the trojan horse: survival tactics of pathogenic mycobacteria in macrophages

The Trojan horse: survival tactics ofpathogenic mycobacteria inmacrophagesLiem Nguyen and Jean Pieters

Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland

Mycobacterium tuberculosis, the causative agent of

tuberculosis, has infected billions of people worldwide.

A key to the success of M. tuberculosis and related

pathogenic mycobacteria lies in their ability to persist

within the hostile environment of the host macrophage.

After internalization by macrophages, most microbes

are rapidly transported to lysosomes in which they are

destroyed. By contrast, pathogenic mycobacteria pre-

vent fusion of phagosomes with lysosomes, thereby

surviving intracellularly. Recent progress in understand-

ing the molecular biology of host–mycobacteria inter-

actions is providing insights into these survival tactics.

Pathogenic mycobacteria: survival within macrophages

The first barrier that amicrobial pathogenmust face wheninfecting multicellular organisms is the innate immunedefense system. A key cell involved in vertebrate immun-ity is the macrophage; this ‘professional phagocyte’recognizes microbes and engulfs them into vacuoles calledphagosomes. Phagosomes then fuse with lysosomes(phagosome maturation), resulting in degradation of thecargo. To survive this primary defense system, intracellu-lar pathogens have developed several different strategies;whereas some pathogens escape macrophage phagocytosisby promoting their uptake by less-bactericidal cells, othersdevelop abilities to tolerate the hostile environmentwithin lysosomes [1].

Pathogenic mycobacteria do not avoid capture bymacrophages or tolerate the lysosomal environment.Instead, they survive the bactericidal milieu of themacrophage by blocking fusion of their intracellularniche – the phagosome – with late endosomes andlysosomes [2–4]. The success at establishing and main-taining a protected niche inside this hostile environmentseems to be crucial for the persistence of pathogenicmycobacteria because they manage to survive for pro-longed periods of time inside host cells.

Phagocytosis of pathogenic mycobacteria

As intracellular pathogens, mycobacteria are activelyinternalized by macrophages. Uptake through phago-cytosis requires recognition of mycobacteria by receptor

Corresponding author: Pieters, J. ([email protected]).Available online 7 April 2005

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molecules exposed at the macrophage cell surface. Theseinclude complement receptors (CRs), the mannose recep-tor, immunoglobulin fragment carrying the constantregion of the heavy chain (Fc) receptors and scavengerreceptors [5]. For certain bacteria, the route of entrydefines their final fate; for example, Salmonella typhi thatenters murine macrophages through CR3 resides withinlysosomes, whereas entering through CR1 enables it tosurvive in phagosomes [6]. Pathogenic mycobacteria,however, are internalized into macrophages throughdifferent receptors without affecting their subsequentsurvival [5,7]. Although the precise uptake mechanismin vivo remains to be established, one of the mostimportant receptors in vitro is CR3 [8–10]. Inhibition ofthis receptor by antibody binding or by blocking its lectinsite leads to strong reduction of phagocytosis of myco-bacteria [11,12].

Phagocytosis of mycobacteria by CR3 requires theaccumulation of the plasma membrane steroid cholesterolat the site of entry [11]. Depletion of cholesterol by amixture of the cholesterol-reducing drug lovastatin andthe cholesterol chelator methyl-b-cyclodextrin results in adefect in uptake of mycobacteria. The role of cholesterol inCR3-mediated uptake is specific for mycobacteria becausecholesterol-depleted macrophages can still internalizeother microorganisms. However, the precise function ofcholesterol remains unknown. It might be involved in thesignal-transduction reactions following the engagementof CR3 [8,13]. Alternatively, the presence of cholesterolmight aid the rearrangement of the membrane–cyto-skeleton network during phagocytosis. Interestingly,cholesterol-rich domains that are formed at the leadingedge of moving endothelial cells change the microviscosityof the membrane at the front, thus enabling efficientformation of the actin network required to push theleading-edge membrane forward [14]. Similarly, choles-terol might help to increase the viscosity of the membranethat is in contact with the hydrophobic mycobacterial cellwall, thereby accelerating phagocytic uptake. Moreover,rearrangements of the actin network at the phagocytosingcup might also help to engulf the bacteria.

