mycobacterium tuberculosis and the environment within the phagosome
TRANSCRIPT
Kyle Rohde
Robin M. Yates
Georgiana E. Purdy
David G. Russell
Authors’ address
Kyle Rohde, Robin M. Yates, Georgiana E. Purdy,
David G. Russell
Department of Microbiology and Immunology,
College of Veterinary Medicine, Cornell Univer-
sity, Ithaca, NY, USA.
Correspondence to:
David G. Russell
Department of Microbiology and Immunology
College of Veterinary Medicine
Cornell University
Ithaca, NY 14853, USA
Tel.: 607 253 3401
Fax: 607 253 4058
E-mail: [email protected]
Acknowledgements
D. G. R. was supported by the following grants from the
National Institutes of Health: AI067027, AI057086,
HL055936. G. E. P. was supported by the Heiser Program
for Research in Leprosy and Tuberculosis.
Immunological Reviews 2007
Vol. 219: 37–54
Printed in Singapore. All rights reserved
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Munksgaard
Immunological Reviews0105-2896
Mycobacterium tuberculosis and the
environment within the phagosome
Summary: Once across the barrier of the epithelium, macrophagesconstitute the primary defense against microbial invasion. For mostmicrobes, the acidic, hydrolytically competent environment of thephagolysosome is sufficient to kill them. Despite our understanding ofthe trafficking events that regulate phagosome maturation, our apprecia-tion of the lumenal environment within the phagosome is only nowbecoming elucidated through real-time functional assays. The assaysquantify pH change, phagosome/lysosome fusion, proteolysis, lipolysis,and b-galactosidase activity. This information is particularly important forunderstanding pathogens that successfully parasitize the endosomal/lysosomal continuum.Mycobacterium tuberculosis infects macrophages througharresting the normal maturation process of the phagosome, retaining itsvacuole at pH 6.4 with many of the characteristics of an early endosome.Current studies are focusing on the transcriptional response of thebacterium to the changing environment in the macrophage phagosome.Manipulation of these environmental cues, such as preventing the pH dropto pH 6.4 with concanamycin A, abrogates the majority of thetranscriptional response in the bacterium, showing that pH is the dominantsignal that the bacterium senses and responds to. These approachesrepresent our ongoing attempts to unravel the discourse that takes placebetween the pathogen and its host cell.
Keywords: Mycobacterium, tuberculosis, macrophage, phagosome, phagocyte
Introduction
The primary function of the macrophage is, as its name
indicates, the engulfment of macromolecular complexes (1).
However, the roles that this process fulfills are actually
functionally distinct, and, among the professional phagocytes,
no cell shows greater plasticity than the tissue macrophage.
There are specific lineages of phagocytes that fulfill specialized
functions: the dendritic cell (DC) samples and presents antigens
to lymphocytes, and the osteoclast absorbs bone for remodeling
and repair. In contrast, the macrophage is called upon to fulfill
many diverse functions dictated predominantly by its interac-
tion with agonists and messengers of the innate and acquired
immune systems.
37
The primary role of a resting, tissue macrophage is one of
homeostasis. This cell roams around one’s tissues internalizing
cellular debris and apoptotic cells. This activity needs to be
achieved in a quiet, non-inflammatory manner to minimize
tissue damage. While this process is clearly critical during
development, it is also a necessary housekeeping activity in
adults. However, the macrophage is also the first line of defense
against microbial pathogens. For the vast majority of microbes,
internalization and exposure to the acidic, hydrolytically active
environment of the phagosome is sufficient to bring about their
demise (2). We like to think of the macrophage like household
disinfectant in that it kills 99.9% of all known household germs
quietly and efficiently. Anti-microbial activity may require
a more aggressive response from the phagocyte, and the cell is
equipped with a full range of Toll-like receptors (TLRs) and
other pattern-recognition receptors capable of recognizing and
inducing a preliminary, inflammatory response against amicro-
bial presence (3–6). The stimulation of TLRs signals the first
step in the transition of themacrophage to its role as an immune
effector cell. While a resting macrophage expresses only low
levels of major histocompatibility complex (MHC) class II
molecules, an activated macrophage upregulates its antigen-
presenting machinery markedly. Macrophage activation is like
a ladder with granulocyte-macrophage (GM) colony-stimulating
factor, TLR agonists, tumor necrosis factor-a, and interferon-g(IFN-g) representing the incremental steps from a resting to
a fully activatedmacrophage (7–10). The activatedmacrophage
shows increasedmicrobicidal activity through the expression of
the inducible nitric oxide synthase (iNOS) (11, 12) and
components of the nicotinamide adenine dinucleotide phos-
phate (NADPH) oxidase complex (NOX2) (13) and is trans-
formed into a potent antigen-presenting cell.
The major functions of the macrophage are all dependent on
the biology of the phagocytic compartment. Biochemical
analysis of the phagocytic compartment shows an increasing
abundance of lysosomal constituents as the phagosomematures
following internalization of particulate cargo (14, 15). This
process governs the degradative capacity of the phagosome and
leads to the death and digestion of most non-pathogenic
microbes. In an activated macrophage, this process is
accompanied by the recruitment of the NOX2 complex and
iNOS to release reactive oxygen and nitrogen intermediates into
the nascent phagosome, increasing its killing capacity.
Probing the intraphagosomal environment
Much of the published data characterizing the vacuoles
containing most intracellular pathogens have used immuno-
fluorescence colocalization methods to probe the differential
distribution of characterized host proteins or markers. This
methodology has been extremely useful and informative in
identifying the relative ‘position’ of bacteria-containing
phagosomes in the endosomal/lysosomal continuum of the
host cell. However, these methods do have their limitations.
First, unless accompanied by quantitation of signal intensity,
the analysis is essentially subjective. Second, the relative
distribution of host proteins does not provide information
about the physiological conditions within the compartment.
For example, the presence of cathepsin D by immunofluores-
cence does not provide information as to the activation status of
the enzyme or its actual activity within that compartment (16).
For these reasons, we have developed a range of quantitative
assays that provide a direct measure of a range of physiological
parameters in the phagosome that are indicative of the
maturation state of that compartment (16–19).
Initially, we measured the kinetics of acidification of
a phagosome following internalization of a particle. In a resting,
bone marrow-derived macrophage, the phagosome reaches
a pH of 5.0 or below within 10–12 min of internalization of an
immunoglobulin G (IgG)-coated particle (17) (Fig. 1A). Using
a fluorescence resonance energy transfer (FRET)-based assay to
measure the accumulation of fluid-phase lysosomal cargo in the
maturing phagosome, we observed that this reaches equilib-
rium approximately 90 min postinternalization (17) (Fig. 1B).
