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3. Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

Author

Radkov, Atanas, Sapiro, Anne L, Flores, Sebastian, Henderson, Corey, Saunders, Hayden, Kim, Rachel, Massa, Steven, Thompson, Samuel, Mateusiak, Chase, Biboy, Jacob, Zhao, Ziyi, Starita, Lea M, Hatleberg, William L, Vollmer, Waldemar, Russell, Alistair B, Simorre, Jean-Pierre, Anthony-Cahill, Spencer, Brzovic, Peter, Hayes, Beth, and Chou, Seemay

Subjects
Biochemistry and Cell Biology, Biological Sciences, Infectious Diseases, Rare Diseases, Aetiology, 2.2 Factors relating to the physical environment, Infection, Amidohydrolases, Anti-Bacterial Agents, Bacterial Proteins, Cell Wall, Peptidoglycan, Pseudomonas aeruginosa, E. coli, biochemistry, chemical biology, cognate immunity, deep mutational scanning, host–microbe interaction, infectious disease, microbiology, peptidoglycan, topology, type VI amidase effector, Biological sciences, Biomedical and clinical sciences, Health sciences
Abstract

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

Published
2022

7. The unique 3D arrangement of macrophage galactose lectin enables Escherichia coli lipopolysaccharide recognition through two distinct interfaces

Author

Abbas, Massilia, primary, Maalej, Meriem, additional, Nieto-Fabregat, Ferran, additional, Thépaut, Michel, additional, Kleman, Jean-Philippe, additional, Ayala, Isabel, additional, Molinaro, Antonio, additional, Simorre, Jean-Pierre, additional, Marchetti, Roberta, additional, Fieschi, Franck, additional, and Laguri, Cedric, additional

Published
2023
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9. The unique three-dimensional arrangement of Macrophage Galactose Lectin enablesE. coliLipoPolySaccharides recognition through two distinct interfaces

Author

Abbas, Massilia, primary, Maalej, Meriem, additional, Nieto-Fabregat, Ferran, additional, Thépaut, Michel, additional, Kleman, Jean-Philippe, additional, Ayala, Isabel, additional, Molinaro, Antonio, additional, Simorre, Jean-Pierre, additional, Marchetti, Roberta, additional, Fieschi, Franck, additional, and Laguri, Cedric, additional

Published
2023
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10. Molecular Recognition of Escherichia Coli Core Lipooligosaccharide by DC-SIGN

Author

Nieto-Fabregat, Ferran, primary, Marseglia, Angela, additional, Thépaut, Michel, additional, Kleman, Jean-Philippe, additional, Abbas, Massilia, additional, Maalej, Meriem, additional, Simorre, Jean-Pierre, additional, Laguri, Cedric, additional, Molinaro, Antonio, additional, Silipo, Alba, additional, Fieschi, Franck, additional, and Marchetti, Roberta, additional

Published
2023
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11. Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide

Author

Falchi, Federica A., primary, Taylor, Rebecca J., additional, Rowe, Sebastian J., additional, Moura, Elisabete C. C. M., additional, Baeta, Tiago, additional, Laguri, Cedric, additional, Simorre, Jean-Pierre, additional, Kahne, Daniel E., additional, Polissi, Alessandra, additional, and Sperandeo, Paola, additional

Published
2022
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13. Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

Author

Radkov, Atanas, primary, Sapiro, Anne L, additional, Flores, Sebastian, additional, Henderson, Corey, additional, Saunders, Hayden, additional, Kim, Rachel, additional, Massa, Steven, additional, Thompson, Samuel, additional, Mateusiak, Chase, additional, Biboy, Jacob, additional, Zhao, Ziyi, additional, Starita, Lea M, additional, Hatleberg, William L, additional, Vollmer, Waldemar, additional, Russell, Alistair B, additional, Simorre, Jean-Pierre, additional, Anthony-Cahill, Spencer, additional, Brzovic, Peter, additional, Hayes, Beth, additional, and Chou, Seemay, additional

Published
2022
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14. Author response: Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