Inhibition of phagosome maturation

Phagosome maturation refers to a process in whichmaterial that is internalized by phagosomes is deliveredto lysosomes, either through a series of membrane

Review TRENDS in Cell Biology Vol.15 No.5 May 2005

. doi:10.1016/j.tcb.2005.03.009

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Box 1. Proteins containing FYVE and PX domains

The FYVE domain is composed of w70 amino acids rich in cysteine

and forms a zinc finger. Its name is derived from the first letters of the

first four proteins found to share the domain (Fab1p, YOTB, Vac1p

and EEA1). This domain binds to PI3P and is required for the

localization of proteins to early-endosomal membranes (Figure I).

The PX domain contains w120 amino acids, with the polyproline

motif PXXP in the middle, and is found primarily in the phagocyte

NADH oxidase (Phox). This domain binds preferentially to PI3P but

can also bind to other phosphoinositides. Many PX-binding proteins

have been reported to have key roles in endosomal trafficking and

protein sorting.

FYVE and PX domain proteins have essential roles in endosomal

fusion. It has been proposed that the requirement of PI3P for these

proteins to bind to the endosomal membrane enables their selective

recruitment to early endosomes because PI3P is present exclusively

on early-endosomal membranes. These proteins, in turn, regulate

the trafficking and sorting of select hydrolases from the trans-Golgi

network to endosomes.

FYVE FYVEZn Zn

PI3PPI3P

Hydrocarboncore

Interface

Figure I. Binding of dimeric Hrs protein to membranes through its interactions

with PI3P. FYVE domains are shown in green; PI3P is shown in white and red;

and phosphate groups are shown in red and yellow.White spheres indicate zinc

molecules. Reproduced, with permission, from Ref. [32].


budding and fusion steps involving vesicular transport orthrough a gradual acquisition of lysosomal markermolecules [15,16]. Pathogenic mycobacteria use severalstrategies to circumvent such lysosomal delivery, and themolecules that are involved in these processes are of bothhost and mycobacterial origin.

Role of host proteins

Cholesterol is essential for association of the host proteincoronin 1, also known as P57 or tryptophan-aspartate-containing coat protein (TACO), to mycobacterial phago-somes [11,17]. Coronin 1 was identified in a proteomicsearch for host proteins that are involved in prevention ofphagosome maturation. This 50-kDa protein contains anN-terminal tryptophan aspartate 40 (WD40)-repeatdomain and a coiled-coil sequence of 30 amino acidsat the C terminus. It belongs to a conserved family ofWD-repeat actin-binding proteins that is found predomin-antly in eukaryotes [18]. Whereas some coronins areexpressed ubiquitously (coronins 2, 3 and 7), others areexpressed in a tissue-specific fashion; coronin 2SE (or pp66)is expressed in epithelial tissues, coronins 4 and 5 areexpressed in the brain, and coronin 1 is expressedexclusively in leukocytes [17,18].

Members of the coronin protein family have beenshown to function in actin-cytoskeleton remodeling,phagocytosis, cell division and polarity, and vesiculartrafficking from the endoplasmic reticulum to the Golgi[19]. In murine macrophages, coronin 1 is retained exclu-sively on phagosomes of macrophages infected with livemycobacteria. In macrophages infected with dead myco-bacteria, coronin 1 rapidly dissociates from phagosomes,followed by fusion of the non-coated phagosome tolysosomes and the subsequent degradation of the myco-bacteria, which suggests an essential role for coronin 1 inprotection of the mycobacterial phagosome from fusion tolysosomes [17,20]. Interestingly, in macrophages that aregenerated by activation with phorbol esters, interferon(IFN)-g or human serum, coronin 1 is retained onlypartially, which is consistent with the majority of themycobacteria internalized by these cells being transferredto lysosomal organelles [21]. Kupffer cells, the majormacrophages in the liver, do not express coronin 1 and candestroy live mycobacteria within lysosomes effectively.Thus, the ability of pathogenic mycobacteria to recruithost components to protect the mycobacterial habitat fromlysosomal fusion is a perfect example of how pathogenicmicroorganisms have evolved to live inside host cells thatare designed to eliminate them.

