scholarly journals Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

2021 ◽  
Author(s):  
Harley King ◽  
Sowmya Ajay Castro ◽  
Amol Arunrao Pohane ◽  
Cynthia M. Scholte ◽  
Vincent A. Fischetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Active Sites ◽  
Group A Streptococcus ◽  
Attractive Alternative ◽  
Cell Wall Polysaccharides ◽  
Cell Wall Binding ◽  
Progeny Phage ◽  
Group A ◽  
A Cell ◽  
Species Specific

Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.

scholarly journals Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

2021 ◽  
Author(s):  
Harley King ◽  
Sowmya Ajay Castro ◽  
Juan Bueren-Calabuig ◽  
Amol Arunrao Pohane ◽  
Cynthia M Scholte ◽  
...  
Keyword(s):  
Cell Wall ◽  
Active Sites ◽  
Group A Streptococcus ◽  
Binding Pocket ◽  
Attractive Alternative ◽  
Side Chains ◽  
Cell Wall Polysaccharides ◽  
Progeny Phage ◽  
Group A ◽  
A Cell

Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively. Molecular dynamics simulations reveal a previously unidentified binding pocket that is regulated by a gatekeeper residue and uncover that a previously reported inactive PlyC mutant is locked into a 'closed' conformation. Docking studies with the short GAC backbone oligosaccharides expose potential protein-carbohydrate interactions and are consistent with PlyCB binding to the unmodified pRha or pRha decorated with the GAC side chains.

Electron microscopy of the replicative events of A25 bacteriophages in group A streptococci

1972 ◽  
Vol 18 (1) ◽  
pp. 93-96 ◽  
Author(s):  
S. E. Read ◽  
R. W. Reed
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Electron Microscope ◽  
Nucleic Acid ◽  
Group A Streptococcus ◽  
Group A Streptococci ◽  
Virulent Phage ◽  
Acid Pool ◽  
Group A ◽  
A Cell

The replicative events of a virulent phage (A25) infection of a group A Streptococcus (T253) were studied using the electron microscope. The first intracellular evidence of phage replication in a cell occurred 30 min after infection with arrest of cell division and increase in the nucleic acid pool. Phage heads were evident in the nucleic acid pool of the cells 45 min after infection. Release of phages occurred by splitting of the cell wall along discrete lines. This appeared to be at sites of active wall synthesis, i.e., near the region of septum formation. Many phage components were released but relatively few complete phages indicating a relatively inefficient replicative system.

scholarly journals The structure of Rv3717 reveals a novel amidase fromMycobacterium tuberculosis

2013 ◽  
Vol 69 (12) ◽  
pp. 2543-2554 ◽  
Author(s):  
Atul Kumar ◽  
Sanjiv Kumar ◽  
Dilip Kumar ◽  
Arpit Mishra ◽  
Rikeshwer P. Dewangan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bartonella Henselae ◽  
General Mechanism ◽  
Isolation And Identification ◽  
Cell Wall Binding ◽  
New Family ◽  
A Cell ◽  
Sad Phasing ◽  
Cell Wall Binding Domain ◽  
Net Positive Charge

BacterialN-acetylmuramoyl-L-alanine amidases are cell-wall hydrolases that hydrolyze the bond betweenN-acetylmuramic acid and L-alanine in cell-wall glycopeptides. Rv3717 ofMycobacterium tuberculosishas been identified as a unique autolysin that lacks a cell-wall-binding domain (CBD) and its structure has been determined to 1.7 Å resolution by the Pt-SAD phasing method. Rv3717 possesses an α/β-fold and is a zinc-dependent hydrolase. The structure reveals a short flexible hairpin turn that partially occludes the active site and may be involved in autoregulation. This type of autoregulation of activity of PG hydrolases has been observed inBartonella henselaeamidase (AmiB) and may be a general mechanism used by some of the redundant amidases to regulate cell-wall hydrolase activity in bacteria. Rv3717 utilizes its net positive charge for substrate binding and exhibits activity towards a broad spectrum of substrate cell walls. The enzymatic activity of Rv3717 was confirmed by isolation and identification of its enzymatic products by LC/MS. These studies indicate that Rv3717, anN-acetylmuramoyl-L-alanine amidase fromM. tuberculosis, represents a new family of lytic amidases that do not have a separate CBD and are regulated conformationally.

