EFFECTIVE CONTRACEPTIVE FOR ALL WOMEN: A GUARANTEED INVESTMENT FOR GEORGIA Abstract Overview of the Problem of Unintended Pregnancy Costs of Unintended pregnancy a. Monetary costs of unintended pregnancy b. Non-monetary costs of unintended pregnancy (teen pregnancy, high school graduation rates, disparities in healthcare, poverty, health risks of unplanned pregnancy, ma
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Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 2004??Review ArticleThe mycobacterial lipoarabinomannan and related moleculesV. Briken, S. A. Porcelli, G. S. Besra and L. Kremer Mycobacterial lipoarabinomannan and related
lipoglycans: from biogenesis to modulation of the
Volker Briken,1 Steven A. Porcelli,1 Gurdyal S. Besra2
progress in the identification of genes involved in the
and Laurent Kremer3*
biosynthesis of LAM is discussed, in particular with
1Department of Microbiology and Immunology, Albert respect to the fact that enzymes controlling the LAM/
Einstein College of Medicine, Bronx, NY 10461, USA. LM balance might represent targets for new antituber-
2School of Biosciences, The University of Birmingham, cular drugs. In addition, inactivation of these genes
may lead to attenuated strains of M. tuberculosis for
3Laboratoire des Mécanismes Moléculaires de la the development of new vaccine candidates.
Pathogénie Microbienne, INSERM U629, Institut Pasteur de Lille/IBL, 1 rue Pr. Calmette, BP245-59019 Lille Cedex, Introduction
Mycobacteria are extraordinarily successful pathogenswith the remarkable ability to persist within the host’s tissues even in the presence of an intact immune system.
The cell wall component lipoarabinomannan (Man-
Pathogenic mycobacteria are predominantly intracellular LAM) from Mycobacterium tuberculosis is involved in
parasites capable of replicating within the normally hos- the inhibition of phagosome maturation, apoptosis
tile environment of macrophages. In this location, the and interferon (IFN)-g signalling in macrophages and
bacillus is protected from many of the immune mecha- interleukin (IL)-12 cytokine secretion of dendritic cells
nisms that normally eliminate bacterial invaders. One (DC). All these processes are important for the host
major challenge that the intracellular bacteria face is to mount an efficient immune response. Conversely,
overcoming cell-mediated mechanisms of immunity that LAM isolated from non-pathogenic mycobacteria
detect signals originating from infected cells. An impor- (PILAM) have the opposite effect, by inducing a potent
tant key to the success of pathogenic mycobacteria is proinflammatory response in macrophages and DCs.
likely to be their unusual cell wall structure and its inter- LAMs from diverse mycobacterial species differ in the
actions with the immune system. This cell envelope con- modification of their terminal arabinose residues. The
sists of a highly complex array of distinctive lipids, strong proinflammatory response induced by PILAM
glycolipids and proteins. It has been intensely scrutinized correlates with the presence of phospho-myo-inositol
as a potential effector in the interaction of Mycobacterium on the terminal arabinose. Interestingly, recent work
tuberculosis with the human host (Glickman and Jacobs, indicates that the biosynthetic precursor of LAM,
2001; Russell et al., 2002; Brennan, 2003; Flynn and lipomannan (LM), which is also present in the cell
wall, displays strong proinflammatory effects, inde-
Lipoarabinomannan (LAM) as well as its related precur- pendently of which mycobacterial species it is iso-
sors, lipomannan (LM) and phosphatidyl-myo-inositol lated from. Results from in vitro assays and knock-
mannosides (PIMs), are found interspersed in the myco- out mice suggest that LM, like PILAM, mediates its
bacterial cell wall. PIMs, LM and LAM are major lipogly- biological activity via Toll-like receptor 2. We hypoth-
cans that are non-covalently attached to the plasma esize that the LAM/LM ratio might be a crucial factor
membrane through their phosphatidyl-myo-inositol anchor in determining the virulence of a mycobacterial spe-
and extend to the exterior of the cell wall (Besra and cies and the outcome of the infection. Recent
Brennan, 1997; Belanger and Inamine, 2000; Nigou et al.,2003). These complex molecules are believed to playimportant roles in the physiology of the bacterium as well Accepted 15 April, 2004. *For correspondence. [email protected]; Tel. (+33) 3 20 87 11 54; Fax (+33) 3 20 87 11 as in the modulation of the host response during infection.
For example, LAM is an important modulator of the V. Briken, S. A. Porcelli, G. S. Besra and L. Kremer immune response in the course of tuberculosis and lep- The size and the degree of branching of the mannan core rosy (Chatterjee and Khoo, 1998; Nigou et al., 2002) and are species dependent. The arabinan polymer of LAM a key ligand in the interaction between M. tuberculosis, consists of a linear a(1Æ5)-linked arabinofuranosyl back- macrophages and dendritic cells (DCs) (Schlesinger bone punctuated with branched hexa-arabinofuranosides et al., 1994; Maeda et al., 2003). In addition, recent stud- (Ara6) and linear tetra-arabinofuranosides (Ara4) (Chatter- ies highlight the potential role of LM in mycobacterial jee et al., 1991; 1993) (Fig. 1).
virulence via its strong proinflammatory and apoptosis- LAM can be classified into three major structural fami- lies according to the capping motifs present on the non- A thorough investigation of the roles of PIMs, LM and reducing termini of the arabinosyl side-chains. The arabi- LAMs in mycobacterial virulence has been hampered by nan termini in the pathogenic strains M. tuberculosis, M. a lack of defined mutants that fail to synthesize these leprae, Mycobacterium avium and M. kansasii are modi- specific cell surface components. Recently, advances in fied with caps consisting of a single Manp, a dimannoside the genetic manipulation of mycobacteria and related act- or a trimannoside, with dimannosides predominating inomycetes, together with the sequencing of the M. tuber- (Nigou et al., 1997; Vercellone et al., 1998; Khoo et al., culosis genome, have allowed several lipoglycan mutants 2001; Guerardel et al., 2003), resulting in molecules des- with defined envelope deficiencies to be generated.