Role of host phosphoinositides

Phagosome maturation requires the biosynthesis ofphosphatidylinositol 3-phosphate (PI3P) [22]. This phos-phoinositide is a membrane-trafficking regulatory lipidessential for phagolysosome biosynthesis. PI3P is syn-thesized by PI3-kinase on early-endosomal and phago-somal membranes and was observed to move on thephagosomal membrane in temporally scheduled waves[23,24]. PI3P functions as a ligand for many proteinscontaining FYVE or phox homology (PX) domains thatmight be involved in phagosome maturation [25,26]

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(Box 1). An example of a PI3P-binding protein ishepatocyte growth factor (HGF)-regulated tyrosine kinasesubstrate (Hrs), which has a role in the regulation oftraffic in the endosomal pathway [27–30]. Depletion of Hrscauses arrest of phagosome maturation, and Hrs associ-ates with phagosomes before fusion to late endosomes orlysosomes [31]. This association requires the interactionof the FYVE domain of Hrs with phagosomal PI3P [32](Box 1). Interestingly, when pathogenic mycobacteriareside within phagosomes, Hrs dissociates [31], possiblybecause of the ability of pathogenic mycobacteria to alterthe wave of PI3P [24].

PI3P is also essential for the elevation of Ca2C

concentration in the cytosol that stimulates the activityof PI3-kinase. This kinase, in turn, raises the concen-tration of PI3P on the phagosomal membrane, whichmodulates phagosomematuration [33]. This feedback-loopcircuit seems to be targeted by pathogenic mycobacteria toprevent the fusion of phagosomes to late endosomes andlysosomes.

Role of the mycobacterial cell wall

Both host-cell and mycobacterial lipids are known tomodulate the intracellular fate of pathogenic myco-bacteria [34,35]. Notably, the lipid-rich cell wall ofmycobacteria has been recognized as providing the bacilli


Review TRENDS in Cell Biology Vol.15 No.5 May 2005 271

with an effective innate immune defense against lyso-somal degradation [34,36]. TheMycobacterium tuberculosisPI3P analog glycosylated phosphatidylinositol lipoarabi-nomannan (ManLAM) has been reported to block phago-some maturation [37]. Phagosomes containing latex beadscovered with ManLAM acquire features of mycobacterialphagosomes, including inhibition of phagosomal acidifica-tion and fission of late-endosomal markers. ManLAMprobably blocks a PI3P-dependent pathway involved inthe transport of cargo between the trans-Golgi networkand phagosomes: a transport step required for phagolyso-some biogenesis [37,38]. The rise in Ca2C concentration inthe macrophage cytosol required for accumulation ofPI3P on the phagosomal membrane is also inhibited byManLAM [39]. ManLAM might exert these activities bycompeting with PI3P for binding to proteins containingFYVE and PX domains, thereby preventing their recruit-ment to phagosomes. Alternatively, ManLAM mightinterfere with the waves of PI3P on the phagosomalmembrane [24].

In contrast to the inhibitory effect of ManLAM onphagosome–lysosome fusion, phosphatidylinositol manno-side (PIM), another mycobacterial cell-wall component,enhances the fusion of phagosomes to early endosomes[40]. Although the molecular partners of the different lipidmoieties that modulate fusion events within the phago-some–lysosome pathway remain to be established, thesemycobacterial cell-wall components could ensure a safeintracellular haven while continuing to provide nutrientsto resident mycobacteria.

