the mycobacterial cell...

The Mycobacterial Cell Envelope—Lipids

Mary Jackson

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, ColoradoState University, Colorado 80523-1682

Correspondence: [email protected]

Mycobacterium tuberculosis (Mtb) lipids are indelibly imprinted in just about every keyaspect of tuberculosis (TB) basic and translational research. Although the interest in thesecompounds originally stemmed from their abundance, structural diversity, and antigenicity,continued research in this field has been driven by their important contribution to TB path-ogenesis and their interest from the perspective of drug, vaccine, diagnostic, and biomarkerdevelopment. This article summarizes what is known of the roles of lipids in the physiologyand pathogenicityof Mtb and the exciting developments that have occurred in recent years inidentifying new lead compounds targeting their biogenesis.

The mycobacterial cell envelope is the domi-nant feature of the biology of Mycobacteri-

um tuberculosis (Mtb) and other mycobacteria,based on sugars and lipids of exceptional struc-ture. Cell envelope lipids constitute �40% of thecell dry mass, although this percentage may varydepending on species/isolate and growth con-ditions (Goren and Brennan 1979; Minnikinet al. 1982). The particular chemical structuresand organization of these lipids between the in-ner and outer membrane of the mycobacterialcell envelope account for much of its imperme-ability to biocides and for the unique stain-ing properties of mycobacteria that aid in thediagnosis of infected specimens (Goren andBrennan 1979; Jarlier and Nikaido 1994; Bren-nan and Nikaido 1995). Being located at theinterface between the bacterium and the host,the outer membrane and capsular lipids of

Mtb also play important roles in directinghost–pathogen interactions. Several lipid com-ponents of Mtb (e.g., isoprenoid lipids, glycero-phospholipids, phosphatidylinositol manno-sides, lipoarabinomannan, mycolic acids, andtrehalose mycolates) are produced by all myco-bacterial species, consistent with their require-ment for growth. The biosynthesis of someof these lipids is the site of action of anti-TBdrugs such as isoniazid (INH) and ethion-amide (ETH), as well as multiple other lead com-pounds under development. Other lipids (e.g.,long-chain polymethyl-branched fatty acid–containing acyltrehaloses and long-chain diols)are not essential for growth but are unique topathogenic mycobacteria and have been in-volved in pathogenesis.

Studies initiated in 1927 by R.J. Andersonand colleagues mark the beginning of the pio-

Editors: Stefan H.E. Kaufmann, Eric J. Rubin, and Alimuddin Zumla

Additional Perspectives on Tuberculosis available at www.perspectivesinmedicine.org

Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a021105

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neering work that has led to the discovery andearly structural definition of some of the mostbiologically relevant lipids of Mtb, includingphosphatidylinositol mannosides, mycolic ac-ids, and branched fatty acids and polyols (Gorenand Brennan 1979). Studies conducted for de-cades to follow by generations of outstandingbiochemists in France, the United Kingdom,and the United States, in particular, have result-ed in a thorough understanding of the chemicalstructures of these lipids. Their detailed descrip-tions have been compiled in several recentbooks and reviews and the reader is referred tothese for further reading on this topic (Brennan1988; Barry et al. 1998; Kaur et al. 2009; Daffeet al. 2014). Developments in the genetic ma-nipulation of mycobacteria in the 1990s andthe release of the first complete genome se-quence of Mtb in 1998 have provided a majorimpetus to the study of the biosynthesis of thesecompounds with the result that many of theenzymes and transporters involved in their bio-genesis have now been identified. Following theclassification of Mtb into six major genetic lin-eages associated with specific geographical re-gions (Gagneux and Small 2007), recent yearshave also seen a return of interest in comparativeanalyses of the lipid content of clinical isolatesfrom different regions of the world in the hopeof identifying correlates of pathogenicity. Inter-estingly, not only are these studies providingnew insights into the biochemical diversity ofMtb and the putative roles of some lipids inhost–pathogen interactions (Daffe et al. 1991;Constant et al. 2002; Reed et al. 2004, 2007;Tsenova et al. 2005; Sinsimer et al. 2008; Huetet al. 2009; Krishnan et al. 2011), they are alsoshedding light on the genetic basis of observa-tions made some 20 to 40 years ago by earlyinvestigators in the field (Goren et al. 1974a;Daffe et al. 1991; Krishnan et al. 2011; Supplyet al. 2013). What is known of the biosynthesisof Mtb lipids has been reviewed recently (Daffeet al. 2014). This review focuses on our currentunderstanding of the roles of cell wall lipids inthe physiology and pathogenicity of Mtb anddescribes ongoing drug discovery efforts aimedat targeting their biosynthesis, export, and reg-ulation.

PHYSICAL ORGANIZATION ANDPHYSIOLOGICAL ROLES OF Mtb CELLENVELOPE LIPIDS

Architecture of the Mtb Cell Envelope

A schematic representation of the cell envelope’sarchitecture of Mtb is presented in Figure 1. Theinnermost layer is the plasma membrane thatseems typical of bacterial membranes. Outsidethe plasma membrane is a massive cell wall“core” comprised of peptidoglycan (PG) cova-lently attached to the heteropolysaccharide ara-binogalactan (AG), in turn esterified at its non-reducing ends to a-alkyl, b-hydroxy long-chain(C60-C90) mycolic acids. Intercalated within thislipid environment are the “free” (noncovalent-ly linked) lipids and lipoglycans that have in-trigued researchers for more than eight decades:the phosphatidylinositol mannosides (PIMs),the phthiocerol dimycocerosates (PDIMs), thephenolic glycolipids (PGLs), a variety of acyltre-haloses, mannose-capped lipoarabinomannan(ManLAM), etc. (Figs. 2 and 3). The covalentlybound mycolic acids of the cell wall core and thefree lipids form the inner and outer leaflets, re-spectively, of a highly impermeable asymmetri-cal bilayer (known as the “mycomembrane” orouter membrane) that confers to mycobacteriatheir characteristic resistance to many therapeu-tic agents (Minnikin et al. 1982; Jarlier and Ni-kaido 1994; Hoffmann et al. 2008; Zuber et al.2008) (Fig. 1). Finally, a loosely attached capsu-lar-like structure outside the outer membrane ofMtb was shown to mainly consist of polysaccha-rides and proteins with only minor amounts oflipids (2%–3% of the material) (Lemassu andDaffe 1994; Ortalo-Magne et al. 1995; Sani et al.2010) (Fig. 1). Thus, in spite of being Gram-positive bacteria, mycobacteria possess a cell en-velope characterized by the presence of an outermembrane and a periplasmic space.