We have also developed a range of readouts that measure the
hydrolytic capacity of the phagosome, assaying bulk proteo-
lysis, cysteine proteinase activity, lipolysis, and b-galactosidaseactivity (17, 18). Not surprisingly, the kinetics of maximal
enzyme activity varied between the different substrates (Fig. 2).
With the exception of the b-galactosidase assay, all the enzyme
readouts reach a point of substrate limitation within a 20–
90 min time frame. Pharmacological agents known to inhibit
phagosome maturation, such as concanamycin A (a Hþ-
adenosine triphosphatase inhibitor) and W7 (a calmodulin
inhibitor), impact phagosome maturation with respect to
acidification, phagosome/lysosome mixing, and the acquisi-
tion of hydrolytic activity.
Biological signals that may modulate phagosome
maturation
TLR signaling and the autonomous phagosome
A recent report detailed that the addition of a TLR ligand to
a particle led to an accelerated maturation of the phagosome
into which the particle was internalized (20), suggesting an
immediate modulation of the phagosome upon formation. The
Rohde et al � Mycobacterium and the intraphagosomal environment
38 Immunological Reviews 219/2007
investigators examined a range of particles with and without
TLR ligands such as apoptotic cells, Staphylococcus aureus, Escherichia
coli, and Salmonella typhimurium. They examined the phagosomes
formed around these particles in wildtype macrophages and
macrophages from TLR2/TLR4 and myeloid differentiation
factor (MyD88)-deficientmice. Using immunofluorescence ana-
lysis with Lysotracker and lysosomal membrane glycoprotein-1
as markers for the lysosome coupled with electron microscopic
visualization of lysosomal structures, the authors concluded that
particles internalized in the absence of TLR signaling show
delayedmaturation of their phagosomes. These data gave rise to
the attractive hypothesis that the fate of a phagosome could be
determined locally and autonomously, and the TLR agonists
provided a localized signal that allowed the phagocyte to
discriminate between benign and potentially pathogenic cargo
(20, 21).
We revisited these experiments with our kinetic assays of
phagosomematuration and used model particles, either with or
without the addition of the TLR agonists lipopolysaccharide
(LPS) (for TLR4) and Pam3Cys (for TLR2), that could be fed to
Fig. 1. Phagosome maturation measured by acidification andphagosome/lysosome fusion. (A) Acidification profiles of phago-cytosed IgG-coupled beads. Fluorescent emission at 520 nm was takenevery 2 s using alternating excitation wavelengths of 450 and 490 nm.Determination of pH of the lumen of the bead-containing phagosomeswas calculated following polynomial regression of the excitation ratiowith a standard curve. Inhibition of acidification was achieved, withthe addition of the V-ATPase-specific inhibitor concanamycin A(100 nM) following binding of the beads. (B) FRET-based phago-some/lysosome fusion profiles. Lysosomal fusion profiles for IgG-
coupled bead-containing phagosomes were generated using theequation FU ¼ FRT/DRT � FBO/DBO (where FU, arbitrary fluorescentunits, FRT, FRET-generated fluorescent emission in real time, DRT,donor emission in real time, FBO, ‘FRET’ signal contribution of thebeads alone, and DBO, donor emission of the beads alone). Fluorescentmeasurements were taken every 2 s for 3 h. Diminished phagosome/lysosome fusion was achieved with the calmodulin inhibitor W7(15 and 50 mM) and the V-ATPase inhibitor concanamycin A(100 nM). Reproduced with permission from (17).
Fig. 2. Phagosome maturation measured by the acquisition ofhydrolytic activities. (A) Cysteine proteinase substrate hydrolysisprofiles for IgG-coated bead-containing phagosomes. Traces weregenerated using the equation FU ¼ R110/AF594 (where FU,arbitrary fluorescent units, R110, real-time rhodamine 110 fluores-cence, and AF594, starting Alexa fluor 594 fluorescence) and wereaveraged over two experiments. Measurements were taken everysecond for 30 min. Manipulation of hydrolytic rates was achievedwith inhibitors concanamycin A (100 nM), W7 (15 mM), and
leupeptin (100 mg/ml). (B) Phagosomal b-galactosidase activityin macrophages. The fluorescence of the calibration fluor (emission at610 nm with excitation at 555 nm) remains constant throughoutthe assay. The fluorescent intensity of the fluorescein-basedsubstrate (emission at 515 nm with excitation at 490 nm) increasesas it is dequenched. b-galactosidase activity can be diminished withthe competitive inhibitor PETG (200 mg/ml) and the V-ATPaseinhibitor concanamycin A (100 nm). PETG, 2-phenylethyl-b-D-thiogalactoside.
Rohde et al � Mycobacterium and the intraphagosomal environment
Immunological Reviews 219/2007 39
macrophages (19). These particles carried the opsonic ligands
IgG [Fc receptor (FcR)] and mannosylated bovine serum
albumin [mannose receptor (ManR)] to target them through
known pathways of internalization. We determined initially
that these particles only activated TLR signaling, assayed by p38
phosphorylation and degradation of inhibitor of nuclear factor
kB (IkB), if a TLR agonist was present on the particle surface.
This finding showed that there were no ‘contaminating’ TLR
agonists on our particles. We observed that the activation of
TLRs during phagocytosis had no detectable effects on either the
rate of acidification or the rate of phagosome/lysosomemixing.
Subsequently, it has been argued that the use of ManR- and FcR-
ligands ‘maxed’ out the signaling pathway, thus obviating the
need for TLR stimulation to accelerate phagosome maturation
(22).However, experiments performedwith phosphotidylserine-
coated C18 silica particles with or without TLR agonists also
failed to show a TLR-dependent component regulating the
kinetics of phagosome maturation (19). We also looked at the
uptake of S. aureus with or without LPS by both wildtype and
TLR2-deficient macrophages (Fig. 3). While activation of TLR
signalingwas observedwith both S. aureus and S. aureus plus LPS in
wildtype macrophages, only the S. aureus plus LPS activated TLR
signaling in the TLR2-deficient macrophages. Most signifi-
cantly, however, the kinetics of phagosome/lysosome mixing
were again identical irrespective of the presence or absence of
TLR signaling. These data suggest strongly that TLR agonists do
not modulate phagosome maturation through TLRs.
