**1. Introduction**

Pyruvylation is a widespread non-carbohydrate modification of monosaccharides found in various classes of glycoconjugates. In most cases, the modification is present as a pyruvate (Pyr) ketal (cyclic acetal/ketal) bridging two hydroxyl groups of a monosaccharide residue and forming a ring structure [1], where pyruvate is most frequently placed across the 2,3-, 4,6-, or 3,4-positions (Figure 1I–III). The best-known example of pyruvylation, however, occurs as enol pyruvate (Figure 1IV), which is elaborated during the biosynthesis of the bacterial cell wall component peptidoglycan [2]. In both modes of pyruvylation, a dedicated pyruvyltransferase catalyses the transfer of the pyruvate moiety to the monosaccharide target.

**Figure 1.** Overview on the most common modes of monosaccharide pyruvylation. Shown is pyruvate ketal at bridging positions 2,3 (**I**), 4,6 (**II**), and 3,4 (**III**), and enol pyruvate (**IV**). Arabic numbers indicate ring positions.

Pyruvate-ketal-modified (henceforth abbreviated as "pyruvylated") glycoconjugates are found in various phylogenetic orders of life, including bacteria, yeast, and algae, but not in humans. Pyruvylated glycoconjugates are typically present in the cell envelope to which they impart a net negative charge; this is necessary for vital biological functions, such as regulation of the cell influx/efflux processes and cell–cell interactions including cell aggregation and pathogenic adhesion. Notably, besides pyruvylation, nature offers various alternate strategies to create anionic cell surfaces, including a wide range of acidic saccharides (e.g., muramic acid, hexuronic acids, sialic acids) and saccharide modifications (e.g., succinate, lactate, phosphate) [3]. These compounds further lead to an increased capability of cells to electrostatically bind cations at the surface, which, in turn, may foster the packing density of the saccharide portion of glycoconjugates [4].

The repertoire of monosaccharide targets of pyruvylation is quite diverse. The most abundant pyruvylated monosaccharide with 59 hits in the Carbohydrate Structure Database (CSDB, http: //csdb.glycoscience.ru/) [5,6] is galactose (Gal). Examples of pyruvylated galactose include, among others, the capsular polysaccharide of *Bacteroides fragilis* [7] and *Streptococcus pneumoniae* [8], *N*-glycans of the fission yeast *Schizosaccharomyces pombe* [9], as well as carragenans [10], and galactans from algae [11–13]. Recently, pyruvylated *N*-acetylmannosamine (ManNAc) has emerged as an important epitope on bacterial "non-classical" secondary cell wall glycopolymers, serving as a cell wall ligand for cell surface (S-) layer proteins, such as those of the pathogen *Bacillus anthracis* and the honeybee saprophyte *Paenibacillus alvei* [14,15]. There are also examples of pyruvylated monosaccharides on capsular polysaccharides serving as an immunostimulatory effector [7,16], or contributor to virulence as in the case of the secondary cell wall polymer of *Bacillus cereus* [17] or the exopolysaccharide xanthan of *Xanthomonas* spp., where pyruvylation is essential for successful colonization and pathogenesis in planta [18]. Pyruvyl groups on terminal glucose (Glc) and *N*-acetylgalactosamine (GalNAc) residues in the lipooligosaccharide of *Pseudomonas stutzeri* OX1, in contrast, are assumed to have biosynthetic implications [4]. All these examples are, among others, discussed in detail below.

Several studies dealing with pyruvylated glycans and their structural elucidation are available in the literature. However, knowledge of the enzymatic machinery governing pyruvylation is scarce. This is mainly due to missing sequencing data of the organisms, which produce pyruvylated glycoconjugates of known structure. Thus, despite their predictably widespread occurrence, pyruvyltransferases are an under-investigated class of enzymes.

This review summarizes the current state of knowledge about pyruvylated glycoconjugates in nature—focusing on bacterial sources—with an emphasis on the pyruvyltransferases involved in their biosynthesis.