Mycolic Acids

The interest in drugs targeting the biosynthesisof mycolic acids is clearly illustrated by the ther-apeutic efficacy of INH and ETH (Table 1). Thisessentially owes to their role as key structuralelements of the outer membrane. The mycolic

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acids covalently linked to AG represent abouthalf of the weight of the cell wall core (Draper1998). Mycolic acids also occur as acyl substit-uents esterifying a variety of outer membrane(glyco)lipids including trehalose mono- and di-mycolates (Fig. 2), glycerol monomycolate, andglucose monomycolate. They play key roles inthe permeability of the cell envelope, the abilityof Mtb to form biofilms (Ojha et al. 2005, 2010;Sambandan et al. 2013), and the pathogenicityof the bacterium during the actively replicatingand persistent stages of the infection (Barryet al. 1998; Daffe and Draper 1998; Yuan et al.1998; Dubnau et al. 2000; Glickman et al. 2000;Sugawara et al. 2002; Vander Beken et al. 2011).

Inner and Outer Membrane Extractable Lipids

Glycerophospholipids, PIM, LM, and ManLAM.Glycerophospholipids (phosphatidylinositol[PI], phosphatidylglycerol, phosphatidylserine[PS], phosphatidylethanolamine [PE], cardio-lipin [CL]), and mannosylated forms of PIknown collectively as PIMs (Fig. 2) are the ma-jor lipid constituents of the plasma membraneof Mtb. In addition, the plasma membrane ofMtb contains major lipoglycans known as lip-omannan (LM) and ManLAM (Fig. 3) (Pit-arque et al. 2008), and a number of isoprenoidsthat play various critical functions in the cell.Among them, decaprenyl phosphate (Dec-P)

TDM

D-mannan

ManLAM

D-arabino-D-mannan

Capsule

Outermembrane

Innermembrane

AG

PG

LM α-D-glucan

TMM PATSLPIM DAT

PhospholipidPDIM/PGL

ManLAM

Figure 1. Schematic representation of the Mtb cell envelope. Many of the classes of (glyco)lipids discussed in thetext are represented schematically and are shown in probable locations in the cell envelope. Light blue symbolsrepresent arabinose residues, red symbols represent galactose residues, brown symbols represent mannoseresidues, and black circles represent glucose residues. D-arabino-D-mannan, D-glucan, and D-mannan are cap-sular polysaccharides. Mycolic acid chains are shown in dark green. LM, lipomannan; ManLAM, mannose-capped lipoarabinomannan; AG, arabinogalactan; PG, peptidoglycan.

Mtb Lipids

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CH

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serves as a carrier of activated sugars in the syn-thesis of essential cell envelope polymers in-cluding PG, AG, LM, and ManLAM (Kaur etal. 2009), and menaquinones are essential com-ponents of the respiratory chain (Weinsteinet al. 2005; Dhiman et al. 2009). Thus, as inother bacteria, plasma membrane lipids play avital role in Mtb, providing a selective perme-ability barrier around the cell and participating

in a number of critical physiological processes,including electron transport, ATP formation,DNA replication, and the biosynthesis of a va-riety of cell envelope components.

Glycerophospholipids (particularly PE),PIM, LM, and ManLAM are also found in theoutermost layers of the cell envelope of Mtb andother Mycobacterium species where they mayinteract with the host (Ortalo-Magne et al.

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Figure 3. Structures of Mtb lipoglycans. Mannose-capped lipoarabinomannan (ManLAM); the mannan moietyof ManLAM consists of �20–30 Manp residues and the arabinan polymers of �60 Araf units; the precisenumber of arabinan chain(s) attached to mannan is still uncertain. Lipomannan (LM) is devoid of the arabinanchains of ManLAM. R1, R2, R3, R4 are tuberculostearic acid, oleic acid, or palmitic acid.

Figure 2. (Continued) (A) Structures of acyltrehaloses: trehalose monomycolate (TMM); trehalose dimycolate(TDM); sulfolipid (SL-I); diacyltrehalose (DAT); polyacyltrehalose (PAT); and the lipooligosaccharide (LOS) ofMtb Canettii (R¼Ac). The major sulfolipid SL-I (2,3,6,60-tetraacyl a-a0-trehalose-20-sulfate) is represented. InSL-I, trehalose is sulfated at the 20 position and esterified with palmitic acid and the multimethyl-branchedphthioceranic and hydroxyphthioceranic acids. In DAT (2,3-di-O-acyltrehalose), trehalose is esterified withpalmitic acid and the multimethyl-branched mycosanoic acid. In PAT, trehalose is esterified with palmitic acidand the multimethyl-branched mycolipenic acids. (B) Structures of the phthiocerol dimycocerosate (PDIM)and phenolic glycolipid (PGL) of Mtb. p, p0 ¼ 3–5; n, n0 ¼ 16–18; m1 ¼ 20–22; m2 ¼ 15–17; R ¼ CH2-CH3

or CH3. (C) Structure of the predominant mannosyl-b-1-phosphomycoketide (MPM) from Mtb H37Rv. (D)Structure of a triacylated phosphatidylinositol dimannoside (PIM2), one of the major forms of PIMs producedby Mtb.

