Effect of naturally occurring variations of the F-type lectin sequence motif on glycan binding: studies on F-type lectin domains with typical and atypical sequence motifs.

The typical F-type lectin domain (FLD) has an L-fucose-binding motif [HX(26)RXDX(4)R/K] with conserved basic residues that mediate hydrogen bonding with alpha-L-fucose. About one-third of the nonredundant FLD sequences in the publicly available databases are "atypical"; they have motifs with substitutions of these critical residues and/or variations in motif length. We addressed the question if atypical FLDs with substitutions of the critical residues retain lectin activity by performing site-directed mutagenesis and assessing the glycan-binding functions of typical and atypical FLDs. Site directed mutagenesis of an L-fucose-binding FLD from Streptosporangium roseum indicated that the critical His residue could be replaced by Ser and the second Arg by Lys without complete loss of lectin activity. Mutagenesis of His to other naturally substituting residues and mutagenesis of the first Arg to the naturally substituting residues, Lys, Ile, Ser, or Cys, resulted in loss of lectin activity. Glycan binding analysis and site-directed mutagenesis of atypical FLDs from Actinomyces turicensis, and Saccharomonospora cyanea confirmed that Ser and Thr can assume the L-fucose-binding role of the critical His, and further suggested that the residue in this position is dispensable in certain FLDs. We identified, by sequence and structural analysis of atypical FLDs, a Glu residue in the complementarity determining region, CDR5 that compensates for a lack of the critical His or other appropriate polar residue in this position. We propose that FLDs lacking a typical FLD sequence motif might nevertheless retain lectin activity through the recruitment of other strategically positioned polar residues in the CDR loops. © 2018 IUBMB Life, 71(3):385-397, 2019.

Replacing the 238th aspartic acid with an arginine impaired the oligomerization activity and inflammation-inducing property of pyolysin.

Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1ß, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO. ABBREVIATIONS: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-ß-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin-Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1ß: interleukin-1ß; IFN-γ: interferon-γ; TGF-ß: transforming growth factor-ß; ELISA: enzyme-linked immunosorbent assay.

Identification of Trueperella pyogenes isolated from bovine mastitis by Fourier transform infrared spectroscopy.

The present study was designed to investigate the potential of Fourier transform infrared (FT-IR) spectroscopy to identify Trueperella (T.) pyogenes isolated from bovine clinical mastitis. FT-IR spectroscopy was applied to 57 isolates obtained from 55 cows in a period from 2009 to 2012. Prior to FT-IR spectroscopy these isolates were identified by phenotypic and genotypic properties, also including the determination of seven potential virulence factor encoding genes. The FT-IR analysis revealed a reliable identification of all 57 isolates as T. pyogenes and a clear separation of this species from the other species of genus Trueperella and from species of genus Arcanobacterium and Actinomyces. The results showed that all 57 isolates were assigned to the correct species indicating that FT-IR spectroscopy could also be efficiently used for identification of this bacterial pathogen.

Comparative chemotaxonomic and phylogenetic studies on the genus Arcanobacterium Collins et al. 1982 emend. Lehnen et al. 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium.

The results of a study comparing the chemotaxonomic characteristics and phylogenetic positions of members of the genus Arcanobacterium indicated that the genus was not monophyletic and, therefore, was in need of taxonomic revision. Phylogenetically, the genus Arcanobacterium consisted of two distinct lines; a group comprising the species Arcanobacterium haemolyticum (the type species of the genus), A. hippocoleae, A. phocae and A. pluranimalium and a robust group consisting of the species A. abortisuis, A. bernardiae, A. bialowiezense, A. bonasi and A. pyogenes. On the basis of 16S rRNA signature nucleotide comparisons and menaquinone and phospholipid compositions, it is proposed that of these nine species only four, A. haemolyticum, A. hippocoleae, A. phocae and A. pluranimalium, should be affiliated with the genus Arcanobacterium and the species A. abortisuis, A. bernardiae, A. bialowiezense, A. bonasi and A. pyogenes should be reclassified as members of a new genus, Trueperella, as Trueperella abortisuis comb. nov., Trueperella bernardiae comb. nov., Trueperella bialowiezensis comb. nov., Trueperella bonasi comb. nov. and Trueperella pyogenes comb. nov. Positive results in Christie-Atkins-Munch-Petersen (CAMP) tests on A. haemolyticum, A. hippocoleae, A. phocae and A. pluranimalium also supported the rearrangement of the nine species in to separate genera. As such, an emended description of the genus Arcanobacterium is provided.

Functional and structural properties of CbpA, a collagen-binding protein from Arcanobacterium pyogenes.