#### **2. Enol-Pyruvylation in Peptidoglycan Biosynthesis**

The UDP-*N-*acetylglucosamine-3-*O*-enol-pyruvyltransferase MurA (EC 2.5.1.7) targets UDP-*N*-acetylglucosamine (UDP-GlcNAc) as an acceptor substrate for enol-pyruvyl transfer from a phosphoenolpyruvate (PEP) substrate, thereby releasing free phosphate and yielding the UDP-activated form of the essential bacterial cell wall compound *N*-acetylmuramic acid (enolpyruvyl-UDP-*N*-acetylglucosamine; MurNAc). This first committed step of peptidoglycan biosynthesis is inhibited by the epoxide antibiotic fosfomycin [19]. As a PEP analogue, fosfomycin binds covalently to the key cysteine residue at position 115/116 (position depending on the source of enzyme) in the active site of MurA, preventing the formation of UDP-MurNAc [19–21]. The co-crystal structure of a Cys (cysteine)-to-Ser (serine) mutant of *Enterobacter cloacae*, MurA, together with its substrates, revealed that the Cys residue is essential for product release and not directly involved in the chemical reaction of enol-pyruvyl transfer. The comparison of the product state with the intermediate state and an unliganded state of MurA indicated that the dissociation of the products is an ordered event, with inorganic phosphate leaving first, followed by conformational changes that lead to the opening of the two-domain structure of MurA and the final release of UDP-MurNAc [20]. A recent study on MurA of the opportunistic pathogen *Acinetobacter baumannii* revealed that the enzyme exists as a monomer in solution and has a pH optimum of 7.5 at 37 ◦C. The Km for UDP-GlcNAc is 1.062 ± 0.09 mM and 1.806 ± 0.23 mM for PEP [21]. The relative enzymatic activity is inhibited approximately threefold in the presence of 50 mM fosfomycin. Superimposition of a model for the *A. baumannii* enzyme with MurA of *Escherichia coli (E. coli)* confirmed the structural similarity in the fosfomycin binding site. Because of the worldwide spread of antimicrobial resistance and the paucity of novel drugs in the development pipeline, there has been a renewed interest in fosfomycin as an alternative option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria [22]. However, it has to be considered that natural MurA mutants exist that render the respective organisms fosfomycin resistant. This includes *Mycobacteria* and *Chlamydia* species, where Cys-to-Asp mutants occur [20].

Interestingly, NikO, another enol-pyruvyltransferase that is structurally closely related to the common MurA enzymes and, consequently, inhibited by fosfomycin, plays an essential role in the biosynthesis of nikkomycins. Nikkomycins are peptide-nucleoside antibiotics, which strongly inhibit chitin synthesis and, therefore, are effective against fungi and insects. NikO was shown to transfer the enol-pyruvyl moiety from PEP to the 3 -hydroxyl group of UMP and to be inactivated by fosfomycin because of alkylation of Cys130. However, the degree of inactivation is not as pronounced as in the case of common MurA enzymes [23].

#### **3. Ketal-Pyruvylated Glycoconjugates**

In this section, an overview of the different classes of pyruvylated glycoconjugates is given, including glycan composition and structure, as well as functional and biosynthetic aspects, when known.

There is a recent interest in understanding the biosynthetic pathways of pyruvylated glycoconjugates from bacterial sources, as these pathways might unravel novel targets for therapeutic intervention. However, the current biosynthesis models for the different classes of glycoconjugates are in most cases only fragmentarily available and, frequently, they consider in silico predictions of involved components without experimental evidence.

#### *3.1. Exopolysaccharides*

Many organisms produce extracellular polysaccharides (exopolysaccharide, EPS) that are actively secreted during growth, including bacteria, yeasts, and microalgae [24]. EPSs are a diverse class of carbohydrate polymers that are composed of either linear or branched repeating units that are connected with varying stereochemistry. Monosaccharide constituents include pentoses (ribose and arabinose—especially in *Mycobacterium* spp.), hexoses (mannose (Man), glucose, fructose, galactose), deoxysugars (rhamnose (Rha), fucose (Fuc), uronic acids (glucuronic and galacturonic acids), and amino

sugars (glucosamine, galactosamine, in several cases modified by *N*-acetylation) [24]. Depending on the monosaccharide composition, homo- or hetero-polymers are differentiated.

EPSs have a "jelly-like" appearance and are part of the glycocalyx—with which the "cellular sugar coat" is referred to [24]; as a common feature, they create a protective matrix around cells. The shielding effect against macromolecules that is conferred by EPS makes some bacteria 1000 times more resistant to antibiotics than their EPS-free counterparts [25].

Given the high application potential of microbial EPSs in medical fields, biomaterials, food applications, and in the replacement of petro-based chemicals [26], these glycoconjugates are currently of high interest.