ignated ManLAM. ManLAM contains about 50 Manp and Progress in the study of mycobacterial glycolipid biosyn- 60 Araf units. A general picture of the M. tuberculosis thesis bears the promise of identifying enzymes that might ManLAM structure is proposed in Fig. 1. In the fast-grow- be essential for the viability and/or virulence of M. tuber- ing non-pathogenic species M. smegmatis, Mycobacte- culosis and targets for future drug development.
rium fortuitum and in an unidentified species, branches of This review article reports the advances made in the the terminal arabinan are terminated by inositol phos- current understanding of PIMs, LM and LAM biosynthesis phate caps (Khoo et al., 1995), characterizing the PILAM and will describe only briefly the structural organization of family. A third LAM family, designated AraLAM, recently the different domains comprising these complex mole- identified in M. chelonae, comprises a LAM molecule cules as this has been the subject of many excellent devoid of both the manno-oligosaccharide and inositol reviews (Chatterjee and Khoo, 1998; Brennan, 2003; phosphate caps (Guerardel et al., 2002).
Nigou et al., 2003). We also discuss recent observationsrelating to the immunomodulatory functions of LAM and Biogenesis of PIMs, LM and LAM
its precursors, in addition to their receptors and intracel-lular signalling pathways. The role of these lipoglycans as Understanding the biosynthesis of PIMs, LM and LAM has antigens presented by the CD1 system, the host’s lipid been the focus of recent genetic and biochemical studies antigen-presenting molecule, has been reviewed recently (Nigou et al., 2003). Enzymes that clearly participate in the elaboration of these complex lipoglycans are repre-sented in Fig. 1.
Structure of mycobacterial LAM and related
PIMs and their multiglycosylated counterparts, LM and Several mannosyltransferases involved in the mannosyla- LAM, are complex lipoglycans that are found ubiquitously tion steps can be distinguished with respect to the man- in the envelopes of all mycobacterial species. PIMs, LM nose donor they use (either GDP-Manp during early steps and LAM all share a conserved mannosyl-phosphatidyl- in PIM biosynthesis, or C35/C50-P-Manp later in LM syn- myo-inositol (MPI) that is presumably used to insert these thesis from PIM precursors). PIM biosynthesis is initiated structures into the plasma membrane (Hunter and Bren- by two distinct mannosyltransferases that use GDP-Manp nan, 1990), suggesting that they are metabolically related as the sugar donor. The first step involves the transfer of (Besra and Brennan, 1997). In addition to the MPI, LAM a mannose residue from GDP-Manp to the 2-position of possesses a mannan core with a branched arabinan poly- the myo-inositol ring of phosphatidyl-myo-inositol (PI) to mer and, in some cases, cap motifs decorate the termini form phosphatidyl-myo-inositol monomannoside (PIM1).
of the branched arabinan (Nigou et al., 2003) (Fig. 1).
This reaction is catalysed by the a-mannosyltransferase The mannan core consists of an a1,6-linked Manp PimA (Kordulakova et al., 2002). The pimA gene of which backbone, which is substituted at C-2 by single Manp is essential, demonstrating that PIM1, and presumably units in numerous species, including M. tuberculosis, higher mannosylated PIMs, are required for cell growth.
Mycobacterium leprae, Mycobacterium kansasii and Interestingly, pimA is the fourth gene in an operon of five Mycobacterium smegmatis, and at C-3 by single Manp genes that are all potentially involved in PIM biosynthesis units in Mycobacterium chelonae (Guerardel et al., 2002).
(Kordulakova et al., 2002). The first gene in this cluster 2004 Blackwell Publishing Ltd, Molecular Microbiology The mycobacterial lipoarabinomannan and related molecules (Rv3793, GT-53)
Fig. 1. General structure of ManLAM from M. tuberculosis and structural relationship between PIMs, LM and LAM. PIM2 is a precursor of the
highly mannosylated LM molecule, which is further extended by the arabinan domain to form LAM. In both LM and LAM, an a1,6-linked Manp
backbone substituted at C-2 by single Manp units constitutes the mannan domain. The arabinan polymer is a linear a(1Æ5)-linked arabinofuranosyl
backbone punctuated with branched hexa-arabinofuranosides: [b-D-Araf-(1Æ2)-a-D-Araf-(1-]2Æ3 and Æ5)-a-D-Araf-(1Æ5)-a-D-ArafÆ and linear
tetra-arabinofuranosides: b-D-Araf-(1Æ2)-a-D-Araf-(1Æ5)-a-D-Araf-(1Æ5)-a-D-ArafÆ. The mannose caps, which terminate the arabinan domain,
consist of a single Manp residue, a dimannoside (a-D-Manp(1Æ2)-a-D-ManpÆ) or a trimannoside (a-D-Manp-(1Æ2)-a-D-Manp-(1Æ2)-a-D-
ManpÆ). R1, R2 and R3 are fatty acyl chains. C35/C50-P-Manp represents a polyprenyl monophosphomannose. The a, b, c and d values are
species specific. Arrows indicate enzymes confirmed to participate in the biosynthesis of these lipoglycans. PimC was found to be present in
M. tuberculosis CDC1551 but absent from M. tuberculosis H37Rv. Classification of the glycosyltransferases by their CAZY family is indicated in
encodes a protein of unknown function, while the second Rv2611c is dispensable in M. smegmatis, although its encodes PgsA1, the PI synthase that catalyses the disruption induces dramatic changes in the PIM content condensation of inositol and the diglyceride of CDP- and a severe growth defect (Kordulakova et al., 2003). The diacylglycerol (Jackson et al., 2000). The third gene last gene of the PIM cluster, Rv2609c, encodes a putative (Rv2611c) of this operon encodes a protein with high GDP-Manp hydrolase that awaits further characterization.
