*3.3. "Non-Classical" Secondary Cell Wall Glycopolymers with Pyruvylated* β*-*d*-ManNAc*

Anionic secondary cell wall glycopolymers (SCWPs)—among which the wall teichoic acids (WTA) and the lipoteichoic acids (LTA) are best known—serve as a rich source for both validated and unexploited pathways that are essential for bacterial virulence and survival [96–98]. Pyruvylated SCPWs are a less-investigated class of peptidoglycan-attached SCWPs that arouse interest because they can be hijacked for a predictably widespread mechanism of protein cell surface display in Gram-positive bacteria [99]. These SCWPs are 5–20 kDa in size, composed of species–specific repeats, but lack repetitive alditol phosphates and phosphodiester bonds typical of WTAs and LTAs [100–102]—hence the terminology "non-classical" SCWPs. Importantly, they contain a 4,6-ketal pyruvylated β-d-ManNAc residue (4,6Pyr-β-d-ManNAc), imparting a negative charge and serving as a specific cell wall ligand for S-layer homology (SLH) domains usually present in triplicate at the termini of cell surface proteins [99,103,104]. Among such proteins are S-layer proteins, which self-assemble into 2D crystalline arrays on the bacterial cell surface [105,106]; they are important for many biological functions such as maintenance of cell integrity, enzyme display, protection to phagocytosis, and interactions with the host and its immune system [107]. Because of their unique 2D crystallization ability, S-layer proteins are of great interest for drug delivery, biomaterial engineering, and vaccine development [108].

Fifty-four thousand specific hits within the conserved protein domain family SLH (pfam00395), showing up most prevalent in the *Firmicutes*, *Cyanobacteria*, and *Actinobacteria* phyla of bacteria, emphasize the prevalence of this protein domain. Several bacteria synthesizing a suite of SLH proteins contain pyruvate in their cell wall and have a pyruvyltransferase CsaB ortholog [99,109], which indicates a functional coupling of SLH domains and SCWP pyruvylation.

The best-known pyruvylated SCWPs are those from *B. anthracis* and *B. cereus* strains [14,110, 111], and from *P. alvei* [15]. The most evident difference between these SCWPs is the presence of 4,6Pyr-β-d-ManNAc exclusively at the terminal repeat in the former SCWPs, while in that of *P. alvei*, the β-d-ManNAc of each repeat is pyruvylated. This might explain the essentiality of the pyruvyltransferase CsaB in the latter organism [109]. It is conceivable to assume that mono- versus poly-pyruvylation of β-d-ManNAc has implications with regard to the biosynthetic pathway of the respective SCWP, especially the mode of activity of the cognate pyruvyltransferase (compare with Section 5.1.1.).

The genetic determinants and assembly reactions of pyruvylated SCWPs are only beginning to be discovered. Pyruvylated SCWPs are peptidoglycan-linked polymers, as are WTAs [97,98], however, they lack experimental evidence of a comparable biosynthetic route.

#### 3.3.1. *Bacillus anthracis* SCWP

The *B. anthracis* SCWP is composed of →4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→ trisaccharide repeats [102] with strain-dependent galactosylation occurring at the GlcNAc residues, and are bound to peptidoglycan via a murein linkage unit [14,112]. Notably this SCWP contains a modified terminal repeat with the structure 4,6-Pyr-β-ManNAc-(1→4)-[3*O*Ac-β-GlcNAc-(1→6)]β-GalN-(1→4) that is pyruvylated and *O*-acetylated [14].

A scenario for the biosynthesis of the *B. anthracis* SCWP has been proposed [113] on the basis of the bioinformatic prediction of four contributing genomic gene clusters and their genetic manipulation, accompanied with analyses of mutant cells. The S-layer gene cluster [99,112–114] encodes, among others, components for pyruvylation (*B. anthracis* CsaB) and *O*-acetylation of the terminal SCWP trisaccharide, and a Wzy-like protein. The other gene clusters play predicted roles in the formation of lipid-linked precursors of the murein linkage unit and the trisaccharide repeat [112,114–116]. The SCWP biosynthesis model proposes the separate assembly of different undp-PP-linked building blocks in the cytoplasm—the murein linkage unit, trisaccharide repeat, and terminal modified trisaccharide [113]—followed by the individual translocation across the cytoplasmic membrane via Wzx, followed by SCWP polymerization at the outer face of the membrane involving Wzy [113]. This model does not explain how the different building block precursors converge and how pyruvylation of β-d-ManNAc is elaborated. A recent study identified PatB1 as *O*-acetyl-transferase in the terminal repeat biosynthesis of *B. cereus* SCWP, proposing an extracellular *O*-acetylation mechanism [117]. Previously, it was surmised that the modifications at the terminal trisaccharide emerge post-polymerization and ligation to peptidoglycan [14].

