Abstract
Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.
Highlights
Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the D-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood
DltA resembles adenylation domains found in nonribosomal peptide synthetases (40 –43), and it has been shown that DltA transfers D-alanine in an ATP-dependent manner to DltC, a peptidyl brane-bound O-acyltransferase; Ghrelin O-acyltransferase (GOAT), ghrelin O-acyltransferase; DDM, n-dodecyl -D-maltoside; IPTG, isopropyl thiogalactoside; bis-Tris, 2-[bis (2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; TSB, tryptic soy broth
Using reverse transcription-PCR with primers spanning dltXdltA and SAOUHSC_00867-dltX, we found that both of these ORFs are cotranscribed with the dltABCD operon (Fig. S1B)
Summary
Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the D-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. WTA is synthesized inside the cell on the undecaprenyl carrier lipid before it is flipped outside and transferred to peptidoglycan [3] Both of these polymers are modified with positively charged D-alanine residues outside the cell through a process that depends on the D-alanyl lipoteichoic acid (DLT) pathway [4]. DltB has been proposed to transfer D-alanine from the carrier protein to undecaprenyl phosphate, the carrier lipid for the biosynthesis of peptidoglycan and wall teichoic acid precursors [36, 39]. DltD is a single-pass membrane protein for which several functions have been proposed These include hydrolyzing misacylated D-Ala-ACP [58], activating DltA catalysis [58], and transferring D-alanine from the undecaprenyl phosphate carrier lipid to teichoic acids outside the cell [38]. Based on active-site features required for DltD’s function, we propose that it forms a covalent intermediate with D-Ala transferred from the acylated product of DltB, which may be a D-Ala-lipid or DltD itself; LTA serves as the acyl acceptor for subsequent transfer of the D-Ala (Fig. 1)
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