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Review

The Emergence of Phenolic Glycans as Virulence Factors in Mycobacterium Tuberculosis Danielle D. Barnes, Mimmi L. E. Lundahl, Ed lavelle, and Eoin M. Scanlan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00394 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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The Emergence of Phenolic Glycans as Virulence Factors in Mycobacterium Tuberculosis Danielle. D. Barnes†, Mimmi L. E. Lundahl†‡, Ed Lavelle‡ and Eoin M. Scanlan†* †School

of Chemistry and Trinity Biomedical Sciences Institute, Trinity College, Pearse St, Dublin 2, Ireland. Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, D02 R590, Dublin 2, Ireland KEYWORDS Tuberculosis, glycolipids, PGLs, carbohydrate vaccines.

‡Adjuvant

ABSTRACT: Tuberculosis is the leading infectious cause of mortality worldwide. The global epidemic, caused by Mycobacterium tuberculosis, has prompted renewed interest in the development of novel vaccines for disease prevention and control. The cell envelope of M. tuberculosis is decorated with an assortment of glycan structures, including glycolipids, that are involved in disease pathogenesis. Phenolic glycolipids and the structurally related para-hydroxybenzoic acid derivatives, display potent immunomodulatory activities and have particular relevance for both understanding the interaction of the bacterium with the host immune system and also in the design of new vaccine and therapeutic candidates. Interest in glycobiology has grown exponentially over the last decade, with advancements paving the way for effective carbohydrate based vaccines. This review highlights recent advances in our understanding of phenolic glycans, including their biosynthesis and role as virulence factors in M. tuberculosis. Recent chemical synthesis approaches and biochemical analysis of synthetic glycans and their conjugates have led to fundamental insights into their roles in host-pathogen interactions. The applications of these synthetic glycans as potential vaccine candidates are discussed.

lenge for the field, however, it is anticipated that the current contributions will pave the way for innovative therapeutics for TB in the future.

Introduction Mycobacterium tuberculosis

The only human vaccine currently available for M. tb is the bacille Calmette-Guérin (BCG) vaccine, which is composed of a live attenuated strain of M. bovis.11 The BCG vaccine was introduced in 1921 and has been employed globally ever since, with 120 million doses administered annually.12 While the vaccine provides a high rate of protection against childhood and disseminated TB, many studies have shown that it displays suboptimal efficacy against pulmonary TB in adult populations, with protection rates being highly variable, ranging from 0-80%.12-15

The global epidemic that is Tuberculosis (TB) is the most prevalent of all potentially fatal bacterial infections worldwide and is caused by Mycobacterium tuberculosis (M. tb). In 2015, M. tb infected 10.5 million people and accounted for 1.8 million deaths worldwide, making it one of the leading causes of death from an infectious agent.1 Multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tb are on the rise,2-4 with an estimated 480,000 new cases of MDR infections being reported in 2015.1 Furthermore, an increase in the frequency of cases involving TB and human immunodeficiency virus (HIV) co-infection has made the treatment of TB more problematic, contributing to the global TB pandemic and high mortality rate associated with the active disease.1-3, 5

A more efficacious vaccine for TB is urgently needed and would represent the most cost-effective way to control the disease. In particular, an effective vaccine would hinder the spread of MDR-tb and TB/HIV co-infection.16 A number of comprehensive reviews covering advances in TB vaccine development have been published;17-19 In order for significant progress to be made, an improved understanding of the interaction between the mycobacterium and the host immune system is needed. In particular, detailed structural analysis of cell wall components and an improved understanding of their biosynthesis is required. Biological evaluation of fully synthetic derivatives enables an improved understanding of how these compounds modulate immune response at a molecular level and provides fascinating prospects for therapeutic and vaccine development. In this review, we focus on the biosyn-

Accordingly, there is an urgent need for the development of new and more effective anti-tubercular therapeutics.6 In 2012, Bedaquiline became the first novel anti-TB drug approved in over 40 years, and is the only drug available for the treatment of MDR-tb.7, 8 A promising anti-TB drug pipeline has recently emerged, with several candidates currently in the Phase II and Phase III clinical trials.8, 9 Notably, Baulard et. al. have reported the discovery of novel drug-like spiroisoxazoline molecules known as Small Molecules Aborting Resistance (SMARt). Remarkably, resistance to the anti-TB drug, ethionamide, was fully reversed upon treatment with SMARt-420.10 The development of new treatments for TB remains a significant chal-

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ACS Chemical Biology cell wall core, and the outer capsule (Fig. 1). This unique multi-layered cell envelope is highly hydrophobic with limited permeability and therefore acts as an effective barrier against antibiotic drugs and chemotheraptic agents. This intricate cell envelope is critical for bacterial survival and infection and has been implicated in the ability of the mycobacterium to develop resistance to several anti-tubercular drugs.29

thesis, chemical synthesis and immunomodulatory properties of mycobacterial cell wall phenolic glycoconjugates (PGLs and pHBADs). We discuss the significant potential of these compounds in the development of novel vaccine candidates.