Role of macrophage activation

Considering the multiple ways in which mycobacteria canremain viable inside macrophage phagosomes, how canthese pathogens be destroyed? One answer to thisquestion lies in host innate immune defense mechanismsthat come into operation after activation of macrophages.One of the most important macrophage-activating mol-ecules involved in combating pathogenic mycobacteria isIFN-g, which is secreted by activated T cells and naturalkiller cells during infection [41,42]. Bothmice and humanswith genetic defects in IFN-g signaling are extremelysusceptible to mycobacterial diseases [43,44]. Manycellular processes such as antigen presentation, leuko-cyte–endothelium cell interactions, cell growth and apop-tosis, and phagosome–lysosome fusion can be modulatedthrough the activity of IFN-g [45–47]. The molecularmechanisms involved in such modulation are, however,largely unknown [48]. Recently, the IFN-g-induced hostguanosine triphosphatase protein LRG-47 was implicatedin the regulation of phagosome maturation; LRG-47associates with phagosomes to promote maturation.Interestingly, LRG-47-depleted macrophages activatedby IFN-g fail to kill infected bacteria because of theirimpaired maturation. Consequently, mice lacking LRG-47cannot control mycobacterial replication, confirming theimportance of phagosome maturation in host control oftuberculosis [49].

In addition to modulation of the adaptive immuneresponse, macrophage activation can be mediated throughactivities of the toll-like receptors (TLRs), of which TLR-2


and TLR-4 have key roles [50,51]. Many mycobacterialcell-wall constituents such as PIM and the non-cappedlipoarabinomannan (AraLAM), and triacetylated peptidesfrom the mycobacterial 19- and 33-kDa lipoproteins havebeen shown to activate TLRs [50,51]. TLR-2, probably incoordination with TLR-1 and TLR-6, recognizes soluble,heat-stable mycobacteria-derived material, whereasTLR-4 recognizes heat-sensitive cell-associated com-ponents [52]. Activation of TLRs leads to the activationof mitogen-activated protein kinases (MAPKs) [53]. Thesekinases initiate signaling cascades to activate transcrip-tion factors, leading to synthesis of the cytokine tumornecrosis factor (TNF)-a, chemokines and other inflamma-tory mediators, in addition to a burst of nitric oxideproduction [54–56]. In turn, these effectors modulate theantibacterial and inflammatory immune responses ofmacrophages, including accelerated phagosome matu-ration [57]. These pathways are likely to be exploited bypathogenic mycobacteria to promote mycobacterial survi-val inside macrophages [56] and, indeed, ManLAM fromM. tuberculosis inhibits the activation of MAPK in humanmonocytes [58].

Mycobacterial protein kinase G

In addition to cell-wall lipids, mycobacterial proteins areinvolved in the arrest of phagosome maturation [59,60]. Acomprehensive screen revealed several proteins that arepotentially involved in this process. Disruption of theseproteins led to defects in preventing acidification ofphagosomes and to attenuation of intracellular survival[59,60]. An independent search for mycobacterial proteinsinvolved in signal-transduction reactions with potential tomodulate phagosome–lysosome fusion suggested thatmycobacterial kinase (or kinases) might be involved inthis modulation. Treatment of Mycobacterium bovisBacillus Calmette–Guerin (BCG) with a eukaryotic pro-tein kinase Ca (PKCa) inhibitor before infection ofmacrophages leads to immediate transport of the myco-bacteria to lysosomes, suggesting the involvement of aPKCa-like kinase activity in blocking lysosomal delivery(Figure 1). Interestingly, 11 different eukaryotic-likeprotein kinases are encoded by pathogenic mycobacteria[61,62], and a ClustalW (http://www.ebi.ac.uk/clustalw/)alignment of these kinases with mammalian PKCarevealed closest homology to mycobacterial protein kinaseG (PknG). Whereas most mycobacterial kinases arepredicted to be transmembrane proteins with an intra-cellular kinase domain, two of these kinases – PknG andprotein kinase K – lack such a transmembrane segment.Interestingly, PknG is retained in the downsized genomeof the obligate intracellular pathogen Mycobacteriumleprae that carries the minimum set of mycobacterialgenes necessary for intracellular survival. This suggeststhat PknG provides an important function for mycobac-terial survival in the host [63].

