Enterococcus NlpC/p60 peptidoglycan hydrolase SagA localizes to sites of cell division and only requires catalytic dyad for protease activity

Peptidoglycan is a vital component of the bacterial cell wall, and its dynamic remodeling by NlpC/p60 hydrolases is crucial for proper cell division and survival. Beyond these essential functions, we previously discovered that Enterococcus species express and secrete the NlpC/p60 hydrolase secreted antigen A (SagA), whose catalytic activity can modulate host immune responses in animal models. However, the localization and peptidoglycan hydrolase activity of SagA in Enterococcus was still unclear. In this study, we show that SagA contributes to a tri-septal structure in dividing enterococci cells and localizes to sites of cell division through its N-terminal coiled-coil domain. Using molecular modeling and site-directed mutagenesis, we identify amino acid residues within the SagA-NlpC/p60 domain that are crucial for catalytic activity and potential substrate binding. Notably, these studies revealed that SagA may function via a catalytic Cys-His dyad instead of the predicted Cys-His-His triad, which is conserved in SagA orthologs from other Enterococcus species. Our results provide key additional insight into peptidoglycan remodeling in Enterococcus by SagA NlpC/p60 hydrolases. D,L-endopeptidase in a catalytic of and how is in remodeling the cell wall in its

[1]  M. Ohliger,et al.  Small molecule sensors targeting the bacterial cell wall. , 2020, ACS infectious diseases.

[2]  H. Hang,et al.  Translation of peptidoglycan metabolites into immunotherapeutics , 2019, Clinical & translational immunology.

[3]  R. Berisio,et al.  The Cell Wall Hydrolytic NlpC/P60 Endopeptidases in Mycobacterial Cytokinesis: A Structural Perspective , 2019, Cells.

[4]  Byungchul Kim,et al.  Enterococcus faecium secreted antigen A generates muropeptides to enhance host immunity and limit bacterial pathogenesis , 2019, eLife.

[5]  D. Underhill,et al.  Peptidoglycan recognition by the innate immune system , 2018, Nature Reviews Immunology.

[6]  Karthik Hullahalli,et al.  Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations , 2017, eLife.

[7]  Yun Lu,et al.  A secreted bacterial peptidoglycan hydrolase enhances tolerance to enteric pathogens , 2016, Science.

[8]  H. Hang,et al.  Exploiting a host-commensal interaction to promote intestinal barrier function and enteric pathogen tolerance , 2016, Science Immunology.

[9]  M. Pavelka,et al.  The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis , 2016, Journal of bacteriology.

[10]  A. Godzik,et al.  Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains , 2015, mBio.

[11]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[12]  R. Berisio,et al.  Mutational and structural study of RipA, a key enzyme in Mycobacterium tuberculosis cell division: evidence for the L-to-D inversion of configuration of the catalytic cysteine. , 2014, Acta crystallographica. Section D, Biological crystallography.

[13]  I. Muñoz,et al.  Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae , 2014, Nature Communications.

[14]  Mitchell D. Miller,et al.  Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase. , 2014, Journal of molecular biology.

[15]  B. Aldridge,et al.  Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria , 2013, PLoS pathogens.

[16]  Jennifer A. Taylor,et al.  Beyond growth: novel functions for bacterial cell wall hydrolases. , 2012, Trends in microbiology.

[17]  S. Mobashery,et al.  Messenger functions of the bacterial cell wall-derived muropeptides. , 2012, Biochemistry.

[18]  G. Schneider,et al.  Peptidoglycan remodeling in Mycobacterium tuberculosis: comparison of structures and catalytic activities of RipA and RipB. , 2011, Journal of molecular biology.

[19]  D. Philpott,et al.  Peptidoglycan: a critical activator of the mammalian immune system during infection and homeostasis , 2011, Immunological reviews.

[20]  R. Berisio,et al.  Structure and functional regulation of RipA, a mycobacterial enzyme essential for daughter cell separation. , 2010, Structure.

[21]  D. Weibel,et al.  Studying the Dynamics of Flagella in Multicellular Communities of Escherichia coli by Using Biarsenical Dyes , 2009, Applied and Environmental Microbiology.

[22]  M. Delepierre,et al.  Impact of peptidoglycan O‐acetylation on autolytic activities of the Enterococcus faecalis N‐acetylglucosaminidase AtlA and N‐acetylmuramidase AtlB , 2009, FEBS letters.

[23]  M. Gross,et al.  Method revealing bacterial cell-wall architecture by time-dependent isotope labeling and quantitative liquid chromatography/mass spectrometry. , 2009, Analytical chemistry.

[24]  M. Gross,et al.  Characterization of structural variations in the peptidoglycan of vancomycin-susceptible Enterococcus faecium: Understanding glycopeptide-antibiotic binding sites using mass spectrometry , 2008, Journal of the American Society for Mass Spectrometry.

[25]  Jessica Y Locke,et al.  Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad. , 2008, Biochemistry.

[26]  M. de Pedro,et al.  Peptidoglycan structure and architecture. , 2008, FEMS microbiology reviews.

[27]  S. Foster,et al.  Bacterial peptidoglycan (murein) hydrolases. , 2008, FEMS microbiology reviews.

[28]  M. Cadene,et al.  MALDI sample preparation: the ultra thin layer method. , 2007, Journal of visualized experiments : JoVE.

[29]  R. Dziarski,et al.  Peptidoglycan recognition proteins: pleiotropic sensors and effectors of antimicrobial defences , 2007, Nature Reviews Microbiology.

[30]  Paul D Lyne,et al.  Accurate prediction of the relative potencies of members of a series of kinase inhibitors using molecular docking and MM-GBSA scoring. , 2006, Journal of medicinal chemistry.

[31]  K. Kazmierczak,et al.  Defective cell wall synthesis in Streptococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator , 2004, Molecular microbiology.

[32]  Hege S. Beard,et al.  Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. , 2004, Journal of medicinal chemistry.

[33]  Matthew P. Repasky,et al.  Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. , 2004, Journal of medicinal chemistry.

[34]  G. Weinstock,et al.  An Enterococcus faecium Secreted Antigen, SagA, Exhibits Broad-Spectrum Binding to Extracellular Matrix Proteins and Appears Essential for E. faecium Growth , 2003, Infection and Immunity.

[35]  Vivek Anantharaman,et al.  Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes , 2003, Genome Biology.

[36]  Robert E Campbell,et al.  New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. , 2002, Journal of the American Chemical Society.

[37]  M. Cadene,et al.  A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins. , 2000, Analytical chemistry.

[38]  Byungchul Kim,et al.  Biochemical analysis of NlpC/p60 peptidoglycan hydrolase activity. , 2020, Methods in enzymology.

[39]  G. Schneider,et al.  RipD (Rv1566c) from Mycobacterium tuberculosis: adaptation of an NlpC/p60 domain to a non-catalytic peptidoglycan-binding function. , 2014, The Biochemical journal.

[40]  Linda,et al.  Supplemental Data Structural Basis of Murein Peptide Specificity of a γ-D-Glutamyl-L-Diamino Acid Endopeptidase , 2009 .

[41]  Mitchell D. Miller,et al.  Structural Biology and Crystallization Communications , 2022 .