M. tb and the host immune system M. tb possesses an extraordinary ability to resist and evade the bactericidal mechanisms of the human immune system. Mycobacteria infect host innate immune cells, prominently macrophages, and subsequently inhibit several host antibacterial processes, allowing them to survive harsh intracellular conditions.20, 21 Various ligands on the mycobacterium cell surface are recognized by a series of receptors on antigen presenting cells of the immune system, such as Toll-like receptors (TLRs), thereby directly triggering the innate immune response.22, 23

The plasma membrane is composed of a symmetrical phospholipid bilayer. The cell wall surrounding this consists of a lower segment made up of peptidoglycan, which is covalently linked to the mycolyl-arabinogalactan (AG) layer via phosphoryl-N-acetylglucosaminosylrhamnosyl.30 The AG layer is composed of the oligosaccharides, galactofuranose and arabinofuranose, and is esterified by α-alkyl and β-hydroxyl long chain fatty acids, known as mycolic acids. Lastly, the upper segment of the cell wall is made up of non-covalently attached glycolipids intercalated into the mycolic acid layer, known as the mycomembrane.30 The outer capsule is composed of noncovalently attached polysaccharides, glycolipids, and proteins, which are secreted across the cell membrane.30

Protective immunity against M. tb is mediated by T-cells.24 By far the most important anti-microbial effectors are orchestrated by T helper 1 (Th1) cells that secrete the pivotal cytokine interferon-γ (IFN-γ). This messenger, amongst other effects, promotes macrophage activation and subsequent bactericidal responses. Inhibition of both T cell proliferation and IFN-γ mediated effects are key components of M. tb. pathogenesis.24, 25 A number of reviews extensively detail and discuss the effects of M. tb on the host immune system, to which the reader is directed.21, 26-28

The surface of the cell wall is decorated with a variety of species-specific complex extractable glycolipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), lipoarabinomannan (LAM), mannose capped lipoarabinomannan (ManLAM), dimycolyl trehalose (TDM), sulfolipids (SL), diacyltrehaloses (DAT), polyacyltrehaloses (PAT) and phenolic glycolipids (PGLs). In other mycobacteria species, extractable glycolipids can include lipooligosaccharides (LOS),31, 32 and glycopeptidolipids (GPLs).33

The cell wall of M. tb The cell envelope of M. tb is a highly complex structure, composed of three major components: the plasma membrane, the

Figure 1. Schematic representation of M. tb cell envelope. The plasma membrane, the cell wall core (peptidoglycan, mycolylarabinogalactan complex), and the outer capsule are depicted along with their comprising biomolecules (LAM, lipoarabinomannan, extractable glycolipids (PGLs, phenolic glycolipids shown) and glycoproteins). The structures and component molecules are not drawn to scale, and the numbers of carbohydrate residues shown are not representative of the actual molecules.

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The outermost components of the cell envelope are the first to interact with a target cell and as such can exert potent immunomodulatory effects. Many of these glycolipids, such as TDM and LAM,34-36 have been implicated as important virulence factors and are involved in the pathogenesis of TB, triggering a downregulation of T-cell proliferation, inhibiting secretion of the pivotal cytokine IFN-γ, and its subsequent activation of macrophages.37

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ed around the cell wall of M. tb and are found in culture isolates of all strains of M. tb and M. bovis BCG.46 The aromatic core is derived from the methyl ester of p-hydroxybenzoic acid (p-HBA) and the glycosyl moieties in both p-HBAD I and p-HBAD II are identical to that of mycoside B and PGL-tb, respectively.47 The production of these glycans by all strains of M. tb, indicates their critical importance in the pathogenesis of the mycobacterium.46

Biological activity of PGLs and p-HBADs

PGLs and p-HBADs

PGLs are involved in many biological activities, for example PGL-1, produced by M. leprae, is implicated in the invasion of Schwann cells and in suppressing the secretion of proinflammatory cytokines by host immune cells.48 Additionally, the PGLs found in M. marinum are known to play a role in cell permeability and in evading the host immune system.49, 50 However, the exact role of PGL-tb is less clear.23

Structure of PGLs and p-HBADs Certain mycobacteria, such as M. leprae, M. kansasii, M. marinum and M. bovis, are known to produce PGLs, with several structural variations depending on the species.38, 39 PGL-tb, synthesised only by a subset of M. tb. strains, was first isolated in 1987 by Daffé et al. from four Canetti strains of M. tb (M. tb “canetti”) and since then interest in the glycolipid has surged.39 Furthermore, their presentation on the cell surface of M. tb makes these compounds ideally located to interact with host immune cells.

A subset of clinical isolates of M. tb, known as the W. Beijing family, are prevalent in southeast Asia and have spread worldwide, causing a number of epidemics.51, 52 These strains were found to have a hypervirulent phenotype in mice; they failed to induce protective Th1 responses in early infection, resulting in a more rapid death.53, 54 Interestingly, most of the W. Beijing family were found to produce PGLs, therefore supporting the hypothesis that these glycolipids may be involved in the hypervirulence associated with these strains of M. tb.43, 55, 56 A study by Reed et al. found that disruption of PGL synthesis in W. Beijing strains lead to a loss in their hypervirulence, without affecting bacterial load during the disease.53 In this study, an increase in the release of pro-inflammatory cytokines; interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) in vivo was also observed upon the loss of PGL production. IL-12 plays a central role in the induction of Th1 responses. Overproduction of PGL also led to the inhibition of the pro-inflammatory mediators in a dose dependent manner, strongly suggesting that the PGLs are involved in suppressing the immediate cytokine response of the host.53

