scholarly journals Preliminary crystallographic study of theStreptococcus agalactiaesortases, sortase A and sortase C1

2010 ◽  
Vol 66 (9) ◽  
pp. 1096-1100 ◽  
Author(s):  
Baldeep Khare ◽  
Alexandra Samal ◽  
Krishnan Vengadesan ◽  
K. R. Rajashankar ◽  
Xin Ma ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Substrate Specificity ◽  
Streptococcus Agalactiae ◽  
Crystal Form ◽  
Sortase A ◽  
Crystal Forms ◽  
Gram Positive Bacteria ◽  
Crystallographic Study ◽  
Surface Adhesins

Sortases are cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. InStreptococcus agalactiae(GBS), the pilin-specific sortase SrtC1 catalyzes the polymerization of pilins encoded by pilus island 1 (PI-1) and the housekeeping sortase SrtA is necessary for cell-wall anchoring of the resulting pilus polymers. These sortases are known to utilize different substrates for pilus polymerization and cell-wall anchoring; however, the structural correlates that dictate their substrate specificity have not yet been clearly defined. This report presents the expression, purification and crystallization of SrtC1 (SAG0647) and SrtA (SAG0961) fromS. agalactiaestrain 2603V/R. The GBS SrtC1 has been crystallized in three crystal forms and the GBS SrtA has been crystallized in one crystal form.

scholarly journals Sortase A Utilizes an Ancillary Protein Anchor for Efficient Cell Wall Anchoring of Pili in Streptococcus agalactiae

2008 ◽  
Vol 76 (8) ◽  
pp. 3550-3560 ◽  
Author(s):  
Angela H. Nobbs ◽  
Roberto Rosini ◽  
C. Daniela Rinaudo ◽  
Domenico Maione ◽  
Guido Grandi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Streptococcus Agalactiae ◽  
Culture Supernatant ◽  
Sortase A ◽  
Deletion Mutants ◽  
Vaccine Candidates ◽  
Detergent Treatment ◽  
Group B ◽  
Pilus Assembly

ABSTRACT Pili are putative virulence factors and promising vaccine candidates in Streptococcus agalactiae (group B Streptococcus [GBS]) infection, a leading cause of neonatal sepsis and meningitis. The genes necessary for pilus synthesis and assembly are clustered in pilus islands (PI). Each gene encodes three structural subunits (a backbone and two ancillary proteins) bearing a C-terminal LPXTG motif and two subfamily C sortases (SrtC) involved in covalent polymerization of the subunits. GBS strains also possess the conserved “housekeeping” sortase A (SrtA), but its role in pilus assembly is unclear. To address this issue, pilus expression and cell wall anchoring were analyzed in srtA deletion mutants. Loss of SrtA did not affect pilus polymerization. However, pilus expression on the cell surface was reduced, and pili accumulated in the culture supernatant. Furthermore, cell-associated pili could be readily released by detergent treatment, indicating that SrtA is involved in covalent anchoring of pili to the cell wall. When each of the genes comprising PI-2a was systematically deleted, only the absence of ancillary subunit GBS150 or the SrtC required for incorporation of GBS150 into pili mimicked the srtA mutant phenotype. Thus, from these data a model for GBS pilus assembly can be proposed in which PI sortases are responsible for polymerization of the pilus structure, while SrtA is required to covalently attach it to the cell wall, utilizing ancillary pilus subunit GBS150 as the anchor protein.

Covalent Sortase A Inhibitor ML346 Prevents Staphylococcus aureus Infection of Galleria mellonella

2021 ◽  
Author(s):  
Xiang-Na Guan ◽  
Tao Zhang ◽  
Teng Yang ◽  
Ze Dong ◽  
Song Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Galleria Mellonella ◽  
Surface Proteins ◽  
Sortase A ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Aureus Infection ◽  
Cell Wall Peptidoglycan

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation...

Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria

2015 ◽  
Vol 43 (5) ◽  
pp. 787-794 ◽  
Author(s):  
Edward N. Baker ◽  
Christopher J. Squire ◽  
Paul G. Young
Keyword(s):  
Cell Surface ◽  
Mechanical Strength ◽  
Cell Walls ◽  
Side Chains ◽  
Structural Studies ◽  
Gram Positive Bacteria ◽  
Intramolecular Reactions ◽  
Isopeptide Bonds ◽  
Cross Links ◽  
Surface Adhesins

The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength.

scholarly journals Novel Sortase A Inhibitors to Counteract Gram-Positive Bacterial Biofilms

Proceedings ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 23
Author(s):  
Maria Valeria Raimondi ◽  
Roberta Listro ◽  
Maria Grazia Cusimano ◽  
Mery La Franca ◽  
Teresa Faddetta ◽  
...  
Keyword(s):  
Cell Wall ◽  
Surface Proteins ◽  
Bacterial Biofilms ◽  
Sortase A ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Membrane Enzyme

Sortase A (SrtA) is a membrane enzyme responsible for the covalent anchoring of surface proteins on the cell wall of Gram-positive bacteria. [...]

The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 643-651 ◽  
Author(s):  
Christina Schäffer ◽  
Paul Messner
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Secondary Cell Wall ◽  
Chemical Study ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Bacterial Cell Surface ◽  
Cell Wall Polymers ◽  
Structural Principles

The cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the past five decades. Outside the cytoplasmic membrane of these organisms the fundamental polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic acids are linked to some of the peptidoglycan chains. In this review these compounds are considered as ‘classical’ cell wall polymers. In the course of recent investigations of bacterial cell surface layers (S-layers) a different class of ‘non-classical’ secondary cell wall polymers (SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell surface. Comparative analyses have shown considerable differences in chemical composition, overall structure and charge behaviour of these SCWPs. This review discusses the progress that has been made in understanding the structural principles of SCWPs, which may have useful applications in S-layer-based ‘supramolecular construction kits' in nanobiotechnology.

scholarly journals AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion

2021 ◽  
Vol 12 ◽  
Author(s):  
Lei Sun ◽  
Gil Rogiers ◽  
Pascal Courtin ◽  
Marie-Pierre Chapot-Chartier ◽  
Hélène Bierne ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Diaminopimelic Acid ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
First Time

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

scholarly journals Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

PLoS ONE ◽  
2011 ◽  
Vol 6 (10) ◽  
pp. e25300 ◽  
Author(s):  
Francesca Necchi ◽  
Vincenzo Nardi-Dei ◽  
Massimiliano Biagini ◽  
Michael Assfalg ◽  
Annalisa Nuccitelli ◽  
...  
Keyword(s):  
Cell Wall ◽  
Substrate Specificity ◽  
Sortase A

scholarly journals C-Terminal WxL Domain Mediates Cell Wall Binding in Enterococcus faecalis and Other Gram-Positive Bacteria

2006 ◽  
Vol 189 (4) ◽  
pp. 1244-1253 ◽  
Author(s):  
Sophie Brinster ◽  
Sylviane Furlan ◽  
Pascale Serror
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Enterococcus Faecalis ◽  
Surface Proteins ◽  
Reporter Protein ◽  
Gram Positive ◽  
Whole Cells ◽  
Gram Positive Bacteria ◽  
Cell Wall Binding ◽  
Genes Encoding

ABSTRACT Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.

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