Author

Radkov, Atanas, primary, Sapiro, Anne L, additional, Flores, Sebastian, additional, Henderson, Corey, additional, Saunders, Hayden, additional, Kim, Rachel, additional, Massa, Steven, additional, Thompson, Samuel, additional, Mateusiak, Chase, additional, Biboy, Jacob, additional, Zhao, Ziyi, additional, Starita, Lea M, additional, Hatleberg, William L, additional, Vollmer, Waldemar, additional, Russell, Alistair B, additional, Simorre, Jean-Pierre, additional, Anthony-Cahill, Spencer, additional, Brzovic, Peter, additional, Hayes, Beth, additional, and Chou, Seemay, additional

Published
2022
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18. Author Reply to Peer Reviews of Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context

Author

Chou, Seemay, primary, Hayes, Beth, additional, Brzovic, Peter, additional, Anthony-Cahill, Spencer, additional, Simorre, Jean-Pierre, additional, Russell, Alistair B., additional, Vollmer, Waldemar, additional, Hatleberg, William, additional, Starita, Lea M., additional, Zhao, Ziyi, additional, Biboy, Jacob, additional, Mateusiak, Chase, additional, Thompson, Samuel, additional, Massa, Steven, additional, Kim, Rachel, additional, Saunders, Hayden, additional, Henderson, Corey, additional, Flores, Sebastian, additional, Sapiro, Anne L, additional, and Radkov, Atanas, additional

Published
2022
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26. SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli

Author

Pazos, Manuel, Peters, Katharina, Boes, Adrien, Safaei, Yalda, Kenward, Calem, Caveney, Nathanael A., Laguri, Cedric, Breukink, Eefjan, Strynadka, Natalie C.J., Simorre, Jean Pierre, Terrak, Mohammed, Vollmer, Waldemar, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, The Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of Northern British Columbia [Prince George] (UNBC), Department of Molecular and Cellular Physiology, Stanford University, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Membrane Biochemistry and Biophysics, Utrecht University [Utrecht], Sub Membrane Biochemistry & Biophysics, and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
cell division, Models, Molecular, Molecular Biology and Physiology, Penicillin binding proteins, Cell division, Protein Conformation, DEDD, Peptidoglycan, medicine.disease_cause, Cefsulodin, Microbiology, Peptidoglycan synthases, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, Virology, medicine, Escherichia coli, Penicillin-Binding Proteins, peptidoglycan synthases, Escherichia coli Infections, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Chemistry, Escherichia coli Proteins, Periplasmic space, QR1-502, 3. Good health, Cell biology, Anti-Bacterial Agents, Enzyme, SPOR domain, Peptidoglycan Glycosyltransferase, Research Article, Protein Binding
Abstract

Escherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division., Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.

Published
2020

27. Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa

Author

Caveney, Nathanael A, Egan, Alexander J F, Ayala, Isabel, Laguri, Cédric, Robb, Craig S, Breukink, Eefjan, Vollmer, Waldemar, Strynadka, Natalie C J, Simorre, Jean-Pierre, Caveney, Nathanael A, Egan, Alexander J F, Ayala, Isabel, Laguri, Cédric, Robb, Craig S, Breukink, Eefjan, Vollmer, Waldemar, Strynadka, Natalie C J, and Simorre, Jean-Pierre

Abstract

Peptidoglycan (PG) is an essential component of the bacterial cell wall and is assembled from a lipid II precursor by glycosyltransferase and transpeptidase reactions catalyzed in particular by bifunctional class A penicillin-binding proteins (aPBPs). In the major clinical pathogen E. coli,

Published
2020

28. Defining the function of OmpA in the Rcs stress response

Author

Dekoninck, Kilian, Létoquart, Juliette, Laguri, Cédric, Demange, Pascal, Bevernaegie, Robin, Simorre, Jean-Pierre, Dehu, Olivia, Iorga, Bogdan, Elias, Benjamin, Cho, Seung-Hyun, Collet, Jean François, Dekoninck, Kilian, Létoquart, Juliette, Laguri, Cédric, Demange, Pascal, Bevernaegie, Robin, Simorre, Jean-Pierre, Dehu, Olivia, Iorga, Bogdan, Elias, Benjamin, Cho, Seung-Hyun, and Collet, Jean François

