scholarly journals An Enterococcus faecium Secreted Antigen, SagA, Exhibits Broad-Spectrum Binding to Extracellular Matrix Proteins and Appears Essential for E. faecium Growth

2003 ◽  
Vol 71 (9) ◽  
pp. 5033-5041 ◽  
Author(s):  
Fang Teng ◽  
Magdalena Kawalec ◽  
George M. Weinstock ◽  
Waleria Hryniewicz ◽  
Barbara E. Murray
Keyword(s):  
Extracellular Matrix ◽  
Cell Wall ◽  
Broad Spectrum ◽  
Enterococcus Faecium ◽  
Collagen Type ◽  
Hydrolase Activity ◽  
Cell Wall Metabolism ◽  
Terminal Domain ◽  
Ecm Proteins ◽  
Cell Wall Hydrolase

ABSTRACT A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.

scholarly journals Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

2003 ◽  
Vol 185 (23) ◽  
pp. 6801-6808 ◽  
Author(s):  
Shannon A. Carroll ◽  
Torsten Hain ◽  
Ulrike Technow ◽  
Ayub Darji ◽  
Philippos Pashalidis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Separation ◽  
Bacterial Species ◽  
Surface Protein ◽  
Wild Type ◽  
Terminal Domain ◽  
Cell Wall Hydrolase ◽  
Characteristic Features ◽  
Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

scholarly journals Differential expression of parietal epithelial cell and podocyte extracellular matrix proteins in focal segmental glomerulosclerosis and diabetic nephropathy

2019 ◽  
Vol 317 (6) ◽  
pp. F1680-F1694 ◽  
Author(s):  
Gek Cher Chan ◽  
Diana G. Eng ◽  
Jeffrey H. Miner ◽  
Charles E. Alpers ◽  
Kelly Hudkins ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Diabetic Nephropathy ◽  
Epithelial Cell ◽  
Focal Segmental Glomerulosclerosis ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Type Iv ◽  
Ecm Proteins ◽  
Segmental Glomerulosclerosis ◽  
Parietal Epithelial Cell

In healthy glomeruli, parietal epithelial cell (PEC)-derived extracellular matrix (ECM) proteins include laminin-β1, perlecan, and collagen type IV-α2 and podocyte-specific ECM proteins include laminin-β2, agrin, and collagen type IV-α4. This study aimed to define individual ECM protein isoform expression by PECs in both experimental and human focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN) and to determine if changes were CD44 dependent. In experimental FSGS induced with a cytotoxic podocyte antibody and in the BTBR ob/ob mouse model of DN, PEC-derived protein staining was significantly increased in PECs. Dual staining also showed de novo expression of the podocyte-specific ECM proteins laminin-β2 and agrin in PECs. Similar findings were observed in biopsies from patients with FSGS and DN. Increases in individual ECM proteins colocalized with CD44 in PECs in disease. To determine the role of CD44, FSGS was induced in CD44−/− and CD44+/+ mice. PEC staining for perlecan, collagen type IV-α2, laminin-β2, and agrin were significantly lower in diseased CD44−/− mice compared with diseased CD44+/+ mice. These results show that in experimental and human FSGS and DN, PECs typically in an activated state, produce both PEC-derived and podocyte-specific ECM protein isoforms, and that the majority of these changes were dependent on CD44.

scholarly journals Morphometric analysis of the human endoneurial extracellular matrix components during aging

2021 ◽  
Vol 73 (1) ◽  
pp. 103-110
Author(s):  
Braca Kundalic ◽  
Sladjana Ugrenovic ◽  
Ivan Jovanovic ◽  
Vladimir Petrovic ◽  
Aleksandar Petrovic ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Linear Regression Analysis ◽  
Collagen Type I ◽  
Nerve Fibers ◽  
Collagen Type Iv ◽  
Type I ◽  
Type Iv ◽  
Ecm Proteins ◽  
Extracellular Matrix Components

The aim of this study was to analyze the expression of extracellular matrix (ECM) proteins in human endoneurium during aging. We harvested 15 cadaveric sural nerves, distributed in 3 age groups (I: 25-44, II: 45-64, III: 65-86 years old). Histological sections were stained immunohistochemically for the presence of collagen type I, type IV and laminin, and the ImageJ processing program was used in morphometrical analysis to determine the percentages of these endoneurial proteins. In two younger groups, the endoneurial matrix of the sural nerve was composed from about equal proportions of these proteins, which may be considered a favorable microenvironment for the regeneration of nerve fibers. Linear regression analysis showed a significant increase in endoneurial collagen type IV with age, while collagen type I and laminin significantly decreased during the aging process. In cases older than 65 years, remodeling of the endoneurial matrix was observed to be significantly higher for the presence of collagen type IV, and lower for the expression of collagen type I and laminin. This age-related imbalance of ECM proteins could represent a disadvantageous microenvironment for nerve fiber regeneration in older adults. Our findings contribute to the development of therapeutic approaches for peripheral nerve regeneration.