PGLs are made up of a lipid core common to that of PDIMs, which are known effectors of virulence in M. tb infection.40-42 The lipid core is composed of a long chain β-diol, naturally occurring as a diester of polymethyl-branched fatty acids/ phenolphthiocerol esterified by two chains of multiple methyl-branched fatty acids, mycocerosic acids (Fig. 2).23, 43 The lipid core is ω-terminated by an aromatic nucleus which is glycosylated with one to four sugar residues, depending on the species producing it, typically O-methylated deoxysugars. In the case of M. tb, the glycosylated domain of the major form of PGL-tb is a trisaccharide substituent, consisting of 2,3,4-triO-methyl-α-L-fucopyranosyl-(1→3)-α-L-rhamnopyranosyl(1→3)-2-O-methyl-α-L-rhamnopyranosyl.44 The minor PGL found in M. tb is mycoside B, also found as the major PGL in M. bovis, whose glycosylated domain consists of 2-O-methyl-α-Lrhamnopyranosyl, with the lipid core identical to that of PGLtb.45

The activity of PGL is postulated to be derived from the saccharide moiety as there is no hypervirulent response associated with the structurally similar monosaccharide derivative, mycoside B.53 Work by Stadthagen et al. found that the glycans, p-HBADs, inhibit pro-inflammatory responses in infected macrophages, thereby having a significant impact on the host response to infection.23 Moreover, failure to produce p-HBADs reduced the ability of the mycobacterium to suppress the innate immune system of the host, which is typically induced by M. tb during infection.23 PGL-tb is synthesized in few M. tb strains, compared to the p-HBADs, which are produced by all strains. Therefore, it is not surprising that the carbohydrate residue is imperative for biological activity.46 Sera from TB patients contain antibodies that specifically recognize PGL-tb, despite the low incidence of PGL producing strains, therefore indicating that the seroactivity is dependent on the carbohydrate moiety (i.e. p-HBADs).46 Significant progress has been made towards elucidating the immunomodulatory role of PGLs, with a large body of work providing supporting evidence that PGL is a vital virulence factor.22, 23, 53, 55 One such study found evidence that PGL may enhance the infectivity of M. tb at the earliest stage of infection, through CC chemokine receptor 2 (CCR2)-mediated recruitment of permissive macrophages.22 It has also been suggested that the lack of PGL production in BCG M. bovis strains

Figure 2. Structures of the major phenolic glycolipid of M. tb (PGL-tb) and structurally related glycans, parahydroxybenzoic acid derivatives I and II, p-HBAD I and pHBAD II; red = phenolphthiocerol; blue = mycocerosic acids. The small carbohydrate molecules, para-hydroxybenzoic acid derivatives (p-HBADs) are structurally related to PGLs and are depicted in Fig. 2 as p-HBAD I and p-HBAD II. They are secret-

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ACS Chemical Biology esterified by mycocerosic acids to yield p-hydroxyphenyl PDIM.

may account for the sub-optimal protection provided by the vaccine.57 However, the exact correlation between the glycolipids and related virulence remains unclear.58

Mycocerosic acids are derived from C16-20 chain fatty acids, which are elongated by successive reactions to incorporate propionate units by the multifunctional enzyme, mycocerosic acid synthase, encoded by mas.64-68 The use of methyl malonyl-CoA as a substrate by this enzyme introduces methyl branches during the fatty acid elongation to form tetra methyl branched mycocerosic acids.64, 69 The fatty-acyl AMP ligase, FadD28, is involved in activating the fatty acid chain precursors to form mycocerosates,70 which are transferred directly onto the diol of the phenolphthiocerol by the acyl transferase, PapA5, in the final esterification step.71, 72

Biogenesis of PGLs and p-HBADs Understanding the biosynthesis of PGLs and related phenolic glycans is critical to the development of an understanding of how these molecules modulate immune response. In addition, potential protein targets for small-molecule inhibitors may be identified through careful elucidation of the genes involved in cell wall biosynthesis. The biosynthesis of PGLs in M. tb is a complex 25 step enzymatic process, with 1.5% of the M. tb genome dedicated to the synthesis and transport of these molecules, providing additional evidence for their essential role in the mycobacterium.30, 59 Consistent with their conserved structures, the PGL-tb and p-HBADs share a similar biosynthetic pathway and common enzymatic steps.60 Sequencing of the M. tb genome, has greatly aided the elucidation of the biosynthetic steps involved in the production of these molecules.61 The genes involved in the biosynthesis are clustered on a 73-kb fragment of the M. tb chromosome with the organization of the locus being highly conserved in all PGL producing strains.30

Biosynthesis of the saccharide residue The synthesis of the saccharide residue of PGL and the pHBADs involves the same set of enzymes; starting from either the p-hydroxyphenyl PDIM or methyl p-hydroxybenzoate precursors, for PGL-tb and p-HBADs respectively (Fig. 3B). Due to the structural similarity between the two, it was initially thought that glycosylation was an early step in the biosynthesis of PGLs and that p-HBADs were simply intermediates in the biosynthetic pathway.46 However, as the p-HBADs are found outside the cell wall and not in the cytosol, where the enzymatic bio-machinery for the PGL synthesis is located, it is unlikely that these glycans are intermediates of PGL biosynthesis. This is further confirmed by the production of p-HBADs in all species of M. tb, including those unable to produce PGLs.46 Moreover, intracellular attachment of the sugar residue to the lipid occurs before translocation of the PGLs to the cell wall. Inactivation of the related transporter proteins, which are involved in transporting the molecules across the cell wall, results in blocked secretion of the PDIMs and PGLs, without affecting their biosynthesis.42