#### 3.1.1. Xanthan

Xanthan is the main EPS produced by *Xanthomonas campestris* and other phytopathogenic *Xanthomonas* spp. that cause various economically important diseases in mono- and di-cotyledonous crops. Xanthan enhances the attachment to plant surfaces through its effect on biofilm formation, promotes pathogenesis by Ca2<sup>+</sup> chelation and, thereby, suppression of the plant defence responses in which Ca2<sup>+</sup> acts as a signal [27]. In practical applications, xanthan is frequently used as a viscosifying agent [28,29].

The pentasaccharide-repeating unit of xanthan consists of two <sup>β</sup>-(1→4) linked <sup>d</sup>-Glc residues as backbone and a trisaccharide side chain, α-(1→3)-linked to every other glucose. The side chain is composed of <sup>α</sup>-d-Man, <sup>β</sup>-d-glucuronic acid (GlcA), and <sup>β</sup>-d-Man, which are <sup>β</sup>-(1→2)- and <sup>β</sup>-(1→4)-linked to another, respectively [30]. In its natural state, the <sup>α</sup>-d-Man residue is acetylated and the β-d-Man is either acetylated or pyruvylated. It was found that the 4,6-ketal-pyruvate (4,6Pyr) specifically and, to a lesser extent, the acetyl groups that decorate the mannose residues are involved in Ca2<sup>+</sup> chelation [27] and affect bacterial adhesion and biofilm architecture and, hence, contribute to the bacterium's virulence [18]. Furthermore, the rheological properties of xanthan are influenced by its pyruvylation and acetylation pattern [28,31].

Xanthan biosynthesis is encoded in a so-called *gum*-cluster. Of the 13 encoded genes, *gumDMHK* are involved in the synthesis of the pentasaccharide repeat, and *gumBCEJ* in polymerization and xanthan export across the outer membrane in a flippase/polymerase (Wzx/Wzy)-dependent pathway. Regarding the modifications of xanthan, the predicted pyruvyltransferase GumL is hypothesised to catalyse pyruvylation of β-d-Man residues, while GumF and GumG are involved in β-d-Man acetylation [28,32]. It remains to be determined at which stage of xanthan biosynthesis the modifications are elaborated; it might be either at the cytoplasmic membrane or in the periplasmic space [32]. To this end, it was shown that GumK, a glucuronic acid transferase, is active on the lipid-linked trisaccharide precursor α-Man-(1→3)-β-Glc-(1→4)-β-Glc-P-P-polyisoprenyl, and shows reduced activity on the acetylated precursor substrate 6-*O*-acetyl-α-Man-(1→3)-β-Glc-(1→4)-β-Glc-PP-polyisoprenyl [33]. This suggests that mannose acetylation occurs after the completion of the trisaccharide side chain [32,33]; this might also hold true for pyruvylation. The xanthan biosynthetic enzymes seem to be highly conserved among different organisms, except for the mannose-transferase GumI and the pyruvyltransferase GumL, for which no homologues are found in other organisms [34].

Interestingly, *Bacillus* sp. strain GL1, which utilizes xanthan for its growth, produces an extracellular xanthan lyase, which catalyses the cleavage of the glycosidic bond between 4,6Pyr-β-d-Man and β-d-GlcA residues in xanthan side chains and, thus, contributes to depolymerisation of xanthan [35,36].

#### 3.1.2. Succinoglycan

Succinoglycan is a pyruvylated EPS that is produced by *Agrobacterium* [37,38], *Alcaligenes*, *Pseudomonas* [39], and *Rhizobium* strains [40], and is of great importance in plant symbiosis.

It is a heteropolymer that is multiply decorated with pyruvate, succinate, and acetate substituents. While the extent of acetylation and succinylation depends on the strain and the cultivation conditions, pyruvate is always found in a stoichiometric manner at the terminal β-Glc residue [32]. The repeat

unit structure of succinoglycan is composed of β-Glc and β-Gal in a molar ratio of 7:1. Nineteen genes are involved in the polymer's biosynthesis, which are referred to as *exo* genes and encoded in a 16 kb gene cluster. The biosynthesis starts with the production of the nucleotide-activated sugars UDP-Glc and UDP-Gal, where ExoC (phosphoglucomutase), ExoB (UDP-glucose-4-epimerase), and ExoN (UDP-pyrophosphorylase) are involved [32,41,42] (Figure 2). The initial step in the biosynthesis is executed by ExoY, a priming galactosyltransferase transferring a single, reducing-end Gal residue onto an undecaprenylphosphate (undp-P) carrier. *ExoA*,*exoL*,*exoM*,*exoO*,*exoU*, and *exoW* encode subsequent glycosyltransferases, which complete the octa-saccharide repeat in a step-wise manner, with each enzyme transferring a single monosaccharide, each, except for ExoW which transfers the subterminal and terminal glucoses. Prior to the export of the octasaccharide via a Wzx-dependent pathway, pyruvylation (at the terminal, non- reducing-end glucose), acetylation, and succinylation reactions catalysed by ExoV, ExoZ, and ExoH, handed over to ExoQ, which is responsible for polymerization of the fully modified repeats [43]. Studies on ExoV, the pyruvyltransferase of *Shinorhizobium* (previously *Rhizobium*) *meliloti*, suggest that pyruvylation is important for polymerization of repeating units and efficient succinoglycan export [44].

