Background

The hallmark of Gram-positive bacteria is a thick cell wall comprised of a peptidoglycan matrix embedded with anionic polyol phosphate polymers collectively called teichoic acids (TA) (Weidenmaier and Peschel, 2008; Silhavy et al., 2010; Brown et al., 2013; Percy and Gründling, 2014). Many Gram-positive bacteria have both wall teichoic acid (WTA) and lipoteichoic acid (LTA) whereby WTA is covalently linked to the peptidoglycan layer and LTA is anchored to the cell membrane via a diacylglycerol moiety. TAs regulate numerous cell envelope-associated processes, including autolysin activity and confer resistance to antibiotics such as daptomycin and β-lactams (Winstel et al., 2014). TA function can be further modulated by a variety of side chain modifications, including D-alanylation (D-ala) and glycosylation (Kovács et al., 2006; Mechler et al., 2016; Rismondo et al., 2018). In Staphylococcus aureus, WTA is comprised of poly-ribitol phosphate repeats whereas its LTA contains polyglycerol phosphate repeats (Swoboda et al., 2010; Percy and Gründling, 2014; Kho and Meredith, 2018). Both polymers are modified with D-ala and α/β-GlcNAc residues, in a manner that can vary between S. aureus strains and environmental culture conditions (Percy and Gründling, 2014; Kho and Meredith, 2018). Methods to rapidly monitor changes in TA structure and identify modifications are thus useful in understanding cell envelope physiology.

Although the S. aureus TA polymer backbones are structurally similar and common methods can be used to analyze their composition, WTA and LTA isolation requires specific extraction methods and care must be taken to prevent cross contamination between polymers. WTA is isolated from the crude peptidoglycan sacculus after thorough washing with detergent to remove LTA. Hydrolysis with either sodium hydroxide or trichloroacetic acid is then used to liberate water soluble WTA from peptidoglycan (Meredith et al., 2008). It is often advisable to extract parallel WTA samples using both methods, as acid/base labile side chain modifications may be inadvertently cleaved during isolation and subsequently overlooked. For instance, D-ala esters are readily cleaved under moderately basic conditions (Morath et al., 2001). To obtain membrane-bound LTA, cells are counter extracted with 1-butanol to disrupt the phospholipid bilayer (Morath et al., 2001; Draing et al., 2006; Gründling and Schneewind, 2007). WTA remains insoluble in the cell remnant pellet, while more hydrophobic phospholipids partition into the organic phase. Once clarified, the aqueous phase is

Either crude WTA or LTA extract can then be initially analyzed by PAGE (Wolters et al., 1990; Pollack and Neuhaus, 1994). While WTA can be loaded directly, LTA PAGE resolution benefits from pretreatment with lipases to deacylate the diacylglycerol anchor (Kho and Meredith, 2018). The distinctive “laddering” of bands in TA-PAGE results from TA chain lengths whereby each subsequent band represents a polymer chain length of n + 1 (Figure 1). The relative abundance and polymer length profile can be evaluated, as well as the presence of heterogeneous side chain modifications which induce an ill-defined smear upon TA visualization. TA can be visualized by immunoblotting with α-TA antibodies (Wörmann et al., 2011), or directly imaged using the cationic dye Alcian blue coupled with modified silver staining for enhanced sensitivity (Meredith et al., 2008; Kho and Meredith, 2018).

To elucidate the TA structure, composition and degree of modifications, extraction protocols can be scaled up and nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) used for subsequent characterization (Bernal et al., 2009; Eugster and Loessner, 2011; Shen et al., 2017). Although purity is often adequate for NMR analysis of hydrolyzed WTA at this stage (Figure 3), hydrophobic interaction chromatography is used for further purification of LTA (Morath et al., 2001). Anion exchange chromatography is a convenient method for further purification of either polymer (Fischer et al., 1993; Kim et al., 2005; Eugster and Loessner, 2011). Purified and intact LTA can be analyzed directly by NMR (Figure 3) but ESI-MS analysis of TA monomers is a particularly sensitive approach for detection of low abundance modifications. Hydrofluoric acid (HF)-catalyzed TA depolymerization through dephosphorylation has been developed for WTA (Eugster and Loessner, 2011; Shen et al., 2017), but can also be utilized to analyze LTA (Figure 4; Kho and Meredith, 2018).

Although the methods described here are routinely used to analyze the TA of Staphylococcus aureus, it should be noted they are most suitable for TA chemotypes harboring phosphodiester repeating units. In contrast, the cell wall polymers of some Gram-positive bacteria such as Streptococcus pyogenes (Group A Streptococcus) are comprised of rhamnose repeating units (Mistou et al., 2016), and may lack the polyanionic charge necessary for certain methods such as TA-PAGE.

Protocols:
Part I:Extraction and analysis of teichoic acids by PAGE

1. Crude wall teichoic acid (WTA) extraction for PAGE
2. Crude lipoteichoic acid (LTA) extraction for PAGE
3. Teichoic acid PAGE and staining
   1. Polyacrylamide gel electrophoresis (PAGE)
   2. Alcian blue–silver staining

1. Wall teichoic acid (WTA) precipitation for nuclear magnetic resonance
2. Lipoteichoic acid (LTA) purification by hydrophobic interaction chromatography
   1. Extraction of crude LTA
   2. Hydrophobic interaction chromatography
   3. Phosphate content analysis of LTA fractions
3. ¹H NMR and ESI-MS analysis of teichoic acids
   1. ¹H Nuclear magnetic resonance
   2. Dephosphorylation and monomerization of LTA for ESI-MS
   3. Electrospray ionization mass spectrometry (ESI-MS)