycobacterium avium complex; LAMP 1, lysosome-associated membrane protein 1; NHS, N-hydroxysuccinimide; MO, bone marrow-derived macrophages; 2m, 2-macroglobulin; NO, nitric oxide; iNOS, inducible nitric oxide synthase.
Received for publication May 21, 1997. Accepted for publication October 9, 1997.
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S. L. Newman, L. Gootee, J. Hilty, and R. E. Morris Human Macrophages Do Not Require Phagosome Acidification to Mediate Fungistatic/Fungicidal Activity against Histoplasma capsulatum J. Immunol., February 1, 2006; 176(3): 1806 - 1813. [Abstract] [Full Text] [PDF]
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 T. M. Doherty and P. Andersen Vaccines for Tuberculosis: Novel Concepts and Recent Progress Clin. Microbiol. Rev., October 1, 2005; 18(4): 687 - 702. [Abstract] [Full Text] [PDF]
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A. Singh, R. Gupta, R. A. Vishwakarma, P. R. Narayanan, C. N. Paramasivan, V. D. Ramanathan, and A. K. Tyagi Requirement of the mymA Operon for Appropriate Cell Wall Ultrastructure and Persistence of Mycobacterium tuberculosis in the Spleens of Guinea Pigs J. Bacteriol., June 15, 2005; 187(12): 4173 - 4186. [Abstract] [Full Text] [PDF]
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M. Santic, M. Molmeret, and Y. Abu Kwaik Maturation of the Legionella pneumophila-Containing Phagosome into a Phagolysosome within Gamma Interferon-Activated Macrophages Infect. Immun., May 1, 2005; 73(5): 3166 - 3171. [Abstract] [Full Text] [PDF]
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L. Ramachandra, J. L. Smialek, S. S. Shank, M. Convery, W. H. Boom, and C. V. Harding Phagosomal Processing of Mycobacterium tuberculosis Antigen 85B Is Modulated Independently of Mycobacterial Viability and Phagosome Maturation Infect. Immun., February 1, 2005; 73(2): 1097 - 1105. [Abstract] [Full Text] [PDF]
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 M. A. Khan, R. Jabeen, T. H. Nasti, and O. Mohammad Enhanced anticryptococcal activity of chloroquine in phosphatidylserine-containing liposomes in a murine model J. Antimicrob. Chemother., February 1, 2005; 55(2): 223 - 228. [Abstract] [Full Text] [PDF]
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D. Wagner, J. Maser, B. Lai, Z. Cai, C. E. Barry III, K. Honer zu Bentrup, D. G. Russell, and L. E. Bermudez Elemental Analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-Containing Phagosomes Indicates Pathogen-Induced Microenvironments within the Host Cell's Endosomal System J. Immunol., February 1, 2005; 174(3): 1491 - 1500. [Abstract] [Full Text] [PDF]
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K. Pethe, D. L. Swenson, S. Alonso, J. Anderson, C. Wang, and D. G. Russell Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation PNAS, September 14, 2004; 101(37): 13642 - 13647. [Abstract] [Full Text] [PDF]
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T. F. Pais and R. Appelberg Induction of Mycobacterium avium growth restriction and inhibition of phagosome-endosome interactions during macrophage activation and apoptosis induction by picolinic acid plus IFN{gamma} Microbiology, May 1, 2004; 150(5): 1507 - 1518. [Abstract] [Full Text] [PDF]
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Z. Hmama, K. Sendide, A. Talal, R. Garcia, K. Dobos, and N. E. Reiner Quantitative analysis of phagolysosome fusion in intact cells: inhibition by mycobacterial lipoarabinomannan and rescue by an 1{alpha},25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway J. Cell Sci., April 15, 2004; 117(10): 2131 - 2140. [Abstract] [Full Text] [PDF]
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H. C. Mwandumba, D. G. Russell, M. H. Nyirenda, J. Anderson, S. A. White, M. E. Molyneux, and S. B. Squire Mycobacterium tuberculosis Resides in Nonacidified Vacuoles in Endocytically Competent Alveolar Macrophages from Patients with Tuberculosis and HIV Infection J. Immunol., April 1, 2004; 172(7): 4592 - 4598. [Abstract] [Full Text] [PDF]
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 K. H. Darwin, S. Ehrt, J.-C. Gutierrez-Ramos, N. Weich, and C. F. Nathan The Proteasome of Mycobacterium tuberculosis Is Required for Resistance to Nitric Oxide Science, December 12, 2003; 302(5652): 1963 - 1966. [Abstract] [Full Text] [PDF]
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J. Timm, F. A. Post, L.-G. Bekker, G. B. Walther, H. C. Wainwright, R. Manganelli, W.-T. Chan, L. Tsenova, B. Gold, I. Smith, et al. Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients PNAS, November 25, 2003; 100(24): 14321 - 14326. [Abstract] [Full Text] [PDF]