One observation that emerged from our studies was that
MyD88-deficient macrophages exhibited depressed levels of
phagosome/lysosome mixing independent of the identity of
Fig. 3. Phagosomes containing S. aureusformed in the presence or absence of TLR2or TLR4 signaling exhibit comparable
phagosome/lysosome fusion profiles,indicating the stimulation of either TLR2
or TLR4 does not impact on phagosomematuration. (A) The FRET-based assay wasused to quantify phagosome/lysosome mix-ing following uptake of formalin-fixed S. aureusþ/� LPS in wildtype (WT) (C57BL/6) andTLR2�/� macrophages. Data are presented asan average over four individual sets of data.(B) Degradation of IkBa and phosphorylationof p38-mitogen-activated protein kinase inthe macrophage were examined by immuno-blotting following phagocytosis of S. aureusparticles with or without incorporated LPS inWT (C57BL/6) and TLR2�/� macrophages.Reproduced with permission from Elsevier (19).
Rohde et al � Mycobacterium and the intraphagosomal environment
40 Immunological Reviews 219/2007
the particle (19). This observation raises the possibility that
MyD88-deficient macrophages have a defect in phagosome
maturation that is independent of an absence of short-term TLR-
mediated signaling. In the earlier report, the investigators did not
explore model particles with and without TLR ligands, but relied
on the genotype of their macrophages to show a role of TLR
signaling (20). It is therefore possible that their interpretation did
not allow for differences acquired bymacrophages developing in
an environment without TLRs (23, 24).
Phagosome maturation in the activated macrophage
The previous section detailed short term, immediate effects of
TLR activation on phagosome maturation; however, macro-
phages stimulated with TLR agonists such as LPS and cytokines
such as IFN-g for a prolonged period (overnight) show
markedly enhanced bactericidal activities and an increased
capacity to present antigen to T lymphocytes. It is unclear how
activation impacts the lumenal environment of the phagosome
to enhance these activities.
Earlier work emphasized the key role that the generation of
reactive oxygen and nitrogen intermediates play in the micro-
bicidal activities within the phagosome in activatedmacrophages
(11, 12, 25), but the effects that activation has on the
physiological parameters inside the phagosome remained to be
determined. To examine this question, we analyzed the rates of
acidification and phagosome/lysosome mixing and the rates of
acquisition of protease, lipase, and b-galactosidase activities inphagosomes in macrophages activated by LPS, IFN-g, or LPS andIFN-g (18). The rates of acidification of phagosomes containing
Man-bovine-serum-albumin (BSA)-coated beads were altered
subtly, with LPS-treated cells exhibiting an accelerated pH drop.
At 2 h postinternalization, the LPS- and LPS plus IFN-g-activatedmacrophage phagosomes were at pH 4.7, while untreated cells
and cells treated with IFN-g had a phagosomal pH of 4.9. The
kinetics of phagosome/lysosomemixing shownbyFRET analysis
also showed subtle alterations. Early on, phagosomes in cells
treated with LPS plus IFN-g showed a delayed acquisition of
lysosomal cargo, but from2 h onwards, all of the phagosomes in
activated macrophages showed enhanced and protracted accu-
mulation of lysosomal cargo.
These data are consistent with increasing the lysosomal
characteristics of the phagosome. It was therefore extremely
surprising when we analyzed the profiles of acquisition of
lysosomal hydrolases by the phagosomes in the activated
macrophages (Fig. 4). The phagosomes showed that the differing
states of activation (LPS versus IFN-g versus LPS plus IFN-g)modulated the acquisitionof protease, lipase, andb-galactosidaseactivities differentially. Protease activitywas depressed by66%by
Fig. 4. Activated macrophages exhibit differential downregulation
of their hydrolytic capacity depending on the activating stimulus.Specific hydrolase activities of phagosomes containing mannosylatedbeads bearing fluorogenic substrates along with a calibration fluor weremeasured in resting and macrophage monolayers activated by overnightincubation with LPS (10 ng/ml) and/or IFNg (100 U/ml). Theincrease in substrate fluorescence relative to the calibration fluor(relative reporter fluorescence) correlates to substrate hydrolysis andwas plotted against time. (A) Phagosomal proteolysis was measuredthrough incorporation of the generic protease substrate DQ greenBodipy BSA. (B) Phagosomal lipolysis was measured throughincorporation of the triglyceride analogue 1-trinitrophenyl-amino-dodecanoyl-2-pyrenedecanoyl-3-O-hexadecyl-sn-glycerol. (C) Phago-somal b-galactosidase activity was measured through the incorporationof the b-galactosidase substrate 5-dodecanoylaminofluorescein di-b-D-galactopyranoside. Reproduced with permission from (18).
Rohde et al � Mycobacterium and the intraphagosomal environment
Immunological Reviews 219/2007 41
IFN-g, lipase activity was depressed by 73% by LPS plus IFN-g,andb-galactosidase activitywas reducedby43%byLPSplus IFN-g.This differential modulation of enzymatic activities indicates
strongly that the effects of activation on the hydrolytic profile of
the phagosome cannot be explained by a global modulation of
either pH or lysosomal fusion.
It is accepted generally that lysosomal hydrolases are acquired
by either fusion with pre-existing, acidified lysosomes, or late
endosomes or through the shuttling of newly synthesized
enzymes, usually in pro-forms, from the trans-Golgi network.
To determine which source was dominating our hydrolase
readouts, we looked at the kinetics of proteolysis in phagosomes
internalized by macrophages treated with brefeldin A (BFA) to
dissolve the Golgi apparatus and block secretory transport (26).
BFA treatment did not change the kinetics of proteolysis,
indicating that the overwhelming bulk of activity observed comes
frommixingwith pre-existing, hydrolytically competent vesicles.
An alternative explanation for the heterogeneity is that the
different activation signals modulate the enzyme profile in pre-
existing, hydrolytically competent lysosomes through either
differential expression or delivery of enzymes. To test this
possibility, we fed macrophages iron dextran, chased this fluid-
phase marker into the lysosomes, and isolated the lysosomal
compartments by magnetic selection (18, 27). Following
solubilization, these lysosomal extracts were characterized for
enzyme activity and found to be comparable and not mirroring
the differences observed in the phagosomes.