**Figure 2.** Scheme of succinoglycan biosynthesis in *Shinorhizobium meliloti* [43]. The pyruvylation step occurs in the cytoplasm at the stage of the undp-PP-linked RU prior to export and polymerization in the periplasmic space. Pyruvylation (ExoV) is indicated by a star. The order of pyruvylation, acetylation (ExoZ), and succinylation (Exo) is unknown. RU: repeating unit. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) [45].

For *Rhizobium leguminosarum*, it was shown that missing pyruvylation on the terminal glucose residue of the succinoglycan impairs the formation of the nitrogen-fixing symbiosis with *Pisum sativum*, supportive of a signalling role of pyruvylation in this process [46]. PssK was identified as the pyruvyltransferase involved in succinoglycan modification of *R. leguminosarum* [46].

#### 3.1.3. Salecan

The salt-tolerant soil bacterium *Agrobacterium* sp. ZX09 is the producer of salecan, a soluble, succinylated, and pyruvylated EPS with a β-(1→3) glucan structure that is of interest because of its multiple bioactivities and unusual rheological properties. Its basic repeating unit structure was initially elucidated as <sup>→</sup>3)-β-d-Glc*p*-(1→3)-[β-d-Glc*p*-(1→3)-β-d-Glc*p*-(1→3)]-α-d-Glc*p*- (1→3)-α-d-Glc*p*-(1<sup>→</sup> [47]. On the basis of amino acid homology with the respective *exo* genes, it can

be concluded that succinyl- and pyruvyl-groups are conferred to salecan upon catalysis of SleA (succinyl-transferase) and SleV (pyruvyltransferase), respectively, both of which are located in a 19.6-kb gene cluster [48]. The exact positions of the salecan modifications remain to be determined.

#### 3.1.4. Colonic Acid

Colonic acid (CA) or M-antigen is another class of pyruvylated EPS mostly found in *Enterobacteriaceae*, including the majority of *Escherichia coli* strains. CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. CA is composed of hexasaccharide repeat units consisting of glucose, two fucoses, two galactoses, and glucuronic acid [49]. Additionally, acetylation is found on fucose or/and galactose, while pyruvylation is found on the terminal galactose only, with both modifications occurring non-stoichiometrically [32,50]. The overall structure of CA is 4,6Pyr-α-Gal*p*-(1→4)-β-Glc*p*A-(1→3)-*O*Ac-α-Gal*p*-(1→3)-α-Fuc*p*-(1→4)-*O*Ac-*O*Ac-α-Fuc*p*-(1→3)-β-Glc-(1→7)-α-Hep*p*-(1→6)-α-Glc*p*-(1→2)-α-Glc*p*-(1→3)-[α-Gal*p*-(1→6]-α-Glc*p*-(1→3)-[α-Hep*pP*-(1→7)] -α-Hep*pP*-(1→3)-[PEtN]α-Hep*p*-(1→5)-αKdo*p*-(1→, where Kdo is 3-deoxy-d-*manno*-oct-2-ulosonic acid.

The genetic determinants for CA biosynthesis reside in a 19-gene *wca* (*cps*) cluster and are tightly regulated by a complex signal transduction cascade [51]. The gene cluster encodes six glycosyl-transferases, named WcaJ, WcaI, WcaE, WcaC, WcaL, and WcaA. Furthermore, a putative pyruvyl-transferase (WcaK) is encoded next to two predicted acetyltransferases (WcaF and WcaB), although there are up to three acetylation positions described in CA [50]. Interestingly, WcaF seems to contribute to biofilm formation of the bacterium, since knocking out of this enzyme led to biofilm disruption under *in vitro* conditions [52].

