Lipoteichoic acid polymer length is determined by competition between free starter units

Abstract

Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (Glc2DAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstitutedStaphylococcus aureusLtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred Glc2DAG starter unit, yet the use of Glc2DAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.

Structure and mechanism of LARGE1 matriglycan polymerase

“…However, the polymerization of glycans is a templatefree process. Branched glycans are produced by the stochastic interaction of precursors and glycosyltransferase (25), whereas the length of linear polysaccharides are potentially governed by factors such as competing reactions (25), capping of polymers (26)(27)(28), substrate availability, allosteric modulators (29)(30)(31)(32) or molecular timers or rulers (33)(34)(35)(36) that are intrinsic to the enzyme-substrate complex. In the case of matriglycan, processive polymerization is predicated by the tandem arrangement of catalytic domains in LARGE1.…”

Insights into the role of lipoteichoic acids and MprF function in Bacillus subtilis

“…In B. subtilis, our analysis shows the severe decrease of LTA produced in Dap R pgsA A64V (Fig 5B), a strain with a reduced PGol content (Hachmann et al, 2011). The function of MprF in LTA synthesis and the new understanding of S. aureus LtaS processivity (Hesser et al, 2020b) can now be linked to the identification of other MRSA Dap R isolates (cls encoding a cardiolipin synthase, pgsA, dlt operon) (Gray and Wenzel, 2020). Together, these findings support the idea that a modification in LTA polymerisation and D-alanylation of teichoic acid (Bertsche et al, 2013, Mishra et al, 2014 could result in a cell envelope less permeable to Dap, as such not necessarily linked to cell wall thickness (Yang et al, 2010).…”

“…Some of the cell wall associated genes mentioned (sigM, ponA, ltaS, yfnI, mprF, pgsA) are differently expressed in cells of B. subtilis BSB1 in LB-glucose (Nicolas et al, 2012, Zhu andStulke, 2018) specificities across species (Slavetinsky et al, 2017, Song et al, 2021 and even synthetise lysyl-glucosyl-DAG glycolipid in Streptococcus agalactiae (Joyce et al, 2021). Recently, it has been shown that when S. aureus LtaS uses PGol as an alternative anchor to the glycolipid produced by UgtP, the concentration of these free lipid starter units regulates the length of the LTA polymerised in vitro (Hesser et al, 2020b). Future works could investigate whether MprF produces a substrate that contributes to LTA initiation/processivity.…”

“…aureus LtaS is processive and that the LTA polymer elongation is directly regulated by the identity and concentration of lipid starter units. The use of Glc2DAG as a preferred starter unit led to the formation of shorter polymer (Hesser et al, 2020b) while the use of PGol resulted in the synthesis of longer polymers. It is not clear if this mechanism might be conserved in B. subtilis however, in absence of B. subtilis LtaS, YfnI expression and LTA length increase (Wormann et al, 2011, Hashimoto et al, 2013.…”

FtsH Sensitizes Methicillin-Resistant Staphylococcus aureus to β-Lactam Antibiotics by Degrading YpfP, a Lipoteichoic Acid Synthesis Enzyme

“…Wall Antibiotics 2021, 10, 1198 2 of 16 teichoic acid (WTA) plays a critical role in the β-lactam resistance of MRSA because it acts as a scaffold to PBP2a by direct binding and is required for the localization of PBP4 [12,13]. Lipoteichoic acid (LTA) is also crucial for MRSA β-lactam resistance [14][15][16][17].…”