Biosynthesis of p-hydroxyphenyl PDIM The common lipid core of PGLs and PDIMs, phenolphthiocerol and phthiocerol respectively, involve similar biosynthetic steps, the genes for which are located on the PDIM/PGL locus of the M. tb chromosome.41 The aromatic core in phenylphthiocerol is derived from chorismate which is converted to p-HBA by a pyruvate-lygase enzyme encoded by Rv2949c (Fig. 3A).47 p-HBA is activated by FadD22, a fatty acid ligase involved in the activation and transfer of long chain fatty acids, to yield p-HBA-S-FadD22, an acyl-S-enzyme covalent intermediate.62 This is subsequently elongated by a type 1 polyketide synthase to give malonyl-CoA units attached to p-hydroxyphenylalkenoate.46 Type 1 polyketide synthase is encoded for by the pks 15/1 gene and it was found that disruption of pks 15/1 in PGL producing strains, eradicated in their ability to produce PGLs.46 Most strains of M. tb naturally contain a pks 15/1 frameshift mutation, which explains why these strains only produce p-HBADs and not PGLs. Interestingly, when pks 15/1 was introduced to a nonPGL producing strain, the ability to produce PGLs was fully restored, highlighting the fact that this gene is vital for the formation of the lipid precursor for PGL, through the elongation of the putative p-HBA precursor.46 Moreover, Tsenova et al. found that disruption of pks 15/1 in PGL producing strains of M. tb resulted in a reduced virulence in the rabbit model of TB.55

The first rhamnose residue is transferred onto phydroxyphenyl PDIM by the glycosyltransferase encoded by Rv2962c,60 followed by the methylation of the 2-OH, catalyzed by the product of Rv2959c.59 A second rhamnose residue is subsequently transferred by the rhamnosyltransferase enzyme encoded by Rv2958c.60 A frameshift mutation in the Rv2958c gene is responsible for the formation of the truncated minor PGL, mycoside B, as well as the accumulation of the monosaccharide p-HBAD I in M. tb.44 Rv2957 encodes for the glycosytransferase which is responsible for the transfer of the terminal fucose residue to form the trisaccharide of PGL-tb or p-HBAD II. The exact order in which the trisaccharide is formed is yet to be fully elucidated. Possibly, the disaccharide is formed first, followed by its transfer to the p-HBAD I and 2O-methyl-rhamnosyl-phenyl PDIM to form p-HBAD II and PGL-tb respectively. Alternatively, the two enzymes may sequentially catalyze the transfer of the rhamnosyl and fucosyl appendages, controlled by a regulation loop, to prevent the accumulation of biosynthetic intermediates containing the disaccharide PGL or p-HBAD, which have never been isolated.60 The final steps in this biosynthetic pathway involve the methylation of the terminal fucose. This occurs after glycosylation, orchestrated by three methyltransferases, encoded by Rv2956, Rv2954c, Rv2955c for the methylation of the 2-OH, 3-OH and 4-OH of fucose respectively. Evidence has suggested that the methylation occurs sequentially, beginning with the 2-OH position, followed by the 4-OH and finally the 3-OH residue.73

The enzyme, FadD29, activates p-hydroxyphenylalkenoate, which is transferred onto the polyketide synthase, ppsA, and elongated with both malonyl-CoA and methyl malonyl-CoA units by ppsA-E to form phenolphthiocerol.46, 49 Finally, the phenolphthiodiolone residue is released from ppsE by type II thioesterase, TesA.63 The enzymes responsible for the unusual methylation of the third hydroxy group of the PDIM are encoded for by two genes, Rv2951c and Rv2952. The reduction of phenolphthiodiolone to phenolphthiotriol is catalyzed by the product of Rv2951c. The subsequent methylation of phenolphthiotriol to form phenolphthiocerol is catalyzed by the product of Rv2952.43, 59 Phenolphthiocerol is subsequently

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these molecules has to date focused on PDIM; however, it is possible that the same transporters are involved in the transport of PGLs across the cell membrane.

Translocation of PGL to the cell wall Both genetic and biochemical strategies have shown that the assembly of PGLs and p-HBADs occurs in the cytosol. Furthermore, some of the enzymes involved in the biosynthesis are known to be located intracellularly (e.g. FadD enzymes); however their presence at the outermost layers of the cell wall and capsule indicate the existence of dedicated PDIM/PGL translocation machinery.74 Most work on the translocation of

The mmpL7 gene encodes for a transporter of the resistancenodulation-division (RND) permease family and is located at the PGL/PDIM locus on the M. tb chromosome; it is implicated in the formation of the outer membrane of M. tb.74, 75 Three genes, drrA, drrB and drrC, encode for an ABC transporter