#### 3.1.5. Unclassified Pyruvylated EPS

Up to now, there are several unclassified types of pyruvylated EPSs. The repeating unit structure of the acidic EPS produced by a mucoid strain of *Burkholderia cepacia* isolated from a cystic fibrosis patient was established as <sup>→</sup>3)-β-d-Gal*p*-(1→3)-4,6Pyr-α-Gal*p*-(1<sup>→</sup> [53].

The freshwater biofilm isolate *Pseudomonas* strain 1.15 produces considerable amounts of an acidic EPS that is composed of repeating units with the structure <sup>→</sup>4)-[4,6Pyr-α-d-Gal*p*-(1→4)-β-d-GlcA*p*- (1→3)-α-d-Gal*p*-(*O*→3]-α-l-Fuc*p*-(1→4)-α-l-Fuc*p*-(1→3)-β-d-Glc*p*-(1→. Furthermore, of the four different *O*-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked galactose and on O-2 of the 4-linked fucose [54].

*Enterobacter amnigenus*, a bacterium isolated from sugar beets, produces an EPS that is rich in l-Fuc and has a terminal, pyruvylated α-d-Man residue [55].

The cystic fibrosis lung pathogen *Inquilinus limosus* produces two EPSs with unique structures—an α-(1→2)-linked mannan and a β-(1→3)-linked glucan—both fully substituted with 4,6-linked pyruvate ketals [56,57]. Cystic fibrosis is an autosomal recessive disorder and its mortality is due to chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection [58]. While *Pseudomonas aeruginosa* is one of the most threatening microbes colonizing cystic fibrosis lungs, the EPS of *I*. *limosus* is suspected to play a role in the pathogenesis of the disease.

The bacterium *Azorhizobium caulinodans* produces a linear homopolysaccharide-type EPS composed of <sup>α</sup>-(1→3)-linked 4,6Pyr-d-Gal residues. The bacterium undergoes a symbiotic interaction with *Sesbania rostrata* as a legume host plant, which results in the development of root nodules, accompanied by a massive production of H2O2. In situ H2O2 localization demonstrated that increased EPS production during early stages of invasion prevents the incorporation of H2O2 inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H2O2 [59].

A special K-antigen-like EPS is found in the marine bacterium *Cobetia marina* DSMZ 4741, with its repeating unit composed of ribose and pyruvylated Kdo [60].

Within the EPS structure of the lactic acid bacterium *Pediococcus pentosaceus* LP28, a pyruvate modification was described to occur on one of the four constituting monosaccharides (Glc, Gal, Man, and GlcNAc) [61]. The EPS biosynthetic gene cluster consists of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide: pyruvyltransferase [61].

EPSs produced by an *Erwinia* spp. in association with the bacterium *Coniothyrium zuluense* are linked to a fungal canker disease of *Eucalyptus* [62]. One of these EPSs is that of *Erwinia stewartii*; another is that of *Erwinia futululu*, whose structures are identical except for the replacement of one terminal Glc residue by 4,6Pyr-Gal*<sup>p</sup>* in the latter, yielding <sup>→</sup>3)-β-d-Gal*p*-(1→3)[4,6Pyr-α-d-Gal*p*-(1→4) <sup>β</sup>-d-Glc*p*A-(1→4)][β-d-Glc*p*-(1→6)]-α-d-Gal*p*-(1→6)-β-d-Glcp-(1<sup>→</sup> [63].

*Agrobacterium radiobacter* (ATCC 53271) produces an anionic EPS that gives aqueous dispersions, exhibiting high viscosity at low concentrations. The *A. radiobacter* EPS is composed of a complex heptadekasaccharide repeating unit, which exposes a subterminal 4,6Pyr-α-d-Glc residue on each of the two identical tetraglycosyl branches [64].

*Methylobacterium* sp. is a slime-forming bacterium isolated from a Finnish paper machine, which is a high EPS producer. Its EPS repeating unit has the structure <sup>→</sup>3)-4,6Pyr-α-d-Gal*p*-(1→3)-4,6Pyr-α-d-Gal*p*-(1→3)-α-d-Gal*p*-(1<sup>→</sup> [65].

The marine bacterium *Alteromonas macleodii* subsp. *fijiensis* isolated from deep-sea hydrothermal vents displays a pyruvylated mannose in its EPS hexasaccharide repeating unit structure <sup>→</sup>4)-β-d-Glc*p*-(1→4)[4,6Pyr-β-d-Manl*p*-(1→4)-β-d-Glc*p*A-(1→3)-α-d-Glc*p*A-(1→3)]α-d-Gal*p*A-(1→4) <sup>α</sup>-d-Gal*p*-(1<sup>→</sup> [66]. Aside from its use in the food industry, this marine polymer has been suggested to be used for the treatment of cardiovascular diseases and bone healing [67].

