Mycobacterium tuberculosis and
the Four-Minute Phagosome
By arresting the maturation of phagosomes, M. tuberculosis
avoids being delivered to lysosomes
David G. Russell, Georgiana E. Purdy, Roisin M. Owens,
Kyle H. Rohde, and Robin M. Yates
acrophages kill many household
microbes with minimal fuss, efficiently producing reactive oxygen and nitrogen intermediates,
and also delivering internalized
microbes to the low-pH, hydrolytically active
environment of the lysosome. However, Mycobacterium tuberculosis parasitizes host macrophages, subverting phagosome maturation and
thus evading this ordinarily effective host defense mechanism.
By arresting maturation of phagosomes, M.
tuberculosis avoids being delivered to lysosomes.
Despite this block, however, the vacuole that
this bacillus inhabits remains fusion competent
and behaves like an early endosome (Fig. 1). Its
pH is 6.4, it derives key nutrients from the rapidly
recycling endosomal system, and it retains the
membrane-fusion machinery associated with early
endosomes. Two big questions are associated
M
• Although Mycobacterium tuberculosis arrests
maturation of phagosomes and thus avoids being
delivered to lysosomes, this bacillus-containing
vacuole remains fusion-competent and behaves
like an early endosome.
• Mycobacterial cell-wall lipids and other bacterial molecules, including enzymes and possibly
isoprenoids, appear to modulate phagosome maturation.
with this phenomenon. How does the microbe
do this, and what are the consequences with
respect to the environment within the phagosome?
Markers of Phagosome Maturation
The differentiation of endosomes and phagosomes is documented extensively. Early endosomes and phagosomes acquire the small
GTPase rab5, which helps to recruit the PI3kinase, Vps34. As PI3-P increases on the membrane, it binds EEA1. EEA1 concentration increases concomitantly with a decrease in rab5
concentration and the appearance of rab7, another small GTPase that is abundant in late
endosomes. The vacuole also acquires lysosome-associated membrane proteins, such as
LAMPs 1 and 2, as well as lysosomal enzymes
that are delivered through fusion with other
lysosomes and also from the trans-Golgi network via the mannose 6-phosphate receptor.
To document the degree of maturation of
microbe-containing phagosomes, researchers typically use antibodies that recognize
some of these protein markers. Despite their
value and wide use, however, these antibody-based methods provide only static
measurements of a dynamic process, are
often employed subjectively, and provide
limited information about the environment
within phagosomes.
• Newer techniques go beyond monitoring pH in
phagosomes to determine how rapidly they acquire hydrolytic capacities, thus providing broadened means for tracking their dynamic responses
to specific pathogens.
Phagosome Modulation by
Mycobacterium: a Two-Step Process?
Since at least the 1970s, researchers suspected that mycobacterial cell wall lipids
David G. Russell is
Professor and
Chair, Georgiana E.
Purdy is a postdoctoral fellow, Roisin
M. Owens is a research associate,
Kyle H. Rohde is a
postdoctoral fellow,
and Robin M. Yates
is a graduate student in the Department of Microbiology and
Immunology, College of Veterinary
Medicine, Cornell
University, Ithaca,
N.Y.
Volume 71, Number 10, 2005 / ASM News Y 459
flux during maturation, M. tuberculosis
appears to inhibit this signaling by suppressing sphingosine kinase activity.
Lipoarabinomannan and other lipids
appear capable of blocking phagosome
maturation, which seems contrary to
the widely held belief that dead bacilli
fail to arrest phagosome/lysosome fusion. However, directly measuring vacuolar pH sheds light on this disparity
(Fig. 2). Phagosomes containing inert
particles such as immunoglobulin G
(IgG) beads are processed rapidly, acidifying to pH 4.5 within 15 minutes of
uptake, while phagosomes that contain
live Mycobacterium bovis BCG equilibrate slowly to pH 6.4. Meanwhile,
phagosomes containing heat-killed M.
bovis BCG stabilize at a pH of about
5.8, suggesting that cell wall lipids influence phagosome maturation but that
other effectors are also required to
modulate this process.
This interpretation is supported by
independent data generated through
analysis of transposon-mutagenized M.
tuberculosis mutants that were isolated
through a genetic screen that enriches
for bacteria defective in arresting
Diagram illustrating the major intracellular trafficking pathways that intersect with the
phagosome/lysosome fusion. Pools of
vacuoles containing live, virulent M. tuberculosis bacilli. These vacuoles are accessed by
transposon-mutagenized bacteria were
the recycling endosomal system, as evidenced by their acquisition of transferrin.
fed to macrophages whose lysosomes
Components of the bacteria released into the bacteria-containing vacuoles traffic out of
the vacuoles and coalesce in dense, lysosomal compartments. Finally, the bacteriawere loaded with iron dextran. Shortly
containing vacuoles, in common with most endosomal/lysosomal stages show intersecafter infection, the macrophages were
tion with delivery vesicles derived from the trans Golgi network of the host cell.
lysed, and the lysosomes isolated by
magnetic selection. Mutants that traffic
modulate membrane fusions inside host cells.
preferentially to these lysosomes were selected
Recent studies, notably by Vojo Deretic of the
through several rounds of enrichment.