So how can this heterogeneity be explained? A similar,
differential acquisition of lysosomal enzymes was observed
previously in both macrophages and DCs, and the authors
invoked heterogeneity among the lysosomal and prelysosomal
compartments and their differential fusion with maturing
phagosomes (28). Data supporting the differential fusion
characteristics of activated versus resting macrophages have
already been generated, mediated by the LPS-regulated gene
(LRG) family of guanosine triphosphatases (GTPases) (29, 30);
however, there is no information available concerning the
nature of such heterogeneous, prelysosomal vesicles. The
possibility also exists that instead of heterogeneous vesicles,
the differential acquisition of hydrolases could be generated by
differential partitioning of lysosomal constituents within
a tubulovesicular lysosome and transient fusion with the
phagosome similar to that invoked previously in the ‘hit and
run’ hypothesis for phagosomal maturation (31) (Fig. 5).
Fig. 5. Generation of a ‘mature’ phagosome that is functionallydistinct from the cell’s lysosomes. The model explores two possiblemechanisms by which a maturing phagosome could acquire some butnot all of the hydrolytic activities represented in the lysosome. Themechanisms proposed would need to be modulated by the immunestatus of the macrophage. The different activities observed could be theproduct of either the differential relative concentrations of hydrolases(lumenal content) or the differential distribution of ion transportersresponsible for the physiological environment that could regulateenzyme activity differentially (membrane components). (A) The
maturing phagosome fuses with a subset of lysosomal or prelysosomalvesicles that contain distinct sets of hydrolytic activities or membranetransporters. (B) The tubularization of the lysosomal network. Fusionwith regions of the lysosome results in selective acquisition of a subsetof hydrolases and/or transporters, consistent with the ‘kiss and run’model. The differential distribution of V-ATPases in the vesicles ortubularized regions of both models could explain the disparity inpH between the ‘total’ lysosome, as defined by bulk enzyme analysisand pH, and the ‘mature’ phagosome. Reproduced with permissionfrom (18).
Rohde et al � Mycobacterium and the intraphagosomal environment
42 Immunological Reviews 219/2007
What is the functional significance of the reduced hydrolytic
capacity when activated macrophages are known to bemore, not
less, microbicidal? As mentioned previously, a resting macro-
phage’s primary role is the quiet, non-inflammatory degradation
of tissue debris. Therefore, this cell ought to be highly
degradative. In contrast, an activated macrophage has a more
ambivalent mission; it is required not only to kill microbes but
also to maximize the efficiency of antigen presentation. If killing
is performed primarily by reactive oxygen and nitrogen
intermediates, it may ‘free’ the lysosomal milieu to focus on
antigen processing. The proteolytic capacity of the DC
phagolysosome is a fraction of that observed in the macrophage
(28). Moreover, the alkalinization of the DC phagosome restricts
this activity even further (32). This finding implies that toomuch
degradation is actually counterproductive to the optimal
generation and half-life of peptide fragments suitable for loading
into MHC class II molecules (33, 34). Activation ofmacrophages
with IFN-g therefore may enhance their immune presence by
both upregulating expression ofMHC class IImolecules and fine-
tuning proteolysis to promote epitope generation.
The endosomal continuum and the Mycobacterium
tuberculosis-containing phagosome
Pathogens capable of surviving within macrophages use a range
of differing strategies to avoid delivery to the lysosome and
subsequent death. Some pathogens such as Listeria and Shigella
escape into the cytosol, others like Leishmania and Coxiella actually
survive and replicate within the lysosomal milieu. Many, like
Legionella, Brucella, Erlichia, and Mycobacterium spp., subvert the
normal progression of their phagosomal compartment and
prevent it from fusingwith ormaturing into an active lysosomal
compartment (2).
The phagosome-containingM. tuberculosis behaves as though it
has been arrested at an early stage of its maturation (35–38). As
such, it appears to retain all the characteristics of a normal
phagosomal compartment shortly after internalization. The
vacuole maintains a lumenal pH of 6.4 (27, 39), it retains Rab5
(40–42), the small GTPase associated with membrane fusion
events in the early endosome, and it remains accessible to the
rapid recycling pathway, as defined by the passage of transferrin
receptor and GM1 ganglioside through the pathogen-containing
compartment (16, 40, 43) (Fig. 6). Several molecules have been
proposed to modulate phagosome maturation (Fig. 6). The cell
wall lipids lipoarabinomannan (44–46), trehalose dimycolate
(47), and the sulfolipids (48) have all been implicated in
blocking phagosome/lysosome fusion. In addition, the bacte-
rial phosphatase SapM (49) and the serine/threonine kinase
PknG (50) are thought capable of regulating phagosome
maturation. Secreted acid phosphatase M (SapM) is proposed to
function through dephosphorylation of phosphatidylinositol 3-
phosphate (PI3P) and protein kinase G (PknG) through the
phosphorylation of an unknown host protein. However, as
M. tuberculosis lacks a type III secretory system, it is unclear how
these enzymes access their respective cytosolic substrates.
This state of arrested maturation appears to correlate with the
viability of the infecting organism. Mutants of M. tuberculosis
defective in achieving full arrest in the maturation of their
phagosomes are delivered into compartments with a lower pH,
pH 5.8, and are incapable of entering into replication (27)
(Fig. 7). This finding is consistent with previous observations
that M. tuberculosis is growth-arrested at lower pH (51). Unlike
Mycobacterium marinum, which is capable of escaping into the
cytosol (52), M. tuberculosis maintains an intravacuolar location
and can be seen in membrane-bound compartments 16 days
postinfection of bone marrow-derived macrophages in culture
(53) (Fig. 8). Therefore, the effective modulation of this
compartment is crucial to the success of the pathogen.
Activation of M. tuberculosis-infected macrophages
Activation of the host macrophage will lead to the death of the
infecting organism, eventually. Activation of the macrophage
with IFN-g prior to infection with M. tuberculosis enables the host
cell to overcome the blockage in phagosome maturation and
deliver the bacterium to an acidic, hydrolytically competent
lysosome (54, 55). This alteration in the fusion capacity of
the bacterium-containing compartment is mediated through the
upregulation of the LRG p47 family of GTPases (29, 30). The
majority of the published literature deals with oxidative killing
mediated through reactive oxygen and nitrogen intermediates,
both in isolation and in combination leading to production of
peroxynitrite. In addition, bacteria that have a reduced capacity
to repair or degrade proteins ‘damaged’ by reactive nitrogen
intermediates show a marked reduction in virulence in mice
showing the key role that reactive nitrogen intermediates (RNI)
play in controlling the tuberculosis infection (56, 57).