#### *3.2. Capsular Polysaccharides*

Capsular polysaccharides (CPSs) are also part of the glycocalyx but, in contrast to EPSs, are covalently connected to the bacterial cell surface via membrane phospholipids [24]. Because of their prominent cellular localization, CPSs are the first interaction zone of bacteria with the host immune system, and thus are important virulence factors of many bacteria. Very often, encapsulated bacteria are pathogenic, whereas capsule-deficient isolates are not [68]. Hence, CPSs are frequently used for the production of polysaccharide conjugate vaccines [69].

Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of CPSs with different structures, and these aid in distinguishing isolates by serotyping [68]. The widespread occurrence and the high structural differences of CPSs are reflected by 84 capsular serotypes (K-antigens) found alone in *E. coli* strains. Essentially, there are four groups of capsules [70]. Group I- and IV-CPS, which are often found in organisms leading to gastrointestinal diseases, use the Wzx/Wzy-dependent export pathway and their biosynthesis proceeds on a polyprenol linker. Capsules from groups II and III use the ABC-transporter export pathway and are frequently present in mucosal pathogens such as *Neisseria meningitidis*. Interestingly, a CPS attached via a novel β-linked poly-3-deoxy-d-*manno*-oct-2-ulosonic acid linker to the phospholipid *lyso*-phosphatidylglycerol is present, which in earlier studies was described as a diacylglycerol because of hydrolysis experiments [68].

#### 3.2.1. *Streptococcus pneumoniae* CPS

*Streptococcus pneumoniae* (pneumococcus) is a leading cause of bacterial-induced pneumonia, meningitis, and bacteraemia globally [71]. Prevnar 13, the most broadly protective pneumococcal conjugate vaccine, is composed of 13 protein-polysaccharide conjugates consisting of pneumococcal CPS serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F—each individually linked to the genetically inactivated diphtheria toxoid CRM197. Currently, approaches on the basis of biocon-jugation and glycosylation engineering are being pursued as manufacturing alternatives to enable the production of vaccines with higher protection rates, especially in children [69,72,73].

*S. pneumonia* CPS serotype 4 (ST4) is a prevalent serotype in vaccine formulations, containing 2,3-pyruvate ketal on the Gal residue of its repeating units—with the structure

<sup>→</sup>3)-β-d-Man*p*NAc-β-(1→3)-α-l-Fuc*p*NAc-(1→3)-Gal*p*NAc-α-(1→4)-2,3Pyr-α-d-Gal-(1→—which has been shown to be the key component of its specific immunogenic motif [74].

Thus, the pyruvate modification is essential for designing minimal synthetic carbohydrate vaccines for ST4, as vaccine formulations without pyruvylation would not recognize the natural CPS [8]. It is, therefore, highly recommended to include the pyruvate ketal epitope in glycoconjugate vaccines [16].

#### 3.2.2. *Acinetobacter baumannii* CPS

A clinically relevant producer of CPS is the opportunistic pathogen *Acinetobacter baumannii*, which triggers infections in immunocompromised patients causing severe nosocomial, bloodstream, pneumonia, urinary tract infections, and septicaemia [75]. Its clinical importance is related to its low susceptibility towards most of the antibiotics commonly used [76].

There are seven different capsule loci—KL1, KL2, KL4 KL6, KL7, KL8, KL9—in *A. baumannii* genomes. Five of these were found in clonal group 2, whereas two were found in clonal group 1, indicating that isolates with developing antibiotic resistance have a lot of variations of these loci [77]. The K4 CPS of isolate D78, which is a multiple antibiotic resistant strain, contains the KL4 cluster. The KL4 CPS backbone repeating structure is composed of a trisaccharide of α-*N*-acetyl-d-quinovosamine (d-Qui*p*NAc), α-*N*-acetyl-d-galactosamine uronic acid (α-d-Gal*p*NAcA), and α-d-Gal*p*NAc, which contains a branching 4,6-pyruvylated GalNAc residue. The trisaccharide structure was elucidated as <sup>→</sup>4)[4,6Pyr-α-d-Gal*p*NAc-(1→6)]-α-d-Gal*p*NAc-(1→4)-α-d-Gal*p*NAcA-(1→3)-α-d-Qui*p*NAc-(1→. The pyruvate ketal is predicted to be transferred by the putative pyruvyltransferase PtrA, however, without biochemical evidence [78].

