scholarly journals Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

2021 ◽  
Vol 22 (17) ◽  
pp. 9536
Author(s):  
Agnieszka Morzywolek ◽  
Magdalena Plotka ◽  
Anna-Karina Kaczorowska ◽  
Monika Szadkowska ◽  
Lukasz P. Kozlowski ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Bioinformatics Analysis ◽  
Analytical Ultracentrifugation ◽  
Lipoteichoic Acid ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Genes Encoding

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

scholarly journals Gram-positive bacteria cell wall-derived lipoteichoic acid induces inflammatory alveolar bone loss through prostaglandin E production in osteoblasts

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsukasa Tominari ◽  
Ayumi Sanada ◽  
Ryota Ichimaru ◽  
Chiho Matsumoto ◽  
Michiko Hirata ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bone Loss ◽  
Alveolar Bone ◽  
Osteoclast Differentiation ◽  
Lipoteichoic Acid ◽  
Alveolar Bone Loss ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

AbstractPeriodontitis is an inflammatory disease associated with severe alveolar bone loss and is dominantly induced by lipopolysaccharide from Gram-negative bacteria; however, the role of Gram-positive bacteria in periodontal bone resorption remains unclear. In this study, we examined the effects of lipoteichoic acid (LTA), a major cell-wall factor of Gram-positive bacteria, on the progression of inflammatory alveolar bone loss in a model of periodontitis. In coculture of mouse primary osteoblasts and bone marrow cells, LTA induced osteoclast differentiation in a dose-dependent manner. LTA enhanced the production of PGE2 accompanying the upregulation of the mRNA expression of mPGES-1, COX-2 and RANKL in osteoblasts. The addition of indomethacin effectively blocked the LTA-induced osteoclast differentiation by suppressing the production of PGE2. Using ex vivo organ cultures of mouse alveolar bone, we found that LTA induced alveolar bone resorption and that this was suppressed by indomethacin. In an experimental model of periodontitis, LTA was locally injected into the mouse lower gingiva, and we clearly detected alveolar bone destruction using 3D-μCT. We herein demonstrate a new concept indicating that Gram-positive bacteria in addition to Gram-negative bacteria are associated with the progression of periodontal bone loss.

scholarly journals Mixed liposomes containing gram-positive bacteria lipids: Lipoteichoic acid (LTA) induced structural changes

2021 ◽  
Vol 199 ◽  
pp. 111551
Author(s):  
Bhavesh Bharatiya ◽  
Gang Wang ◽  
Sarah E. Rogers ◽  
Jan Skov Pedersen ◽  
Stephen Mann ◽  
...  
Keyword(s):  
Structural Changes ◽  
Lipoteichoic Acid ◽  
Gram Positive ◽  
Gram Positive Bacteria

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

scholarly journals Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

2006 ◽  
Vol 401 (1) ◽  
pp. 279-285 ◽  
Author(s):  
Ana L. Stern ◽  
Emmanuel Burgos ◽  
Laurent Salmon ◽  
Juan J. Cazzulo
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Type B ◽  
Nucleotide Synthesis ◽  
Gene Coding ◽  
Genes Encoding

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

scholarly journals Closed Genome Sequence of Clostridium botulinum Strain CFSAN064329 (62A)

2018 ◽  
Vol 6 (26) ◽  
Author(s):  
Travis G. Wentz ◽  
Kuan Yao ◽  
Kristin M. Schill ◽  
N. Rukma Reddy ◽  
Guy E. Skinner ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Food Challenge ◽  
Strictly Anaerobic ◽  
Botulinum Neurotoxin A ◽  
Gram Positive ◽  
Content Type

Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies.

scholarly journals The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

2007 ◽  
Vol 79 (4) ◽  
pp. 649-663 ◽  
Author(s):  
Mariana Igoillo-Esteve ◽  
Dante Maugeri ◽  
Ana L. Stern ◽  
Paula Beluardi ◽  
Juan J. Cazzulo
Keyword(s):  
Oxidative Stress ◽  
Trypanosoma Cruzi ◽  
Pentose Phosphate Pathway ◽  
Single Copy ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Haploid Genome ◽  
Salt Bridges ◽  
Phosphogluconate Dehydrogenase ◽  
Genes Encoding

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

scholarly journals Gram Positive Bacterial Lipoteichoic Acid Role in a Root Canal Infection – A Literature Review

2021 ◽  
Author(s):  
Vinoo Subramaniam Ramachandran ◽  
Mensudar Rathakrishnan ◽  
Malathy Balaraman Ravindrran ◽  
Alargarsamy Venkatesh ◽  
Vidhya Shankari Shanmugasundaram ◽  
...  
Keyword(s):  
Cell Wall ◽  
Immune Surveillance ◽  
Lipoteichoic Acid ◽  
The Body ◽  
Gram Positive ◽  
Gram Negative ◽  
Cell Wall Components ◽  
Root Canals ◽  
Endodontic Infection ◽  
Wall Components

Bacteria and its by-products are found to be the main cause of pulpal and periapical infection of tooth. Infected root canals of tooth harbours a wide variation of microbial flora that includes both Gram-positive and Gram-negative microorganisms. Bacterial components such as Lipopolysaccharide (LPS) of gram negative bacteria and Lipoteichoic Acid (LTA) of gram positive bacteria have the potential to enter the peri-apical tissue of tooth and initiate the inflammatory process. After microbial death that occurs either due to body’s defence cells or by antibiotic action, bacterial cell wall components such as LTA are released which can persist inside macrophages for prolonged periods causing chronic inflammation. Once these cell-wall components are recognized by the body immune surveillance cells, numerous inflammatory mediators are released leading to inflammation and subsequent pathological consequences. The purpose of this review is intend to summarize the role of gram positive bacterial component LTA in causing endodontic infection and use of potential therapeutic agents against LTA.

scholarly journals A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

2021 ◽  
Vol 11 ◽  
Author(s):  
Orla M. Dunne ◽  
Xin Gao ◽  
Ruodan Nan ◽  
Jayesh Gor ◽  
Penelope J. Adamson ◽  
...  
Keyword(s):  
Host Cell ◽  
Analytical Ultracentrifugation ◽  
Dissociation Constants ◽  
Alternative Pathway ◽  
Factor H ◽  
Regulatory Control ◽  
Size Exclusion ◽  
Complement Factor ◽  
Dimer Formation ◽  
Self Association

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

scholarly journals Functional CBS modules make IMPDHs octameric

2014 ◽  
Vol 70 (a1) ◽  
pp. C1283-C1283
Author(s):  
Gilles Labesse ◽  
Thomas Alexandre ◽  
Laurène Vaupré ◽  
Isabelle Salard-Arnaud ◽  
Joséphine Lai Kee Him ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Binding Pockets ◽  
Essential Enzyme ◽  
Cbs Domains

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

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