Mycobacterium tuberculosis and the environment within the phagosome

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.


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).



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).



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).



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



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.



(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).



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).



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).



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.



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.



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.



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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Mycobacterium tuberculosis and the environment within the phagosome