Mtb Lipids


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Table 1. TB drug discovery pipeline showing the inhibitors targeting lipid biosynthesis

Pathway

targeted

(Proposed)

target protein

Inhibitor

Discovery

Preclinical

development

Clinical

development

Mycolic acidsInhA CD117

(Vilcheze et al. 2011)Pyridomycin

(Hartkoorn et al. 2012)Triclosan derivatives

(Pan and Tonge 2012; North et al.2013)

Novel structural classes(GlaxoSmithKline)

HadABC ISO and TAC analogs(Phetsuksiri et al. 1999; Bhowruthet al. 2006; Alahari et al. 2007; Shahabet al. 2010; Coxon et al. 2013)

NAS-21, NAS-91 and analogs(Bhowruth et al. 2008; Gratraud et al.2008)

KasA/KasB Thiolactomycin and analogs(Pan and Tonge 2012; North et al.2013)

Pks13 Thiophene compounds; Compound 30

(Ioerger et al. 2013; Wilson et al. 2013)FadD32 4,6-diaryl-5,7-dimethyl coumarins

(Stanley et al. 2013)Antigen 85

A, B, andC

Ebselen(Favrot et al. 2013)

I3-AG85(Warrier et al. 2012)

MmpL3 Adamantyl ureas(Grzegorzewicz et al. 2012a)

BM212 and analogs(La Rosa et al. 2012; Poce et al. 2013)

C215(Stanley et al. 2012)

SPIRO2 and THPP1(Remuinan et al. 2013)

Compound 2(Ioerger et al. 2013)

SQ109(Tahlan et al.2012)(Sequella)

Unknown(Inhibition ofoxygenatedmycolic acidbiosynthesis)

PA-824(Stover et al.2000)

OPC-67683(Matsumotoet al. 2006)

Continued

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Table 1. Continued

Pathway

targeted

(Proposed)

target protein

Inhibitor

Discovery

Preclinical

development

Clinical

development

EthR ETH “boosters”: BDM 31343 andanalogs (Willand et al. 2009;Villemagne et al. 2012)

Decaprenyl phosphateIspC Fosfomidomycin analogs

(Obiol-Pardo et al. 2011; Uh et al.2011)

Isoprenoid-based biosynthetic precursorsPG biosynthesis MraY Capuramycin analogs

(Sequella)SQ641

(lead capuramycinanalog) (Sequella)

PG biosynthesis WecA Caprazamycin derivatives (CPZEN-45)(Ishizaki et al. 2013)

LAM (and AG)biosynthesis

DprE1 DNB1(Christophe et al. 2009)

VI-9376(Magnet et al. 2010)

377790(Stanley et al. 2012)

TCA-1(Wang et al. 2013)

Several synthetic compounds(TB Alliance, Scripps)

Benzothiazinones(BTZ043) (Makarovet al. 2009)

MenaquinonesMenA Aurachin RE, Ro 48-8071 and analogs

(Dhiman et al. 2009; Debnath et al.2012)

MenB 1,4-benzoxazines; 4-oxo-4-phenylbut-2-enoates(Li et al. 2010; 2011)

MenE Sulfonyladenosine analogs(Lu et al. 2008; 2012)

PGLFadD22 50-O-[N-(4-

hydroxybenzoyl)sulfamoyl]-adenosine(Ferreras et al. 2008)

Ser/Thr kinasesPknAPknBPknG

Several chemical scaffolds(Vertex Pharmaceuticals Inc.)(Magnet et al. 2010; Danilenko et al.2011; Lougheed et al. 2011;Chapman et al. 2012)

See text and the Working Group for New Drugs, Stop TB Partnership (http://www.newtbdrugs.org) for details.

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1996; Pitarque et al. 2008; Dhiman et al. 2011).Whether Mtb requires phosphatidylglycerol,CL, PS, and PE for growth is at present notknown. PI, PIM, LM, and ManLAM, in contrast,are all required for the viability of Mtb in vitro(Kaur et al. 2009; Guerin et al. 2010). Emergingdata indicate that PIM play important roles inthe permeability of the cell envelope, innermembrane integrity, and regulation of cell sep-tation and division (Parish et al. 1997; Kordu-lakova et al. 2002; Patterson et al. 2003; Moritaet al. 2005, 2006). Likewise, structural defectsin LM and ManLAM were shown to affect theb-lactam susceptibility and acid-fast stainingproperties of mycobacterial cells, indicatingan equally important role of these moleculesin cell envelope integrity (Fukuda et al. 2013).

Trehalose esters, PDIM, and PGL. Other outermembrane/surface-exposed lipids include tre-halose monomycolates (TMM) and trehalosedimycolates (TDM), PDIM, sulfolipids (SL), di-acyltrehaloses (DAT), polyacyltrehaloses (PAT),phenolic glycolipids (PGL), and lipooligosac-charides (LOS) (Fig. 2). In addition to servingas a mycolic acid donor in the formation ofTDM and the cell wall core, TMM was recentlyidentified as the likely form under which my-colic acids, which are synthesized in the cyto-plasm, translocate to the periplasm or outermembrane (Grzegorzewicz et al. 2012a). Con-sistently, compounds that prevent TMM trans-location are potent inhibitors of Mtb (Grzegor-zewicz et al. 2012a; Tahlan et al. 2012; Stanleyet al. 2012; La Rosa et al. 2012; Remuinan et al.2013). In contrast to TDM and TMM, which areconsidered essential lipid components of thecell envelope of all mycobacteria, SL, DAT,PAT, and PDIM are more restricted in their dis-tribution and are not essential for the growth ofMtb in vitro, although they have been involvedin pathogenicity (see the section Roles of MtbLipids in Pathogenicity). PDIM and closely re-lated phthiocerol diphthioceranates are essen-tially confined to pathogenic Mycobacteriumspecies (Daffe and Laneelle 1988). Consistentwith their important roles in the permeabilityof the cell envelope (Camacho et al. 2001) andpathogenicity, PDIMs are apparently producedby all virulent clinical isolates of Mtb. DAT and