There are, however, other avenues leading to bacterial
killing. Recent data show that the induction of autophagy in
infected macrophages leads to the delivery of M. tuberculosis to
lysosomes and their subsequent demise. This killing appears
independent of iNOS or NOX2 activity, although the process
also appears to be mediated by the LRG p47 family of GTPases
and can be upregulated either by exposure to IFN-g or by classicstimulators of autophagy, such as serum starvation or treatment
with rapamycin (58). In recent experiments, we showed that
Rohde et al � Mycobacterium and the intraphagosomal environment
Immunological Reviews 219/2007 43
isolated macrophage lysosomes, when solubilized in medium
withM. tuberculosis, led to death of the bacteria (59) (Fig. 9). This
finding indicated that macrophage activation or induction of
autophagy could lead to the delivery of the bacterium to the
lysosome, which contained an activity capable of killing the
bacterium directly. Fractionation of the lysosomal extract and
analysis by mass spectrometry identified ubiquitin-derived
peptides as the active component. We found that purified
ubiquitin obtained commercially had no bactericidal activity;
however, preincubation of the ubiquitin with lysosomal
hydrolases, such as cathepsins B or L, led to bacterial killing
through the liberation of bactericidal peptides (the hydrolases
themselves had no effect). Moreover, the addition of ubiquitin
to isolated lysosomal extracts augmented the microbicidal
activity. Finally, M. tuberculosis could be killed by a synthetic
peptide based on the ubiquitin sequence; this peptide had been
shown previously to bemicrobicidal to fungi andGram-positive
bacteria (60). These data imply that lysosomal degradation of
ubiquitin releases peptides capable of killing M. tuberculosis.
How does ubiquitin get into the lysosome?
Ubiquitinated proteins are usually thought of as being tar-
geted for proteosomal degradation within the cytoplasm
Fig. 6. Diagrams of the Mycobacterium-containing phagosome ina resting macrophage (A), and the proposed mechanisms that
modulate the behavior of the vacuole (B–E). (A) The vacuolecontaining viable bacteria has a pH of pH 6.4, generated by a smallnumber of V-ATPase complexes. The vacuole remains accessible to therapid recycling pathway, as defined by the trafficking of transferring,but fails to fuse with lysosomes. The vacuolar membrane retains Rab5GTPase and the phosphatidylinositol 3-kinase Vps34 but acquires littlePI3P and consequently does not accumulate EEA. (A) LAM on particlesarrests the maturation of the phagosome. LAM inhibits the PI3P kinaseVps34, thus avoiding PI3P accumulation and inhibiting recruitment ofEEA1. It remains to be determined how LAM in the lumen of thephagosome inhibits an enzyme on the cytosolic face of the vacuole.(B) The mycobacterial phosphatase SapM dephosphorylates PI3P and is
thought to work in concert with LAM to minimize PI3P accumulationon the cytosolic face of the phagosome. However, PI3P is generated onthe cytoplasmic face, and it is unclear if SapM needs to access thecytoplasm or if PI3P flips in the bilayer, which given the charge of PI3Pwould have to be a facilitated process. (C) The serine/threonine kinasePknG is thought to phosphorylate a host substrate that regulatesphagosome maturation; however, it is unclear how this proteinaccesses the host cell cytoplasm. (D) Recent experiments onMycobacterium avium indicate that the close physical association formedbetween the bacterial cell wall and the phagosomal membrane is crucialto maintaining the phagosome in its arrested state. Disruption of thisclose relationship by cholesterol depletion directs the bacterium to thelysosome. EEA, early endosome antigen; LAM, lipoarabinomannan;ATP, adenosine triphosphate.
Rohde et al � Mycobacterium and the intraphagosomal environment
44 Immunological Reviews 219/2007
(61); however, integral membrane proteins that are mono-
ubiquitinated are delivered to multivesicular bodies (MVBs) in
the early endosomal system (62, 63). In addition, ubiquitinated
proteins that denature and form aggregates in the cytosol cannot
be degraded by the proteosome and are sequestered into
autophagosomes prior to delivery to the vesicular late endosome
(64–66). Thus, there are two potential sources of ubiquitin in
the cell (Fig. 10). Immunoelectron microscopy with an anti-
ubiquitin antibody that recognizes only protein-conjugated
ubiquitin showed a significant degree of label associatedwith the
internal membranes in structures resembling either MVBs or late
endosomes and within the dense luminal matrix of lysosomes
(59) (Fig. 11). These data support the hypothesis that ubiquitin
gains access to the lysosomal system of macrophages.
The MVB is generated by the endosomal sorting complex
required for transport (ESCRT) sorting machinery, requires
ubiquitination of membrane constituents for its formation, and
is the site of delivery of ubiquitinated membrane proteins
destined for degradation in the lysosome (62, 63). Budding of
internal vesicles is thought to take place preferentially in the
early endosome. Integral membrane proteins are reported to be
de-ubiquitinated prior to their delivery to internal vesicles.
However, our immunoelectron microscopic data showing
ubiquitin on themembranes of these internal vesicles argue that
de-ubiquitination is incomplete or can be regulated (59).
Therefore, theMVB represents one potential source of ubiquitin
that could access the lysosome.
Cytosolic proteins that are ubiquitinated but aggregate prior
to degradation by the proteosome are sequestered into
Fig. 9. Bactericidal activity of lysosomal SF on Mycobacteriumsmegmatis and M. tuberculosis. (A) Bacteria were incubated with buffer(squares) or with SF at 50 mg/ml (open squares) or 100 mg/ml(triangle). At indicated time points, the viable bacteria were determinedby plating colony-forming units. (B) The bactericidal activity oflysosomal SF is protease sensitive. M. smegmatis was incubated overnightat 37 �C with buffer, SF, or with SF preincubated with trypsin, and thenthe viable bacteria were determined. (C) SF-treated mycobacteriadisplay membrane damage. M. smegmatis was incubated overnight withbuffer or SF and then analyzed by electron microscopy. An arrowindicates the compromised cell wall in the treated sample. SF, solublefraction. Reproduced with permission from (59).
Fig. 8. An electron micrograph of a macrophage infected 16 days
previously with M. tuberculosis (CDC1551). The micrograph shows theaccumulation of membranous whorls within infected macrophages.The bacteria in the macrophage clearly remain intravacuolar, even after16 days in culture. Reproduced with permission from (53).