To analyze its role in mycobacterial survival inside hostcells, PknG was disrupted in M. bovis BCG using phage-mediated homologous recombination. The resultingM. bovis BCG pknG-deleted mutant was immediatelytransferred to lysosomes, and the mutant mycobacteriacould not survive inside macrophages [59] (Figure 2).

http://www.ebi.ac.uk/clustalw/


(a) (b)

Figure 1. Involvement of kinase activity in lysosomal delivery of pathogenic mycobacteria. (a) Live untreated Mycobacterium bovis BCG. (b) Effect of the PKCa inhibitor

chelerythrine on lysosomal delivery of M. bovis BCG. Incubation of mycobacteria [expressing green fluorescent protein (GFP)] with chelerythrine leads to enhanced

lysosomal delivery comparedwith that in (a). Lysosomeswere visualized using antibodies against the lysosomalmarker lysosomal-associatedmembrane protein 1 (LAMP1),

followed by secondary antibodies coupled to Alexa Fluor 568.


Subcellular-localization studies suggest that PknG ispresent in the phagosomal lumen, in addition to in thecytosol of infected macrophages, suggesting that patho-genic mycobacteria actively secrete PknG. These datasuggest that PknG is a mycobacterial virulence factor thatis delivered into the cytosol of host cells and mightinterfere with the regulation of phagosome–lysosometransfer (Figure 3).

Notably, PknG lacks signal sequences required for itstranslocation by the classical Sec secretion system (Box 2).However, many pathogenic microbes deliver virulencefactors into host cells by specialized secretion mechanisms[64]. The type III secretion system (Box 2), derived fromthe ancestral flagellum, is used by many Gram-negativepathogenic bacteria to transfer into host cells effectorsthat, in turn, prevent the host response to infection by

LAMP–568

M. bovis BCG

M. bovis BCG ∆pknG

Figure 2. Effect of PknG deletion on mycobacterial intracellular trafficking. Bone marrow

pknG-deletedmutant (DpknG) expressing GFP for one hour, followed by a two-hour chas

the lysosomalmarker LAMP1, followed by secondary antibodies coupled to Alexa Fluor 5

colocalized mostly within LAMP1-positive compartments. Scale barZ10 mm. Reproduce


inhibiting signaling pathways [65]. Conjugation systems,which were designed for genetic exchange, have also beenexploited by Gram-negative bacteria to deliver virulencefactors into hosts [66]. Similarly, the Gram-positivepathogen Streptococcus pyogenes has been shown todeliver its toxin inside the host through a cytolysin-mediated process [67]. Also, pathogenic mycobacteriapossess specialized secretion systems required for theirvirulence, such as proteins encoded in the RD1 (region ofdeletion in M. bovis BCG) region of the M. tuberculosisgenome. These proteins comprise a secretion apparatusthat is essential for the secretion of the major T-cellantigenic proteins ESAT-6 and CFP-10, which have nodetectable signal sequence [68,69]. In the non-pathogenicMycobacterium smegmatis, the RD1 proteins form aDNA-conjugation system, which suggests that pathogenic

GFP Merge

macrophages were infected with Mycobacterium bovis BCG or an M. bovis BCG

e. Cells were processed as described in Ref. [59] and stained with antibodies against

68 (LAMP–568). WhereasM. bovis BCG resided in LAMP1-negative vacuoles,DpknG

d, with permission, from Ref. [59]. q (2004) AAAS (http://www.sciencemag.org).