PAT are confined to virulent isolates of the My-cobacterium tuberculosis complex (Goren andBrennan 1979; Jackson et al. 2007), and SL areexclusively found in the human pathogen Mtb(Goren 1990). Structural similarities betweenSL, DAT, and PAT and, to some extent PDIM,particularly the fact that they all are esterifiedwith long-chain polymethyl-branched fatty ac-ids, is suggestive of at least partially redundantfunctions in the cells. One of them is to alleviatethe propionate-mediated stress undergone byMtb when the bacterium switches to host cho-lesterol and fatty acids as major carbon sources(Singh et al. 2009; Lee et al. 2013). The propio-nyl-CoA generated upon b-oxidation of cho-lesterol is converted to methylmalonyl-CoA bythe propionyl-CoA carboxylase and then usedby dedicated polyketide synthases in the elon-gation of the polymethyl-branched fatty acidsesterifying PDIM, SL, DAT, and PAT. Indepen-dent from this metabolic function and sugges-tive of their collective role in the permeabilityof the cell envelope, Mtb mutants deficientin the synthesis of more than one polymethyl-branched fatty acid–containing lipid (eitherPDIM and SL or DAT, PAT, and SL) but notmutants lacking only one lipid species, losethe ability to stain with neutral red (Cardonaet al. 2006). Finally, DAT and PAT have beeninvolved in the retention of the capsular mate-rial at the surface of Mtb (Rousseau et al. 2003a).

PGL and LOS are other lipids acylated bypolymethyl-branched fatty acids produced byMtb and related tubercle bacilli, albeit not alllineages (Fig. 2). LOS have thus far only beenfound in Mycobacterium canettii (Daffe et al.1991), a representative of smooth tubercle bacillithat seem to have originated from the same poolof ancestors as Mtb but rarely causes humandisease (Supply et al. 2013). Based on studiesperformed in Mycobacterium marinum (Renet al. 2007), the presence of LOS in the cell en-velope of M. canettii may account for the par-ticular smooth colonial morphotype of thisstrain. The ability to synthesize PGL, on the oth-er hand, has only been retained by M. canettiiand some Mtb isolates of the East Asian/Beijinglineage (Daffe et al. 1987; Constant et al. 2002;Reed et al. 2004; Huet et al. 2009). PGL have

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been involved in pathogenicity (see the sectionRoles of Mtb Lipids in Pathogenicity). In an at-tempt to correlate the lipid content with thevirulence of Mtb isolates, Goren et al. character-ized a methoxylated phenolphthiocerol (a non-glycosylated variant of PGL), the so-called “at-tenuation indicator lipid” (Goren et al. 1974a).This lipid and its unmethylated form were de-tected in East Asian/Beijing isolates and foundto accumulate in all Indo-Oceanic Mtb strainsexamined (Krishnan et al. 2011). Similarly,Beijing strains were reported to accumulate var-iants of PDIM and eventually PGL, knownas phthiotriol and glycosylated phenolphthio-triol dimycocerosates (Huet et al. 2009). Thecorrelation between the occurrence of these lip-ids and variations in virulence remains, how-ever, unclear (Huet et al. 2009; Krishnan et al.2011). PGL and LOS are clearly not essentialfor growth but they are potent B-cell antigensmaking them potentially useful, albeit line-age-specific, serodiagnostics (Brennan 1988;He et al. 1991; Vera-Cabrera et al. 1994; Simon-ney et al. 1995, 1996; Constant et al. 2002; Julianet al. 2002).

Other Lipids

Another family of lipids known as the manno-syl-b-1-phosphomycoketides (MPM) are pro-duced by slow growing mycobacteria includingMtb, Mycobacterium avium, and M. marinum(Fig. 2). They are found inside the cells andreleased in the culture medium. They are notessential for the growth of Mtb but are thoughtto be involved in pathogenicity (Matsunaga andSugita 2012).

Triglycerides (TAG) are considered intra-cellular lipids of Mtb. They represent the mainapolar lipid when glycerol serves as the majorcarbon source in the medium (Brennan 1988).TAGs are thought to serve as an energy reservefor the long-term survival of Mtb during thepersistence phase of infection (Brennan 1988;Daniel et al. 2004), as well as a means by whichfree fatty acids are detoxified. Mtb Beijing familyisolates accumulate large quantities of TAG,possibly as a result of the up-regulation of theTAG synthase, Rv3130c (Reed et al. 2007).

Regulation of Lipid Synthesis

The physiological status of mycobacterial bacilli(i.e., age of the culture, actively replicatingvs. persistent) and a number of environmentalfactors including temperature, oxygen tension,and nitrogen and carbon sources are known toimpact their lipid composition, particularlyphospholipids, TAG, PIM, LM, ManLAM, my-colic acid–based glycolipids, and polymethyl-branched fatty acid–containing lipids (Brennan1988; Barry et al. 1998; Daffe and Draper 1998;Singh et al. 2009; Dhiman et al. 2011; Lee et al.2013; Yang et al. 2013). Less is known of theregulation of lipid synthesis during host infec-tion. PDIM and mycolic acids are apparentlyproduced in abundant quantities by in vivo–grown tubercle bacilli (Kanai et al. 1970; Bha-midi et al. 2012). Yet, variations in the acid-fastcharacteristics of Mtb during mouse and guineapig infection suggest that the bacterium under-goes drastic adaptative changes of its lipid com-position in vivo (Ryan et al. 2010). Increasedproduction of SL and DAT during host in-fection is supported by the up-regulation ofbiosynthetic genes on phagocytosis of Mtb byhuman primary macrophages or during pro-gressive pulmonary TB infection in mice (Gra-ham and Clark-Curtiss 1999; Rodriguez et al.2013). The virulence-associated two-compo-nent regulator PhoP-PhoR may be involved inthis up-regulation as it has been found to coor-dinately and positively regulate the synthesis ofSL, DAT, and PAT in Mtb (Gonzalo-Asencio etal. 2006; Walters et al. 2006). The metabolicswitching of Mtb to host lipids as carbon sourc-es during infection and the associated increasein the production of PDIM, SL, DAT, and PATwere shown to be facilitated by the regulatoryprotein WhiB3 (Singh et al. 2009). In recentyears, PknA, PknB, and other members of theSer/Thr kinase family were found to regulatethe activity of multiple enzymes and transport-ers involved in the biosynthesis of mycolic acids,ManLAMs, and PDIMs (Molle and Kremer2010). It is thus apparent that lipid synthesisin Mtb is tightly controlled and that regulationoccurs both at the transcriptional and post-translational levels.