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Figure 3. Biosynthetic pathway of M. tb PGLs and p-HBADs. A) biosynthetic pathway and intermediates in the synthesis of PDIM; B) biosynthetic pathway and intermediates in the synthesis of PGLs and p-HBADs; m = 12-16; n = 18-20; R = H, Me; orange box = enzymes; blue box = genes; purple box = acyl carrier proteins; PDIM, phthiocerol dimycoserosate; PGL-tb = phenolic glycolipid of M. tb; p-HBAD I, para-hydroxybenzoic acid derivative I; p-HBAD II, para-hydroxybenzoic acid derivative II PGL-tb glycan epitope conjugated to Bovine Serum Albumin (BSA) by Fujiwara in 1991, who demonstrated that the glycan could be used as a potential tool for serodiagnosis of M. tb.87 In the same year, Viswanadam et al. also published the synthesis of trisaccharide segment of PGL-tb.88

found in the cell membrane, with the subunit encoded for by drrC being required for the proper localisation of PDIMs.74 Inactivation of these transporters blocks the transport of both PDIM and PGL across the cell membrane, while the biosynthesis of the glycolipids remains unaffected.42, 74 Interestingly, the A domain of the mmpL7 transporter was found to interact biochemically with ppsE, the polyketide synthase involved in the synthesis of PDIM and PGL, suggesting that the synthesis and transport of these molecules may be related processes.76 Furthermore, the mutants of these genes, which were unable to properly transport the PDIMs, showed increased cell wall permeability, indicating that these molecules may play an integral role in the architecture and permeability of the cell wall, in addition to being important virulence factors.63, 74

Impressively, the total chemical synthesis of PGL-tb was reported by Minnaard et al. in 2012 and represents a landmark in glycoconjugate synthesis, marking the first fully synthetic PGL compound.89 The synthetic approach utilized a Sonogashira coupling to conjugate the phenolic trisaccharide onto the phthiocerol. Asymmetric Cu-catalyzed 1,4-additions to unsaturated thioesters and cyclic enones were employed to introduce the methyl groups. Minnaard et al. subsequently published the total chemical synthesis of mycoside B the following year.90 In 2013 the same group notably reported the synthesis of p-HBAD I, starting from L-rhamnose, in only five synthetic steps.90 In 2014, Scanlan et al. published the chemical synthesis of both p-HBAD I and p-HBAD II, accompanied by biological studies on the immunological effects of the molecules. This was the first time that the p-HBADs were studied in isolation from the parent bacterium; remarkably, they were found to down-regulate cytokine production by host immune cells.91 More recently, both Lowary et al. and Astarie-Dequeker and co-workers reported the synthesis and biological evaluation of the trisaccharide moiety of PGL.

Finally, the lipoprotein encoded by LppX is required for the transport of PDIMs from the periplasm to the outer layers of the cell wall. The crystal structure of this protein revealed a large hydrophobic cavity suitable for accommodating a PDIM molecule. Lipid analysis revealed that mutants of LppX fails to release PDIMs, without affecting their biosynthesis.77

Chemical synthesis and biological studies of PGLs and p-HBADs As outlined above, PGLs and p-HBADs are important virulence factors in M. tb infection as they modulate the host immune response. Due to the complex pattern and variable ratios of these molecules in vivo, isolation of appreciable amounts of the pure compounds has proven to be extremely challenging. Therefore, chemical synthesis is necessary to provide sufficient quantities of pure material in order to fully elucidate the immune response in isolation from the parent bacterium and to develop potential vaccine candidates. However, these complex molecules represent challenging synthetic targets, encompassing lengthy synthetic routes and intricate protecting group strategies. Moreover, a major obstacle to be considered in the chemical synthesis of these compounds is the selective formation of the α-glycoside linkages, which are found in the natural products.78

Lowary and co-workers prepared a methoxy derivative of the p-HBAD II trisaccharide using a convergent synthesis, starting from three monosaccharide building blocks (1-3, Fig. 4). These building blocks were sequentially added, starting from the reducing end to the non-reducing end of the molecule to assemble the trisaccharide. The rhamnose building blocks, 1 and 2, were both prepared from per-acetylated rhamnose in eight synthetic steps. The tri-methylated thiofucoside starting material, 3, was synthesized from L-fucose via a four-step synthetic approach.

Since the elucidation of the molecular structures of the PGL family, several elegant approaches have been reported regarding their total chemical synthesis. As the biological specificity of the PGLs is thought to be related to the carbohydrate residue, much of this work has focused on obtaining synthetic analogues of the saccharide domain. The synthesis of the trisaccharide epitope of PGL-1 found in M. leprae, was first described by Brennan et al. in 1984.79 Since then, other derivatives of the PGL-1 carbohydrate have since been published by Brennan,80, 81 and Pinto.82 Synthesis of the glycan segment of the PGL found in M. kanasii, was initially published by Reddy et al. in 1992.83 More recently, Lowary and co-workers described the synthesis of an epitope of this PGL saccharide accompanied by extensive immunological studies.84, 85 The synthesis of the trisaccharide unit of the PGL found in M. tb was first reported in 1990 by Van Boom, who introduced the α-glycosyl linkages using an iodonium ion-promoted glycosylation approach in order to achieve the desired stereoselectivity.86 This was subsequently followed by the synthesis of a

