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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. Unexpectedly, only weak interaction was detected with parent neutral β-galactosides including LN-LN-LN and branched (LN)

Work over the past two decades clearly defined a significant role of glycosyltransferase effectors in the infection strategy of the Gram-negative, respiratory pathogen

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. difficile. Our further study on VirB4- and VirD4-like products encoded by CTn4 revealed that both proteins possess Mg(2+)-dependent ATPase activity, form oligomers (most likely hexamers) in aqueous solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. The DNA-binding activity of VirD4 strongly decreased with the W241A variant. Mutations in the nucleotide sequences encoding presumable Walker A and Walker B motifs decreased the stability of the oligomers and significantly but not completely attenuated the enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs neither attenuated the enzymatic activity of the protein nor influenced the oligomerization state of the ATPase. IMPORTANCE C. difficile is a Gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include the toxins TcdA, TcdB, and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered possible virulence factors of C. difficile. In the current paper, we describe the structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to its significance for basic science, can potentially aid the development of antivirulence drugs suitable for the treatment of C. difficile infection.

The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and which has attracted intense interest due to its roles in the pathogenesis of infectious diseases. During the current investigation we uncovered three distinct gene clusters in Clostridioides difficile strain 630 coding for 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 shows that VirB4- and VirD4-like proteins of CTn4 locus, on one hand, and those of CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in physiology of C. difficile. Our further study on VirB4- and VirD4-like products coded by CTn4 revealed that both proteins possess Mg2+-dependent ATPase activity, form oligomers (most probably, hexamers) in water solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. Its DNA binding activity strongly decreased with the W241A variant. Mutations in the nucleotide sequences coding for presumable Walker A and Walker B motifs decreased stability of the oligomers and significantly but not completely attenuated enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs resulted neither in attenuation of enzymatic activity of the protein nor influenced the oligomerization state of the ATPase.ImportanceC. difficile is a Gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include toxins TcdA, TcdB and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered as possible virulence factors of C. difficile. In the current paper we describe structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to significance for basic science, can potentially aid development of anti-virulence drugs suitable for treatment of C. difficile infection.

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. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

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. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l -methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.

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. Exchange of Trp36 to alanine reduced the enzyme activity of SetA. All three amino acids were crucial for the cytotoxic effects of SetA in yeast. We observed that phosphatidylinositol-3-phosphate (PI3P) increased the glucosyltransferase activity of SetA severalfold, while the glucohydrolase activity was not affected. In the presence of PI3P, we observed the glucosylation of actin, vimentin and the chaperonin CCT5 in the cytosolic fraction of target cells. Studies on the functional consequences of glucosylation of skeletal muscle α-actin in vitro revealed inhibition of actin polymerization by glucosylation.

Background:Staphylococcus aureusis a Gram-positive bacterium that causes severe illnesses in the human population. The capacity ofS. aureusstrains 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 concentratedS. aureusliquid culture significantly decreased the development of staphylococcal biofilmin vitro. To discover the corresponding staphylococcal antigen, we used mass-spectrometry and molecular cloning and identified three major immunodominant proteins. They included α-haemolysin, serine proteinase SplB andS. aureussurface protein G, known as adhesin SasG.Results:Although according to literature data, all these proteins represent virulence factors ofS. aureusand play diverse and important roles in the pathogenesis of staphylococcal diseases, only SasG can be directly implicated into the biofilm formation because of its surface location on a staphylococcal cell. Indeed, rabbit serum directed against purified recombinant SasG, similar to serum against crude staphylococcal liquid culture, prevented the formation of a biofilm.Conclusion:SasG can be considered as a target in an anti-biofilm drug development and a component of the vaccine or immunotherapeutic preparations directed against staphylococcal infections in humans.

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 kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL-1 , 4.4 U mL-1 , 1.2 U mL-1 , and 3.4 U mL-1 , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

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. This effector modifies serine-53 in eukaryotic elongation factor 1A (eEF1A) by mono-O-glucosylation. Modification of eEF1A results in inhibition of protein synthesis and death of the eukaryotic cell, processes which are thought to contribute to Legionella infection. Here we describe a protocol for isolation of the glucosyltransferase Lgt1 from L. pneumophila culture followed by assaying its enzymatic activity using

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. This effector modifies serine-53 in eukaryotic elongation factor 1A (eEF1A) by mono-O-glucosylation. Modification of eEF1A results in inhibition of protein synthesis and death of the eukaryotic cell, processes which are thought to contribute to Legionella infection. Here we describe a protocol for isolation of the glucosyltransferase Lgt1 from L. pneumophila culture followed by assaying its enzymatic activity using 14C-UDP-glucose autoradiography.

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. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

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. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the alpha amino group of mono-L-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe, but not in various other eukaryotic organisms tested despite strict conservation of the Glu-45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification, which appears to influence protein translation.

Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast., 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. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62–K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62–K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62–K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.