similarity to bacterial acyltransferases. This protein has The second mannosylation step, catalysed by PimB, been shown to be responsible for the acylation of the 6- allows the transfer of another Manp residue to the 6- position of the Manp residue linked to position 2 of the position of the myo-inositol ring of PIM1, leading to PIM2 myo-inositol in PIM1 and PIM2, with the mono-mannosy- (Schaeffer et al., 1999). A third Manp unit is finally intro- lated lipid acceptor being the primary substrate of the duced on to the growing molecule to form PIM3 in a reac- enzyme (Kordulakova et al., 2003). In contrast to pimA or tion carried out by the product of the pimC gene identified pgsA1, which are both essential, the acyltransferase in M. tuberculosis CDC1551 (Kremer et al., 2002). How- 2004 Blackwell Publishing Ltd, Molecular Microbiology V. Briken, S. A. Porcelli, G. S. Besra and L. Kremer ever, inactivation of pimC in Mycobacterium bovis BCG less, one exception is the LM of M. chelonae, which has did not affect cell growth and did not alter the PIM/LM/ a(1Æ3)-linked Manp residues (Guerardel et al., 2002).
LAM composition of the mutant. This suggests the pres- None of the specific genes encoding these branching ence of an alternative synthesis pathway present in M. mannosyltransferases has been identified.
bovis BCG and M. tuberculosis CDC1551, a hypothesisthat is supported by the fact that pimC is not found in M. Biogenesis of the arabinan domain in LAM tuberculosis H37Rv (Kremer et al., 2002).
The mannose unit at the position 6 of PIM3 is then The ‘mature’ LM is then subsequently glycosylated with further elongated with mannose residues to generate arabinan to form LAM. Until very recently, little was known PIM4-6. However, mannosyltransferases participating in about the genetics of arabinan biosynthesis. Two forms of this elongation process have not been identified.
arabinans are found in the mycobacterial cell wall: one ispart of the heteropolysaccharide arabinogalactan (AG)and the other is part of LAM. The two forms of D-arabinan differ in that mycolic acids esterify arabinan in AG, thus Besra et al. (1997) established that PIMs are extended constituting the basis of the lipid barrier of mycobacteria.
with additional Manp residues from the alkali-stable C35/ In contrast, in M. tuberculosis LAM, the arabinan moiety C50 polyprenyl monophosphomannose (C35/C50-P-Manp) is further capped with mannose residues responsible for donor to form ‘linear’ LMs, containing only a(1Æ6) Manp some of its biological functions. Therefore, arabinan rep- residues. C35/C50-P-Manp is synthesized from GDP-Manp resents a valid target for the generation of antimycobac- and polyprenols by the polyprenol monophosphomannose terial drugs because blocking of its biosynthesis would led (ppm) synthase, encoded by the ppm1 gene (Gurcha to dual disruption of both the mycolyl–AG–peptidoglycan et al., 2002). Disruption of the ppm synthase gene in Corynebacterium glutamicum, identified on the basis of As mentioned above, the arabinan domain consists of homology searches, induced a complex phenotype includ- a linear a(1Æ5)-Araf backbone substituted by two kinds ing altered cell growth rates and inability to synthesize of arrangements, linear tetra-arabinofuranosides (Ara4) C55-P-Manp (Gibson et al., 2003a). This mutant was also and hexa-arabinofuranosides (Ara6). In both cases, the unable to produce any ‘mature’ lipoglycans, such as LM non-reducing end is characterized by the disaccharide or LAM, but could still produce PIMs, highlighting the key unit b-D-Araf-(1Æ2)-a-D-Araf-(1Æ) (Besra and Brennan, role of ppm synthase in LM/LAM synthesis (Gibson et al., The only Araf sugar donor identified so far is the C35/ The ppm synthase-dependent a(1Æ6)-mannosyltrans- C50 polyprenyl monophosphoarabinose (C35/C50-P-Araf) ferase involved in the polymerization step leading to the (Wolucka et al., 1994), which is synthesized from 5- linear mannan core is currently unknown. Interestingly, phospho-D-ribose pyrophosphate (Scherman et al., prenyl-linked benzophenone photoreactive probes have 1996). Initially identified as the major target of ethambutol recently been shown to be excellent substrates for the (an effective antimycobacterial drug) in M. avium recombinant ppm synthase. Furthermore, photoactivation (Belanger et al., 1996) and M. tuberculosis (Telenti et al., abolishes the enzymatic activity of ppm synthase in vitro 1997), the two homologue proteins EmbA and EmbB have (Guy et al., 2004). More importantly, unique mannosy- been reported to participate in the formation of the proper lated derivatives of these photoreactive probes were all Ara6 motif in AG (Escuyer et al., 2001). These two proteins mannose donors through a ppm synthase-dependent have been proposed to catalyse a1,3-arabinosyltrans- a(1Æ6)-mannosyltransferase to a synthetic Manp-Manp ferase activity in the arabinan of AG. M. smegmatis dissacharide acceptor using M. smegmatis membranes.
mutants lacking embA or embB are viable, probably In addition, photoactivation of these mannosylated probes because the two gene products partially compensate for led to specific inhibition of the ppm synthase-dependent each other. Although arabinosylation of AG was dramati- a(1Æ6)-mannosyltransferase activity (M. R. Guy, P. A.
cally diminished, arabinosylation of LAM remained unaf- Illarionov, S. S. Gurcha, K. J. C. Gibson, P. W. Smith, D.