*B. anthracis* CsaB is not essential for survival, but it is important for the pathogenesis of infection; Δ*csaB* mutants lacking SCWP pyruvylation fail to retain SLH-domain containing proteins in the cell wall, leading to an atypical cell morphology [99,118]. Of note, in addition to SCWP [14], the *B. anthracis* cell wall contains a polyglycerol phosphate LTA [119] and a poly-γ-d-glutamic acid capsule [101], which could support the cell wall integrity in a strain devoid of pyruvylation providing the anionic character.

#### 3.3.2. *Paenibacillus alvei* SCWP

The SCWP of *P. alvei* consists of <sup>→</sup>3)-β-d-ManNAc-(1→4)-β-d-GlcNAc-(1<sup>→</sup> repeats, where β-d-Man*p*NAc of each disaccharide is modified with 4,6-linked pyruvate ketal [15,120]. Notably, an identical SCWP composition was found in the S-layer carrying *Lysinibacillus sphaericus* CCM 2177, where on average, every second β-d-ManNAc residue contains a 4,6Pyr-modification [121].

*P. alvei* possesses a polycistronic SCWP biosynthesis gene cluster comprised of *csaB*, *tagA, tagO*, two SLH-protein encoding genes—*slhA* and *spaA*—and two ORFs of unknown function [109,122]. For SCWP assembly in *P. alvei*, distinct enzymatic steps have been investigated [109]. The bacterium utilizes the subsequent TagO [123] and TagA [124,125] catalysed reactions—typically involved in WTA biosynthesis to produce the undp-PP-bound murein linkage unit [97,126,127] for the biosynthesis of the lipid-linked disaccharide substrate needed to generate the repeat unit backbone of its SCWP [15]. Pyruvylation with PEP as donor substrate was experimentally determined *in vitro* at the stage of the lipid-linked disaccharide repeat precursor [109] (see Section 5.1.1.).

In *P. alvei*, no viable deletion mutant could be obtained of either *tagO*, *tagA*, or *csaB* [109]—each of which is located on the *P. alvei* genome as a single copy—indicating essentiality of the pyruvylated SCWP for the bacterium. This might be explained by the presence of pyruvylated SCWP as exclusive anionic SCWP in *P. alvei* and may be supportive of the necessity of at least one anionic polymer in the Gram-positive cell wall [128,129].

#### 3.3.3. Cell Wall Polysaccharide of *Paenibacillus polymyxa*

*Paenibacillus* (previously *Bacillus*) *polymyxa* AHU 1385 was among the first bacteria for which a pyruvylated ManNAc residue was described [130]. The pyruvylated epitope is contained in a →3)-4,6Pyr-ManNAc-(1→4)-GlcNAc-(1→-repeating unit of an SCWP that is presumably peptidoglycan-linked. However, its clear that assignment to a specific SCWP class has not yet been reported, nor have any functional implications such as protein binding. Notably, *P. polymyxa* does not possess an S-layer.

#### *3.4. SCWPs with Other Pyruvylated Sugar Epitopes*

Some SCWPs containing pyruvylated epitopes other than β-d-ManNAc have been reported from the phylum *Actinobacteria*. There, sugar pyruvylation mainly serves as a chemotaxonomic marker of distinct strains, without further knowledge of putative associated functions.

#### 3.4.1. SCWPs from the Genus *Promicromonospora*

Two strains of the genus *Promicromonospora* are recently uncovered examples of bacteria, which possess non-phosphorylated anionic glycopolymers ("non-classical" SCWPs) with pyruvic acid acetals of *R*-configuration in their cell wall [131]. Members of this genus produce a mycelium that fragments into rod-shaped or coccoid elements and are characterised according to different genus-specific chemotaxonomic markers, including the peptidoglycan of the A4α type [132].

