Artykuły w czasopismach na temat „Peptidoglycan polymerization”
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Arthur, Michel. "Regulation of Bacterial Peptidoglycan Polymerization". Trends in Microbiology 24, nr 7 (lipiec 2016): 519–21. http://dx.doi.org/10.1016/j.tim.2016.05.003.
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Vasudevan, Pradeep, Jessica McElligott, Christa Attkisson, Michael Betteken i David L. Popham. "Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism". Journal of Bacteriology 191, nr 19 (31.07.2009): 6012–19. http://dx.doi.org/10.1128/jb.00604-09.
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ABSTRACT Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes (yabM, ykvU, and ytgP) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.
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Chan, Yvonne G. Y., Matthew B. Frankel, Dominique Missiakas i Olaf Schneewind. "SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion". Journal of Bacteriology 198, nr 7 (25.01.2016): 1123–36. http://dx.doi.org/10.1128/jb.00983-15.
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ABSTRACTThe envelope ofStaphylococcus aureusis comprised of peptidoglycan and its attached secondary polymers, teichoic acid, capsular polysaccharide, and protein. Peptidoglycan synthesis involves polymerization of lipid II precursors into glycan strands that are cross-linked at wall peptides. It is not clear whether peptidoglycan structure is principally determined during polymerization or whether processive enzymes affect cell wall structure and function, for example, by generating conduits for protein secretion. We show here thatS. aureuslacking SagB, a membrane-associatedN-acetylglucosaminidase, displays growth and cell-morphological defects caused by the exaggerated length of peptidoglycan strands. SagB cleaves polymerized glycan strands to their physiological length and modulates antibiotic resistance in methicillin-resistantS. aureus(MRSA). Deletion ofsagBperturbs protein trafficking into and across the envelope, conferring defects in cell wall anchoring and secretion, as well as aberrant excretion of cytoplasmic proteins.IMPORTANCEStaphylococcus aureusis thought to secrete proteins across the plasma membrane via the Sec pathway; however, protein transport across the cell wall envelope has heretofore not been studied. We report thatS. aureus sagBmutants generate elongated peptidoglycan strands and display defects in protein secretion as well as aberrant excretion of cytoplasmic proteins. These results suggest that the thick peptidoglycan layer of staphylococci presents a barrier for protein secretion and that SagB appears to extend the Sec pathway across the cell wall envelope.
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Zuber, Benoît, Marisa Haenni, Tânia Ribeiro, Kathrin Minnig, Fátima Lopes, Philippe Moreillon i Jacques Dubochet. "Granular Layer in the Periplasmic Space of Gram-Positive Bacteria and Fine Structures of Enterococcus gallinarum and Streptococcus gordonii Septa Revealed by Cryo-Electron Microscopy of Vitreous Sections". Journal of Bacteriology 188, nr 18 (15.09.2006): 6652–60. http://dx.doi.org/10.1128/jb.00391-06.
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ABSTRACT High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.
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Ruiz, Natividad. "Bioinformatics identification of MurJ (MviN) as the peptidoglycan lipid II flippase inEscherichia coli". Proceedings of the National Academy of Sciences 105, nr 40 (1.10.2008): 15553–57. http://dx.doi.org/10.1073/pnas.0808352105.
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Peptidoglycan is a cell-wall glycopeptide polymer that protects bacteria from osmotic lysis. Whereas in Gram-positive bacteria it also serves as scaffold for many virulence factors, in Gram-negative bacteria, peptidoglycan is an anchor for the outer membrane. For years, we have known the enzymes required for the biosynthesis of peptidoglycan; what was missing was the flippase that translocates the lipid-anchored precursors across the cytoplasmic membrane before their polymerization into mature peptidoglycan. Using a reductionist bioinformatics approach, I have identified the essential inner-membrane protein MviN (renamed MurJ) as a likely candidate for the peptidoglycan flippase inEscherichia coli. Here, I present genetic and biochemical data that confirm the requirement of MurJ for peptidoglycan biosynthesis and that are in agreement with a role of MurJ as a flippase. Because of its essential nature, MurJ could serve as a target in the continuing search for antimicrobial compounds.
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Yagi, Tetsuya, Sebabrata Mahapatra, Katarína Mikušová, Dean C. Crick i Patrick J. Brennan. "Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan". Journal of Biological Chemistry 278, nr 29 (28.04.2003): 26497–504. http://dx.doi.org/10.1074/jbc.m302216200.
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Arbeloa, Ana, Heidi Segal, Jean-Emmanuel Hugonnet, Nathalie Josseaume, Lionnel Dubost, Jean-Paul Brouard, Laurent Gutmann, Dominique Mengin-Lecreulx i Michel Arthur. "Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance in Enterococcus faecalis". Journal of Bacteriology 186, nr 5 (1.03.2004): 1221–28. http://dx.doi.org/10.1128/jb.186.5.1221-1228.2004.
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ABSTRACT Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 ΔponA ΔpbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.
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Rice, Louis B., Lenore L. Carias, Susan Rudin, Rebecca Hutton, Steven Marshall, Medhat Hassan, Nathalie Josseaume, Lionel Dubost, Arul Marie i Michel Arthur. "Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium". Journal of Bacteriology 191, nr 11 (20.03.2009): 3649–56. http://dx.doi.org/10.1128/jb.01834-08.
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ABSTRACT Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of β-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the ΔpbpF ΔponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the l,d-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the ΔpbpF ΔponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different β-lactam antibiotics differed as a function of its partner glycosyltransferase.
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Allen, N. E., J. N. Hobbs i T. I. Nicas. "Inhibition of peptidoglycan biosynthesis in vancomycin-susceptible and -resistant bacteria by a semisynthetic glycopeptide antibiotic." Antimicrobial Agents and Chemotherapy 40, nr 10 (październik 1996): 2356–62. http://dx.doi.org/10.1128/aac.40.10.2356.