Effect of wounding on cell wall hydrolase activity in tomato fruit

2006 ◽  
Vol 40 (3) ◽  
pp. 250-255 ◽  
Author(s):  
Thanh Tu Chung ◽  
Gillian West ◽  
Gregory A. Tucker
Keyword(s):  
Cell Wall ◽  
Tomato Fruit ◽  
Hydrolase Activity ◽  
Cell Wall Hydrolase

scholarly journals Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e31588 ◽  
Author(s):  
Ingmar J. J. Claes ◽  
Geert Schoofs ◽  
Krzysztof Regulski ◽  
Pascal Courtin ◽  
Marie-Pierre Chapot-Chartier ◽  
...  
Keyword(s):  
Cell Wall ◽  
Biochemical Characterization ◽  
Lactobacillus Rhamnosus ◽  
Hydrolase Activity ◽  
Secreted Protein ◽  
Lactobacillus Rhamnosus Gg ◽  
Cell Wall Hydrolase

scholarly journals Enterococcus faecalis Adhesin, Ace, Mediates Attachment to Extracellular Matrix Proteins Collagen Type IV and Laminin as well as Collagen Type I

2000 ◽  
Vol 68 (9) ◽  
pp. 5218-5224 ◽  
Author(s):  
Sreedhar R. Nallapareddy ◽  
Xiang Qin ◽  
George M. Weinstock ◽  
Magnus Höök ◽  
Barbara E. Murray
Keyword(s):  
Extracellular Matrix ◽  
Enterococcus Faecalis ◽  
Collagen Type ◽  
Collagen Type I ◽  
Immune Serum ◽  
Collagen Type Iv ◽  
Type I ◽  
Collagen Types ◽  
Type Iv ◽  
Ecm Proteins

ABSTRACT Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ∼105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF ace mutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 μg/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.

scholarly journals Role of the Emp Pilus Subunits of Enterococcus faecium in Biofilm Formation, Adherence to Host Extracellular Matrix Components, and Experimental Infection

2016 ◽  
Vol 84 (5) ◽  
pp. 1491-1500 ◽  
Author(s):  
Maria Camila Montealegre ◽  
Kavindra V. Singh ◽  
Sudha R. Somarajan ◽  
Puja Yadav ◽  
Chungyu Chang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Urinary Tract ◽  
Infective Endocarditis ◽  
Enterococcus Faecium ◽  
Biofilm Formation ◽  
Type I ◽  
Content Type ◽  
Ecm Proteins ◽  
Extracellular Matrix Components ◽  
Tract Infections

Enterococcus faeciumis an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, includingE. faecium. We previously demonstrated that a nonpiliated ΔempABC::catderivative ofE. faeciumTX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion ofempAreduced biofilm formation to the same level as deletion of theempABCoperon, a phenotype that was restored by reconstitutingin situtheempAgene. Deletion ofempBalso caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion ofempAandempB, while deletion ofempChad no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model (P< 0.001 to 0.048), that reconstitution ofempArestored virulence in the UTI model, and that deletion ofempAalso resulted in attenuation in an infective endocarditis model (P= 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important forE. faeciumto cause infection in the urinary tract.

scholarly journals Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of Lactococcus lactis subsp. cremoris

2002 ◽  
Vol 68 (9) ◽  
pp. 4357-4363 ◽  
Author(s):  
A. S. Malone ◽  
T. H. Shellhammer ◽  
P. D. Courtney
Keyword(s):  
High Pressure ◽  
Cell Wall ◽  
Lactococcus Lactis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Suspensions ◽  
Hydrolase Activity ◽  
Cell Extracts ◽  
Cell Wall Hydrolase ◽  
Sds Page

ABSTRACT Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity.

scholarly journals Analysis of the C‐terminal domain of MtlA and its role in cell wall metabolism in the fungus Aspergillus nidulans

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Stewart Nichols ◽  
Matthew M. Cannavo ◽  
Loretta M. Jackson‐Hayes ◽  
Darlene M. Loprete
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Cell Wall Metabolism ◽  
Terminal Domain

scholarly journals A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 245
Author(s):  
Hiroshi Sekiya ◽  
Maho Okada ◽  
Eiji Tamai ◽  
Toshi Shimamoto ◽  
Tadashi Shimamoto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Clostridium Perfringens ◽  
Antimicrobial Agents ◽  
Catalytic Domain ◽  
Food Poisoning ◽  
Binding Activity ◽  
Lytic Activity ◽  
Intestinal Bacterium ◽  
Terminal Domain ◽  
Cell Wall Binding

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

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