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J. P. Lopez, E. Clark, and V. L. Shepherd Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages J. Leukoc. Biol., October 1, 2003; 74(4): 523 - 530. [Abstract] [Full Text] [PDF]
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 D. Schnappinger, S. Ehrt, M. I. Voskuil, Y. Liu, J. A. Mangan, I. M. Monahan, G. Dolganov, B. Efron, P. D. Butcher, C. Nathan, et al. Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment J. Exp. Med., September 2, 2003; 198(5): 693 - 704. [Abstract] [Full Text] [PDF]
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V. Venketaraman, Y. K. Dayaram, A. G. Amin, R. Ngo, R. M. Green, M. T. Talaue, J. Mann, and N. D. Connell Role of Glutathione in Macrophage Control of Mycobacteria Infect. Immun., April 1, 2003; 71(4): 1864 - 1871. [Abstract] [Full Text]
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U. E. Schaible, H. L. Collins, F. Priem, and S. H.E. Kaufmann Correction of the Iron Overload Defect in {beta}-2-Microglobulin Knockout Mice by Lactoferrin Abolishes Their Increased Susceptibility to Tuberculosis J. Exp. Med., December 2, 2002; 196(11): 1507 - 1513. [Abstract] [Full Text] [PDF]
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E. Ghigo, C. Capo, C.-H. Tung, D. Raoult, J.-P. Gorvel, and J.-L. Mege Coxiellaburnetii Survival in THP-1 Monocytes Involves the Impairment of Phagosome Maturation: IFN-{gamma} Mediates its Restoration and Bacterial Killing J. Immunol., October 15, 2002; 169(8): 4488 - 4495. [Abstract] [Full Text] [PDF]
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T. R. M. da Silva, J. R. de Freitas, Q. C. Silva, C. P. Figueira, E. Roxo, S. C. Leao, L. A. R. de Freitas, and P. S. T. Veras Virulent Mycobacterium fortuitum Restricts NO Production by a Gamma Interferon-Activated J774 Cell Line and Phagosome-Lysosome Fusion Infect. Immun., October 1, 2002; 70(10): 5628 - 5634. [Abstract] [Full Text] [PDF]
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K. Mukherjee, S. Parashuraman, G. Krishnamurthy, J. Majumdar, A. Yadav, R. Kumar, S. K. Basu, and A. Mukhopadhyay Diverting intracellular trafficking of Salmonella to the lysosome through activation of the late endocytic Rab7 by intracellular delivery of muramyl dipeptide J. Cell Sci., September 15, 2002; 115(18): 3693 - 3701. [Abstract] [Full Text] [PDF]
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Y.-H. Sun, A. B. den Hartigh, R. d. L. Santos, L. G. Adams, and R. M. Tsolis virB-Mediated Survival of Brucella abortus in Mice and Macrophages Is Independent of a Functional Inducible Nitric Oxide Synthase or NADPH Oxidase in Macrophages Infect. Immun., September 1, 2002; 70(9): 4826 - 4832. [Abstract] [Full Text] [PDF]
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 D. G. Russell, H. C. Mwandumba, and E. E. Rhoades Mycobacterium and the coat of many lipids J. Cell Biol., August 5, 2002; 158(3): 421 - 426. [Abstract] [Full Text] [PDF]
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D. M. Bouley, N. Ghori, K. L. Mercer, S. Falkow, and L. Ramakrishnan Dynamic Nature of Host-Pathogen Interactions in Mycobacterium marinum Granulomas Infect. Immun., December 1, 2001; 69(12): 7820 - 7831. [Abstract] [Full Text] [PDF]
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L. Ramachandra, E. Noss, W. H. Boom, and C. V. Harding Processing of Mycobacterium tuberculosis Antigen 85B Involves Intraphagosomal Formation of Peptide-Major Histocompatibility Complex II Complexes and Is Inhibited by Live Bacilli that Decrease Phagosome Maturation J. Exp. Med., November 12, 2001; 194(10): 1421 - 1432. [Abstract] [Full Text] [PDF]
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K. Fischer, D. Chatterjee, J. Torrelles, P. J. Brennan, S. H. E. Kaufmann, and U. E. Schaible Mycobacterial Lysocardiolipin Is Exported from Phagosomes upon Cleavage of Cardiolipin by a Macrophage-Derived Lysosomal Phospholipase A2 J. Immunol., August 15, 2001; 167(4): 2187 - 2192. [Abstract] [Full Text] [PDF]
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L.-G. Bekker, S. Freeman, P. J. Murray, B. Ryffel, and G. Kaplan TNF-{{alpha}} Controls Intracellular Mycobacterial Growth by Both Inducible Nitric Oxide Synthase-Dependent and Inducible Nitric Oxide Synthase-Independent Pathways J. Immunol., June 1, 2001; 166(11): 6728 - 6734. [Abstract] [Full Text] [PDF]
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C. B. Stober, D. A. Lammas, C. M. Li, D. S. Kumararatne, S. L. Lightman, and C. A. McArdle ATP-Mediated Killing of Mycobacterium bovis Bacille Calmette-Guerin Within Human Macrophages Is Calcium Dependent and Associated with the Acidification of Mycobacteria-Containing Phagosomes J. Immunol., May 15, 2001; 166(10): 6276 - 6286. [Abstract] [Full Text] [PDF]