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LY191145 is a p-chlorobenzyl derivative of LY264826 (A82846B) with activity against both vancomycin-susceptible and -resistant enterococci. Incorporation of L-[14C]lysine into peptidoglycan of intact vancomycin-susceptible and -resistant Enterococcus faecium was inhibited by LY191145 (50% inhibitory concentrations of 1 and 5 microgram/ml, respectively). Inhibition was accompanied by accumulation of UDP-muramyl-peptide precursors in the cytoplasm. This agent inhibited late-stage steps in peptidoglycan biosynthesis in permeabilized E. faecium when either the UDP-muramyl-pentapeptide precursor from vancomycin-susceptible E. faecium or the UDP-muramyl-pentadepsipeptide precursor from vancomycin-resistant E. faecium was used as a substrate. Inhibition of late-stage steps led to accumulation of an N-acetyl-[14C]glucosamine-labeled lipid intermediate indicative of inhibition of the transglycosylation step. Inhibition of peptidoglycan polymerization without affecting cross-linking in a particulate membrane-plus-wall-fragment assay from Aerococcus viridans was consistent with this explanation. The fact that inhibition of peptidoglycan biosynthesis by LY191145 was not readily antagonized by an excess of free acyl-D-alanyl-D-alanine or acyl-D-alanyl-D-lactate ligands indicates that the manner in which this compound inhibits transglycosylation may not be identical to that of vancomycin.
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Braddick, Darren, Sandeep Sandhu, David I. Roper, Michael J. Chappell i Timothy D. H. Bugg. "Observation of the time-course for peptidoglycan lipid intermediate II polymerization by Staphylococcus aureus monofunctional transglycosylase". Microbiology 160, nr 8 (1.08.2014): 1628–36. http://dx.doi.org/10.1099/mic.0.079442-0.
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The polymerization of lipid intermediate II by the transglycosylase activity of penicillin-binding proteins (PBPs) represents an important target for antibacterial action, but limited methods are available for quantitative assay of this reaction, or screening potential inhibitors. A new labelling method for lipid II polymerization products using Sanger’s reagent (fluoro-2,4-dinitrobenzene), followed by gel permeation HPLC analysis, has permitted the observation of intermediate polymerization products for Staphylococcus aureus monofunctional transglycosylase MGT. Peak formation is inhibited by 6 µM ramoplanin or enduracidin. Characterization by mass spectrometry indicates the formation of tetrasaccharide and octasaccharide intermediates, but not a hexasaccharide intermediate, suggesting a dimerization of a lipid-linked tetrasaccharide. Numerical modelling of the time-course data supports a kinetic model involving addition to lipid-linked tetrasaccharide of either lipid II or lipid-linked tetrasaccharide. Observation of free octasaccharide suggests that hydrolysis of the undecaprenyl diphosphate lipid carrier occurs at this stage in peptidoglycan transglycosylation.
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Zheng, Sanduo, Lok-To Sham, Frederick A. Rubino, Kelly P. Brock, William P. Robins, John J. Mekalanos, Debora S. Marks, Thomas G. Bernhardt i Andrew C. Kruse. "Structure and mutagenic analysis of the lipid II flippase MurJ fromEscherichia coli". Proceedings of the National Academy of Sciences 115, nr 26 (11.06.2018): 6709–14. http://dx.doi.org/10.1073/pnas.1802192115.
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The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophileThermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organismEscherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability ofE. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.
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Yunck, Rachel, Hongbaek Cho i Thomas G. Bernhardt. "Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria". Molecular Microbiology 99, nr 4 (19.11.2015): 700–718. http://dx.doi.org/10.1111/mmi.13258.
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van Heijenoort, Jean. "Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan". Microbiology and Molecular Biology Reviews 71, nr 4 (grudzień 2007): 620–35. http://dx.doi.org/10.1128/mmbr.00016-07.
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SUMMARY This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.
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Ali Hasan, Waseem. "Differential Study of Antimicrobial Activity of Vancomycin and Teicoplanin (Targocid) against Strains of Staphylococcus aureus and Streptococci sp." Tikrit Journal of Pharmaceutical Sciences 5, nr 2 (13.04.2023): 203–7. http://dx.doi.org/10.25130/tjphs.2009.5.2.11.203.207.
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The glycopeptide antibiotics vancomycin and teicoplanin (targocid) are widely used in the treatment of infections caused by gram positive bacteria. Vancomyc inhibits both transglycosylation and transpeptidation reaction during peptidoglycan assembly, targocid inhibits peptidoglycan polymerization, resulting in inhibition of bacterial cell wall synthesis and cell death. In this study twenty strains of streptococcus pyogenes and streptococcus pneumoniae and twenty five strains of staphylococcus aureus were collected from different infections. MI Cs and MBCs of vancomycin and targocid were determined against the bacterial strains, the MICs of vancomycin for the streptococcus strains were 4-8 µg /ml compared with 8-16 µg/ml for targocid and MI Cs of targocid for staphylococcus aureus were 4-16 µg/ml MBC for the same strains were 8-32 µg/ml. the conclusion of this study was that targocid was less active than vancomycin against staphylococci but equal or more active against streptococci specifically it presents excellent in vitro activity against streptococcus pneumonia strains.
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Sjodt, Megan, Patricia D. A. Rohs, Morgan S. A. Gilman, Sarah C. Erlandson, Sanduo Zheng, Anna G. Green, Kelly P. Brock i in. "Structural coordination of polymerization and crosslinking by a SEDS–bPBP peptidoglycan synthase complex". Nature Microbiology 5, nr 6 (9.03.2020): 813–20. http://dx.doi.org/10.1038/s41564-020-0687-z.
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Plocinski, P., M. Ziolkiewicz, M. Kiran, S. I. Vadrevu, H. B. Nguyen, J. Hugonnet, C. Veckerle i in. "Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes". Journal of Bacteriology 193, nr 13 (29.04.2011): 3246–56. http://dx.doi.org/10.1128/jb.00188-11.