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ROLES OF Mtb LIPIDS IN PATHOGENICITY

Several excellent reviews have been publishedabout the biological activities of Mtb lipids, in-cluding very recently (Daffe and Draper 1998;Russell et al. 2002; Karakousis et al. 2004; Ber-tozzi and Schelle 2008; Britton and Triccas 2008;Gilleron et al. 2008; Glickman 2008; Guilhotet al. 2008; Guenin-Mace et al. 2009; Neyrollesand Guilhot 2011; Russell 2011; Daffe et al.2014). Thus, only a brief overview of what isknown and remains to be investigated aboutthe involvement of lipids in the pathogenesisof TB will be presented here.

The existence of a possible correlation be-tween the lipid content of Mtb clinical isolatesand their pathogenicity was noted early on byMiddlebrook and Goren among others. Thesestudies led to the identification of SL, TDM,and PDIM and derivatives as lipids of potentialrelevance to TB immunopathogenesis (Bloch1950; Goren et al. 1974a,b; Daffe and Draper1998). Further work on the biological activitiesof purified lipids in vitro and in vivo followed,in the postgenomic era, by an examination ofthe virulence phenotype and immune responsesinduced by Mtb mutants deficient in variousaspects of their biosynthesis lent further sup-port to the involvement of lipids in host–path-ogen interactions. Their contribution to the in-fection process accompanies every step of thelife cycle of the bacterium. The entry of Mtbinside host macrophages and dendritic cells isessentially mediated by specialized phagocyticreceptors including complement receptors,scavenger receptors, and C-type lectins, such asthe mannose receptor, DC-SIGN, and Mincle.PIM, TDM, and ManLAM are among the pre-dominant glycolipids mediating Mtb’s interac-tions with these receptors (Villeneuve et al.2005; Schlesinger et al. 2008; Ishikawa et al.2009; Neyrolles and Guilhot 2011). Once insidephagocytic cells, Mtb resides in a phagosomethat fails to fuse with lysozymes. The isopren-oid-derived compounds, isotuberculosinol andtuberculosinol, ManLAM, TDM, PDIM, SL,and DATall have been implicated in the impair-ment of phagosome maturation, although themechanisms involved and amount of support-

ing data varies for each compound (Daffe andDraper 1998; Brodin et al. 2010; Neyrolles andGuilhot 2011; Russell 2011; Mann and Peters2012). Inside the cells, Mtb releases significantamounts of proteins and lipids, among themTMM, TDM, PIM, PGL, PE, phosphatidylgly-cerol, CL, and ManLAM, that traffic within thecell and may be released through exocytosis(Russell et al. 2002; Russell 2011). These mole-cules are not only taken up by bystander anti-gen-presenting cells, they also act as modula-tors of the function of the host cell andsurrounding tissue, impacting the secretion ofpro- and anti-inflammatory cytokines (e.g.,PGL, TDM, mycolic acids, SL, PIM, LM,ManLAM, and lipoproteins), apoptosis (lipo-proteins, PIM, ManLAM, and TDM), T-cellfunctions (ManLAM, DAT, and PAT), the in-duction of foamy macrophages (oxygenatedmycolates and TDM), and granuloma forma-tion (TMM, TDM, and ManLAM) (Daffe andDraper 1998; Jackson et al. 2007; Gilleron et al.2008; Peyron et al. 2008; Guenin-Mace et al.2009; Neyrolles and Guilhot 2011; Rajni et al.2011; Russell 2011; Vander Beken et al. 2011; Liet al. 2012; Sakamoto et al. 2013; Vir et al. 2013).Importantly, several Mtb lipids, including diac-ylated forms of SL, PIM, ManLAM, MPM, andmycolyl lipids, represent themselves as antigensfor immune recognition and are presented to Tlymphocytes by MHC-I-like molecules of theCD1 family (Matsunaga and Sugita 2012; Aroraet al. 2013; Ly et al. 2013; Van Rhijn et al. 2013).Some of these lipids may show potential as sub-unit vaccines (Guiard et al. 2009; Arora et al.2013). Finally, ManLAM, SL, TDM, PGL, LOS,DAT, and lipoproteins are also potent inducersof humoral immune responses.

Although the critical involvement of PDIMand TDM in TB pathogenesis is now supportedby a wealth of in vitro and in vivo data (Guilhotet al. 2008; Neyrolles and Guilhot 2011; Rajniet al. 2011; Li et al. 2012), the role(s) played byother prominent (glyco)lipids including (iso)-tuberculosinol, SL, DAT, PAT, PGL, some PDIMvariants, and ManLAM during host infectionare far from being as clear. Their roles in path-ogenicity typically come into question whentheir demonstrated biological activities in vitro