Abstract

OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of β-barrel proteins for several decades. In Escherichia coli ,OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal β-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results suggest a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response., info:eu-repo/semantics/published

Published
2020

29. SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli

Author

Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Pazos, Manuel, Peters, Katharina, Boes, Adrien, Safaei, Yalda, Kenward, Calem, Caveney, Nathanael A., Laguri, Cedric, Breukink, Eefjan, Strynadka, Natalie C.J., Simorre, Jean Pierre, Terrak, Mohammed, Vollmer, Waldemar, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Pazos, Manuel, Peters, Katharina, Boes, Adrien, Safaei, Yalda, Kenward, Calem, Caveney, Nathanael A., Laguri, Cedric, Breukink, Eefjan, Strynadka, Natalie C.J., Simorre, Jean Pierre, Terrak, Mohammed, and Vollmer, Waldemar

Published
2020

30. Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa

Author

Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Caveney, Nathanael A, Egan, Alexander J F, Ayala, Isabel, Laguri, Cédric, Robb, Craig S, Breukink, Eefjan, Vollmer, Waldemar, Strynadka, Natalie C J, Simorre, Jean-Pierre, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Caveney, Nathanael A, Egan, Alexander J F, Ayala, Isabel, Laguri, Cédric, Robb, Craig S, Breukink, Eefjan, Vollmer, Waldemar, Strynadka, Natalie C J, and Simorre, Jean-Pierre

Published
2020

35. Interaction of the mitotic inhibitor monastrol with human kinesin Eg5

Author

DeBonis, Salvatore, Simorre Jean-Pierre, Crevel, Isabelle, lebeau, Luc, Skoufias, Dimitrios A., Blangy, Anne, Ebel, Christine, Gans, Pierre, Cross, Robert, Hackney, David D., Wade, Richard H., and Kozielski, Frank

Subjects
Adenosine triphosphatase -- Physiological aspects, Kinesin -- Physiological aspects, Enzyme activation -- Analysis, Biological sciences, Chemistry
Abstract

Results discusses the monastrol inhibition of different human kinesin Eg5 constructs in terms of monastrol activation of ATPase, mantADP release, and motility. Data indicate that monastrol is very effective in arresting mitotic cells in vivo but not as effective in in vitro.

Published
2003

37. Macromolecular import into Eschericha coli: the TolA C-terminal domain changes conformation when interacting with the colicin A toxin

Author

Deprez, Christophe, Blanchard, Laurence, Guerlesquin, Francoise, Gavioli, Marthe, Simorre, Jean-Pierre, Lazdunski, Claude, Marion, Dominique, and Lloubes, Roland

Subjects
Biochemistry -- Research, Toxins -- Physiological aspects, Escherichia coli -- Physiological aspects, Macromolecules -- Physiological aspects, Biological sciences, Chemistry
Abstract

Research has been conducted on the macromolecules which parasite Escherichia coli envelope proteins and infect bacteria. Results suggest that these macromolecules affect E. coli via different interactions between the proteins and between colicin translocation domain and a protein.

Published
2002

39. Refinement of local and long-range structural order in theophylline-binding RNA using C(super 13)-H(super 1) residual dipolar couplings and restrained molecular dynamics

Author

Sibille, Nathalie, Pardi, Arthur, Simorre, Jean-Pierre, and Blackledge, Martin

Subjects
Molecular dynamics -- Analysis, RNA processing -- Usage, Theophylline -- Research, Theophylline -- Structure, Theophylline -- Chemical properties, Chemistry
Abstract

The C(super13)-H(super 1) residual dipolar couplings are measured from theophylline-RNA complex. The semi rigid-body molecular dynamics calculations were used to reorient the refined helical regions to an orientation consistent with a common alignment tensor.