#### 3.2.3. *Klebsiella* CPSs

Pyrogenic liver abscess-causing *Klebsiella pneumoniae* produces a CPS, which is composed of trisaccharide repeating units with the structure <sup>→</sup>4)-β-d-Glc-(1→4)-2,3(*S*)Pyr-β-d-GlcA-(1→4)-β-l-Fuc-(1→, in which each glucuronic acid residue is pyruvylated and additional acetylation of the fucose residue occurs at the C2-OH or C3-OH [79]. The CPS induces secretion of tumour necrosis factor and interleukin-6 by macrophages through the Toll-like receptor 4 dependent pathway, which is abandoned when pyruvylation is missing in the trisaccharide. This finding indicates that pyruvylation on glycoconjugates may be relevant for immune system stimulation [80]. Previously, the recognition of pyruvylated CPS from *K. pneumoniae* by IgM antibodies has been described [81].

The structures of several other pyruvylated *Klebsiella* CPS structures have been elucidated, however, without any functional information.

The structure of the CPS from *Klebsiella* serotype K14 was the first report on the rare case of a *Klebsiella* polysaccharide to contain a Gal*f* residue. The repeating hexasaccharide structure was shown to terminate with a glucose residue carrying a 4,6Pyr modification—→4)-β-d-Glc*p*A-(1→3) <sup>β</sup>-d-Gal*f*-(1→3)-β-d-Glc*p*-(1→4)[4,6Pyr-β-d-Glc*p*-(1→2)][α-l-Rha(1→3)]β-d-Man*p*-(1<sup>→</sup> [82].

Also in the doubly pyruvylated CPS of *Klebsiella* K12, Gal*f* residues are found; its repeating unit has the structure 5,6Pyr-β-d-Gal*f*-(1→4)-β-d-Glc*p*A-(1→3)-β-d-Gal*f*-(1→6)-β-d-Glc*p*-(1→3)-α-l-Rha- (1→3)-α-d-Gal*p*-(1→2)[5,6Pyr-β-d-Gal*f*-(1→4)-β-d-Glc*p*A-(1→3)]-β-d-Gal*f*-(1→6)-β-d-Glc*p*-(1→3)-α-<sup>l</sup> -Rha-(1→3)-α-d-Gal*<sup>p</sup>* [83].

The structure of the CPS from *Klebsiella* serotype K70 is composed of linear hexasaccharide repeating units that contain a Pyr group attached to a (1→2)-linked <sup>α</sup>-l-Rha residue in every second repeating unit. The full structure of the*Klebsiella* K70 CPS is→4)-β-d-Glc*p*A-(1→4)-α-l-Rha*p*-(1→2)-α-l-Rha*p*-(1→2)-α-d-Glc*p*-(1→3)-β-d-Gal*p*-(1→2)-3,4Pyr-α-l-Rhap(1<sup>→</sup> [84].

The *Klebsiella* serotype K64 CPS consists of hexasaccharide repeating units, composed of a <sup>→</sup>4)-α-d-Glc*p*A-(1→3)-α-d-Man*p*-(1→3)-β-d-Gal*p*-(1→4)-α-d-Man*p*-(1<sup>→</sup> backbone with a 4,6Pyr-β-d-Gal*<sup>p</sup>* and a <sup>l</sup>-Rha residue attached to the (1→4)-linked <sup>α</sup>-d-Man*<sup>p</sup>* residue at O-2 and O-3, respectively; the repeating unit further contains one *O*-acetyl substituent [85].

The structure of the CPS from *Klebsiella* type K46 consists of a hexasaccharide repeating unit, which is unique in having a 4,6Pyr residue on a lateral, but non-terminal sugar residue—[β-d-Glc*p*- (1→3)-4,6Pyr-β-d-Man*p*-(1→4)]→3)-α-d-Glc*p*A-(1→3)-α-d-Man*p*-(1→3)-α-d-Gal*p*-(1<sup>→</sup> [86].

The *Klebsiella* K33 CPS revealed to be a tetrasaccharide alditol with the structure <sup>β</sup>-d-Glc*p*-(1→4)[3,4Pyr-β-d-Gal*p*-(1→6)]-β-d-Man*p*-(1→2)-Ery-ol, where Ery-ol is erythritol [87].