Arcanobacterium pyogenes, an opportunistic pathogen of economically important food animals, is the causative agent of liver abscesses in feedlot cattle, osteomyelitis in turkeys, and pneumonia and arthritis in pigs. Previous studies identified the first A. pyogenes adhesin, CbpA, a protein located on the bacterial surface which has the ability to bind collagen and promotes adhesion to the host cells. The protein has an N-terminal ligand-binding region (region A) and a C-terminal repetitive domain (region B). In this study we found that CbpA bound to almost all the collagen types tested but not to other proteins, and it displayed a propensity to interact with several collagenous peptides derived by CNBr cleavage of type I and II collagens. The K(D) values of CbpA for type I and II collagens and collagen peptides determined by solid-phase binding assay and intrinsic tryptophan fluorescence were in the range of 1-15 nM. It was also found that CbpA and its A region bound fibronectin, and that collagen and fibronectin interacted with distinct subsites. Anti-CbpA antibodies were effective at inhibiting both binding of isolated CbpA and bacterial adhesion to immobilized collagen, suggesting that CbpA is a functional collagen-binding adhesin. Analysis of the immunological cross-reactivity of CbpA with antibodies against other bacterial collagen-binding proteins indicated that CbpA is immunologically related to ACE from Enterococcus faecalis but not to CNA from Staphylococcus aureus or Acm from Enterococcus faecium. Far-UV and near-UV circular dichroism spectra showed that full-length CbpA and its region A are mainly composed of beta-sheet with only a minor alpha-helical component and that both the proteins have a well-defined tertiary structure.

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo.

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

Arcanobacterium bialowiezense sp. nov. and Arcanobacterium bonasi sp. nov., isolated from the prepuce of European bison bulls (Bison bonasus) suffering from balanoposthitis, and emended description of the genus Arcanobacterium Collins et al. 1983.

A taxonomic study was performed on 13 bacterial strains isolated from preputial swabs of European bison (Bison bonasus) bulls suffering from balanoposthitis. The isolates were Gram-positive, non-motile, facultatively anaerobic, diphtheroid-shaped cells. Based on biochemical profiles and BOX-PCR-generated genomic fingerprints, the isolates were grouped into two clusters represented by four and nine strains, respectively. Strains 1(W3/01)T and 2(W106/04)T, selected as representatives of the two clusters, shared 97.2 % 16S rRNA gene sequence similarity. The highest gene sequence similarities found (95.5-96.4 %) were to Arcanobacterium pyogenes DSM 20630T and Arcanobacterium bernardiae DSM 9152T, demonstrating that the novel strains are members of the genus Arcanobacterium, but are not members of a recognized species. The polar lipid profiles of the two novel strains displayed the major characteristics also found in A. pyogenes DSM 20630T and Arcanobacterium haemolyticum DSM 20595T. Detection of a quinone system with MK-10(H4) as the predominant compound confirmed phylogenetic relatedness of the novel strains to A. pyogenes and separated them from the type species of the genus, A. haemolyticum, which contains MK-9(H4) as the predominant quinone. Results from DNA-DNA hybridizations clearly demonstrated that strains 1(W3/01)T and 2(W106/04)T represent separate species. Based on these data, two novel species of the genus Arcanobacterium are described, for which the names Arcanobacterium bialowiezense sp. nov. [type strain 1(W3/01)T = DSM 17162T = NCTC 13354T] and Arcanobacterium bonasi sp. nov. [type strain 2(W106/04)T = DSM 17163T = NCTC 13355T] are proposed.

Siderophore mediated plutonium accumulation by Microbacterium flavescens (JG-9).

Uptake of plutonium and uranium mediated by the siderophore desferrioxamine-B (DFOB) has been studied for the common soil aerobe Microbacterium flavescens(JG-9). M. flavescens does not bind or take up nitrilotriacetic acid (NTA) complexes of U(VI), Fe(III), or Pu(IV) or U(VI)-DFOB but does take up Fe(III)-DFOB and Pu(IV)-DFOB. Pu(IV)-DFOB and Fe(III)-DFOB accumulations are similar: only living and metabolically active bacteria take up these metal-siderophore complexes. The Fe(III)-DFOB and Pu(IV)-DFOB complexes mutually inhibit uptake of the other, indicating that they compete for shared binding sites or uptake proteins. However, Pu uptake is much slower than Fe uptake, and cumulative Pu uptake is less than Fe, 1.0 nmol of Fe vs 0.25 nmol of Pu per mg of dry weight bacteria. The Pu(IV)-DFOB interactions with M. flavescens suggest that Pu-siderophore complexes could generally be recognized by Fe-siderophore uptake systems of many bacteria, fungi, or plants, thereby affecting Pu environmental mobility and distribution. The results also suggest that the siderophore complexes of tetravalent metals can be recognized by Fe-siderophore uptake proteins.