Published
2001

40. Solution structure of 2-(pyrido[1,2-e]purin-4-yl)amino-ethanol intercalated in the DNA duplex d(CGATCG) (sub)2

Author

Favier, Adrien, Blackledge, Martin, Simorre, Jean-Pierre, Crouzy, Serge, Dabouis, Vincent, Gueiffier, Alain, Marion, Dominique, and Debouzy, Jean-Claude

Subjects
Biochemistry -- Research, Amino compounds -- Physiological aspects, Solution (Chemistry) -- Physiological aspects, DNA -- Research, Nuclear magnetic resonance spectroscopy -- Usage, Biological sciences, Chemistry
Abstract

Research has been conducted on the amino-ethanol complex. The solution structure of this complex has been resolved via NMR spectroscopy and restrained molecular dynamic simulations.

Published
2001

41. Defining the function of OmpA in the Rcs stress response

Author

Dekoninck, Kilian, primary, Létoquart, Juliette, additional, Laguri, Cédric, additional, Demange, Pascal, additional, Bevernaegie, Robin, additional, Simorre, Jean-Pierre, additional, Dehu, Olivia, additional, Iorga, Bogdan I, additional, Elias, Benjamin, additional, Cho, Seung-Hyun, additional, and Collet, Jean-Francois, additional

Published
2020
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42. Author response: Defining the function of OmpA in the Rcs stress response

Author

Dekoninck, Kilian, primary, Létoquart, Juliette, additional, Laguri, Cédric, additional, Demange, Pascal, additional, Bevernaegie, Robin, additional, Simorre, Jean-Pierre, additional, Dehu, Olivia, additional, Iorga, Bogdan I, additional, Elias, Benjamin, additional, Cho, Seung-Hyun, additional, and Collet, Jean-Francois, additional

Published
2020
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43. Defining the function of OmpA in the Rcs stress response

Author

Létoquart, Juliette, primary, Dekoninck, Kilian, additional, Laguri, Cédric, additional, Demange, Pascal, additional, Bevernaegie, Robin, additional, Simorre, Jean-Pierre, additional, Dehu, Olivia, additional, Iorga, Bogdan I., additional, Elias, Benjamin, additional, Cho, Seung-Hyun, additional, and Collet, Jean-François, additional

Published
2020
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47. Combined structural and biochemical analysis of the H-T complex in the glycine decarboxylase cycle: evidence for a destabilization mechanism of the H-protein

Author

Guilhaudis, Laure, Simorre, Jean-Pierre, Blackledge, Martin, Marion, Dominique, Gans, Pierre, Neuburger, Michel, and Douce, Roland

Subjects
Biochemistry -- Research, Glycine -- Research, Decarboxylases -- Research, Proteins -- Research, Catalysis -- Analysis, Biological sciences, Chemistry
Abstract

Research has been conducted on the lipoate containing H-proteins. The role of the lipoate in the glycine decarboxylase complex's catalytic cycle has ben investigated.

Published
2000

50. Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

Author

Maya-Martinez, Roberto, Alexander, J Andrew N, Otten, Christian, Ayala, Isabel, Vollmer, Daniela, Gray, Joe, Bougault, Catherine M, Burt, Alister, Laguri, Cédric, Fonvielle, Matthieu, Arthur, Michel, Strynadka, Natalie, Vollmer, Waldemar, Simorre, Jean-Pierre, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of Northern British Columbia [Prince George] (UNBC), The Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Structure bactérienne et résistance aux antibiotiques (CRC - Inserm U1138), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), ANR-11-BSV5-0024,CarbaTub,Mécanisme d'inhibition d'une nouvelle cible des antibiotiques appartenant à la famille des bêta-lactamines(2011), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), University of Northern British Columbia (UNBC), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), ANR: CarbaTub,ANR-2011-BSV5-024-04, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), École Pratique des Hautes Études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-École Pratique des Hautes Études (EPHE)

Subjects
Microbiology (medical), Glycan, Staphylococcus aureus, penicillin-binding protein 4, Mutant, lcsh:QR1-502, Peptide, peptidoglycan, medicine.disease_cause, Microbiology, lcsh:Microbiology, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, Hydrolase, medicine, Original Research, 030304 developmental biology, X-ray crystallography, chemistry.chemical_classification, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, NMR, cyclic muropeptides, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], chemistry, Biochemistry, biology.protein, Peptidoglycan, Cell envelope
Abstract

International audience; Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to β-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of β-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to β-lactams in S. aureus.

Published
2019

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