Revealing O-acetylhomoserine sulfhydrylase involved in direct sulfhydrylation pathway in Clostridium tetani.

Bacterial methionine biosynthesis is an attractive target for research due to its central role in cellular metabolism, as most steps of this pathway are missing in mammals. Up to now little is known about sulfur metabolism in pathogenic Clostridia species, making the study of the enzymes of Cys/Met metabolism in Clostridium tetani particularly relevant. Analysis of the C.

Structural insight into recognition of Clostridioides difficile toxin A by novel neutralizing nanobodies targeting QTIN-like motifs within its receptor-binding domain.

Clostridioides difficile causes a large proportion of nosocomial colon infections by producing toxins TcdA and TcdB as key virulence factors. TcdA and TcdB have analogous domain structures with a receptor-binding domain containing C-terminal combined repetitive oligopeptides (CROPs), an attractive target for the development of therapeutic antibodies. Here, we identify and characterize two potent neutralizing single-domain camelid anti-CROPsA antibodies, C4.

Oligomerization and Adjuvant Activity of Peptides Derived from the VirB4-like ATPase of .

In a previous study, we demonstrated that the VirB4-like ATPase forms oligomers in vitro. In the current investigation, to study the observed phenomenon in more detail, we prepared a library of VirB4-derived peptides (delVirB4s) fused to a carrier maltose-binding protein (MBP). Using gel chromatography and polyacrylamide gel electrophoresis, we found a set of overlapping fragments that contribute most significantly to protein aggregation, which were represented as water-soluble oligomers with molecular masses ranging from ~300 kD to several megadaltons.

Research: Still Many Miles to Go.

is a widespread Gram-negative bacterium occurring in water reservoirs and soils [...

Specificity of viscumin revised. As probed with a printed glycan array.

Viscumin, a lectin used in anti-cancer therapy, was originally considered as βGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galβ1-4GlcNAc, was observed.

Glycosylating Effectors of Finding the Sweet Spots for Host Cell Subversion.

Work over the past two decades clearly defined a significant role of glycosyltransferase effectors in the infection strategy of the Gram-negative, respiratory pathogen . Identification of the glucosyltransferase effectors Lgt1-3, specifically modifying elongation factor eEF1A, disclosed a novel mechanism of host protein synthesis manipulation by pathogens and illuminated its impact on the physiological state of the target cell, in particular cell cycle progression and immune and stress responses. Recent characterization of SetA as a general O-glucosyltransferase with a wide range of targets including the proteins Rab1 and Snx1, mediators of membrane transport processes, and the discovery of new types of glycosyltransferases such as LtpM and SidI indicate that the vast effector arsenal might still hold more so-far unrecognized family members with new catalytic features and substrates.

VirB4- and VirD4-Like ATPases, Components of a Putative Type 4C Secretion System in Clostridioides difficile.

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and has attracted intense interest due to its roles in the pathogenesis of infectious diseases. In the current investigation, we uncovered three distinct gene clusters in Clostridioides difficile strain 630 encoding proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis revealed that VirB4- and VirD4-like proteins of the CTn4 locus, on the one hand, and those of the CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in the physiology of C.

O-acetylhomoserine sulfhydrylase from Clostridium novyi. Cloning, expression of the gene and characterization of the enzyme.

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit.

Targeting Eukaryotic mRNA Translation by .

is a gram-negative microorganism and an infectious agent of pneumonia in humans. It is an intracellular pathogen and multiplies in different eukaryotic cells like amoebae, ciliated protozoa, macrophages, monocytes, and lung epithelial cells. Proliferation of in eukaryotic cells depends on its type 4 secretion system, which delivers an arsenal of bacterial effector proteins to cytoplasm of its host.

Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa.

Purification and Analysis of Effector Glucosyltransferase Lgt1 from Legionella pneumophila.

Legionella pneumophila is a facultative intracellular pathogen responsible for legionellosis, a severe lung disease in humans. This bacterium uses a type 4b secretion system to deliver various effector proteins into the cytoplasm of a eukaryotic target cell. Among those is the glucosyltransferase Lgt1.

Characterization of the glucosyltransferase activity of Legionella pneumophila effector SetA.

Legionella pneumophila glucosyltransferase SetA, which is introduced into target cells by a type IV secretion system, affects the intracellular traffic of host cells. Here, we characterized the enzyme activity of the Legionella effector. We report that Asp118 and Arg121 of SetA are essential for glucohydrolase and glucotransferase activities.

Surface Protein G is An Immunodominant Protein and a Possible Target in An Anti-Biofilm Drug Development.

Background: is a Gram-positive bacterium that causes severe illnesses in the human population. The capacity of strains to form biofilms on biotic and abiotic surfaces creates serious problems for treatment of hospital infections and has stimulated efforts to develop new means of specific protection or immunotherapy.

Material And Methods: We found that rabbit serum raised against crude concentrated liquid culture significantly decreased the development of staphylococcal biofilm .

Protein glutaminylation is a yeast-specific posttranslational modification of elongation factor 1A.

Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified.

Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits k and k /K for methionine γ-elimination reaction that are 2.

Ribosomal Protein Rps26 Influences 80S Ribosome Assembly in Saccharomyces cerevisiae.

The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5' untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62-K70) motif.

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction.

Bacterial glycosyltransferase toxins.

Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia.

Cytotoxic glucosyltransferases of Legionella pneumophila.

Legionella is a gram-negative bacterium and the causative pathogen of legionellosis-a severe pneumonia in humans. A large number of Legionella effectors interfere with numerous host cell functions, including intracellular vacuole trafficking and maturation, phospholipid metabolism, protein ubiquitination, pro-/anti-apoptotic balances or inflammatory responses. Moreover, eukaryotic protein synthesis is affected by L.

Elongation factor 1A is the target of growth inhibition in yeast caused by Legionella pneumophila glucosyltransferase Lgt1.

Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death.

Domain organization of Legionella effector SetA.

Legionella pneumophila is a human pathogen causing severe pneumonia called Legionnaires' disease. Multiple Legionella effectors are type IV-secreted into the host cell to establish a specific vesicular compartment for pathogen replication. Recently, it has been reported that the Legionella effector SetA shares sequence similarity with glycosyltransferases and interferes with vesicular trafficking of host cells.

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases.

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis.

Effector glycosyltransferases in legionella.

Legionella causes severe pneumonia in humans. The pathogen produces an array of effectors, which interfere with host cell functions. Among them are the glucosyltransferases Lgt1, Lgt2 and Lgt3 from L.

Structural basis of the action of glucosyltransferase Lgt1 from Legionella pneumophila.

The glucosyltransferase Lgt1 is one of three glucosylating toxins of Legionella pneumophila, the causative agent of Legionnaires disease. It acts through specific glucosylation of a serine residue (S53) in the eukaryotic elongation factor 1A and belongs to type A glycosyltransferases. High-resolution crystal structures of Lgt1 show an elongated shape of the protein, with the binding site for uridine disphosphate glucose at the bottom of a deep cleft.

Bacterial toxin and effector glycosyltransferases.

Clostridial glucosylating cytotoxins, including Clostridium difficile toxins A and B, Clostridium novyi alpha-toxin, and Clostridium sordellii lethal toxin, are major virulence factors and causative agents of human diseases. These toxins mono-O-glucosylate (or mono-O-GlcNAcylate) a specific threonine residue of Rho/Ras-proteins, which is essential for the function of the molecular switches. Recently, a related group of glucosyltransferases from Legionella pneumophila has been identified.