Summary 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 gly- cosyltransferases and interferes with vesicular trafficking of host cells. Here we show that SetA possesses glycohydrolase and mono-O- glucosyltransferase activity by using UDP-glucose as a donor substrate. Whereas the catalytic activity is located at the N terminus of SetA, the C terminus (amino acids 401-644) is essential for guidance of SetA to vesicular compartments of host cells. EGFP-SetA expressed in HeLa cells localizes to early endosomes by interacting with phosphati- dylinositol 3-phosphate. EGFP-SetA, transiently expressed in RAW 264.7 macrophages, associates with early phagosomes after infection with Escherichia coli and L. pneumophila. Only the combined expression of the C- and N-terminal domains induces growth defects in yeast similar to full-length SetA. The data indicate that SetA is a multidomain protein with an N-terminal glucosyl- transferase domain and a C-terminal phosphati- dylinositol 3-phosphate-binding domain, which guides the Legionella effector to the surface of the Legionella-containing vacuole. Both, the localiza- tion and the glucosyltransferase domains of SetA are crucial for cellular functions.

Aggregatibacter actinomycetemcomitans is a gram negative bacterium and an infectious agent of human diseases with severe oral and extra oral manifestations. One of the major virulence factors in this microorganism is cytolethal distending toxin (CDT). This toxin consists of three subunits—A, B, and C and is able to disrupt cell cycle by intrinsic DNAse activity of its B-subunit. Due to the fact that this protein can represent an important component of diagnostic, prophylactic and therapeutic preparations, production of CDT subunits in preparative quantities is of considerable practical importance. In the current study we demonstrated that deletion of NH2-terminal regions from the molecules of CDT-A, -B, or -C resulted in 20–400-fold increase in production of the corresponding subunits. These truncated molecules were used as immunogens to raise monospecific sera, which were shown in western blot to react specifically with the homologous subunits of cytolethal distending toxin.

Lgt1 is one of the glucosyltransferases produced by the Gram-negative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide 50GKGSFKYAWV59 of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A′ of eEF1A and is part of the first turn of helix A′. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide 50GKGSFKYAWV59 with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBankTM search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment (303GKASFAYAWV312) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an α-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.

Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the ∼60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila , which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella -host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella .

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. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

Legionella pneumophila , the causal agent of Legionnaires' disease, is an intracellular parasite and invades and proliferates within different eukaryotic cells, including human alveolar macrophages. After several 100-fold multiplication within host cells, the pathogens are released for new invasion by induction of apoptosis or necrosis. Here we report that L. pneumophila produces a glucosyltransferase, which selectively modifies an ≈50-kDa mammalian protein by using UDP-glucose as a cosubstrate. MS analysis identified the protein substrate as the mammalian elongation factor (EF)1A. Legionella glucosyltransferase modifies its eukaryotic protein substrate at serine-53, which is located in the GTPase domain of the EF. Glucosylation of EF1A results in inhibition of eukaryotic protein synthesis and death of target cells. Our findings show a mode of inhibition of protein synthesis by microbial pathogens and offer a perspective for understanding of the host-pathogen interaction of L. 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. pneumophila glucosyltransferases Lgt1, Lgt2, and Lgt3. Structurally, these enzymes are similar to large clostridial cytotoxins, use UDP-glucose as a co-substrate and modify a conserved serine residue (Ser-53) in elongation factor 1A (eEF1A). The ternary complex consisting of eEF1A, GTP, and aminoacylated-tRNA seems to be the substrate for Lgts. Studies with Saccharomyces cerevisiae corroborated that eEF1A is the major target responsible for Lgt-induced cytotoxic activity. In addition to Lgt proteins, Legionella produces other effector glycosyltransferase, including the modularly composed protein SetA, which displays tropism for early endosomal compartments, subverts host cell vesicle trafficking and demonstrates toxic activities toward yeast and mammalian cells. Here, our current knowledge about both groups of L. pneumophila glycosylating effectors is reviewed.

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. pneumophila glucosyltransferases Lgt1, Lgt2, and Lgt3. Structurally, these enzymes are similar to large clostridial cytotoxins, use UDP-glucose as a co-substrate and modify a conserved serine residue (Ser-53) in elongation factor 1A (eEF1A). The ternary complex consisting of eEF1A, GTP, and aminoacylated-tRNA seems to be the substrate for Lgts. Studies with Saccharomyces cerevisiae corroborated that eEF1A is the major target responsible for Lgt-induced cytotoxic activity. In addition to Lgt proteins, Legionella produces other effector glycosyltransferase, including the modularly composed protein SetA, which displays tropism for early endosomal compartments, subverts host cell vesicle trafficking and demonstrates toxic activities toward yeast and mammalian cells. Here, our current knowledge about both groups of L. pneumophila glycosylating effectors is reviewed.

The steady-state kinetic parameters of pyridoxal 5-phosphate-dependent recombinant methionine -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in - and -elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the -elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine -lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

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. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.