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Punekar, Avinash S., Firdaus Samsudin, Adrian J. Lloyd, Christopher G. Dowson, David J. Scott, Syma Khalid i David I. Roper. "The role of the jaw subdomain of peptidoglycan glycosyltransferases for lipid II polymerization". Cell Surface 2 (czerwiec 2018): 54–66. http://dx.doi.org/10.1016/j.tcsw.2018.06.002.
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Perlstein, Deborah L., Tsung-Shing Andrew Wang, Emma H. Doud, Daniel Kahne i Suzanne Walker. "The Role of the Substrate Lipid in Processive Glycan Polymerization by the Peptidoglycan Glycosyltransferases". Journal of the American Chemical Society 132, nr 1 (13.01.2010): 48–49. http://dx.doi.org/10.1021/ja909325m.
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Zawadzka-Skomiał, Joanna, Zdzislaw Markiewicz, Martine Nguyen-Distèche, Bart Devreese, Jean-Marie Frère i Mohammed Terrak. "Characterization of the Bifunctional Glycosyltransferase/Acyltransferase Penicillin-Binding Protein 4 of Listeria monocytogenes". Journal of Bacteriology 188, nr 5 (1.03.2006): 1875–81. http://dx.doi.org/10.1128/jb.188.5.1875-1881.2006.
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ABSTRACT Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M−1 s−1 from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to β-lactam antibiotics but did increase the resistance of the mutant to moenomycin.
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Qiao, Lei, i John C. Vederas. "Synthesis of a C-phosphonate disaccharide as a potential inhibitor of peptidoglycan polymerization by transglycosylase". Journal of Organic Chemistry 58, nr 13 (czerwiec 1993): 3480–82. http://dx.doi.org/10.1021/jo00065a004.
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Schaefer, Kaitlin, Tristan W. Owens, Julia E. Page, Marina Santiago, Daniel Kahne i Suzanne Walker. "Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization". Nature Microbiology 6, nr 1 (9.11.2020): 34–43. http://dx.doi.org/10.1038/s41564-020-00808-5.
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Varma, Archana, Miguel A. de Pedro i Kevin D. Young. "FtsZ Directs a Second Mode of Peptidoglycan Synthesis in Escherichia coli". Journal of Bacteriology 189, nr 15 (18.05.2007): 5692–704. http://dx.doi.org/10.1128/jb.00455-07.
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ABSTRACT Certain penicillin binding protein mutants of Escherichia coli grow with spirillum-like morphologies when the FtsZ protein is inhibited, suggesting that FtsZ might govern aspects of cell wall growth other than those strictly associated with septation. While investigating the mechanism of spiral cell formation, we discovered conditions for visualizing this second function of FtsZ. Normally, inhibiting the cytoskeleton protein MreB forces E. coli cells to grow as smoothly enlarging spheres from which the poles disappear, yielding coccoid or lemon-shaped forms. However, when FtsZ and MreB were inhibited simultaneously in a strain lacking PBP 5 and PBP 7, the resulting cells ballooned outward but retained conspicuous rod-shaped extensions at sites representing the original poles. This visual phenotype was paralleled by the biochemistry of sacculus growth. Muropeptides are usually inserted homogeneously into the lateral cell walls, but when FtsZ polymerization was inhibited, the incorporation of new material occurred mainly in the central regions of cells and was significantly lower in those portions of side walls abutting a pole. Thus, reduced precursor incorporation into side walls near the poles explained why these regions retained their rod-like morphology while the rest of the cell grew spherically. Also, inhibiting FtsZ increased the amount of pentapeptides in sacculi by about one-third. Finally, the MreB protein directed the helical or diagonal incorporation of new peptidoglycan into the wall, but the location of that incorporation depended on whether FtsZ was active. In sum, the results indicate that in addition to nucleating cell septation in E. coli, FtsZ can direct the insertion of new peptidoglycan into portions of the lateral wall.
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Hamilton, Andrea, David L. Popham, David J. Carl, Xavier Lauth, Victor Nizet i Amanda L. Jones. "Penicillin-Binding Protein 1a Promotes Resistance of Group B Streptococcus to Antimicrobial Peptides". Infection and Immunity 74, nr 11 (listopad 2006): 6179–87. http://dx.doi.org/10.1128/iai.00895-06.
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ABSTRACT Evasion of host immune defenses is critical for the progression of invasive infections caused by the leading neonatal pathogen, group B streptococcus (GBS). Upon characterizing the factors required for virulence in a neonatal rat sepsis model, we found that a surface-associated penicillin-binding protein (PBP1a), encoded by ponA, played an essential role in resistance of GBS to phagocytic clearance. In order to elucidate how PBP1a promotes resistance to innate immunity, we compared the susceptibility of wild-type GBS and an isogenic ponA mutant to the bactericidal components of human neutrophils. The isogenic strains were found to be equally capable of blocking complement activation on the bacterial surface and equally associated with phagocytes and susceptible to oxidative killing. In contrast, the ponA mutant was significantly more susceptible to killing by cationic antimicrobial peptides (AMPs) of the cathelicidin and defensin families, which are now recognized as integral components of innate host defense against invasive bacterial infection. These observations may help explain the sensitivity to phagocytic killing and attenuated virulence of the ponA mutant. This novel function for PBP1a in promoting resistance of GBS to AMP did not involve an alteration in bacterial surface charge or peptidoglycan cross-linking. While the peptidoglycan polymerization and cross-linking activity of PBPs are essential for bacterial survival, our study is the first to identify a role for a PBP in resistance to host AMPs.
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Markovski, Monica, Jessica L. Bohrhunter, Tania J. Lupoli, Tsuyoshi Uehara, Suzanne Walker, Daniel E. Kahne i Thomas G. Bernhardt. "Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity". Proceedings of the National Academy of Sciences 113, nr 17 (11.04.2016): 4788–93. http://dx.doi.org/10.1073/pnas.1524538113.