http://www.sciencemag.org


TRENDS in Cell Biology

pknG mutantmycobacteria

Pathogenic mycobacteria

MycobacterialphagosomePhagosomal

vacuole

PknG

Lysosome

Other microbes

Figure 3. Involvement of PknG in mycobacterial trafficking in macrophages. Whereas most microbes and non-pathogenic mycobacteria are rapidly transferred to lysosomes,

pathogenic mycobacteria remain within phagosomes. PknG is secreted by pathogenic mycobacteria into the phagosomal lumen and cytosol to block the fusion of

phagosomes to lysosomes. Deletion of the pknG gene results in rapid lysosomal transfer of the resulting mutant bacteria.


mycobacteria have adapted an ancestral conjugationsystem for protein secretion [70]. This evolutionaryconservation has also been observed for type IVsecretion systems found in pathogenic Gram-negativebacteria [64]. Whereas the RD1 region is absent fromthe genome of M. bovis BCG, five RD1-homologousregions are distributed among different loci in thegenome of M. tuberculosis [71]. It remains to be

Box 2. Bacterial secretion systems

To perform extracellular activities, bacteria have developed several

systems for secreting their proteins to the surrounding environment.

At least five major protein-secretion systems have been described.

Type I protein-secretion systemA secretion system composed of three proteins forming a channel that

spans the inner membrane, periplasmic space and outer membrane. It

is a one-step continuous secretion. The secretion signal is present in

the C-terminal region of the secreted protein but secretion does not

involve proteolytic processing of secreted proteins. In Escherichia coli,

the most-studied type I secreted protein is a-haemolysin [80,81].

Type II protein-secretion systemA secretion system that employs a two-step mechanism and is

dependent on the Sec machinery. The Sec machinery is composed of

an ATPase, SecA, several integral inner-membrane proteins (SecD,

SecE, SecF, SecG and SecY) and a signal peptidase. First, secreted

proteins pass across the inner membrane by the Sec machinery,

during which the cleavage of a signal sequence at the N terminus

occurs. Subsequently, crossing the outer membrane is mediated by a

dedicated secretion apparatus composed of multiple proteins. The

type II protein-secretion system is exemplified by the secretion of a

starch-hydrolyzing lipoprotein called pullulanase from Klebsiella

oxytoca [82].

Type III protein-secretion systemA system in which secretion occurs in a continuous process without


investigated whether or not PknG is secreted throughan RD1-like secretion machinery.

The ability of PknG to block lysosomal deliverysuggests that this kinase might be a valuable target inthe development of compounds that could induce myco-bacterial death inside macrophages. A screen for PknGinhibitors identified a tetrahydrobenzothiophene thatspecifically inhibits the kinase activity of PknG. When

the presence of periplasmic intermediates. This secretion does not

involve proteolytic processing of secreted proteins. The system

involves at least 20 proteins, including cytoplasmic, inner-membrane

and outer-membrane proteins, many of which have homologs

involved in flagellar biogenesis. This secretion system is used by

pathogens to inject their proteins into eukaryotic cells. The type III

secretion system was identified in pathogenic Yersinia spp. for the

secretion of Yop proteins [83].

Type IV protein-secretion system

A secretion system related to the bacterial DNA conjugative transfer

system that enables the transfer of nucleoprotein DNA-conjugation

intermediates or proteins into the extracellular milieu or directly into

host cells. Crossing the outer membrane involves pore formation by

the C-terminal region of the proteins involved. The prototypical type IV

secretion system is that of the Agrobacterium tumefaciens nucleo-

protein tDNA-transfer system [84].

Type V protein-secretion system

A secretion system also referred to as the auto-transporter system. The

auto-transporter proteins have a tripartite unifying structure: the

N-terminal leader sequence, the secreted mature protein (passenger

domain) and a C-terminal domain that forms a b-barrel pore to enable

secretion of the passenger protein. Proteins secreted in thismanner are

implicated as putative virulence factors in many pathogens. The first

type V protein-secretion system to be described was that of gonococcal

immunoglobulin A1 protease [85].



added to infected macrophages, this compound inducesthe fusion of phagosomes to lysosomes and mediates thekilling of mycobacteria inside macrophages [59]. Onepossible advantage of targeting PknG is that this willenable the macrophage to carry out its natural anti-bacterial activity of redirecting intracellularly survivingmycobacteria to lysosomes. Also, by targeting a secretedmolecule such as PknG, transport of antimycobacterialcompounds through the extremely impermeable myco-bacterial cell wall can be circumvented, greatly improvingthe accessibility of the compounds to their target.