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methoxy group at the rhamnose 2-OH and the fucose 3-OH’’ position displayed a substantial loss in the inhibitory activity, compared to that of the naturally methylated analogue, 7. Furthermore, Lowary et. al. demonstrated that the inhibitory activity of these synthetic PGL analogues is mediated through Toll-like receptor 2 (TLR2), and not Toll-like receptor 4 (TLR4).92 Astarie-Dequeker and co-workers have recently published the synthesis of a methyl analogue of the p-HBAD II trisaccharide using a convergent synthetic route. This was achieved using three monosaccharide starting materials, 8-10, depicted in Fig 5. The rhamnose starting materials 8 and 9 were both synthesized from the orthoester of L-rhamnose via nine and five step synthetic routes, respectively. In a similar approach to the synthetic route used by Lowary the fucose building block, 10, was a tri-methylated thiofucoside. However, in contrast, AstarieDequeker introduced the pivotal methyl group at the 2-OH of 8 prior to the initial glycosylation step.93 The rhamnosyl acceptor, 8, was coupled to the trichloroacetimidate donor, 9, by NIS-TMSOTf -promoted glycosylation. The stereoselectivity of the glycosylation was achieved by the neighbouring group participation of the adjacent acetyl (Ac) protecting group, to furnish the α-linked disaccharide, 11, exclusively. The 3’-O-Bn protecting group of the non-reducing rhamnose unit was removed using hydrogenation conditions to give alcohol 12 and the terminal fucose residue was introduced upon NIS-TMSOTf-promoted glycosylation with 10 to yield the trisaccharide 13. Finally, deprotection of the Ac protecting group using Zemplén conditions, followed by the removal of the tert-butyldimethylsilyl (TBS) protecting groups using NBu4F furnished the final compound 14.

Figure 4. Synthesis of methoxy derivative of the p-HBAD II trisaccharide reported by Lowary and co-workers92; a) NIS, AgOTf, CH2Cl2, -20 °C, 30 min, 87%; b) NH2NH2·HOAc, CH2Cl2, 4 h, 77%; c) 3, NIS, AgOTf, CH2Cl2, -40 °C, 30 min; d) NaOCH3, CH3OH/CH2Cl2 (1:1), 5 h, 70% over 2 steps; e) CH3I, NaH, DMF, 1 h; f) Pd/C, H2, CH3OH/ CH2Cl2 (1:1), 72 h, 71% over 2 steps. The initial glycosylation of 1 and thioglycoside 2 was achieved using NIS-AgOTf-promoted conditions to furnish the α-linked disaccharide. Typically, in the synthesis of p-HBAD molecules, the methyl group of the 2-OH of 1 is introduced prior to glycosylation with the 2 residue.91, 93 However, in this case, it was possible to introduce the methyl functionality after the formation of the trisaccharide. The use of a benzoyl (Bz) protecting group provided the α-linked disaccharide exclusively, via neighbouring group participation. The levulinoyl (Lev) protecting group was selectively removed using hydrazine acetate to furnish acceptor 5. This was subsequently coupled to thiofucoside 3 by an NIS-AgOTf-promoted glycosylation, using an “inverse glycosylation” procedure, to provide the α-linked trisaccharide exclusively. Schmidt and coworkers introduced the concept of “inverse glycosylation” whereby the acceptor and activator are maintained at a higher concentration than that of the donor, thus providing higher yielding reactions with greater selectivity.94 The benzoyl group was subsequently removed using Zemplén deacetylation conditions and the resulting 2-OH was methylated on treatment with sodium hydride and methyl iodide. The final compound, 7, was obtained following global deprotection of 6 via hydrogenolytic cleavage of the benzyl (Bn) ethers. This compound was subsequently tested in THP-1 (human acute leukemic monocyte/macrophage) cells. They were found to inhibit the release of nitric oxide (NO) and the cytokines; TNF-α, interleukin 1β (IL-1β) and IL-6, in a concentration dependent manner, supporting previous biological studies on these compounds.91 In this study, further testing with other analogues of p-HBAD II showed that the correct methylation pattern on these molecules is essential. Derivatives lacking the

Figure 5. Synthetic route used by Astarie-Dequeker et al. for the synthesis of methyl derivative of the p-HBAD II93; a) TMSOTf, CH2Cl2, -20 °C to 0 °C, 2 h, 89%; b) Pd/C, H2, CH3OH/CH2Cl2 (1:1), rt, 72 h, 95%; c) 10, NIS, TMSOTf, CH2Cl2, -20 °C to 0 °C, 2 h; d) NaOCH3, CH3OH, rt, 5 h; e) NBu4F, THF, rt, 5 h, 69% over 2 steps.

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ACS Chemical Biology tial of blocking Man-LAM by an antagonistic/neutralizing aptamer to increase the protective effect of the BCG vaccine against M. tb.103 Furthermore, both PIMs and LAM have recently been shown to be promising candidates for a potential TB vaccine.104, 105 Recently, Zhu and co-workers have synthesised a PGL-tb epitope conjugated to the protein carrier, CRM197.106 Interestingly, this monovalent glycoconjugate has been found to induce high antigen-specific IgG levels in the serum of mice, rabbits and guinea pigs and has highlighted the utility of the PGL-tb glycan as a valuable tool for the generation of PGL specific monoclonal antibodies. Furthermore, the immunoprotective effect associated with this PGL-tb epitope has reinforced the potential of a synthetic PGL based vaccine for the treatment of TB.106

This synthetic epitope of PGL-tb was subsequently utilized to investigate the molecular mechanisms by which the PGLs act on the host immune system. From this study, it was shown that the trisaccharide moiety of the PGL-tb acts as a pathogenassociated molecular pattern (PAMP) recognized by the host immune system. This enables the mycobacterium to exploit host pattern recognition receptors (PRRs) and consequently modulate the immune response. The trisaccharide epitope is directly responsible for the specificity of binding to TLR2, and the common lipid core of the PGL family may be involved in enhancing the affinity of the receptor for the glycan antigen. Binding of the glycan to TLR2 causes the inhibition of TLR2dependant signaling cascades and the secretion of inflammatory cytokines (TNF-γ), resulting in an overall dampening of the host immune response.93 This study also demonstrated that the expression of PGLs on the surface of the mycobacterium enhances infectivity and furthermore provided important insight into the mechanism of action of PGLs, which may aid the future development of improved therapeutics.