Fig. 7. A graph illustrating the survival curves of mutants defectivein arresting phagosome maturation following infection of macro-
phages. There is a reasonable consensus between the acidification of thephagosomes and the survival of mutants in macrophages. The survivalprofile of the mutants Rv0986, Rv3377c, Rv3378c, and MT3491.1, allof which went into phagosomes of pH 5.8, in comparison withwildtype CDC1551, which resided in phagosomes of pH 6.4.Reproduced with permission from (27).
Rohde et al � Mycobacterium and the intraphagosomal environment
Immunological Reviews 219/2007 45
autophagosomes (65–68). Cells that are deficient inmembers of
the Atg family of autophagy-mediating proteins show enhanced
accumulation of ubiquitinated aggregates in the cytoplasm,
indicating that this process occurs continuously within cells and
that the autophagous pathway is required to regulate the
‘damage’ (69, 70). It is generally accepted that the autophago-
some is formedwhen endoplasmic reticulum (ER)membrane is
laid down around a region of the cytoplasm or a cytoplasmic
organelle. LC3, or Atg8, is a lipidated, membrane-associated
component essential to the formation of the autophagosome
(71, 72). LC3 has been shown to bind to ubiquitinated protein
aggregates by means of a bridging protein, p62 (64, 67). It is
proposed that this mechanism ensures the incorporation of
protein aggregates into the autophagosome. The autophago-
some then fuses with a late endosomal compartment, delivering
its cargo into the endosomal/lysosomal continuum.
We have shown previously that fluid-phase markers, such as
Texas red- or digoxigenin-labeled dextran, introduced into the
cytosol of macrophages by scrape loading, are transferred into
the vesicular late endosome through a process that is sensitive to
inhibitors or promoters of autophagy (73). Early on, the
cytosol-derived label lay predominantly inside the internal
vesicles in multivesicular compartments. These internal struc-
tures are comparable with the ubiquitin-positive, internal
vesicles observed in autophagous macrophages; moreover, the
induction of autophagy enhanced the delivery of ubiquitin to
the lysosomes. Immunofluorescent analysis of macrophages
treated by serum starvation showed a strong colocalization of
ubiquitin conjugates and the autophagosome protein LC3 (59).
In Drosophila, proteins involved in the fusion of multivesicular
endosomes with lysosomes, such as Vps18, also mediate fusion
of autophagosomes with lysosomes, indicating that the two
pathways converge at the level of the late endosome (74). This
finding implies that the internal vesicles in the multivesicular
late endosome/lysosome may be derived from two different
cellular processes. The inter-relationship between the MVBs,
autophagosomes, and the late endosomal compartments needs
to be defined further to clarify the pathway(s) of delivery of
ubiquitin to the lysosome.
Most significantly, ubiquitin was observed in vacuoles
containing M. tuberculosis following activation of the macro-
phages with IFN-g or by serum starvation (Fig. 12). These data
Fig. 10. Diagram of the possible route(s) taken by ubiquitin toaccess the lysosome and the M. tuberculosis-containing vacuole.
Mono-ubiquitinated integral membrane proteins are sequestered inendosomes that are integrated in the MVB through the activity of theESCRT machinery. The MVB delivers its cargo to the late endosome,which is hydrolytically active. In addition, poly-ubiquitinated proteinsthat aggregate in the cytoplasm associate with p62, which binds to thekey component of the autophagous pathway, LC3 or Atg8. It ishypothesized that this ensures sequestration of the denatured,
ubiquitinated cargo into the autophagosome. The autophagosomes,like the MVB, will deliver its cargo into the hydrolytic environment ofthe late endosome, leading to the degradation of ubiquitin and theliberation of the microbicidal peptides. Activation of the macrophageand the induction of autophagy will drive the M. tuberculosis-containingvacuole to fuse with the lysosome, but it also enhances theconcentration of ubiquitin in the lysosomal compartment augmentingthe killing activity (59).
Rohde et al � Mycobacterium and the intraphagosomal environment
46 Immunological Reviews 219/2007
argue persuasively that autophagy facilitates delivery of
M. tuberculosis to a compartment that contains hydrolyzed
ubiquitin (59). Furthermore, the link to the autophagosome
as a route of delivery of ubiquitin to the lysosome provides
a killing mechanism that may explain how the induction of
autophagy leads to the demise of a range of bacterial pathogens
(58, 75–77).
Predicting the conditions within the M. tuberculosis-
containing phagosome
Although it is generally assumed that the intracellular
environment of the macrophage phagosome is hostile, there
are few direct data. Previously, we postulated that the
M. tuberculosis-containing phagosome was comparable physio-
logically to a ‘4-min’ IgG-bead-containing phagosome (37).
This hypothesis was based on the logic that the M. tuberculosis-
containing phagosome has a pH of 6.4 and retains the fusion
characteristic of an early endosome, implying that the
compartment is not altered functionally but merely ‘immortal-
ized’. An IgG-bead-containing phagosome attains a pH of 6.4
4 min postinternalization; therefore, we predicted that the
M. tuberculosis-containing phagosome would share the other
physiological characteristics of the 4 min phagosome (Fig. 13).
At this time point in a phagosome, there is minimal proteolytic
or lipolytic activity, and the degree of phagosome/lysosome
mixing is extremely low. However, this is merely a hypothesis;
these conditions remain to be determined experimentally.
Bacterial responses to the changing environment within
the phagosome
Given our increasing knowledge of the physiology of the
phagosome, we have initiated analysis of the ‘other side of the
equation’, namely the bacterial responses to the environments
encountered by the bacterium during the infection process. The
uptake of M. tuberculosis and the establishment of an intra-
macrophage infection represent the transition between two
different environments, and it is intriguing to consider the
environmental cues detected by the bacterium during this
journey and how the bacterium responds to these cues. There
are several published studies that address the changing
transcriptional response of intracellular versus extracellular
bacilli through promoter traps, differential transcript screens,
proteomics, or microarray analysis (78–84). These methods
have identified a range of transcripts and proteins that are
upregulated in the intracellular environment, some of which
have been shown subsequently to be important for survival
inside the host cell (85–87). However, what is absent from
these studies is a link between the actual environmental change
or cue and the transcriptional response.
To address this particular issue, we decided to perform
a temporal dissection of the transcriptional response of
M. tuberculosis through the first 2 h of the infection process, from
the time the bacterium bound to the macrophage surface to
a time point 2 h later, when the vacuole had stabilized to a pH of
6.4 (K. H. Rohde and D. G. Russell, manuscript submitted).