fected in these mutants (Escuyer et al., 2001). In M. E. Minnikin, and G. S. Besra, submitted). We will use tuberculosis, the Emb proteins are encoded by a cluster these powerful tools by simply modifying the mannosy- of three genes, embC, embA and embB (Cole et al., lated probes through inclusion of a radiolabelled tag in 1998). Zhang et al. (2003) found recently that inactivation order to identify the a(1Æ6)-mannosyltransferase(s) via a of the remaining embC gene in M. smegmatis abolished arabinosylation of LAM, but not AG. The three Emb pro- In M. tuberculosis and almost all other mycobacteria teins are predicted to contain 13 membrane-spanning analysed to date, the ‘mature’ LM consists of ‘linear’ LM segments in their N-terminal region and a globular bearing single a(1Æ2)-linked Manp residues. Neverthe- C-terminal domain. It has been proposed that the N- 2004 Blackwell Publishing Ltd, Molecular Microbiology The mycobacterial lipoarabinomannan and related molecules terminus of EmbC participates in the recognition of the ciated with ManLAM. In this regard, it is conceivable that LM as a precursor of LAM and that the C-terminus is inhibitors such as ethambutol may modulate the immune responsible for arabinosylation (Zhang et al., 2003). The interactions of M. tuberculosis with the host, although this transmembrane segments of the Emb proteins are very remains to be demonstrated further. Genes participating likely to be involved in translocating the arabinan in the synthesis of these caps have not been reported, component across the plasma membrane. However, and the identification of mannosyltransferases involved in whether the C-terminal domain is able to synthesize full- this reaction remains a challenge. Pathak et al. (2004) length arabinan is not known. It remains possible that reported the synthesis of two a(1Æ6)- and a(1Æ2)-linked arabinan motifs might be preassembled on carrier Manp-Manp disaccharides as photoaffinity probes for molecules, polymerized and attached to the LM acceptor active-site labelling studies. Photoaffinity probe technol- molecule, a scenario that would suggest the requirement ogy offers new avenues for the identification of putative mannosyltransferases involved in the synthesis of the EmbR belongs to the Streptomyces coelicolor antibiotic a(1Æ6)-mannan core and mannose caps.
regulatory protein (SARP) family (Wietzorrek and Bibb, All known sequences of glycosyltransferases have been 1997), known to regulate genes involved in the synthesis of secondary metabolites. Belanger et al. (1996) proposed afmb.cnrs-mrs.fr/CAZY/). It was reported recently that M. that EmbR influences the expression of the M. avium tuberculosis H37Rv contains 37 putative glycosyltrans- embAB operon. M. smegmatis membranes carrying the ferases, but the precise reaction catalysed by most of M. avium embAB and embR genes retain significantly them has not been determined experimentally. Classifica- more arabinosyltransferase activity than membranes tion of glycosyltransferases with functions that have been originating from M. smegmatis carrying only the embAB confirmed shows that they belong to the GT-2 (Ppm1), GT- cluster, when treated with similar amounts of ethambutol 4 (PimA, PimB, PimC) and GT-53 (EmbC) CAZY family (Belanger et al., 1996). The M. avium embR gene is (Wimmerova et al., 2003). Although glycosyltransferases located immediately upstream of embAB, while the embR share little sequence similarity, they are proposed to adopt gene of M. tuberculosis is elsewhere in the genome (Telenti only two different folds, BGT and SpsA, according to the et al., 1997; Cole et al., 1998). It was demonstrated first structure solved in each case. For instance, Ppm1 recently that PknH, a newly described Ser/Thr kinase from has been proposed to contain an SpsA fold, and PimA M. tuberculosis, phosphorylates EmbR through recogni- and PimB a BGT fold (Wimmerova et al., 2003).
tion of a FHA (forkhead-associated) domain (Molle et al.,2003). Arg-312, Ser-326 and Asn-348 in the EmbR FHA Modulation of the immune response by PIM/LM/LAM
are key residues in the interaction between EmbR and PknH. However, it remains to be established whether phos-phorylation of EmbR by PknH plays a role in the transcrip- Historically, most studies analysing the effect of LAM on tional regulation of the embCAB cluster and in ethambutol the induction of an inflammatory response by macroph- resistance in M. tuberculosis. Whether the PknH/EmbR ages or DCs have been performed using ManLAM from pair regulates the arabinosyltransferase activity of EmbC M. tuberculosis or M. bovis BCG and PILAM from an in vivo, ultimately leading to arabinan synthesis of LAM, unidentified, fast-growing mycobacterial species (previ- ously named AraLAM) that is structurally very similar toPILAM of M. smegmatis. Results from these studies dem-onstrated that treatment of macrophages with PILAM induced the secretion of various cytokines [interleukin LAM is modified further by either manno-oligosaccharides (IL)-8, IL-12, tumour necrosis factor (TNF)-a] and apopto- or phospho-inositol caps, according to the species, result- sis, whereas ManLAM did not or did so only weakly (Chat- ing in ManLAM or PILAM respectively. It is noteworthy terjee et al., 1992; Roach et al., 1993; Zhang et al., 1995; that, although ethambutol was shown to affect the com- Riedel and Kaufmann, 1997; Yoshida and Koide, 1997; plete elaboration of the arabinan in PILAM from an etham- Ghosh et al., 1998). These observations led to the hypoth- butol-resistant M. smegmatis mutant (Khoo et al., 1996), esis that the presence of mannose caps on LAM (such as it has also been suggested that ethambutol inhibits the in ManLAM) inhibit its proinflammatory activity. Unfortu- extent of mannose capping of ManLAM in M. tuberculosis nately, uncapped LAM was not included in these studies strains grown in the presence of subminimal inhibitory for direct comparison of the biological effects of ManLAM drug concentrations (Khoo et al., 2001). As mannose cap- and PILAM. Therefore, some of the biological effects ping is a major structural entity engaged in receptor bind- associated with PILAM could also be attributed to their ing and subsequent immunopathogenesis, inhibition of phospho-myo-inositol caps. The recent isolation and char- this motif may directly affect the biological functions asso- acterization of LAM (AraLAM) from the facultative patho- 2004 Blackwell Publishing Ltd, Molecular Microbiology V. Briken, S. A. Porcelli, G. S. Besra and L. Kremer genic M. chelonae revealed that it lacks both the manno- molecules, thus revealing the proinflammatory activity of oligosaccharide and phosphoinositol caps on its terminal the LM core (Vignal et al., 2003).