The downstream targets of PknG are currentlyunknown. It is, however, interesting to note that mam-malian PKCa, besides being associated with phagosomalmembranes itself, modulates the association of p57, thehuman homolog of coronin 1, to phagosomes [72,73].Considering the importance of PKCa in membrane-trafficking events and its possible involvement in phago-some maturation, PknG might modulate signalingpathways involved in phagolysosome biogenesis by com-peting with host PKCa for substrate binding.

Mycobacterial resistance to destruction

Lysosomal delivery and subsequent degradation by resi-dent hydrolytic enzymes do not constitute the onlymechanism for degrading internalized microbes. Reduc-tion in pH and increase in the local concentrations ofreactive oxygen and nitrogen species such as nitric oxidethat accompany lysosomal cargo delivery also aid microbedegradation. In macrophages, nitric oxide is generated byinducible nitric oxide synthase (iNOS), which is a cytosolicenzyme that catalyzes the conversion of L-arginine toL-citrulline and nitric oxide. Nitric oxide is extremelybactericidal [74], and mice lacking iNOS succumb rapidlyto M. tuberculosis [75]. Not surprisingly, pathogenicmycobacteria have evolved ways of countering thedestructive effects of nitric oxide and preventing theassembly of iNOS with phagosomes, possibly throughrearrangement of the actin cytoskeleton, and this mightlead to a reduction in the local concentration of nitricoxide [76].

Unexpectedly, potential damage by nitric oxide can alsobe countered by the mycobacterial proteasome [77]. Theproteasome is a multisubunit molecular machine that ishighly conserved from archaebacteria to humans and isresponsible for the proteolysis of cytoplasmic proteins.Whereas the proteasome in eukaryotes is involved inmultiple pathways such as the cell cycle, development anddifferentiation, immune and inflammatory responses, andsignal transduction, the function of the proteasome inprokaryotes is less clear [78]. M. tuberculosis has adaptedthe proteasome machinery to protect itself from the killingeffect of nitric oxide [77,79]. Deletion of genes that encodeproteins involved in the formation of the proteasomecauses hypersensitivity of the bacilli to nitric oxide. Itremains to be elucidated whether protease activity isinvolved in the resistance to reactive nitrogen intermedi-ates; because the precise nitric-oxide-mediated bacteri-cidal mechanisms are unknown, the demonstration thatdeletion of a putative proteasome subunit attenuatesvirulent M. tuberculosis might, therefore, provide clues to


the destruction mechanisms involved in nitric-oxide-induced mycobacterial cell death.

Concluding remarks

Interactions between pathogenic mycobacteria andmacro-phages represent an interesting example of the co-evolution of infectious microbial agents with their hosts.Mycobacteria, probably evolved from saprophytes in soil tobecome pathogens, have gained several mechanisms topersist inside macrophages. Besides directing mycobac-terial entry into macrophages, these tactics include signal-transduction-mediated modulation of fusion–fission reac-tions to ensure residence within the safe haven of themycobacterial phagosome.

It is becoming clear that, for a full understanding ofthese host-evasion strategies, a better understanding ofmycobacterial physiology is needed, both inside andoutside host macrophages. Knowledge of the mycobacter-ial tactics for survival inside macrophages might help notonly to control tuberculosis and other mycobacterialdiseases but also to uncover novel aspects of host cellbiology.

Acknowledgements

We thank Giorgio Ferrari for expert technical assistance, and Guy R.Cornelis for critical comments about the manuscript. Current support isprovided by grants from the Swiss National Science Foundation and theWorld Health Organization to J.P.

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