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or in vivo (e.g., inhibition of phagosome matu-ration and immunomodulatory activities), usu-ally as purified molecules, fail to translate into aclear “virulence” phenotype in Mtb strains ge-netically engineered to produce or, on the con-trary, lose the ability to produce them. Classicalexamples of this include, for instance, SL, whoseplethora of biological activities (Goren andBrennan 1979; Goren 1990; Daffe and Draper1998; Bertozzi and Schelle 2008) fail to translateinto a clear replication or persistence advantagein infected mice or guinea pigs (Rousseau et al.2003b); DAT and PAT, whose absence from MtbH37Rv does not hamper the ability of this strainto grow in mice (Rousseau et al. 2003a); or Man-LAM, the suppression of the bioactive mannosecaps of which fails to translate into any detect-able phenotype in macrophages and animalmodels of infection (Afonso-Barroso et al.2012). A combination of factors may accountfor this. First, no pathogenic trait of Mtb is at-tributable to a single virulence factor and socompensatory mechanisms within certain lipidfamilies (e.g., polymethyl-branched fatty acid–containing lipids) or cell ligands (e.g., Man-LAMs and mannosylated glycoproteins) areknown to exist (Pitarque et al. 2005; Passemaret al. 2014). Second, an inherent flaw associatedwith studies that use purified molecules is thatthey may not accurately mimic qualitativelyand/or quantitatively the way these moleculesare presented to the host when carried by wholebacilli. Furthermore, the genetic background ofthe Mtb strain in which they are produced islikely to affect the way they interact with thehost and their subsequent contribution to path-ogenicity (e.g., PGL) (Sinsimer et al. 2008).Third, lipids such as SL and (iso)tuberculosinolare restricted to the human pathogen, Mtb, andmay only show a clear phenotype in this specifichost. The facts that the ability of Mtb to producesome PDIM variants and that PGL is relativelyrestricted to East-Asian isolates (Constant et al.2002; Reed et al. 2004; Huet et al. 2009) suggestthat the different Mtb lineages may even haveevolved regionally to tailor their lipid composi-tion to the genetic background of their humanhost (Neyrolles and Guilhot 2011). From amechanistic standpoint, concluding to the di-

rect effect of a lipid on a specific host functioncan be challenging. This owes to the roles thatlipids often play in the structure and permeabil-ity of the cell envelope in addition to potentiallyinteracting with the host. Thus, a deficiency intheir production may not only cause Mtb tobecome more susceptible (i.e., more permeable)to host defense mechanisms (e.g., PDIMs andtheir impact on the resistance of Mtb to oxida-tive stress) (Rousseau et al. 2004), it may alsoprofoundly alter the way other immunomodu-latory antigens are presented at the cell surfaceand interact with host functions.

Mtb LIPIDS IN THE CONTEXT OF DRUGDISCOVERY

General Considerations

The previous sections have highlighted thediversity, unique structures, and essential rolesplayed by cell envelope lipids in the physiologyand pathogenicity of Mtb. These features ac-count for the specificity and therapeutic efficacyof first- and second-line TB drugs such as INHand ETH, which inhibit the biosynthesis of my-colic acids (Banerjee et al. 1994; Vilcheze andJacobs 2007; Grzegorzewicz et al. 2012b) (Fig.4), and those of ethambutol (EMB), which in-hibits the formation of the arabinan domains ofManLAM (Fig. 3) and AG (Mikusova et al.1995). In addition, pyrazinamide was reportedto be an inhibitor of the fatty acid synthase I(FAS-I) (Zimhony et al. 2000). With multidrugresistance on the increase, recent years have seena marked intensification of TB drug discoveryefforts with the result that many new lead com-pounds are now at various stages of the drugdiscovery and preclinical development pipeline.Interestingly, these efforts keep pointing at cellenvelope lipids as one of the Achille’s heels ofMtb (Table 1) (Jackson et al. 2013). As impor-tantly, they are leading the TB field to revisitearlier impressions that drug targeting the bio-genesis of the cell envelope lipids may not besynergistic with other drugs, useful againstMRD-Mtb isolates, or active against persistentbacilli. This section summarizes recent develop-ments that have occurred in the discovery ofdrugs targeting lipid synthesis in Mtb.

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Drugs Targeting Mycolic Acid Biosynthesisand Export

The biosynthesis of mycolic acids is shown sche-matically in Figure 4. In the past 10 years, bothtarget-based and whole cell–based approacheshave been used toward the identification anddevelopment of inhibitors acting on virtuallyall portions of this biosynthetic pathway (Fig.4 and Table 1). A comprehensive review of theseefforts was recently published by North et al.(2013), and we will here only briefly describesome of the most exciting developments thathave occurred in the last few years. Target-basedscreening uses biochemical and structural ap-proaches to design, screen, and further evaluatea compound’s activity against a specific molec-ular target. This type of approach has been usedextensively to rationally optimize inhibitors ofthe FAS-II elongation cycle—in particular, an-alogs of thiolactomycin as inhibitors of KasA/KasB and triclosan derivatives as inhibitors ofInhA (Pan and Tonge 2012). The screeningof inhibitors showing activity against the Plas-modium falciparum enoyl reductase and b-hydroxyacyl-ACP dehydratase, FabZ, identifiedcompounds that appear to target the cor-responding FAS-II enzymes in Mtb and dis-play an MIC against whole mycobacterial cells(Bhowruth et al. 2008; Gratraud et al. 2008;Vilcheze et al. 2011). Pyridomycin, a cyclic dep-sipeptide isolated from Streptomyces with anMIC value of 0.4 mg/mL against Mtb, is nowknown to also target InhA (Haatkoorn et al.2012), as are novel structural classes of inhibi-tors in the lead-optimization phase reportedby the Working Group on New TB Drugs (Table

1). Incidentally, the recent elucidation of themode of action of isoxyl (ISO) and thiacetazone(TAC), two prodrugs formerly used in the clin-ical treatment of Mtb, revealed that they bothinhibit the dehydration step of the FAS-II cyclecatalyzed by HadAB and HadBC (Grzegorze-wicz et al. 2012b). The finding that relativelysimple structural modifications of the parentISO and TAC compounds can increase the po-tency of analogs more than 20-fold is encour-aging in that it suggests that it may be possible toidentify compounds that could be administeredat a lower dose, thereby reducing side effects(Phetsuksiri et al. 1999; Bhowruth et al. 2006;Alahari et al. 2007; Shahab et al. 2010; Coxonet al. 2013).