The type strain *Promicromonospora citrea* 665T contains two "non-classical" SCWPs, namely a 2-keto-3-deoxy-d-*glycero*-d-*galacto*-nononic acid (Kdn)-teichulosonic acid containing polymer with the repeating unit structure <sup>→</sup>6)-α-d-Glc*p*(1→6)-α-d-Glc*p*3SO3 <sup>−</sup>-(1→4)-α-7,9Pyr-Kdn-(2→, where the Kdn residue is 7,9-pyruvylated, and a galactan with the repeating unit structure <sup>→</sup>3)-α-4,6Pyr-d-Gal*p-*2OAc-(1→, including 4,6-pyruvylation of Gal.

The cell wall of *Promicromonospora* sp. VKM Ac-1028 contains a teichuronic acid-like structure with the repeating unit <sup>→</sup>6)-α-d-Glc*p*-(1→4)-β-2,3Pyr-d-Glc*p*A-(1→, where glucuronic acid is 2,3-pyruvylated [131].

#### 3.4.2. Teichoic Acids from the Genus *Nocardiopsis*

The first description of a pyruvate ketal modification on a classical teichoic acid (TA) was reported in *Nocardiopsis* strains, a widespread group among the *Actinobacteria* [133]. The genus *Nocardiopsis* is of pharmaceutical and biotechnological interest because of its ability to produce a variety of secondary metabolites—accounting for its wide range of biological activities—and, thus, holds promises as a source of novel bioactive compounds [134].

The major TA of *Nocardiopsis metallicus* VKM Ac-2522T is a 1,5-poly(ribitol phosphate) TA, with each ribitol unit carrying a pyruvate ketal group at positions 2 and 4. The major TA of *N. halotolerans* is a poly(glycerol phosphate-*N*-acetyl-β-galactosaminylglycerol phosphate) structure in which the GalNAc residue carries a 4,6-ketal pyruvate modification.

#### 3.4.3. Teichoic Acids of *Brevibacterium iodinum*

*Brevibacterium iodinum* VKM Ac-2106 produces two distinct WTAs, namely a mannitol-WTA and a glycerol-TA, present in minor amounts. Mannitol-WTA is a 1,6-poly(mannitol phosphate) bearing β-d-Glc*p* residues at the C-2 of mannitol (Man-ol) and, optionally, 4,5-*S*-Pyr residues. Glycerol-WTA is a 1,3-poly(glycerol phosphate) substituted at the C-2 of glycerol by α-d-Gal*p*NAc residues bearing 4,6-*R*-Pyr [135].

#### *3.5. Lipopolysaccharides and Lipooligosaccharides*

Lipopolysaccharides (LPSs) of Gram-negative bacteria are a unique family of glycolipids based on a highly conserved lipid moiety known as lipid A. These molecules are produced by most Gram-negative bacteria, in which they play important roles in the integrity of the outer-membrane permeability barrier and participate extensively in the host–pathogen interplay [136,137]. Complete LPSs have a three-domain molecule architecture; the two-domain variants without an O-antigenic polysaccharide (O-PS) are termed lipooligosaccharides (LOSs) [138]. Lipid A is the hydrophobic anchor of LPSs; it is a unique phosphoglycolipid containing glucosamine (GlcN) residues, which are present as β-(1→6)-linked dimers. The disaccharide contains phosphoryl groups and (*R*)-3-hydroxy fatty acids in ester and amide linkages. Variations in the fine structure can arise from the type of hexosamine present, the degree of phosphorylation, the presence of phosphate substituents, and, importantly, in the nature, chain length, number, and position of the acyl groups. Lipid A is glycosylated with a core oligosaccharide (core OS)—typically containing Kdo, a signature molecule of LPS [139], and heptose residues, which may provide an attachment site for a long-chain O-PS of varying repeating unit composition. The O-PS provides a major cellular antigen (O-antigen) used for serological typing of clinical isolates of a given species. Notably, the O-antigen is expressed by most of the clinically relevant strains and is an important phage receptor; LOS, in contrast has been found to be expressed by a group of Gram-negatives that colonize genital and respiratory mucosal surfaces [140].