University of New Mexico in Albuquerque and
The majority of these mutants are attenuated
his collaborators, suggest that the lipid lifor intracellular survival and show enhanced
poarabinomannan inhibits Vps34 activity, limfusion with late endosomes and lysosomes coniting production of PI3-P and arresting maturataining colloidol gold. Of particular signifition of the phagosome prior to its accumulation
cance, the most-attenuated mutants sequester in
of EEA1. Pharmacologic studies further implivacuoles at about pH 5.7. The overlap in phagocate calcium, calmodulin, calmodulin-depensome conditions between dead bacteria and
dent kinase II, and sphingosine kinase as reguthese mutants reinforces the idea that cell wall
lating maturation of phagosomes. For instance,
lipids go so far, but require further help to attain
David Kusner of the University of Iowa has
full modulation.
shown that treating infected macrophages with a
What Other Effectors Might Modulate
calcium ionophore recruits calmodulin, activates
Maturation?
calmodulin-dependent kinase II, and enhances
maturation of bacterium-containing vacuoles.
What molecules other than lipids modulate
Although ordinary phagosomes induce calcium
phagosomes? One possibility comes from study-
460 Y ASM News / Volume 71, Number 10, 2005
ing mutants defective in producing the
serine/threonine kinase PknG; these
mutants cannot arrest maturation of
their phagosomes and show reduced
survival in macrophages, according to
Jean Pieters of the University of Basel in
Switzerland and his collaborators. They
report that the kinase gains access to the
cytosol of the host cell and interferes
with the signaling cascade that drives
maturation. How the kinase PknG
moves from the lumen of the phagosome across the vacuolar membrane
and into the cell cytosol is a mystery,
because Mycobacterium spp. do not
have type III secretion systems.
Vojo Deretic and his collaborators
identified a secreted phosphatase, SapM,
that is capable of dephosphorylating
PI3-P, the phosphoinositol species required for recruiting EEA1 in maturing
phagosomes. Their studies indicate that
this enzyme can augment the activity
pH profiles generated by phagosomes containing IgG-beads, and live or heat-killed
of lipoarabinomannan, maintaining
Mycobacterium bovis BCG. The IgG bead-containing phagosomes show rapid acidification and attain a pH below pH 5.0 within 10 –12 minutes of internalization. In contrast, live
low PI3-P levels in the MycobacteriumM. bovis BCG-containing phagosomes equilibrate gradually to a pH of pH 6.3– 4.
containing phagosome and therefore
Surprisingly, dead bacteria behave like neither particle. They generate an intermediate
avoiding accumulation of EEA1. Beprofile and reach equilibium around pH5.9.
cause the substrate of SapM is a membrane component, the enzyme could
certainly work on the lipid within the phagomembrane-associated proteins that mediate the
some. Whether PI3-P is mobile within the lipid
membrane-fusion events required for passage to
bilayer and whether the phosphorylated form of
the lysosomes.
the phosphatidylinositol flips to the inner face of
In recognition of the dynamic nature of
the membrane are unaddressed questions.
phagosome
maturation, we developed several
Our transposon mutants include several geassays
to
quantify
functional characteristics that
notypes yet to be characterized. Although the
are
key
to
the
biology
of these compartments
explanation for some of these phenotypes may
(Fig.
3).
For
instance,
we
use fluorescence energy
be fitness and not mechanism dependent, at least
resonance
transfer
(FRET)
microscopy to meaone is involved in synthesizing a geranylgeranyl
sure
phagosome/lysosome
fusion.
pyrophosphate-based isoprenoid, which could
This is achieved by allowing macrophages to
act as an effector molecule. This possibility repinocytose
an acceptor fluorochrome that is
mains to be explored.
concentrated in lysosomes. Later, we add particles labeled with a donor fluorochrome that the
New Assays Help To Explore the
macrophages phagocytose, then we use the
Environment within Phagosomes
FRET signal to measure the degree of mixing of
the phagosomes with lysosomal contents— exDespite knowing the pH of phagosomes conciting the fluorochrome at the donor wavelength
taining pathogenic Mycobacterium spp., we
and measuring the signal at the acceptor wavehave few other direct physical measurements to
length. This assay provides a dynamic readout
describe changes in the lumen of such phagoover a period of 90 minutes before mixing, or
somes as they mature. In general, cell biologists
phagolysosomal fusion, reaches steady state.
have studied how this process is regulated,
Compounds that inhibit phagosome/lysosome
focusing almost exclusively on cytosolic and
Volume 71, Number 10, 2005 / ASM News Y 461
vide dynamic readouts over 15 and 60
minutes, respectively.
