The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.
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This work was supported by NIH grants R01 AI143690 from the NIAID and R01 DE028916 from the NIDCR and (to N.K.), the Swedish Research Council (no. 2017-03703) and The Knut and Alice Wallenberg Foundation (to G.W.). The Swedish NMR Centre at University of Gothenburg is acknowledged for support. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894).
Atomic coordinates and structure factors of PplD crystal structure generated in this study have been deposited in the Protein Data Bank under accession code 6DQ3. All data generated during this study are included in the article and Supplementary information files. Source data are provided with this paper.
Rush, Jeffrey S.; Parajuli, Prakash; Ruda, Alessandro; Li, Jian; Pohane, Amol Arunrao; Zamakhaeva, Svetlana; Rahman, Mohammad M.; Chang, Jennifer C.; Gogos, Artemis; Kenner, Cameron W.; Lambeau, Gérard; Federle, Michael J.; Korotkov, Konstantin V.; Widmalm, Göran; and Korotkova, Natalia, "PplD Is a De-N-Acetylase of the Cell Wall Linkage Unit of Streptococcal Rhamnopolysaccharides" (2022). Molecular and Cellular Biochemistry Faculty Publications. 195.