scholarly journals A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall

2004 ◽  
Vol 186 (17) ◽  
pp. 5865-5875 ◽  
Author(s):  
Timothy C. Barnett ◽  
Aman R. Patel ◽  
June R. Scott
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Surface Protein ◽  
Surface Proteins ◽  
Human Pathogen ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Group A ◽  
Covalently Linked

ABSTRACT The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

scholarly journals STUDIES OF L FORMS AND PROTOPLASTS OF GROUP A STREPTOCOCCI

1963 ◽  
Vol 117 (3) ◽  
pp. 377-399 ◽  
Author(s):  
Earl H. Freimer
Keyword(s):  
Cell Wall ◽  
Membrane Fraction ◽  
Group A Streptococci ◽  
Differential Centrifugation ◽  
Bacterial Membranes ◽  
Group A ◽  
Antigenic Material ◽  
A Cell ◽  
Membrane Antigens ◽  
Gram Positive Cocci

Intact bacterial membranes have been isolated from protoplasts prepared from Group A streptococci by a cell wall-dissolving enzyme. A membrane fraction with identical chemical and serological properties has been obtained by differential centrifugation of mechanically disrupted streptococci. The membrane is chemically distinct from the cell wall and is composed of 72 per cent protein, 26 per cent lipid, and 2 per cent carbohydrate. Capillary precipitin tests and analysis by microdiffusion have demonstrated that the membrane contains antigens distinct from those of the cell wall and from those of the cytoplasm which it envelops. Evidence is presented which demonstrates that this antigenic material is common to the membranes of Group A streptococci, and that it can be distinguished by immunodiffusion from related antigenic substances present in membranes of several other serological groups of hemolytic streptococci. This antigenic material does not cross-react with the membrane antigens of other Gram-positive cocci.

scholarly journals Species-Specific Immunological Reactivities Depend on the Cell-Wall Organization of the Two Aspergillus, Aspergillus fumigatus and A. flavus

2021 ◽  
Vol 11 ◽  
Author(s):  
Sarah Sze Wah Wong ◽  
Lakshmi Prabha Venugopalan ◽  
Audrey Beaussart ◽  
Anupama Karnam ◽  
Mohammed Razeeth Shait Mohammed ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Inflammatory Responses ◽  
Treatment Strategies ◽  
Specific Treatment ◽  
Antifungal Drugs ◽  
Cell Wall Polysaccharides ◽  
Superficial Infection ◽  
Species Specific

Although belong to the same genus, Aspergillus fumigatus is primarily involved in invasive pulmonary infection, whereas Aspergillus flavus is a common cause of superficial infection. In this study, we compared conidia (the infective propagules) of these two Aspergillus species. In immunocompetent mice, intranasal inoculation with conidia of A. flavus resulted in significantly higher inflammatory responses in the lungs compared to mice inoculated with A. fumigatus conidia. In vitro assays revealed that the dormant conidia of A. flavus, unlike A. fumigatus dormant conidia, are immunostimulatory. The conidial surface of A. fumigatus was covered by a rodlet-layer, while that of A. flavus were presented with exposed polysaccharides. A. flavus harbored significantly higher number of proteins in its conidial cell wall compared to A. fumigatus conidia. Notably, β-1,3-glucan in the A. flavus conidial cell-wall showed significantly higher percentage of branching compared to that of A. fumigatus. The polysaccharides ensemble of A. flavus conidial cell wall stimulated the secretion of proinflammatory cytokines, and conidial cell wall associated proteins specifically stimulated IL-8 secretion from the host immune cells. Furthermore, the two species exhibited different sensitivities to antifungal drugs targeting cell wall polysaccharides, proposing the efficacy of species-specific treatment strategies. Overall, the species-specific organization of the conidial cell wall could be important in establishing infection by the two Aspergillus species.

scholarly journals PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides

2021 ◽  
Author(s):  
Jeffrey S. Rush ◽  
Prakash Parajuli ◽  
Alessandro Ruda ◽  
Jian Li ◽  
Amol A. Pohane ◽  
...  
Keyword(s):  
Cell Wall ◽  
Group A Streptococcus ◽  
Bacterial Pathogen ◽  
Antimicrobial Proteins ◽  
Promising Target ◽  
Highly Sensitive ◽  
Streptococcal Species ◽  
Group A ◽  
Base Mechanism ◽  
Nmr Methods

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

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