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To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.
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Rohs, Patricia D. A., Jackson Buss, Sue I. Sim, Georgia R. Squyres, Veerasak Srisuknimit, Mandy Smith, Hongbaek Cho i in. "A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery". PLOS Genetics 14, nr 10 (18.10.2018): e1007726. http://dx.doi.org/10.1371/journal.pgen.1007726.
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Cremniter, Julie, Jean-Luc Mainardi, Nathalie Josseaume, Jean-Charles Quincampoix, Lionel Dubost, Jean-Emmanuel Hugonnet, Arul Marie, Laurent Gutmann, Louis B. Rice i Michel Arthur. "Novel Mechanism of Resistance to Glycopeptide Antibiotics in Enterococcus faecium". Journal of Biological Chemistry 281, nr 43 (29.08.2006): 32254–62. http://dx.doi.org/10.1074/jbc.m606920200.
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Glycopeptides and β-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-d-Ala4-d-Ala5 extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and d,d-transpeptidase activities of the penicillin-binding proteins (PBPs). The β-lactams are structural analogues of d-Ala4-d-Ala5 and act as suicide substrates of the d,d-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described β-lactam-insensitive l,d-transpeptidase from Enterococcus faecium (Ldtfm) can lead to high level resistance to glycopeptides and β-lactams. Cross-resistance was selected by glycopeptides alone or serially by β-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of d-Ala5, thereby providing the substrate of Ldtfm. Complete elimination of d-Ala5, a residue essential for glycopeptide binding, was possible because Ldtfm uses the energy of the l-Lys3-d-Ala4 peptide bond for cross-link formation in contrast to PBPs, which use the energy of the d-Ala4-d-Ala5 bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of d-Ala5 by d-Ser or d-lactate.
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Chang, Chungyu, Chenggang Wu, Jerzy Osipiuk, Sara D. Siegel, Shiwei Zhu, Xiangan Liu, Andrzej Joachimiak, Robert T. Clubb, Asis Das i Hung Ton-That. "Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes". Proceedings of the National Academy of Sciences 116, nr 36 (19.08.2019): 18041–49. http://dx.doi.org/10.1073/pnas.1907733116.
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Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.
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Di Guilmi, Anne Marie, Andréa Dessen, Otto Dideberg i Thierry Vernet. "The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains". Journal of Bacteriology 185, nr 15 (1.08.2003): 4418–23. http://dx.doi.org/10.1128/jb.185.15.4418-4423.2003.
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ABSTRACT The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat/K m were measured at 40.6 μM (± 15.5) and 1 × 10−3 M−1 s−1, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.
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Marmont, Lindsey S., i Thomas G. Bernhardt. "A conserved subcomplex within the bacterial cytokinetic ring activates cell wall synthesis by the FtsW-FtsI synthase". Proceedings of the National Academy of Sciences 117, nr 38 (9.09.2020): 23879–85. http://dx.doi.org/10.1073/pnas.2004598117.
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Cell division in bacteria is mediated by a multiprotein assembly called the divisome. A major function of this machinery is the synthesis of the peptidoglycan (PG) cell wall that caps the daughter poles and prevents osmotic lysis of the newborn cells. Recent studies have implicated a complex of FtsW and FtsI (FtsWI) as the essential PG synthase within the divisome; however, how PG polymerization by this synthase is regulated and coordinated with other activities within the machinery is not well understood. Previous results have implicated a conserved subcomplex of division proteins composed of FtsQ, FtsL, and FtsB (FtsQLB) in the regulation of FtsWI, but whether these proteins act directly as positive or negative regulators of the synthase has been unclear. To address this question, we purified a five-memberPseudomonas aeruginosadivision complex consisting of FtsQLB-FtsWI. The PG polymerase activity of this complex was found to be greatly stimulated relative to FtsWI alone. Purification of complexes lacking individual components indicated that FtsL and FtsB are sufficient for FtsW activation. Furthermore, support for this activity being important for the cellular function of FtsQLB was provided by the identification of two division-defective variants of FtsL that still form normal FtsQLB-FtsWI complexes but fail to activate PG synthesis. Thus, our results indicate that the conserved FtsQLB complex is a direct activator of PG polymerization by the FtsWI synthase and thereby define an essential regulatory step in the process of bacterial cell division.
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Allen, N. E., D. L. LeTourneau i J. N. Hobbs. "Molecular interactions of a semisynthetic glycopeptide antibiotic with D-alanyl-D-alanine and D-alanyl-D-lactate residues." Antimicrobial Agents and Chemotherapy 41, nr 1 (styczeń 1997): 66–71. http://dx.doi.org/10.1128/aac.41.1.66.
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LY191145 is an N-alkylated glycopeptide antibiotic (the p-chlorobenzyl derivative of LY264826) with activity against vancomycin-susceptible and -resistant bacteria. Similar to vancomycin, LY191145 inhibited polymerization of peptidoglycan when muramyl pentapeptide served as a substrate but not when muramyl tetrapeptide was used, signifying a substrate-dependent mechanism of inhibition. Examination of ligand binding affinities for LY191145 and the effects of this agent on R39 D,D-carboxypeptidase action showed that, similar to vancomycin, LY191145 had an 800-fold greater affinity for N,N'-diacetyl-L-Lys-D-Ala-D-Ala than for N,N'-diacetyl-L-Lys-D-Ala-D-Lac. The antibacterial activity of LY191145 was antagonized by N,N'-diacetyl-L-Lys-D-Ala-D-Ala, but the molar excess required for complete suppression exceeded that needed to suppress inhibition by vancomycin. LY191145 is strongly dimerized and the p-chlorobenzyl side chain facilitates interactions with bacterial membranes. These findings are consistent with a mechanism of inhibition where interactions between antibiotic and D-Ala-D-Ala or D-Ala-D-Lac residues depend on intramolecular effects occurring at the subcellular target site.