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J. E. Wigginton and D. Kirschner A Model to Predict Cell-Mediated Immune Regulatory Mechanisms During Human Infection with Mycobacterium tuberculosis J. Immunol., February 1, 2001; 166(3): 1951 - 1967. [Abstract] [Full Text] [PDF]
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A. M. Cooper, J. E. Pearl, J. V. Brooks, S. Ehlers, and I. M. Orme Expression of the Nitric Oxide Synthase 2 Gene Is Not Essential for Early Control of Mycobacterium tuberculosis in the Murine Lung Infect. Immun., December 1, 2000; 68(12): 6879 - 6882. [Abstract] [Full Text] [PDF]
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H.-J. Ullrich, W. L. Beatty, and D. G. Russell Interaction of Mycobacterium avium-Containing Phagosomes with the Antigen Presentation Pathway J. Immunol., December 1, 2000; 165(11): 6073 - 6080. [Abstract] [Full Text] [PDF]
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P. Chen, R. E. Ruiz, Q. Li, R. F. Silver, and W. R. Bishai Construction and Characterization of a Mycobacterium tuberculosis Mutant Lacking the Alternate Sigma Factor Gene, sigF Infect. Immun., October 1, 2000; 68(10): 5575 - 5580. [Abstract] [Full Text] [PDF]
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N. Mohagheghpour, A. van Vollenhoven, J. Goodman, and L. E. Bermudez Interaction of Mycobacterium avium with Human Monocyte-Derived Dendritic Cells Infect. Immun., October 1, 2000; 68(10): 5824 - 5829. [Abstract] [Full Text] [PDF]
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A. W. Tsang, K. Oestergaard, J. T. Myers, and J. A. Swanson Altered membrane trafficking in activated bone marrow-derived macrophages J. Leukoc. Biol., October 1, 2000; 68(4): 487 - 494. [Abstract] [Full Text]
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U. E. Schaible, K. Hagens, K. Fischer, H. L. Collins, and S. H. E. Kaufmann Intersection of Group I CD1 Molecules and Mycobacteria in Different Intracellular Compartments of Dendritic Cells J. Immunol., May 1, 2000; 164(9): 4843 - 4852. [Abstract] [Full Text] [PDF]
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A. M. Cooper, B. H. Segal, A. A. Frank, S. M. Holland, and I. M. Orme Transient Loss of Resistance to Pulmonary Tuberculosis in p47phox-/- Mice Infect. Immun., March 1, 2000; 68(3): 1231 - 1234. [Abstract] [Full Text] [PDF]
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M. S. Gomes, S. Paul, A. L. Moreira, R. Appelberg, M. Rabinovitch, and G. Kaplan Survival of Mycobacterium avium and Mycobacterium tuberculosis in Acidified Vacuoles of Murine Macrophages Infect. Immun., July 1, 1999; 67(7): 3199 - 3206. [Abstract] [Full Text] [PDF]
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M. S. Gomes, M. Florido, T. F. Pais, and R. Appelberg Improved Clearance of Mycobacterium avium Upon Disruption of the Inducible Nitric Oxide Synthase Gene J. Immunol., June 1, 1999; 162(11): 6734 - 6739. [Abstract] [Full Text] [PDF]
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S. M. Levitz, S.-H. Nong, K. F. Seetoo, T. S. Harrison, R. A. Speizer, and E. R. Simons Cryptococcus neoformans Resides in an Acidic Phagolysosome of Human Macrophages Infect. Immun., February 1, 1999; 67(2): 885 - 890. [Abstract] [Full Text] [PDF]
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T. R. Garbe, N. S. Hibler, and V. Deretic Response to Reactive Nitrogen Intermediates in Mycobacterium tuberculosis: Induction of the 16-Kilodalton alpha -Crystallin Homolog by Exposure to Nitric Oxide Donors Infect. Immun., January 1, 1999; 67(1): 460 - 465. [Abstract] [Full Text] [PDF]
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R. Teitelbaum, A. Glatman-Freedman, B. Chen, J. B. Robbins, E. Unanue, A. Casadevall, and B. R. Bloom A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival PNAS, December 22, 1998; 95(26): 15688 - 15693. [Abstract] [Full Text] [PDF]
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S Searle, N. Bright, T. Roach, P. Atkinson, C. Barton, R. Meloen, and J. Blackwell Localisation of Nramp1 in macrophages: modulation with activation and infection J. Cell Sci., January 10, 1998; 111(19): 2855 - 2866. [Abstract] [PDF]
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 A. Prada-Delgado, E. Carrasco-Marin, G. M. Bokoch, and C. Alvarez-Dominguez Interferon-gamma Listericidal Action Is Mediated by Novel Rab5a Functions at the Phagosomal Environment J. Biol. Chem., May 25, 2001; 276(22): 19059 - 19065. [Abstract] [Full Text] [PDF]
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G. St. John, N. Brot, J. Ruan, H. Erdjument-Bromage, P. Tempst, H. Weissbach, and C. Nathan Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates PNAS, August 14, 2001; 98(17): 9901 - 9906. [Abstract] [Full Text] [PDF]
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