While inhibitors of the V-ATPase
complex block acidification and suppress both phagosome/lysosome fusion
and cysteine proteinase activity, they
increase the lipolysis rate. Whether this
is due to an increased delivery of lipases
or to a higher pH optimum for this
enzyme remains to be determined. In
any case, these assays demonstrate the
rates by which phagosomes acquire hydrolytic activities, which indicates how
quickly the compartment becomes hostile to intracellular pathogens.
M. tuberculosis and the Concept
of the Four-Minute Phagosome
Now that we have the tools to measure
the hydrolytic capacity of the M. tuberculosis-containing vacuole, it is interNovel assays for probing the lumenal environment of IgG-bead-containing phagosomes
esting to extrapolate from the above
have implications for the conditions within the M. tuberculosis-containing vacuole. (A) An
studies to predict what this environIgG-bead-containing phagosome reaches a pH of 6.4, the same pH as a M. tuberculosisment might be. For instance, an IgGcontaining vacuole, within four minutes of internalization by the macrophage. Therefore
the other conditions of a four-minute IgG-bead containing phagosome may be compabead phagosome reaches a pH of about
rable to the pathogen-containing compartment. (B) Mixing with lysosomal contents
6.5, the same pH as the M. tuberculosismeasured by FRET analysis indicates that, although progress is exponential, minimal
containing phagosome, within four
fusion with lysosomes has taken place. (C) Hydrolysis of the cathepsin L substrate
(Biotin-FR)2-Rhodamine 110 has barely moved from baseline at four minutes. (D)
minutes of internalization.
Similarly, hydrolysis of the lipase substrate, 1-trinitrophenyl-amino-dodecanoyl-2-pyreneAt four minutes, the FRET profile
decanoyl-3-O-hexadecyl-sn-glycerol, is minimal at four minutes postinternalization. These
indicates that phagosome/lysosome fudata imply that the M. tuberculosis-containing vacuole does not generate an environment
that is particularly hostile for the bacterium.
sion is just entering a phase of exponential increase, meaning that there is
still very little mixing with lysosomal
fusion, such as concanamycin A (V-ATPase),
contents. The hydrolytic capacity of a phagoW7 (calmodulin), and SB 203580 (MAP Kisome depends on several factors, including the
nase), depress the FRET signal.
concentrations, activation status, and pH opWe also developed an assay to measure ditima of its hydrolytic enzymes. In four-minute
rectly the hydrolytic capacity of phagosomes
phagosomes, both the lipase and cysteine prothat take up particles. To visualize protease acteinase substrates register minimal activity, pretivity, we load ligand-bearing particles with the
dicting that this environment will not be particfluorogenic substrate (biotin-Phe-Arg)2-rhodaularly hostile to M. tuberculosis.
mine 110, which is recognized by the cysteine
The reporter fluorochromes and fluorogenic
proteinase cathepsin L. Alternatively, to detect
substrates can be linked to the surface of M.
lipase activity we load particles with the lipase
tuberculosis, with no impact on viability, paving
substrate 1-trinitrophenyl-amino-dodecanoylthe way for direct experimental analysis of the
2-pyrenedecanoyl-3-O-hexadecyl-sn-glycerol. In
phagosomal environment. Moreover, we can
both cases, the fluorescent moiety, rhodamine 110
also test the compartment to which dead bacteand pyrene, respectively, remains quenched until
ria and transposon-mutagenized bacteria gain
hydrolyzed by an appropriate enzyme. The cysaccess. Finally, our longer-term goal is to use
teine proteinase substrate is processed more rappharmacological agents and the maturation aridly than the lipase substrate, and the assays prorest-defective mutants to manipulate the phago-
462 Y ASM News / Volume 71, Number 10, 2005
somes containing M. tuberculosis and to perform microarray transcriptional analysis to
build a picture of the differential responses induced by the environments experienced by Mycobacterium during intramacrophage infection.
So, while it is important to identify the mecha-
nism(s) responsible for arresting the maturation
of the phagosome, we feel that the information
gained from those studies can be exploited further to assemble a much broader understanding
of the intravacuolar environment and how the
bacterium detects and responds to its host.
SUGGESTED READING
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endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J. 15:6960 – 6968.
Sturgill-Koszycki, S., P. H. Schlesinger, P. Chakraborty, P. L. Haddix, H. L. Collins, A. K. Fok, R. D. Allen, S. L. Gluck,
J. Heuser, and D. G. Russell. 1994. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular
proton-ATPase. Science 263:678 – 681.
Thompson, C. R., S. S. Iyer, N. Melrose, R. VanOosten, K. Johnson, S. M. Pitson, L. M. Obeid, and D. J. Kusner. 2005.
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Vergne, I., J. Chua, and V. Deretic. 2003. Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2⫹/
calmodulin-PI3K hVPS34 cascade. J. Exp. Med. 198:653– 659.
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Wagner, D., J. Maser, B. Lai, Z. Cai, C. E. Barry, 3rd, K. Honer Zu Bentrup, D. G. Russell, and L. E. Bermudez. 2005.
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