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Boll, Joseph M., Alexander A. Crofts, Katharina Peters, Vincent Cattoir, Waldemar Vollmer, Bryan W. Davies i M. Stephen Trent. "A penicillin-binding protein inhibits selection of colistin-resistant, lipooligosaccharide-deficientAcinetobacter baumannii". Proceedings of the National Academy of Sciences 113, nr 41 (28.09.2016): E6228—E6237. http://dx.doi.org/10.1073/pnas.1611594113.
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The Gram-negative bacterial outer membrane fortifies the cell against environmental toxins including antibiotics. Unique glycolipids called lipopolysaccharide/lipooligosaccharide (LPS/LOS) are enriched in the cell-surface monolayer of the outer membrane and promote antimicrobial resistance. Colistin, which targets the lipid A domain of LPS/LOS to lyse the cell, is the last-line treatment for multidrug-resistant Gram-negative infections. Lipid A is essential for the survival of most Gram-negative bacteria, but colistin-resistantAcinetobacter baumanniilacking lipid A were isolated after colistin exposure. Previously, strain ATCC 19606 was the onlyA. baumanniistrain demonstrated to subsist without lipid A. Here, we show that otherA. baumanniistrains can also survive without lipid A, but some cannot, affording a unique model to study endotoxin essentiality. We assessed the capacity of 15 clinicalA. baumanniiisolates including 9 recent clinical isolates to develop colistin resistance through inactivation of the lipid A biosynthetic pathway, the products of which assemble the LOS precursor. Our investigation determined that expression of the well-conserved penicillin-binding protein (PBP) 1A, prevented LOS-deficient colony isolation. The glycosyltransferase activity of PBP1A, which aids in the polymerization of the peptidoglycan cell wall, was lethal to LOS-deficientA. baumannii. Global transcriptomic analysis of a PBP1A-deficient mutant and four LOS-deficientA. baumanniistrains showed a concomitant increase in transcription of lipoproteins and their transporters. Examination of the LOS-deficientA. baumanniicell surface demonstrated that specific lipoproteins were overexpressed and decorated the cell surface, potentially compensating for LOS removal. This work expands our knowledge of lipid A essentiality and elucidates a drug resistance mechanism.
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Terrak, Mohammed, i Martine Nguyen-Distèche. "Kinetic Characterization of the Monofunctional Glycosyltransferase from Staphylococcus aureus". Journal of Bacteriology 188, nr 7 (1.04.2006): 2528–32. http://dx.doi.org/10.1128/jb.188.7.2528-2532.2006.
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ABSTRACT The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of ∼5,800 M−1 s−1 and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2+. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.
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Steed, Molly E., Céline Vidaillac i Michael J. Rybak. "Evaluation of Telavancin Activity versus Daptomycin and Vancomycin against Daptomycin-Nonsusceptible Staphylococcus aureus in anIn VitroPharmacokinetic/Pharmacodynamic Model". Antimicrobial Agents and Chemotherapy 56, nr 2 (28.11.2011): 955–59. http://dx.doi.org/10.1128/aac.05849-11.
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ABSTRACTDaptomycin-nonsusceptible (DNS)Staphylococcus aureusstrains have been reported over the last several years. Telavancin is a lipoglycopeptide with a dual mechanism of action, as it inhibits peptidoglycan polymerization/cross-linking and disrupts the membrane potential. Three clinical DNSS. aureusstrains, CB1814, R6212, and SA-684, were evaluated in anin vitropharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations (starting inoculum, 108.5CFU/g) for 120 h. Simulated regimens included telavancin at 10 mg/kg every 24 h (q24h; peak, 87.5 mg/liter;t1/2, 7.5 h), daptomycin at 6 mg/kg q24h (peak, 95.7 mg/liter;t1/2, 8 h), and vancomycin at 1 g q12h (peak, 30 mg/liter;t1/2, 6 h). Differences in CFU/g between regimens at 24 through 120 h were evaluated by analysis of variance with a Tukey'spost hoctest. Bactericidal activity was defined as a ≥3-log10CFU/g decrease in colony count from the initial inoculum. MIC values were 1, 0.25, and 0.5 mg/liter (telavancin), 4, 2, and 2 mg/liter (daptomycin), and 2, 2, and 2 mg/liter (vancomycin) for CB1814, R6212, and SA-684, respectively. Telavancin displayed bactericidal activities against R6212 (32 to 120 h; −4.31 log10CFU/g), SA-684 (56 to 120 h; −3.06 log10CFU/g), and CB1814 (48 to 120 h; −4.9 log10CFU/g). Daptomycin displayed initial bactericidal activity followed by regrowth with all three strains. Vancomycin did not exhibit sustained bactericidal activity against any strain. At 120 h, telavancin was significantly better at reducing colony counts than vancomycin against all three tested strains and better than daptomycin against CB1814 (P< 0.05). Telavancin displayed bactericidal activityin vitroagainst DNSS. aureusisolates.
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Xayarath, Bobbi, i Janet Yother. "Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-Dependent Streptococcus pneumoniae Capsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall". Journal of Bacteriology 189, nr 9 (23.02.2007): 3369–81. http://dx.doi.org/10.1128/jb.01938-06.
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ABSTRACT Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.
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Voedts, Henri, Sean P. Kennedy, Guennadi Sezonov, Michel Arthur i Jean-Emmanuel Hugonnet. "Genome-wide identification of genes required for alternative peptidoglycan cross-linking in Escherichia coli revealed unexpected impacts of β-lactams". Nature Communications 13, nr 1 (27.12.2022). http://dx.doi.org/10.1038/s41467-022-35528-3.