Because of their pivotal roles in the finalassembly of mycolic acids and their transfer tocell envelope acceptors, Pks13, FadD32, and themycoloyltransferases of the antigen 85 com-plex are also receiving a lot of attention fromthe perspective of drug development (Fig. 4).High-throughput screening assays were report-ed for FadD32 (Galandrin et al. 2013) and theantigens 85 (Boucau et al. 2009; Elamin et al.2009; Sanki et al. 2009; Favrot et al. 2013). Thelatter assays led to the identification of a numberof inhibitors of antigens 85, one of them, knownas ebselen, also shows activity against Mtb inculture (MIC of 20 mg/mL). The molecularmechanism of inhibition of antigens 85 by eb-selen was elucidated and found to be particular-ly interesting in the sense that it is unlikely tolead to the selection of drug-resistant isolates(Favrot et al. 2013). Small molecule binders ofantigen 85C identified by magnetic resonancespectroscopy also showed activity against Mtb in

Figure 4. (Continued) After reduction by the b-keto-acyl-ACP reductase MabA, dehydration by the (3R)-hydroxyl-dehydratases HadAB and HadBC and reduction by the enoyl-CoA reductase InhA, either the b-ketoacyl-ACP synthase KasA or KasB catalyzes the condensation of the resulting product with malonyl-ACPunits, thereby initiating the next round of elongation. Methyltransferases and other as yet unknown enzymesmodify the meromycolyl chain introducing double bonds, cyclopropyl, hydroxy, methoxy, and keto function-alities. These two chains are coupled together via Claisen condensation by the acyl-AMP ligase FadD32 and thepolyketide synthase Pks13. Upon release from Pks13, reduction by CmrA yields mycolic acids, which are thentranslocated to the periplasm under the form of TMM by partially elucidated translocation machinery involvingthe essential integral membrane transporter, MmpL3, and attached to AG or another molecule of TMM to formTDM, by the antigen 85 complex. The sites of action of Mtb inhibitors are indicated. Drugs presently or formerlyused in the clinical treatment of TB are in red; inhibitors under development are in blue (see Table 1). THL,thiolactomycin; PZA, pyrazinamide.

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vitro and inside macrophages in the 100-mM

range (Warrier et al. 2012). The return of inter-est in whole cell–based screening that the TBfield has witnessed in recent years has led tothe identification of thiophenes and other com-pounds active against Pks13 (Ioerger et al. 2013;Wilson et al. 2013), diaryl coumarin–basedcompounds active against FadD32 (Stanley etal. 2013), and diverse chemotypes active againstthe TMM transporter, MmpL3 (Grzegorzewiczet al. 2012a; Stanley et al. 2012; Ioerger et al.2013; Remuinan et al. 2013). BM212 and deriv-atives (La Rosa et al. 2012; Poce et al. 2013) andSQ109, a drug candidate originally designedto be an EMB analog and currently undergo-ing phase II clinical trials (Sacksteder et al.2012; Tahlan et al. 2012), were also shown tokill Mtb through the inhibition of MmpL3.The reason why so many different chemical scaf-folds apparently inhibit the MmpL3-mediatedtranslocation of TMM is unclear. Establishing avalidated assay for the direct measurement ofMmpL3 inhibition is a high priority, becauseit would greatly facilitate the further develop-ment of these inhibitors and could potentiallyreveal new scaffolds for development.

In summary, recent drug discovery effortshave yielded a number of chemotypes inhibit-ing various aspects of the synthesis of mycolicacid, some of which are promising candidatesfor further development. Inhibiting the FAS-IIsystem and MmpL3 currently appear to be themost attractive/druggable targets for TB drugdiscovery in this pathway.

Isoprenoids and Related Lipids

Inhibitors of the MEP pathway. As a lipid carrierof activated saccharide subunits, polyisoprenylphosphate (Dec-P) is involved in the biosynthe-sis of the arabinan portion of AG, the mannanand arabinan domains of arabinomannan, LM,and ManLAM, the lipid I and lipid II precursorsof PG, and the biosynthesis of the “linker unit”between AG and PG (Kaur et al. 2009; Daffe et al.2014) (Fig. 1). The discovery that Dec-P synthesisin Mtb proceeds through the methylerythritolphosphate pathway, which has no homolog inhumans, has provided stimulus for the charac-

terization and identification of inhibitors of therelevant enzymes. Fosmidomycin, which is cur-rently in clinical studies for the treatment ofmalaria, is a competitive inhibitor of the secondenzyme of the pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC). Severalderivatives of this antibiotic have been synthe-sized, some of which have shown promising re-sults against Mtb (Jackson et al. 2013) (Table 1).

Inhibitors of lipid-linked biosynthetic precur-sors. The phospho-N-acetylmuramyl pentapep-tide translocase (MraY) catalyzes the exchangeof UDP-N-acetylmuramic acid pentapeptidefrom UDP to Dec-P thereby generating lipid Iin PG biosynthesis. The synthesis of capuramy-cin analogs as inhibitors of this enzyme has ledto the identification of several compounds with2- to 4-mg/mL MICs against drug-susceptibleand MDR Mtb isolates (Jackson et al. 2013).Although its mode of action has yet to be con-firmed inwhole Mtb cells, one of them, SQ641, isat the stage of preclinical development (Table 1).

Very recently, the caprazamycin derivativeCPZEN-45 was shown to be an inhibitor ofMtb’s decaprenyl-phosphate-GlcNAc-1-phos-phate transferase, WecA, in the biosynthesis ofAG (Ishizaki et al. 2013).

Decaprenylphosphoryl arabinose (DPA) isthe only known arabinose donor in the build-ing of the arabinan domains of the two essen-tial cell envelope (lipo)polysaccharides, AG, andManLAM. In the last 4 years, whole cell–basedscreening of compounds against Mtb has pro-duced several inhibitors of the essential epim-erase DprE1 required for the formation of DPA(Table 1). The molecular mechanism of actionof some of these compounds has been thor-oughly investigated and several DPA inhibitorsare now reported to be in the hit-to-lead orpreclinical development phases (Table 1) (Jack-son et al. 2013).