Conclusion PGLs and the related glycans, p-HBADs, play a key role in M. tb pathogenesis. They are located on the outermost layers of the cell wall, where they act as important virulence factors, interacting with the host immune system. A combination of genetic and biochemical studies have enabled the elucidation of the key steps in the biogenesis of these molecules. These complex molecules represent challenging synthetic targets for chemists, with a number of impressive total chemical syntheses of PGL and p-HBAD analogues reported. Chemical synthesis has provided, for the first time, access to pure samples of these important biomolecules and has been crucial in probing their biological and immunomodulatory activities. Recent studies have focused on the application of synthetic cell wall glycolipids as potential vaccine candidates for M. tb and it is anticipated that the development of a novel carbohydrate conjugate vaccine candidate for M. tb will continue to remain an active area of interest into the future. Ultimately, access to pure samples of these glycolipids and their conjugates will be essential for the realization of PGL/p-HBAD based vaccines.

The Development of TB Vaccines Based on Cell Wall Carbohydrates There is significant precedent for the application of carbohydrates in vaccine development, especially those found on the cell surface of pathogens.13, 95, 96 Carbohydrates can play key roles in the initial stages of many pathogenic infections and are therefore often located on the surface of the cell, making them ideal targets for vaccine design. However, saccharides alone are generally T-cell independent antigens, rendering them poorly immunogenic, facilitating bacterial immune evasion by a mechanism known as “glycan shielding”.95 The application of chemical synthesis towards the preparation of PGLs and related compounds has provided significant benefit to the field of mycobacterial glycobiology. The chemical synthesis approach enables the preparation of homogeneous compounds suitable for immunological evaluation, protein conjugation, labelling and further modification. The biological evaluation of these compounds in isolation from the host bacterium has provided fundamental new insights into the roles of these compounds in modulating immune response and offers a template for the development of novel vaccine candidates. One well established technique used to overcome the low immunogenicity of carbohydrate antigens is to conjugate the glycan to a carrier protein; this can elicit a T-cell immune response, aiding B cells, allowing for class switching and conferring long lasting protection against the pathogen. One of the earliest conjugate vaccines developed was for Haemophilus influenza (Hib).97 Prevnar is a carbohydrate-protein conjugate vaccine against Streptococcus pneumoniae.98 Other carbohydrate based vaccines include the meningococcal C conjugate vaccine.99 Another method that can increase the immunogenicity of carbohydrates is the “glycoside cluster effect”; this involves the multivalent presentation of the carbohydrates and has been employed in the virosomal vaccine for Hepatitis A, Epaxal.96, 100 Various methods have been utilized to achieve this multivalent glycan display, such as gold nanoparticles and liposomes, allowing for substantial progress to be made towards the development of a more effective carbohydrate based vaccine for Streptococcus pneumoniae.101, 102

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interest

ACKNOWLEDGMENTS The authors would like to acknowledge Trinity College Dublin for postgraduate studentships (to D. Barnes and M. Lundahl). E. Scanlan is funded by Science Foundation Ireland (SFI) under Grant number 15/CDA/3310. E. Lavelle is funded by Science Foundation Ireland (SFI) under Grant number 12/IA/1421 and the SFI Research Centre, Advanced Materials and BioEngineering Research (AMBER) under Grant number SFI/12/RC/2278.

ABBREVIATIONS Ac, acetyl; AG, arabinogalactan; BCG, Bacille-Calmette-Guérin; Bn, benzyl; Bz, benzoyl; CCR2, CC chemokine receptor 2; DAT, diacyltrehaloses; GPL, glycopeptidolipids; HIV, Human immunodeficiency virus; IL-1β, Interleukin 1β; ; IL-6, Interleukin 6; IL-12, Interleukin 12; IFN-γ, Interferon-γ; LAM, lipoarabinomannan; Lev, levulinoyl; LM, lipomannan; LOS, lipooligosaccharide; Man-LAM, mannose capped lipoarabinomannan; MDR, multi-drug resistant; M. tb, Mycobacterium tuberculosis; NO,

The success of these carbohydrate based vaccines has generated considerable interest in utilizing the immunomodulatory glycolipids at the cell surface of M. tb as potential vaccine candidates.13 Recently, a study by Sun et al. highlighted the poten-