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AbstractThe d,d-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of β-lactam antibiotics that block peptidoglycan polymerization. β-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a β-lactam-insensitive l,d-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by β-lactams in actively dividing bacteria. The functions of the 179 conditionally essential genes identified by this approach extend far beyond l,d-transpeptidase partners for peptidoglycan polymerization to include proteins involved in stress response and in the assembly of outer membrane polymers. The unsuspected effects of β-lactams include loss of the lipoprotein-mediated covalent bond that links the outer membrane to the peptidoglycan, destabilization of the cell envelope in spite of effective peptidoglycan cross-linking, and increased permeability of the outer membrane. The latter effect indicates that the mode of action of β-lactams involves self-promoted penetration through the outer membrane.
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Atze, Heiner, Yucheng Liang, Jean-Emmanuel Hugonnet, Arnaud Gutierrez, Filippo Rusconi i Michel Arthur. "Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs". eLife 11 (9.06.2022). http://dx.doi.org/10.7554/elife.72863.
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Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this undocumented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.
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Sichel, Sophie R., Benjamin P. Bratton i Nina Reda Salama. "Distinct regions of H. pylori's bactofilin CcmA regulate protein-protein interactions to control helical cell shape". eLife 11 (8.09.2022). http://dx.doi.org/10.7554/elife.80111.
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The helical shape of H. pylori cells promotes robust stomach colonization, however, how the helical shape of H. pylori cells is determined is unresolved. Previous work identified helical-cell-shape-promoting protein complexes containing a peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homologue of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal bacterial proteins. We sought to understand CcmA's function in generating the helical shape of H. pylori cells. Using CcmA deletion analysis, in vitro polymerization, and in vivo co-immunoprecipitation experiments we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with Csd5 and Csd7. We also found that CcmA's N-terminal region inhibits interaction with Csd7. Deleting the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Thus, CcmA helps organize cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate cell wall modification and synthesis, promoting the curvature required to build a helical cell.
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Garde, Shambhavi, Pavan Kumar Chodisetti i Manjula Reddy. "Peptidoglycan: Structure, Synthesis, and Regulation". EcoSal Plus, 20.01.2021. http://dx.doi.org/10.1128/ecosalplus.esp-0010-2020.
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ABSTRACT Peptidoglycan is a defining feature of the bacterial cell wall. Initially identified as a target of the revolutionary beta-lactam antibiotics, peptidoglycan has become a subject of much interest for its biology, its potential for the discovery of novel antibiotic targets, and its role in infection. Peptidoglycan is a large polymer that forms a mesh-like scaffold around the bacterial cytoplasmic membrane. Peptidoglycan synthesis is vital at several stages of the bacterial cell cycle: for expansion of the scaffold during cell elongation and for formation of a septum during cell division. It is a complex multifactorial process that includes formation of monomeric precursors in the cytoplasm, their transport to the periplasm, and polymerization to form a functional peptidoglycan sacculus. These processes require spatio-temporal regulation for successful assembly of a robust sacculus to protect the cell from turgor and determine cell shape. A century of research has uncovered the fundamentals of peptidoglycan biology, and recent studies employing advanced technologies have shed new light on the molecular interactions that govern peptidoglycan synthesis. Here, we describe the peptidoglycan structure, synthesis, and regulation in rod-shaped bacteria, particularly Escherichia coli , with a few examples from Salmonella and other diverse organisms. We focus on the pathway of peptidoglycan sacculus elongation, with special emphasis on discoveries of the past decade that have shaped our understanding of peptidoglycan biology.
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Shlosman, Irina, Elayne M. Fivenson, Morgan S. A. Gilman, Tyler A. Sisley, Suzanne Walker, Thomas G. Bernhardt, Andrew C. Kruse i Joseph J. Loparo. "Allosteric activation of cell wall synthesis during bacterial growth". Nature Communications 14, nr 1 (10.06.2023). http://dx.doi.org/10.1038/s41467-023-39037-9.
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AbstractThe peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division.
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Nygaard, Rie, Chris L. B. Graham, Meagan Belcher Dufrisne, Jonathan D. Colburn, Joseph Pepe, Molly A. Hydorn, Silvia Corradi i in. "Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex". Nature Communications 14, nr 1 (24.08.2023). http://dx.doi.org/10.1038/s41467-023-40483-8.
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AbstractPeptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.
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Li, Franco K. K., Liam J. Worrall, Robert T. Gale, Eric D. Brown i Natalie C. J. Strynadka. "Cryo-EM analysis of S. aureus TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance". Science Advances 10, nr 9 (marzec 2024). http://dx.doi.org/10.1126/sciadv.adj3864.
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Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo–electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate–ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.
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Hugonnet, Jean-Emmanuel, Dominique Mengin-Lecreulx, Alejandro Monton, Tanneke den Blaauwen, Etienne Carbonnelle, Carole Veckerlé, Yves, V. Brun i in. "Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli". eLife 5 (21.10.2016). http://dx.doi.org/10.7554/elife.19469.
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The target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB.
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Teh, Hui Wen, Marimuthu Citartan, Hazrina Yusof Hamdani, Mohamad Zaki Salleh, Lay Kek Teh, Mohd Nur Fakhruzzaman Noorizhab i Thean-Hock Tang. "Identification of potential mutations associated with multidrug resistance among isolates of Mycobacterium tuberculosis in Malaysia by in silico screening". Asia Pacific Journal of Molecular Biology and Biotechnology, 27.12.2023, 49–58. http://dx.doi.org/10.35118/apjmbb.2023.031.4.06.