arabinose residues (Guerardel et al., 2002). Interestingly, Deciphering the complex molecular basis of LAM/LM only PILAM, but not ManLAM or AraLAM, significantly activities could greatly benefit from the increasing charac- induces IL-12 expression and apoptosis (Dao et al., terization of new structural LAM variants. Lipoglycans 2004). PILAM, but neither ManLAM nor AraLAM, consis- related to mycobacterial LAM have been described in tently induces the secretion of the proinflammatory cytok- several actinomycetes, including Rhodococcus (Garton ines IL-8 and TNF-a (Guerardel et al., 2002; Vignal et al., et al., 2002), Corynebacteria (Sutcliffe, 1995), Gordonia 2003). These results support the hypothesis that mannose (Flaherty and Sutcliffe, 1999) and Amycolatopsis (Gibson caps do not inhibit the proinflammatory activities of LAM, et al., 2003b). The LAM-like molecule from the intracellu- but rather that the phosphoinositol caps of PILAM are lar pathogen Rhodococcus equi consists of a linear (a1– potent proinflammatory constituents. However, this does 6)-mannan backbone substituted by 2-linked single Manp not diminish the potential importance of mannose caps residues (Garton et al., 2002). In contrast to mycobacte- with respect to their capacity to inhibit proinflammatory rial LAM, there are no extensive arabinan domains but signals engaged by other ligands, as discussed below, single terminal a-D-Araf residues capping the 2-linked a- which is most likely to be an important activity in the D-Manp. This ‘simpler’ LAM molecule, which resembles an context of infection of macrophages or DCs. The availabil- LM-like molecule, was found to induce an early macroph- ity of AraLAM makes it feasible to address this hypothesis age proinflammatory response (Garton et al., 2002), sup- porting the notion that an extended arabinan domain may Characterization of LAM from the facultative pathogenic hinder the LM-dependent inflammatory response.
mycobacteria M. kansasii and M. chelonae enabled us to LAM from Tsukamurella paurometabola was recently analyse the effects of their precursors on the induction of demonstrated to induce the secretion of TNF-a in murine proinflammatory cytokines and apoptosis in macroph- and human macrophages (Gibson et al., 2004). Interest- ages. Interestingly, whereas neither ManLAM from M. ingly, this activity was dramatically increased after removal kansasii nor AraLAM from M. chelonae had any activity, of the arabinan chains by mild acidic treatment, which the addition of LM from either species induced potent exposed the LM core. These observations are consistent secretion of IL-8 and TNF-a (Vignal et al., 2003) and sig- with the results analysing mycobacterial LM/LAM, and nificant expression of IL-12 and apoptosis (Dao et al., therefore reinforce our hypothesis that the LM-mediated 2004). LM purified from M. smegmatis, M. tuberculosis proinflammatory activity is obstructed by the arabinan and M. bovis BCG also induced proinflammatory responses (Dao et al., 2004). Moreover, LM but not the As a consequence, enzymes modifying the LM core by corresponding LAM induced macrophage activation char- the addition of arabinose residues should be important acterized by cell surface expression of CD40 and CD86, targets for the creation of attenuated strains of M. tuber- as well as NO secretion (Quesniaux et al., 2004).
culosis and for the discovery of new antitubercular drugs.
Therefore, LMs of mycobacteria in general are strong One attractive gene candidate for inactivation in M. tuber- proinflammatory factors and, as LAM and LM are part of culosis is embC, which has been shown to participate in the cell wall, one could argue that it is important for viru- the arabinosylation of LM in M. smegmatis (Zhang et al., lent mycobacteria to minimize the amount of LM present 2003). Deletion of this gene should strongly increase the in the cell wall in order to reduce the host’s proinflamma- amount of LM in the cell wall and should affect the viru- tory response. Consequently, one might expect a direct correlation between mycobacterial virulence and a high Several reports demonstrated that PIMs isolated from LAM/LM ratio. Analysis of the LAM/LM ratio in the cell M. tuberculosis are able to induce TNF-a and IL-8 secre- walls of different virulent, facultative pathogenic and non- tion by human and murine macrophages (Barnes et al., pathogenic mycobacteria would address this hypothesis.
1992; Zhang et al., 1995; Jones et al., 2001). Highly puri- Alternatively, differences in the structural organization of fied PIM2 and PIM6 were also found to induce similar but the cell wall between bacteria may also lead to different very low levels of TNF-a secretion (Gilleron et al., 2003).
accessibility of LM for its interaction with TLR-2 on In contrast, a number of studies failed to detect significant induction of IL-8, IL-12 and TNF-a secretion and found no The arabinan domain of LAM inhibits the proinflamma- increased induction of apoptosis upon treatment of mac- tory activity of LM on macrophages, presumably by mask- rophages with PIMs isolated from M. tuberculosis, M. ing the mannan core of LAM (Fig. 1). Consistently, gradual kansasii or M. chelonae compared with treatment of cells chemical reduction in the amount of arabinan domain of with equal molar amounts of PILAM or LM (Guerardel the M. kansasii ManLAM correlated with increased proin- et al., 2002; 2003; Vignal et al., 2003; Dao et al., 2004).
flammatory cytokine expression of the truncated LAM Interestingly, the two studies (Barnes et al., 1992; Zhang 2004 Blackwell Publishing Ltd, Molecular Microbiology The mycobacterial lipoarabinomannan and related molecules et al., 1995) reporting the strongest activity of PIMs on with ManLAM inhibit phagosome–lysosome fusion (Fratti cytokine secretion used either primary human peripheral et al., 2001; 2003), suggesting that ManLAM is an impor- blood mononuclear cells or primary human alveolar mac- tant mediator of the inhibition of phagosome maturation rophages respectively. In contrast, the activity of PIMs on in the context of infection with live bacteria.