Interestingly, there are two human monoclonal macroglobulins, IgMWEA and IgMMAY [88], which show specificity for *Klebsiella* polysaccharides containing 3,4-(K30, K33) and 4,6-(K21, K11) pyruvylated d-Gal in a pH-dependent manner and with differences in co-precipitation in dependence of the number of the CPS repeating units (i.e., CPS length) [81]. Of note, agar, which has an internal 4,6Pyr-Gal residue in its repeating unit, cross-reacts with IgMWEA [81].

*Klebsiella rhinoscleromatis* is a heavily capsulated bacterium that possesses a K3-type capsule. The repeating unit of K3 is a pentasaccharide with the structure <sup>→</sup>2)-4,6-*S-*Pyr-α-d-Man-(1→4)-α-d-GalA-(1→3)-α-d-Man-(1→2)-α-d-Man-(1→3)-β-d-Gal-(1<sup>→</sup> [89]. The *Klebsiella* K3 capsule has been shown to be one of the few *Klebsiella* K types that are able to bind to the eukaryotic mannose receptor [90].

### 3.2.4. *Bacteroides fragilis* CPS A

*Bacteroides fragilis* is an opportunistic anaerobe, most frequently isolated from intra-abdominal abscesses [7,91–93]. Its most prominent CPS—CPS A—is composed of tetrasaccharide repeating units with the structure <sup>→</sup>4)-α-d-2-*N*-acetylamido-4-amino-galactopyranose (AAdGal*p*)-(1→3)-4,6PyrGal*p*-(1→3)-[β-d-Gal*f*-(1→3)]α-d-Gal*p*NAc-(1→) [94]. CPS A has been shown to have a tremendous effect on the immune system of a mammalian host and to be internalized by antigen-presenting cells [7]. Upon genetic deletion of CPS A, the abscess-inducing capability of the bacterium was drastically reduced [93]. CPS A from *B. fragilis* caught recent interest as a carbohydrate antigen to be used in vaccine formulations instead of conventional cationic proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KHL) [92].

CPS A biosynthesis is encoded by a single ~10.7-kb gene locus on the *B. fragilis* genome [93], and predictably employs a Wzx/Wzy-dependent pathway, on the basis of genomic evidence (Figure 3).

The gene locus encodes four transferases (WcfN, WcfP, WcfQ, and WcfS), where WcfS is responsible for the transfer of the AAdGal*p* residue from its nucleotide activator to an undp-P-lipid carrier as the first step in the synthesis of the CPS A repeating unit, and WcfR is responsible for the prior transfer of the amino group on the AdGal*p* residue to yield AAdGal*p*, which is crucial for virulence. A recent *in vitro* study of the individual enzymatic steps involved in the repeating unit biosynthesis of CPS A yielded first insight into the sugar pyruvylation reaction, with phosphoenolpyruvate (PEP) serving as a donor substrate. There is evidence that pyruvylation occurs on the undp-PP-linked disaccharide repeat unit precursor prior to tetrasaccharide repeat completion, export, and polymerization by a Wzx/Wzy-dependent system [7]. The pyruvyltransferase WcfO from the CPS A biosynthesis of *B. fragilis* [7] is one of the few biochemically characterized enzyme ketal-pyruvyltransferases (for details, see Section 5.1.2).

**Figure 3.** Scheme of capsular polysaccharide (CPS) A biosynthesis in *Bacteroides fragilis*. The pyruvylation step occurs in the cytoplasm at the stage of the lipid-PP-linked RU precursor. Pyruvylation (WcfO) is indicated by a star. Notably, in contrast to succinoglycan biosynthesis (Figure 2), pyruvylation of the internal Gal*p* of the RU needs to proceed prior to completion of the lipid-PP-linked tetrasaccharide repeat. RU: repeating unit. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) [45].

#### 3.2.5. *Rhodococcus equi* CPS

The bacterial horse pathogen *Rhodococcus equi* elaborates a serotype-specific CPS that functions as a potential virulence factor [94]. This CPS is a high-molecular-weight acidic polymer composed of d-Glc, d-Man, pyruvate, and 5-amino-3,5-dideoxynonulosonic acid (rhodaminic acid, Rho) in the molar ratio of 2:1:1:1. Structural analysis revealed that the CPS consists of linear pyruvylated tetra-saccharide repeats with the structure <sup>→</sup>3)-β-d-Man*p*-(1→4)-β-d-Glc*p*-(1→4)-α-d-Glc*p*-(1→4)-7,9Pyr-α-RhoAmAc-(2<sup>→</sup> [95].