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The emergence of multidrug resistance tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis (MTB) adaptation to survive in the presence of antibiotic, that were contributed by mutations in the MDR-associated genes. Previous research has indicated that the gene expression knockdown of fhaA leads to an accumulation of peptidoglycan (PG) precursors at the bacillary septum and poles, which suggest a possible deficiency in PG biosynthesis. Consequently, the cell wall becomes resistant to antibiotics, leading to multidrug resistance (MDR). In this study, bioinformatics analyses were performed on MDR-TB isolates from 24 clinical samples to search for novel mutations that contribute to antibiotic resistance. We found out a potential deletion of nucleotides encoding 6 amino acids in all 12 samples, particularly in fhaA gene (RV0020c). Our subsequent structural analysis shows that the deletion is at the position 243-248, causing conformational change of the native FhaA protein. We postulated that the deletion will potentially cause the loss of its binding affinity to MviN (precursor) and STPK (protein kinase), resulting in the inhibition and blockage of the peptidoglycan polymerization, causing MDR in MTB. In the future, experimental validation is necessitated to substantiate the association of these mutations with MDR.
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Sütterlin, Laetitia, Zainab Edoo, Jean-Emmanuel Hugonnet, Jean-Luc Mainardi i Michel Arthur. "Peptidoglycan Cross-Linking Activity of L,D-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams". Antimicrobial Agents and Chemotherapy 62, nr 1 (23.10.2017). http://dx.doi.org/10.1128/aac.01607-17.
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ABSTRACT In most bacteria, the essential targets of β-lactam antibiotics are the d , d -transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l , d -transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l , d -transpeptidase paralogues Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 , which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt Cd2 and Ldt Cd3 catalyzed the formation of 3→3 cross-links ( l , d -transpeptidase activity), the hydrolysis of the C-terminal d -Ala residue of the disaccharide-tetrapeptide substrate ( l , d -carboxypeptidase activity), and the exchange of the C-terminal d -Ala for d -Met. Ldt Cd1 displayed only l , d -carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt Cd1 and Ldt Cd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt Cd3 by these β-lactams was not detected. The acylation efficacy of Ldt Cd1 and Ldt Cd2 was higher for the carbapenems (480 to 6,600 M −1 s −1 ) than for ampicillin and ceftriaxone (3.9 to 82 M −1 s −1 ). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt Cd1 and Ldt Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt Cd1 and Ldt Cd2 are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.
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Stamsås, Gro Anita, Marine Restelli, Adrien Ducret, Céline Freton, Pierre Simon Garcia, Leiv Sigve Håvarstein, Daniel Straume, Christophe Grangeasse i Morten Kjos. "A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae". mBio 11, nr 5 (27.10.2020). http://dx.doi.org/10.1128/mbio.02461-20.
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ABSTRACT Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae. This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape. IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.
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Vélez, Marisela. "How Does the Spatial Confinement of FtsZ to a Membrane Surface Affect Its Polymerization Properties and Function?" Frontiers in Microbiology 13 (3.05.2022). http://dx.doi.org/10.3389/fmicb.2022.757711.
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FtsZ is the cytoskeletal protein that organizes the formation of the septal ring and orchestrates bacterial cell division. Its association to the membrane is essential for its function. In this mini-review I will address the question of how this association can interfere with the structure and dynamic properties of the filaments and argue that its dynamics could also remodel the underlying lipid membrane through its activity. Thus, lipid rearrangement might need to be considered when trying to understand FtsZ’s function. This new element could help understand how FtsZ assembly coordinates positioning and recruitment of the proteins forming the septal ring inside the cell with the activity of the machinery involved in peptidoglycan synthesis located in the periplasmic space.
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Midonet, Caroline, Sean Bisset, Irina Shlosman, Felipe Cava, David Z. Rudner i Thomas G. Bernhardt. "MacP bypass variants of Streptococcus pneumoniae PBP2a suggest a conserved mechanism for the activation of bifunctional cell wall synthases". mBio, 17.10.2023. http://dx.doi.org/10.1128/mbio.02390-23.
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ABSTRACT The peptidoglycan (PG) layer protects bacteria from osmotic lysis and defines their shape. The class A penicillin-binding proteins (aPBPs) are PG synthases that possess both glycan polymerization and crosslinking activities needed for PG biogenesis. In Gram-negative bacteria, aPBPs require activation by outer membrane lipoproteins, which are thought to stimulate their cognate synthase by inducing conformational changes that promote polymerase function. How aPBPs are controlled in Gram-positive bacteria is less clear. One of the few known regulators is MacP in Streptococcus pneumoniae ( Sp ). MacP is required for the activity of Sp PBP2a, but its mode of action has been obscure. We therefore selected for PBP2a variants capable of functioning in the absence of MacP. Amino acid substitutions that bypassed the MacP requirement for PBP2a function in vivo also activated its polymerase activity in vitro . Many of these changes mapped to the interface between the transmembrane (TM) helix and polymerase domain in a model PBP2a structure. This region is conformationally flexible in the experimentally determined structures of aPBPs and undergoes a structural transition upon binding the substrate-mimicking drug moenomycin. Our findings suggest that MacP promotes PG polymerization by altering the TM-polymerase domain interface in PBP2a and that this mechanism for aPBP activation may be broadly conserved. Furthermore, Sp cells expressing an activated PBP2a variant displayed heterogeneous shapes, highlighting the importance of proper aPBP regulation in cell morphogenesis. IMPORTANCE Class A penicillin-binding proteins (aPBPs) play critical roles in bacterial cell wall biogenesis. As the targets of penicillin, they are among the most important drug targets in history. Although the biochemical activities of these enzymes have been well studied, little is known about how they are regulated in cells to control when and where peptidoglycan is made. In this report, we isolate variants of the Streptococcus pneumoniae enzyme PBP2a that function in cells without MacP, a partner normally required for its activity. The amino acid substitutions activate the cell wall synthase activity of PBP2a, and their location in a model structure suggests an activation mechanism for this enzyme that is shared with aPBPs from distantly related organisms with distinct activators.
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Sacco, Emmanuelle, Mélanie Cortes, Nathalie Josseaume, Louis B. Rice, Jean-Luc Mainardi i Michel Arthur. "Serine/Threonine Protein Phosphatase-Mediated Control of the Peptidoglycan Cross-Linking l,d-Transpeptidase Pathway in Enterococcus faecium". mBio 5, nr 4 (8.07.2014). http://dx.doi.org/10.1128/mbio.01446-14.