Fig. 11. Immunoelectron microscopy of Mycobacterium tuberculosis-infected macrophages. Cells were probed with mouse anti-ubiquitin(12 nm gold) and rat anti-lysosomal membrane glycoprotein-1 (anti-LAMP1) (6 nm gold). An untreated, infected macrophage showing thatthe bacteria-containing vacuoles have minimal ubiquitin signal. Theubiquitin signal is associated predominantly with the limitingmembranes or LAMP1-positive membranous material inside the lumenof LAMP1-positive vesicles. Reproduced with permission from (59).
Fig. 12. Immunoelectron microscopy of M. tuberculosis-infectedmacrophages. Cells were probed with mouse anti-ubiquitin (12 nmgold) and rat anti-lysosomal membrane glycoprotein-1 (anti-LAMP1)(6 nm gold). In cells treated by starvation for 2 h, M. tuberculosis can beseen in vacuoles with flocculent lysosomal matrix that is positive forubiquitin and is also positive for LAMP1. Reproduced with permissionfrom (59).
Rohde et al � Mycobacterium and the intraphagosomal environment
Immunological Reviews 219/2007 47
Host cell contact is known to induce a transcriptional response
in awide range of bacterial pathogens including Yersinia,Neisseria,
Pseudomonas, Porphyromonas, Salmonella, and Actinobacillus (88–93).
Indeed, bacteria that rely on the release of effectors through
a type III secretory apparatus need to sense the host cell to be
able to inoculate these effectors into the cell cytosol early in their
interaction. In contrast, M. tuberculosis accesses macrophages
through phagocytosis and does not necessarily require
modulation of the host cell for entry. Usingmicroarray analysis,
we examined the transcriptional response ofM. tuberculosis bound
to the surface of macrophages in the presence of cytochalasin D
to prevent internalization (K. H. Rohde and D. G. Russell,
manuscript submitted). In contrast to the pathogens listed
above, we failed to detect any movement in the M. tuberculosis
transcriptome under these conditions, indicating that the
bacterium was unaware of its location and could not detect
the host cell by contact alone.
We then allowed the macrophages to internalize the bound
bacteria and sampled the bacterial RNA at 5, 20, 40, 60, 80,
100, and 120 min postinternalization to generate a high-
resolution temporal profile of bacterial transcription during
the entry process. Rather than relying solely on an arbitrary
cutoff based on fold induction of individual transcript levels,
we used extraction and analysis of differential gene expression
(EDGE) software to detect transcriptional change (94). This
software preserves the data linkages between time points and
expanded the list of upregulated genes. At 2 h, 68 genes were
identified as upregulated by cutoff (>1.5-fold, P< 0.05), and
a further 75 genes were identified by EDGE analysis, showing
a sustained increase in transcriptional abundance over the 2 h
infection.
Among the genes upregulated, several patterns or unifying
themes emerged, illustrated in the gene trees in Fig. 14. Several
members of the WhiB family of putative transcriptional
Fig. 13. Assays for probing the lumenal environment of IgG-bead-containing phagosomes have implications for the conditions within
the M. tuberculosis-containing vacuole. (A). An IgG-bead-containingphagosome reaches a pH of 6.4, the same pH as a M. tuberculosis-containing vacuole, within 4 min of internalization by the macro-phage. Therefore, the other conditions of a 4 min IgG-bead-containingphagosome may be comparable with the pathogen-containingcompartment. (B). Mixing with lysosomal contents measured by FRET
analysis indicates that, although progress is exponential, minimalfusion with lysosomes has taken place. (C). Hydrolysis of the cathepsinL substrate (Biotin-FR)2-Rhodamine 110 has barely moved frombaseline at 4 min. (D). Similarly, hydrolysis of the lipase substrate,1-trinitrophenyl-amino-dodecanoyl-2-pyrenedecanoyl-3-O-hexadecyl-sn-glycerol, is minimal at 4 min postinternalization. These data implythat the M. tuberculosis-containing vacuole does not generate anenvironment that is directly hostile to the bacterium.
Rohde et al � Mycobacterium and the intraphagosomal environment
48 Immunological Reviews 219/2007
regulators unique to actinomycetes (95–97), notably whib3,
whib6, and whib7, were upregulated. WhiB7 is upregulated
markedly by exposure of bacteria to aminoglycosides, and
mutants deficient in WhiB7 expression showed increased
sensitivity to the drug. Therefore, it was proposed that WhiB7
regulates drug-resistance mechanisms in Mycobacterium (98).
Recently, however, it has been shown that heat shock, iron
starvation, growth phase, and exposure to toxic fatty acids all
induce increased expression of whiB7, implying that the gene
responds to a broad range of noxious stimuli (99).WhiB3 binds
to the sigma factor RpoV and is thought to play a role in bacterial
survival and tissue pathology late in infection (100, 101). Both
WhiB3 and WhiB6 expression are induced to some degree by
aminoglycosides, ethanol, oxidants, low pH, sodium dodecyl
sulfate, and heat shock, again indicating that a range of stressful
stimuli impact on their expression levels (99). This family of
proteins has an extremely interesting structural feature: they all
possess an iron-sulfur (FE-S) cluster. Electron paramagnetic
resonance and ultraviolet visible spectroscopy analysis of
reduced WhiB3 indicates that the cluster is sensitive to O2,
which leads to the conversion of a [4Fe-4S]2þ active form of the
protein to a [3Fe-4S]1þ inert form (102). This mechanism of
cluster degradation is also observed in the fumerate nitrate
regulator (Fnr) of E. coli, which regulates the transcription
of >100 genes in response to oxygen and nitric oxide (103).
Both Fnr andWhiB3 are acutely sensitive to reactive oxygen and
nitrogen intermediates, such as superoxide and nitric oxide.
This sensitivity suggests that this family of proteins may be
extremely responsive sensors for redox changes experienced
during the infection process.
We also observed increased expression of a number of genes
that are members of the DosR regulon. The two-component
signal transduction system DosSR comprises a membrane-
bound histidine kinase sensor (DosS) and a cytoplasmic
response regulator (DosR). Genes lying downstream of this
regulator are upregulated in experimental models that induce
dormancy in M. tuberculosis and upon exposure of the bacterium
to NO or hypoxia, both of which reduce aerobic respiration
(104–107). We also noted the upregulation of genes belonging
to the regulon of another two-component signal transduction
system, PhoPR. At 2 h postinfection, 25 of the 44 genes
reported to be controlled by PhoPR were upregulated. The
M. tuberculosismutant lacking PhoPR showmarked attenuation in
survival in both macrophages and mice (108–110). Among the
genes upregulated are two operons required for the synthesis of
deacyltrehaloses, polyacyltrehaloses, and sulfolipids, all impor-
tant components of the mycobacterial cell wall, suggesting that
the bacteria surface is modified postinvasion of the macro-
phage. The homologous two-component signal transduction
system in Salmonella was thought to respond to intracellular
[Mg2þ] (111); however, recent data suggest that it is primarily
an intracellular pH sensor (112).