cytokine secretion, reported by Jones et al. (2001) andGilleron et al. (2003), was modest compared with the Receptors involved in inhibition and activation processes activity of PILAM, LM or LPS and was conducted usingmurine macrophages. Therefore, it appears that PIMs dis- Toll-like receptors are important initiators of the innate play a residual proinflammatory activity, which becomes immune response that are specific for pathogen- more or less apparent depending on the sensitivity of the associated molecular patterns, such as CpG-oligodeoxy- target cells (primary human cells versus murine cells) and nucleotides, lipoteichoic acid, peptidoglycan and flagellin the detection assay [reverse transcription polymerase (Kopp and Medzhitov, 2003). Interaction of agonists with chain reaction (RT-PCR) versus enzyme-linked immun- TLR-2 induces IL-12 secretion and apoptosis by the cell.
osorbent assay (ELISA)] used. In addition, the purity of PILAMs purified from rapidly growing mycobacteria, but the PIM fraction is critical as a crude preparation of PIM not ManLAM from M. tuberculosis, have been shown to would contain ‘higher’ PIMs with multiple mannose resi- interact with TLR-2 (Heldwein and Fenton, 2002). Inter- dues (such as PIM6), which may explain their biological estingly, LM isolated from M. kansasii, M. chelonae or M. activity as these structures start to resemble LM.
tuberculosis all interact with TLR-2, but not with TLR-4, asdetermined by TLR-induced CD25 expression in trans-fected Chinese hamster ovary cells (Dao et al., 2004).
These results were also confirmed in in vitro assays The first demonstration of the capacity of LAM to inhibit a on bone marrow-derived macrophages isolated from host response involved in defence against bacterial infec- TLR-2–/– or TLR-4–/– mice, showing that LM had no activity tion was conducted by Sibley et al. (1988), who reported in the former but had normal cytokine-inducing activity in the inhibition of the interferon (IFN)-g response of mac- the latter (Quesniaux et al., 2004). Moreover, macrophage rophages by ManLAM. Subsequently, live M. tuberculosis activation by LM was also found to be mediated through infection was shown to inhibit IFN-g signalling, as demon- the adaptor protein myeloid differentiation factor 88 strated by the reduction in the IFN-g-mediated cell surface (MyD88), but independent of either TLR-4 or TLR-6 rec- expression of MHC class II and receptors for the Fc por- ognition (Quesniaux et al., 2004). PIMs were shown to be tion of IgG after infection of macrophages with M. tuber- TLR-2 agonists, which may explain their biological activity culosis (Hmama et al., 1998; Hussain et al., 1999; Ting observed by some investigators (Jones et al., 2001; et al., 1999; Pai et al., 2003). Furthermore, ManLAM from M. tuberculosis inhibited the M. tuberculosis infection- Two receptors have been implicated to date in the inhib- induced apoptosis of macrophages (Rojas et al., 1997; itory activity of ManLAM. ManLAM can inhibit the LPS- Rojas et al., 2000) and the secretion of IL-12 induced by induced IL-12 secretion of human DCs (Nigou et al., lipopolysaccharide (LPS) in DCs (Nigou et al., 2001) and 2001). This activity was abolished by enzymatic removal macrophages (Knutson et al., 1998). The activity of Man- of the mannose caps or by treatment with antimannose LAM reflects the capacity of whole M. tuberculosis bacte- receptor (MR) antibodies, and was mimicked by the addi- ria to inhibit infection-induced apoptosis (Keane et al., tion of mannan from Saccharomyces cerevisiae, a known 2000) and Il-12 secretion of macrophages (Giacomini agonist of the MR, suggesting that the MR is the receptor et al., 2001; Hickman et al., 2002; Li et al., 2002). Contra- that mediates the inhibition. Nevertheless, subsequent dictory results show that, in DCs, M. tuberculosis seems studies showed that anti-MR antibodies did not block bind- either to induce secretion of IL-12 (Giacomini et al., 2001) ing of ManLAM to DCs, in contrast to antibodies directed or to inhibit IL-12 production (Johansson et al., 2001; against DC-specific intracellular adhesion molecule-3- Demangel et al., 2002). One of the hallmarks of the host– grabbing non-integrin (DC-SIGN) (Geijtenbeek et al., pathogen interaction between macrophages and M. tuber- 2003; Tailleux et al., 2003). Furthermore, the binding of culosis is the ability of M. tuberculosis to inhibit the fusion ManLAM to DC-SIGN on DCs induced the secretion of IL- of phagosomes with lysosomes (Armstrong and Hart, 10, a known inhibitor of IL-12 secretion (Geijtenbeek 1971). Lysosomes have a low pH and contain a multitude et al., 2003). Thus, DC-SIGN appears as a major mediator of lytic enzymes that are meant to lyse any bacterial or of IL-12-inhibition by ManLAM on DCs.