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ABSTRACTThe last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of β-lactam antibiotics.d,d-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteinel,d-transpeptidases that use tetrapeptides inMycobacterium tuberculosis,Clostridium difficile, and β-lactam-resistant mutants ofEnterococcus faecium. We have investigated reprogramming of theE. faeciumpeptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs byl,d-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of thel,d-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfmat the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of thed,d-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfmalso requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of thel,d-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes.IMPORTANCEThe mechanism of acquisition of high-level ampicillin resistance involving bypass of the penicillin-binding proteins (PBPs) byl,d-transpeptidase Ldtfmwas incompletely understood, as production of tetrapeptide precursors following transcriptional activation of theddclocus by the DdcRS two-component regulatory system was necessary but not sufficient for full activation of thel,d-transpeptidation pathway. Here, we identified the release of a negative control of Ser/Thr protein phosphorylation mediated by phosphatase StpA as the additional factor essential for ampicillin resistance. Thus, bypass of PBPs by Ldtfmrequires the modification of signal transduction regulatory systems without any gain of function by mutational alteration of peptidoglycan biosynthetic enzymes. In contrast, previously characterized mechanisms of antibiotic resistance involve horizontal gene transfer and mutational alteration of drug targets. Activation of thel,d-transpeptidation pathway reported in this study is an unprecedented mechanism of emergence of a new metabolic pathway since it involved the recruitment of preexisting functions following modifications of regulatory circuits.
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Sadecki, Patric W., Alexander M. Justen, Jordan S. Ho i Laura L. Kiessling. "Regiospecificity of Galactan Polymerization by Divergent GlfT2 Orthologs". FASEB Journal 31, S1 (kwiecień 2017). http://dx.doi.org/10.1096/fasebj.31.1_supplement.951.2.
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Despite their ubiquity of polysaccharides, we lack detailed mechanistic understanding toward how carbohydrate polymers are assembled. Unlike the predictable linkage patterns used in other major molecules, polysaccharides can draw upon a large bevy of diverse glycosidic linkages to form complex polymers. To better understand polysaccharide assembly, our research group has turned toward using the mycobacterial galactan as a model. Composed of galactofuranose (Galf) residues, this essential polymer serves as a covalent connector between the mycolic acids and peptidoglycan of mycolated bacteria. The processive glycosyltransferase GlfT2 uses the activated form of galactofuranose, UDP‐Galf, to continuously add residues onto the galactan until reaching a mature length.Due to its virulence, one of the most extensively studied mycobacteria is Mycobacterium tuberculosis (M. tb). M. tb GlfT2 makes alternating β‐(1→5) and β‐(1→6) linkages in the galactan biosynthesis. Previous work from our research group sought to evaluate whether M. tb GlfT2 strictly adheres to this linkage pattern. When given UDP‐Galf with a floro‐ substituted 5 or 6 position, the enzyme is unable to elongate the polymer past two units.1 This suggests that the alternating β‐(1→5), β‐(1→6) linkage pattern is maintained with high fidelity. Curiously, linkage patterns within galactan isolated from other mycolated bacterium, such as Nocardia and Rhodococcus species differ from those in Mycobacterium. Data suggests that Nocardia brasiliensis (N. bras) only synthesizes β‐(1→5) linkages and Rhodococcus equi (R. equi) synthesizes β‐(1→3), β‐(1→5), and β‐(1→6) linkages.2 Since GlfT2 is responsible for much of galactan biosynthesis, we propose that GlfT2 from these organisms contain differences from M. tb GlfT2, allowing them to synthesize galactan with different glycosidic linkages.Our research group is keenly interested in gaining mechanistic insight toward what variation in the active site of GlfT2 leads to control over glycosidic linkage formation. We are specifically interested in understanding differences and similarities to the mechanism used by M. tb GlfT2. Since our research group has demonstrated two ‘DXD’ motifs are critical to GlfT2 catalysis, we seek to examine the active site of Nocardia brasiliensis and Rhodococcus equi GlfT2 by mutating these motifs. We hope that these studies will better illuminate which features of glycosyltransferases guide glycosidic linkage formation.
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Madeswaran, Arumugam, i Premavathi Gunasekaran Midhuna. "In Silico Evaluation Of Some Commercially Available Flavonoids As Galactofuranoyltransferase-2 Inhibitors In The Management Of Tuberculosis". Letters in Drug Design & Discovery 19 (2.02.2022). http://dx.doi.org/10.2174/1570180819666220202155320.
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Background: Background: Galactofuranoyltransferase-2 (GlfT2) enzyme involved in the galactan polymerization of the arabinogalactan (AG) region of the mycolylarabinogalactan-peptidoglycan (mAGP) complex, an important component of the mycobacterial cell wall Objective: Objective: With the existing challenge the study focused into identifying certain commercially available flavonoids through molecular docking studies against the Galactofuranoyltransferase-2 enzyme. Methods: Methods: The initial pharmacokinetic screening was carried out using Lipinski’s rule of 5 with the help of Molinspiration software. In this perspective, Apigenin, Kaempferol, Rutin, Silibinin and Vitexicarpin were selected for the current study. Except for rutin all other selected flavonoids did not show any violations and thereby selected for the docking studies using AutoDock 4.2. Results: Results: The docking results showed that the selected flavonoids have excellent binding energy values between −8.98 to −6.58 kcal/mol against the GlfT2 enzyme. The theoretical inhibition constant was found to be in the range of 260.90 nM to 15.13 µM which coincides with the binding energies of the selected compounds. Conclusion: Conclusion: From the selected flavonoids, Silibinin showed excellent binding scores and it has the potential to inhibit the GlfT2 enzyme. Silibinin could act as a novel GlfT2 inhibitor with promising therapeutic activity with low toxicity profile against tuberculosis