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10. Blondiaux, N., Moune, M., Desroses, M., Frita, R., Flipo, M., Mathys, V., Soetaert, K., Kiass, M., Delorme, V., Djaout, K., Trebosc, V., Kemmer, C., Wintjens, R., Wohlkönig, A., Antoine, R., Huot, L., Hot, D., Coscolla, M., Feldmann, J., Gagneux, S., Locht, C., Brodin, P., Gitzinger, M., Déprez, B., Willand, N., and Baulard, A. R. (2017) Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420, Science 355, 1206. 11. Kaushal, D., Foreman, T. W., Gautam, U. S., Alvarez, X., Adekambi, T., Rangel-Moreno, J., Golden, N. A., Johnson, A. M., Phillips, B. L., Ahsan, M. H., Russell-Lodrigue, K. E., Doyle, L. A., Roy, C. J., Didier, P. J., Blanchard, J. L., Rengarajan, J., Lackner, A. A., Khader, S. A., and Mehra, S. (2015) Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis, Nat. Commun. 6, 8533. 12. Ritz, N., Hanekom, W. A., Robins-Browne, R., Britton, W. J., and Curtis, N. (2008) Influence of BCG vaccine strain on the immune response and protection against tuberculosis, FEMS Microbiol. Rev. 32, 821-841. 13. Moliva, J. I., Turner, J., and Torrelles, J. B. (2015) Prospects in Mycobacterium bovis Bacille Calmette et Guerin (BCG) vaccine diversity and delivery: why does BCG fail to protect against tuberculosis?, Vaccine 33, 5035-5041. 14. Ponnighaus, J. M., Msosa, E., Gruer, P. J. K., Liomba, N. G., Fine, P. E. M., Sterne, J. A. C., Wilson, R. J., Bliss, L., Jenkins, P. A., and Lucas, S. B. (1992) Efficacy of BCG vaccine against leprosy and tuberculosis in northern Malawi, Lancet 339, 636-639. 15. Aronson, N. E., Comstock, G. W., Howard, R. S., Moulton, L. H., Rhoades, E. R., and Harrison, L. H. (2004) Long-term Efficacy of BCG Vaccine in American Indians and Alaska Natives, J. Amer. Med. Assoc. 291, 2086. 16. Parida, S. K., Axelsson-Robertson, R., Rao, M. V., Singh, N., Master, I., Lutckii, A., Keshavjee, S., Andersson, J., Zumla, A., and Maeurer, M. (2015) Totally drug-resistant tuberculosis and adjunct therapies, J. Intern. Med. 277, 388-405. 17. Orme, I. M. (2013) Vaccine development for tuberculosis: current progress, Drugs 73, 1015-1024. 18. Tang, J., Yam, W. C., and Chen, Z. (2016) Mycobacterium tuberculosis infection and vaccine development, Tuberculosis 98, 30-41. 19. Young, D., and Dye, C. (2006) The development and impact of tuberculosis vaccines, Cell 124, 683-687. 20. Richardson, E. T., Shukla, S., Sweet, D. R., Wearsch, P. A., Tsichlis, P. N., Boom, W. H., and Harding, C. V. (2015) Toll-Like Receptor 2-Dependent Extracellular Signal-Regulated Kinase Signaling in Mycobacterium tuberculosis-Infected Macrophages Drives Anti-Inflammatory Responses and Inhibits Th1 Polarization of Responding T Cells, Infect. Immun. 83, 2242-2254. 21. Kaufmann, S. H. E. (2001) How can immunology contribute to the control of tuberculosis?, Nat. Rev. Immunol. 1, 20-30. 22. Cambier, C. J., Takaki, K. K., Larson, R. P., Hernandez, R. E., Tobin, D. M., Urdahl, K. B., Cosma, C. L., and Ramakrishnan, L. (2014) Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids, Nature 505, 218-222. 23. Stadthagen, G., Jackson, M., Charles, P., Boudou, F., Barilone, N., Huerre, M., Constant, P., Liav, A., Bottova, I., Nigou, J., Brando, T., Puzo, G., Daffé, M., Benjamin, P., Coade, S., Buxton, R. S., Tascon, R. E., Rae, A., Robertson, B. D., Lowrie, D. B., Young, D. B., Gicquel, B., and Griffin, R. (2006) Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives, Microbes Infect. 8, 2245-2253. 24. Cooper, A. M., Dalton, D. K., Stewart, T. A., Griffin, J. P., Russell, D. G., and Orme, I. M. (1993) Disseminated tuberculosis in interferon gamma gene-disrupted mice, J. Exp. Med. 178, 22432247.

nitric oxide; PAMP, pathogen-associated molecular pattern; PAT, polyacyltrehaloses; PDIM, phthiocerol dimycoserosate; pHBA, para-hydroxybenzoic acid; p-HBADs, parahydroxybenzoic acid derivatives; p-HBAD I, parahydroxybenzoic acid derivative I; p-HBAD II, parahydroxybenzoic acid derivative II; PGL, phenolic glycolipid; PIMs, phosphatidylinositol mannosides; PRR, host pattern recognition receptor; SL, sulfolipids; SMARt, Small Molecules Aborting Resistance; TB, tuberculosis; TBS, tertbutyldimethylsilyl; TDM, dimycolyl trehalose; TLR, Toll like receptor; TLR2, Toll like receptor 2; TLR4, Toll like receptor 4; TNF-α, tumor necrosis factor α; RND, Resistance-nodulationdivision; XDR, Extensively-drug resistant.

KEY WORDS Tuberculosis: an infectious bacterial disease caused by Mycobacterium tuberculosis (M. tb); Glycolipid: a lipid connected to one or more sugar residues linked by a glycosidic bond; Vaccine: an antigenic substance prepared from the causative agent of a disease or a synthetic substitute, used to provide protective immunity against one or several diseases; Glycosylation: the addition of a glycan onto a protein, lipid or other organic molecule; Hypervirulent: a substance that is capable of causing severe illness; Pathogenesis: the cellular events and reactions and other pathologic mechanisms occurring in the development of disease; Cytokine: a cell signaling molecule that aids cell to cell communication during immune response and stimulates the movement of cells towards sites of inflammation and infection; L-Rhamnose: a naturally occurring 6deoxy sugar

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Mycobacterium tuberculosis - American Chemical Society

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