parasitic invaders that have been phagocytosed by themacrophages. Therefore, the capacity of M. tuberculosis Intracellular mediators of inhibition to inhibit the fusion of its phagosome with lysosomes iscrucial for its intracellular survival. Latex beads coated Very little is known about the signalling components that 2004 Blackwell Publishing Ltd, Molecular Microbiology V. Briken, S. A. Porcelli, G. S. Besra and L. Kremer connect DC-SIGN and/or the MR after binding of ManLAM factor (protein/lipid/glycolipid) that has a fast turnover and to the intracellular effectors that have been reported to be therefore requires the continuous bacterial transcription triggered by ManLAM binding. We propose that the M. and translation machinery. Alternatively, the difference tuberculosis-mediated inhibition of the increase in cytoso- between live and dead bacteria may result from the lic Ca2+ ([Ca2+]c) (Fig. 2), which is usually associated with requirement for specific genes that are only induced dur- phagocytosis of bacteria, is a central mediator of the inhi- ing phagocytosis of the bacteria by the macrophage. In bition of three important macrophage responses to infec- either case, the molecular mechanism by which live M. tion: phagosome maturation, macrophage apoptosis and tuberculosis mediate the inhibition of SK1 activation First, how does M. tuberculosis or ManLAM inhibit the How does M. tuberculosis- or ManLAM-mediated inhi- cellular [Ca2+]c response? Recent work demonstrated that bition of the cellular [Ca2+]c response arrest the phago- live, but not dead, M. tuberculosis inhibit sphingosine some maturation? Initially, the inhibition of [Ca2+]c by live kinase 1 (SK1) activity (Malik et al., 2003) (Fig. 2). This M. tuberculosis, but not dead M. tuberculosis, was enzyme converts sphingosine to sphingosine-1-phos- reported as important only for inhibiting phagosome mat- phate (S1P). Increased concentrations of S1P induce an uration (Malik et al., 2000). Further characterization of the increase in [Ca2+]c levels through the release of Ca2+ from signalling pathway demonstrated that phagosomes con- the endoplasmic reticulum by an unknown mechanism taining live M. tuberculosis contained less of the [Ca2+]c- that is independent of the inositol triphosphate pathway dependent effector protein calmodulin (CaM) compared (Malik et al., 2003). It remains to be established whether with phagosomes containing dead M. tuberculosis (Malik this activity of M. tuberculosis on SK1 can also be repro- et al., 2001). This results in lower activation of the CaM- duced using purified ManLAM. The comparison between dependent protein kinase II (CaMKII) on the phagosome live and dead (heat killed or irradiated) bacteria suggests membrane. Interestingly, the same characteristics could that the inhibition of SK1 activity is mediated through a also be attributed to phagosomes containing ManLAM-coated latex beads compared with uncoated beads (Frattiet al., 2001; 2003; Vergne et al., 2003a). The lack of activated CaMKII seems to decrease the recruitment ofphosphoinositol-3-kinase (PI3K) on the phagosome,thereby inhibiting the increase in phosphoinositol-3 phos- phate (PI3P) in the membranes (Vergne et al., 2003b).
The amount of PI3P is crucial for recruitment of earlyendosomal antigen 1 (EEA1) to phagosomes (Fratti et al.,2001; Vergne et al., 2003b). Furthermore, beads coated with ManLAM, but not PIMs, inhibited the recruitment ofthe intracellular markers syntaxin 6 and cathepsin D to theengulfing phagosome as a result of inhibition of EEA1 recruitment (Fratti et al., 2003). The importance of the lipidcomposition of the phagosome membrane for its intracel-lular trafficking has been clearly demonstrated by Anes et al. (2003). In an elegant in vitro assay, these authorscharacterized various lipids that either accelerated orinhibited phagosome maturation (Anes et al., 2003). Fur- No change
thermore, the addition of these lipids to cells infected with cytosolic Ca2+
M. tuberculosis had the same effect on phagosome mat-uration, which subsequently resulted in either accelerated Fig. 2. Induction of elevation of cytosolic Ca2+ in macrophages by
dead but not live Mycobacterium tuberculosis (Mtb). Complement
killing or prolonged survival of the intracellular bacteria opsonized bacteria are phagocytosed by the complement receptor-3 (Anes et al., 2003). These studies were the first to dem- (CR3). This interaction activates phospholipase D, which in the case onstrate the relationship between phagosomal lipid com- of dead Mtb leads to activation of the sphingosine-kinase-1 (SK-1), converting sphingosine to sphingosine-1-phosphate (S1P). The rise position, intracellular trafficking and the survival of in S1P induces the release of Ca2+ from the endoplasmic reticulum mycobacteria within this compartment.
by an unknown mechanism. This signalling pathway is clearly inter- Regulation of programmed cell death via calcium fluxes rupted at the level of SK-1 activation in the case of interaction of live Mtb with CR3, possibly through increased dephosphorylation medi- has been reviewed recently (Mattson and Chan, 2003; ated by the phosphatase SHP-1, which is to be activated after Mtb Orrenius et al., 2003), and one report provides evidence infection. Although phospholipase D is activated by infection of live of a possible link between the activity of ManLAM in inhib- and dead Mtb, it remains unclear whether its subcellular localization is the same, which might affect the activation of SK-1.
iting infection-induced apoptosis and its capacity to inhibit 2004 Blackwell Publishing Ltd, Molecular Microbiology The mycobacterial lipoarabinomannan and related molecules Phagosome maturation
Fig. 3. Effect of increased [Ca2+]c on the phagosome maturation, apoptosis and IFN-g signalling in macrophages.
I. Rise in [Ca2+]c allows the association with calmodulin (CaM) on the phagosome membrane, which induces the activation of CaM kinase II
(CaMKII) and phosphoinositol-3-kinase (PI3K). Subsequently, the PI3K increases the amount of phosphoinositol-3-phosphate (PI3P) in the
phagosome membrane, which allows the recruitment of the early endosomal antigen 1 (EEA1) and syntaxin 6. The latter are part of the vesicular
fusion complex that mediates the fusion of phagosomes with late endosomes and subsequently with lysosomes.
II. Apoptosis can be induced by increases in [Ca2+]c in multiple ways. The association of [Ca2+]c with CaM allows the activation of the phosphatase
calcineurin, which induces dephosphorylation of the proapoptotic protein Bad. Activated Bad induces the release of cytochrome C from
mitochondria into the cytosol, which is a central signal for the cell to undergo apoptosis. In addition, Ca2+ displaces cytochrome C from its
association with the phospholipid cardiolipin in the mitochondria, which induces the rise in reactive oxygen species (ROS), leading to oxidation
of the mitochondrial membrane proteins and lipids and, as a result, increased membrane permeability. This allows the free cytochrome C to
diffuse into the cytosol and to induce apoptosis.