Menaquinones. The lipoquinones involvedin the respiratory chains of mycobacteria consistof menaquinones (2-methyl-3-polyprenyl-1,4-naphthoquinones) (Daffe et al. 2014). Evidencebased on transcriptomics profiling and the in-hibitory activity of menaquinone inhibitorsagainst Mtb in culture has highlighted the re-quirement of this bacterium for electron trans-

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port and, therefore, menaquinone synthesisboth during active replication and microaero-philic persistence (Dhiman et al. 2009). Accord-ingly, three key enzymes of the menaquinonebiosynthetic pathway of Mtb, MenB (1,4-dihy-droxy-2-napthoic acid synthase), MenE (O-succinylbenzoyl-CoA synthase), and MenA(1,4-dihydroxy-2-napthoic acid octaprenyltransferase) have been studied as potential drugtargets and a number of promising inhibitorssynthesized (Table 1).

Inhibition of PIM, LM, and ManLAMBiosynthesis

The essential character of PIM, LM, and Man-LAM, their restricted distribution to mycobac-teria and closely related Actinomycetes, andtheir demonstrated impact on the structureand permeability of the cell envelope of Mtbmake the biosynthetic enzymes of these mole-cules attractive candidates for the developmentof specific Mtb inhibitors with the potentialto synergize with or potentiate the activity ofother drugs used in combination. Accordingly,target-to-drug approaches are pursuing vari-ous essential enzymes acting both at early (e.g.,PimA, PimB’) and late (e.g., Emb proteinsand other lipid-linked sugar-utilizing glycosyl-transferases) stages of the pathway. Innovativehigh-throughput screening assays have been de-veloped for some of these enzymes (Zhang et al.2010, 2011). The most advanced ManLAM in-hibitors to date are those targeting DPA synthe-sis, as described in the previous section.

Targeting Lipids Involved in the Replicationand Persistence of Mtb In Vivo

Targeting TAG synthesis through inhibition ofthe primary TAG synthase of Mtb, Rv3130c(Daniel et al. 2004; Sirakova et al. 2006), mayrepresent a viable approach to the eradicationof persistent bacilli for the key role TAG is likelyto play as an energy reserve for the long-termsurvival of Mtb during the persistence phase ofinfection.

As detailed in the previous section, PDIMsare critical components of the permeability bar-

rier surrounding Mtb and contribute to a sig-nificant extent to the survival and ability of thebacterium to replicate in vivo. To the best of ourknowledge, no specific PDIM inhibitors haveyet been reported but promising compoundstargeting the Ser/Thr kinases of Mtb that regu-late their biosynthesis (among other physiolog-ical processes) are under development (Jacksonet al. 2013) (Table 1). Another regulatory sys-tem of interest in the context of drug develop-ment is the two-component transcriptional reg-ulator PhoP-PhoR, which regulates multiplevirulence-associated processes in Mtb, includ-ing the biosynthesis of SL, DAT, and PAT (Gon-zalo-Asencio et al. 2006). Finally, a compoundinhibiting the production of PGL in whole Mtbcells has been reported (Ferreras et al. 2008).

CONCLUDING REMARKS

Mtb lipids have been for the last eight decadesand still remain today the object of much re-search activity. One of the highlights of thisresearch in the last five years is undoubtedlythe discovery of a variety of novel chemical scaf-folds inhibiting various aspects of lipid syn-thesis, particularly mycolic acids, ManLAM,and AG. Beyond their therapeutic applications,lipids involved in the immunopathogenesis ofTB may also represent useful targets for futurepreventive interventions, as illustrated by cur-rent studies aimed at investigating the vaccinepotential of CD1-restricted lipid antigens (GPuzo, pers. comm.) and that of an attenuatedphoP-based Mtb knockout mutant (Nambiaret al. 2012). Although the interest in developingTB serodiagnostics based on Mtb lipids seems tohave somewhat waned in the last decade, impor-tant efforts are currently focusing on their po-tential as biomarkers to monitor TB reactiva-tion, as well as the efficacy of treatment andvaccination (Shui et al. 2012; Wallis et al. 2013;Chan et al. 2013).

ACKNOWLEDGMENTS

Vicki Jones is gratefully acknowledged for herhelp with the preparation of the figures. Theauthor’s research on cell envelope lipids and

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inhibitors is supported through the NationalInstitutes of Health/National Institute of Aller-gy and Infectious Diseases grants AI064798and AI063054.

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August 7, 20142014; doi: 10.1101/cshperspect.a021105 originally published onlineCold Spring Harb Perspect Med

Mary Jackson

Lipids−−The Mycobacterial Cell Envelope

Subject Collection Tuberculosis

of TuberculosisTransmission and Institutional Infection Control

Edward A. Nardell

Clinical Aspects of Adult Tuberculosis

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InfectionMycobacterium tuberculosisto Innate and Adaptive Cellular Immune Responses

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Advances in Diagnostic Assays for TuberculosisStephen D. Lawn

and Noncommunicable DiseasesTuberculosis Comorbidity with Communicable

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AbubakarLaura Muñoz, Helen R. Stagg and Ibrahim

Host-Directed Therapies for TuberculosisDavid M. Tobin

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Peptidoglycan and−−The Mycobacterial Cell Wall

Lloyd, et al.Luke J. Alderwick, James Harrison, Georgina S.

Drug DevelopmentGenetic Approaches to Facilitate Antibacterial

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Tuberculosis and HIV Coinfection

Gunilla KälleniusJudith Bruchfeld, Margarida Correia-Neves and

TargetsDevelopment Pipeline and Emerging Drug The Tuberculosis Drug Discovery and

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Imaging in Tuberculosis

Gulati, et al.Jamshed B. Bomanji, Narainder Gupta, Parveen

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