Fig. 14. Gene trees illustrating the relative levels ofexpression of genes in the WhiB family and in the
PhoPR and DosSR regulons at 2 and 24 h post-infection. Both the PhoPR and the DosSR regulonsshow broad upregulation that increases from 2 to24 h. The expression levels are relative to theextracellular bacteria in culture medium, that ist ¼ 0. The red–blue spectrum shows the dynamicrange of the expression profiles from zerofold tofourfold.
Rohde et al � Mycobacterium and the intraphagosomal environment
Immunological Reviews 219/2007 49
In addition to these linked regulators and regulons, we also
observed the upregulation of multiple genes linked to fatty acid
metabolism in the b-oxidation pathway, several fadD and fadE
genes, as well as icl1, the gating enzyme into the glyoxylate shunt.
The glyoxylate shunt is mobilized by bacteria and plants
exploiting fatty acids as their primary carbon source and avoids
the loss of carbon as carbon dioxide generated by flux through
the Krebs cycle (113, 114). This involvement is consistent with
previous observations that lipid metabolism is critical to the
survival ofM. tuberculosis inside themacrophage. Mutants deficient
in icl1or in both icl1 and icl2 showan impaired ability to survive in
activated macrophages or a complete inability to infect macro-
phages, respectively (85, 86). What remains to be determined is
whether the fatty acids are acquired directly from the host or
mobilized from triacylglycerol stores present in the bacteria.
Linking transcriptional response to the physiological
cue in the phagosome
The transcriptional response of M. tuberculosis as it enters the
macrophage is likely triggered by multiple, inter-dependent
cues such as pH, ionic balance, and nutritional and oxidative
stresses. Existing studies have examined the pH response of
M. tuberculosis by adjusting the pH of the bacterial medium in vitro
(115). This treatment, however, denies the contribution of
other, undefined cues in the phagosome. To dissect the relative
contribution of some of these cues, we adopted a subtractive
approach by blocking acidification of M. tuberculosis-containing
phagosomes through treatment with concanamycin A, which
inhibits Hþ-ATPase activity. This treatment prevents the
M. tuberculosis-containing vacuole from acidifying from pH 7.2,
the pH of the external medium, to pH 6.4. Although this pH
shift is relatively minor, considerably less than the shift that
would be experienced on translocation to the lysosome, we
found that preventing acidification to pH 6.4 abrogated the
upregulation of around 80% of the genes normally upregulated
on macrophage entry (K. H. Rohde and D. G. Russell,
manuscript submitted) (Fig. 15). This included almost all the
genes under regulation of PhoPR and DosRS. Intriguingly, the
list of pH-sensitive genes generated within the phagosomal
environment showed only partial overlapwith the acid-induced
transcriptome reported for bacteria in culture (115).
Clearly, much work remains to determine the other
physiological cues that the bacterium detects and responds to
within the phagosome. However, the availability of NOX2- and
iNOS-deficientmice, among other knockoutmouse strains, will
allow us to adopt a similar subtractive approach to the
contributions of reactive oxygen and nitrogen intermediates.
These data will be comparedwith existing studies conducted on
macrophages from these mice (82).
The M. tuberculosis transcriptome 24 h postinfection
The data discussed previously relate to the first 2 h of infection,
the early response. Published studies have focused primarily on
the transcriptional response at later time points (81, 82, 116).
Our 2 h postinfection profile is considerably less complex than
previous studies; however, if we look at 24 h postinfection, we
observe approximately 135 upregulated genes, a number much
more comparable with those detailed previously. Moreover, the
majority of regulons upregulated at 2 h show enhanced
transcriptional activity at 24 h (Fig. 14).What is the significance
of the increased flux in the transcriptome? To understand the
significance, one needs to accommodate two contributory
factors. First, the bacterium is still adapting to its environment or
adapting the environment to its liking. Second,when one infects
cells with M. tuberculosis, a significant fraction of the initial
inoculum will be killed, which means that during this process,
until the ‘failed’ bacteria are cleared, the transcriptome will
reflect a heterogeneous population of bacteria. This heteroge-
neity will reduce the power of transcriptional profiling to
resolve the genes important for intracellular growth. To address
both issues, we are extending our temporal analysis to several
days postinfection to identify the point at which the
transcriptome stabilizes. This stabilization will be indicative of
a return to homogeneity in which most of the bacteria will have
Fig. 15. Behavior of CDC1551 2 h upregulated genes (>1.5�, P <0.05, shown in red) in the neutral pH of the concanamycin A-
treated phagosome. Positive expression ratio indicates genes expressedhigher in untreated (pH 6.4) versus concanamycin A-treated (pH 7.0)macrophage. A subset of genes in the 2 h transcriptional response areseen as concanamycin A sensitive because they require the pH shift topH 6.4 during invasion of the macrophage to induce their increasedexpression.
Rohde et al � Mycobacterium and the intraphagosomal environment
50 Immunological Reviews 219/2007
entered into a replicative phase, allowing us to identify the ‘core’
metabolic transcriptome required for a productive infection.
Concluding remarks
These transcriptional studies provide insights into the bacterium’s
responses to the environments within the host cell. Our
interpretations are based on the supposition that an alteration in
the transcriptional profile is preceded by the detection of an
environmental change and its transduction by the bacterium’s
sensor/effector systems. Currently, our appreciation of the
physiological changes taking place within the lumen of the
phagosome is limited. However, the intraphagosomal pH
measurements and the real-time hydrolytic assays afford us an
invaluable glimpse into how the physiology of the phagosomal
changes during maturation and with the immune status of the
macrophage. Closing the loop between the phagosomal environ-
ment and the bacterial response is a challenge but one that is
considerably more accessible with these new methods. The
transfer of these phagosomal reporters to the surface of
M. tuberculosis should facilitate analysis of the bacterium-containing
vacuoles by a confocalmicroscopewith a spectral analyzer. Couple
this analysis with the expression of fluorescent proteins driven by
promoters specific to defined intracellular cues, and you can start
to see how real-time readouts of bacteria fitness or stress can be
developed. The system is more accessible than it has ever been.
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