III. The same Ca2+-CaM/CaMKII pathway induced by the rise in [Ca2+]c described in (I) might also induce the phosphorylation of Stat1 on its Ser-
727 by CaMKII. This allows the efficient association of Stat1 with the CBP/p300 complex, and only this ternary complex is capable of initiating
the transcription of IFN-g-inducible genes.
[Ca2+]c accumulation in macrophages (Rojas et al., 2000).
Bad/Akt signalling pathway and thus promotes cell sur- Effector mechanisms by which [Ca2+]c accumulation might vival (Maiti et al., 2001). In addition, ManLAM increases lead to apoptosis include the induction of increased mem- the activity of the Src homology 2-containing tyrosine brane permeability of the mitochondria, which leads to phosphatase 1 (SHP-1) (Knutson et al., 1998), which cytochrome C release into the cytosol (Fig. 3). Increased inhibits IFN-g signalling by inducing dephosphorylation of cytosolic cytochrome C leads to the formation of the apo- the IFN-g receptor-associated JAK kinases (Starr and Hil- ptosome in which caspases are activated. Next, activated ton, 1999). Moreover, the ability of ManLAM to inhibit caspases and nucleases finalize the apoptosis process by apoptosis of macrophages is absent from macrophages digesting proteins and DNA respectively (Mattson and isolated from mice deficient in SHP-1 expression (Rojas Chan, 2003; Orrenius et al., 2003).
et al., 2002). In addition, SHP-1 activity might also be The connection between the Ca2+-CaM pathway and involved in the inhibition of the [Ca2+]c response usually IFN-g-mediated upregulation of MHC II on macrophages associated with complement receptor 3 (CR3)-mediated was first demonstrated using a calmodulin antagonist phagocytosis by inducing dephosphorylation of tyrosine (W7) that inhibited MHC II expression, whereas an inhib- kinases that are important for the signal transduction upon itor of the protein kinase C had no effect (Ina et al., 1987; binding of mycobacteria to CR3 (Fig. 3).
Koide et al., 1988). Furthermore, CaMKII is known tomediate phosphorylation of residue S727 of Stat1, a crit- Conclusion
ical event in IFN-g-induced gene activation (Nair et al.,2002), presumably because phosphorylation of Stat1 at Considerable strides have been made in identifying and this position allows its interaction with the transcription characterizing genes that are required for PIMs, LM and factors CBP and p300 (Fig. 3). Thus, an important part of LAM biosynthesis, but there is still much to be learned.
the inhibition of IFN-g signalling by M. tuberculosis is Genetic strategies have shown that genes involved in the mediated through the inhibition of [Ca2+]c.
early steps of PIM biosynthesis appear to be essential for Finally, ManLAM can probably mediate inhibition of cel- mycobacterial growth. Recent work demonstrated that it lular responses in addition to inhibition of the cellular is now feasible to generate LAM-deficient strains of C. [Ca2+]c response. Indeed, ManLAM directly activates the glutamicum or M. smegmatis, and that LAM, in contrast 2004 Blackwell Publishing Ltd, Molecular Microbiology V. Briken, S. A. Porcelli, G. S. Besra and L. Kremer to PIM, is not a requisite for in vitro growth. This also the cell wall of Mycobacterium tuberculosis. Tuberculosis suggests that it will be possible to generate similar 83: 91–97.
mutants in M. tuberculosis in the near future, which will Chatterjee, D., and Khoo, K.H. (1998) Mycobacterial lipoara- binomannan: an extraordinary lipoheteroglycan with pro- be essential in order to establish the biological importance found physiological effects. Glycobiology 8: 113–120.
of LM/LAM in mycobacterial virulence, persistence and Chatterjee, D., Bozic, C.M., McNeil, M., and Brennan, P.J.
replication in the infected host. Such a genetic approach (1991) Structural features of the arabinan component of will demonstrate a causal relationship between the multi- the lipoarabinomannan of Mycobacterium tuberculosis. J tudes of biological activities attributed to isolated LAM and Biol Chem 266: 9652–9660.
LM and the effect of bacterial infection on macrophages Chatterjee, D., Roberts, A.D., Lowell, K., Brennan, P.J., and and DCs. In view of the vast array of effects mediated by Orme, I.M. (1992) Structural basis of capacity of lipoarabi-nomannan to induce secretion of tumor necrosis factor.
LAM, some of these mutant strains should be strongly Infect Immun 60: 1249–1253.
attenuated in animal models of tuberculosis and might Chatterjee, D., Khoo, K.H., McNeil, M.R., Dell, A., Morris, therefore be interesting vaccine candidates. These H.R., and Brennan, P.J. (1993) Structural definition of the mutants will also help to define targets for new tuberculo- non-reducing termini of mannose-capped LAM from Myco- bacterium tuberculosis through selective enzymatic degra-
dation and fast atom bombardment-mass spectrometry.
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Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., et al. (1998) Deciphering the biology of G.S.B. acknowledges support as a Lister Institute–Jenner Mycobacterium tuberculosis from the complete genome Research Fellow, from the Medical Research Council and the sequence. Nature 393: 537–544.
Wellcome Trust. L.K. is supported by INSERM. V.B. is sup- Dao, D.N., Kremer, L., Guerardel, Y., Molano, A., Jacobs, ported by NIH grant AI51696-01, and S.A.P. by NIH grants W.R., Jr, Porcelli, S.A., and Briken, V. (2004) Mycobacte- AI48933 and AI45889. We would like to thank Dr David J.
rium tuberculosis lipomannan induces apoptosis and IL-12 Kusner for critical reading of the manuscript and helpful production in macrophages. Infect Immun 72: 2067–2074.
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Psychiatric Alert for Psychiatrists on Prader-Willi Syndrome Janice L. Forster, MD—Child and Adolescent Psychiatrist and Linda M. Gourash, MD—Developmental Pediatrician Reasons for Consultation and Referral meal plan, restricted food access, a plan for daily activities, Prader-Willi syndrome (PWS) is a rare chromosomal disorder mandatory exercise, opportunities for sensory experience,