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. Here we show that SetA possesses glycohydrolase and mono-O-glucosyltransferase activity by using UDP-glucose as a donor substrate. Whereas the catalytic activity is located at the N terminus of SetA, the C terminus (amino acids 401-644) is essential for guidance of SetA to vesicular compartments of host cells. EGFP-SetA expressed in HeLa cells localizes to early endosomes by interacting with phosphatidylinositol 3-phosphate. EGFP-SetA, transiently expressed in RAW 264.7 macrophages, associates with early phagosomes after infection with Escherichia coli and L. pneumophila. Only the combined expression of the C- and N-terminal domains induces growth defects in yeast similar to full-length SetA. The data indicate that SetA is a multidomain protein with an N-terminal glucosyltransferase domain and a C-terminal phosphatidylinositol 3-phosphate-binding domain, which guides the Legionella effector to the surface of the Legionella-containing vacuole. Both, the localization and the glucosyltransferase domains of SetA are crucial for cellular functions.

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. pneumophila. Lgt1 and Lgt2 are produced predominately in the post-exponential phase of bacterial growth, while synthesis of Lgt3 is induced mainly in the lag-phase before intracellular replication of bacteria starts. Lgt glucosyltransferases are structurally similar to clostridial glucosylating toxins. The enzymes use UDP-glucose as a donor substrate and modify eukaryotic elongation factor eEF1A at serine-53. This modification results in inhibition of protein synthesis and death of target cells.In addition to Lgts, Legionella genomes disclose several genes, coding for effector proteins likely to possess glycosyltransferase activities, including SetA (subversion of eukaryotic vesicle trafficking A), which influences vesicular trafficking in the yeast model system and displays tropism for late endosomal/lysosomal compartments of mammalian cells. This review mainly discusses recent results on the structure-function relationship of Lgt glucosyltransferases.

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. Lgt1 shows only a low sequence identity with other type A glycosyltransferases, and structural conservation is limited to a central folding core that is usually observed within this family of proteins. Domains and protrusions added to the core motif represent determinants for the specific recognition and binding of the target. Manual docking experiments based on the crystal structures of toxin and target protein suggest an obvious mode of binding to the target that allows for efficient transfer of a glucose moiety.

Lgt1 is one of the glucosyltransferases produced by the Gram-negative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide (50)GKGSFKYAWV(59) of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A' of eEF1A and is part of the first turn of helix A'. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide (50)GKGSFKYAWV(59) with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBank search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment ((303)GKASFAYAWV(312)) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an alpha-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.

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. These Legionella glucosyltransferases modify the large GTPase elongation factor eEF1A at a serine residue by mono-O-glucosylation, thereby inhibiting protein synthesis of target cells. Recent results on structures, functions and biological roles of both groups of bacterial toxin glucosyltransferases will be discussed.

A panel of mAb (IgG1, IgG3, IgM) against Legionella pneumophila cytolysin (CL)-protease of 37 kDa was obtained. Subtyping of L. pneumophila strains of serogroup 1 by using mAb against CL (mAb-CL) was carried out. The results of comparative analysis of the specificity of mAb-CL and the panel of mAb kindly provided by Dr. J. M. Barbaree (Centers for Disease Control, Atlanta, GA) allowed us to recommend mAb-CL to be used as a diagnostic tool to reveal the pathogenicity of L. pneumophila strains of serogroup 1. Hybridomas were also raised in a syngenic system which produced anti-idiotypic mAb (mAb2) against anti-CL mAb B6/1. The Ab2 belonged to Ab2 gamma type: 1) Ab2 reacted with B6/1 Id only, 2) Ab2 inhibited the interaction of B6/1 Ab1 with CL, and 3) CL inhibited the reaction of Ab2 with Ab1. The use of Ab2 allowed us to show that B6/1 Id is expressed in 4 to 32% of serum antibodies during the primary and secondary immune responses of BALB/c mice to CL. Ab2 induced the production of anti-anti-idiotypic antibodies (Ab3) in BALB/c mice, and some of them reacted with CL. Thus, we have demonstrated the possibility of inducing an antibody response to CL (one of the main L. pneumophila pathogenic factors) in intact syngenic mice with anti-idiotypic antibodies.

Bacteria of the genus Legionella are intracellular parasites and major human pathogens. They bind to surface receptors, penetrate eukaryotic cells and initiate complex disorders during phagocytosis. These disorders include inhibition of oxidative burst, a decrease in phagosome acidification, the blocking of phagosome maturation and changes in organelle trafficking. As a result, the microorganisms prevent the bactericidal activity of the phagocyte and transform the phagosome into a niche for their replication. Biological, biochemical and molecular-genetic approaches have been used to identify a panel of bacterial products that may be involved in Legionella virulence. They include cytotoxins, several enzymes and a set of genes thought to encode proteins of the export machinery. However, despite distinct progress in research, the molecular mechanisms underlying intracellular parasitism in Legionella are unclear.