The role of ABC transporters in antibiotic-producing organisms: drug secretion and resistance mechanisms.

Knowledge about biosynthetic gene clusters from antibiotic-producing actinomycetes is continuously increasing and the presence of an ABC transporter system is a fairly general phenomenon in most of these clusters. These transporters are involved in the secretion of the antibiotic through the cell membrane and also contribute to self resistance to the produced antibiotic.

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica.

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50,805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

ABC transporters in antibiotic-producing actinomycetes.

Many antibiotic-producing actinomycetes possess at least one ABC (ATP-binding cassette) transporter which forms part of the antibiotic biosynthetic pathway and in most cases confers resistance to the drug in an heterologous host. Three types of antibiotic ABC transporters have been so far described in producer organisms. In Type I two genes are involved, one encoding a hydrophilic ATP-binding protein with one nucleotide-binding domain and the other encoding a hydrophobic membrane protein. In Type II transporters only a gene encoding the hydrophilic ATP-binding protein with two nucleotide-binding domains is present and no gene encoding a hydrophobic membrane protein has been found. In Type III only one gene is involved which encodes both the hydrophilic and hydrophobic components. Possibly these ABC transporters are responsible for secretion of the antibiotics outside the cells. A comparative analysis of the ATP-binding components of the different antibiotic ABC transporters and analysis of the amino acid distances between the so-called Walker motifs suggests that the three types of transporters have probably evolved from a common ancestor containing a single nucleotide-binding domain.

Selection of suitable marker genes for the development of cloning vectors and electroporation in different strains of Amycolatopsis mediterranei.

To select suitable genetic markers for optimizing electroporation efficiency in Amycolatopsis mediterranei, thiostrepton (tsr), erythromycin (ermE) and apramycin (am) resistance genes were used. Although tsr could not be suitably expressed in A. mediterranei, the cloning of ermE in pRL1 or its derivative (containing am) resulted in the development of cloning vectors pRLM20, pRLM30 and pRL90. In contrast to tsr and km (kanamycin resistance gene), ermE and am were suitably expressed in A. mediterranei strains and no spontaneous mutants were observed among transformants. Under optimum conditions, maximum electroporation efficiency of 1.2 x 10(4) transformants/micrograms DNA was achieved for A. mediterranei DSM 40,773. These plasmids could also be effectively transferred in other strains of A. mediterranei including F1/24 and T-195. With the cloning of ermE and am and their expression in different strains of Amycolatopsis, we have overcome the problem of the choice of suitable selectable markers for A. mediterranei and related species.

Amphistin, a new melanogenesis inhibitor, produced by an actinomycete.

A new melanogenesis inhibitor, named amphistin, was isolated from the fermentation broth of an actinomycete strain KP-3052. Amphistin was purified from the culture filtrate by the combination of cation exchange, gel filtration, and aminosilyl silica gel chromatographic methods. The structure of amphistin was elucidated as gamma-(beta-histidinoalanino)homoalanine by NMR experiments including 1H-15N HMBC experiment and other spectroscopic analyses. Amphistin inhibited the melanogenesis of B16 melanoma cells at concentration of 6.8 microM.

Demetria terragena gen. nov., sp. nov., a new genus of actinomycetes isolated from compost soil.

A novel actinomycete was isolated from compost soil and was studied taxonomically and phylogenetically. Cells of this organism were gram positive, not acid fast, nonmotile, nonsporulating, irregular coccoid to short rod shaped, and microaerophilic. The cell wall peptidoglycan contained lysine and was cross-linked via an L-Lys<--L-Ser<--D-Asp interpeptide bridge. The major menaquinone was MK-8(H4). The polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and two unknown phospholipids. Mycolic acids were absent. The cellular fatty acid profile was complex, with large amounts of saturated and monounsaturated straight-chain acids and smaller amounts of iso and anteiso branched-chain acids. The G+C content of the DNA was 66 mol%. Comparative 16S ribosomal DNA studies revealed that strain HKI 0089T represents a novel lineage within Actinobacteria (32) distinct from all previously described genera and most closely related to members of the genera Kytococcus, Dermacoccus, and Dermatophilus of the family Dermatophilaceae. On the basis of our results, we suggest that strain HKI 0089 should be classified in a new genus and species, for which we propose the name Demetria terragena. The type strain and the only strain of the genus and species is HKI 0089 (DSM 11295).

Terracoccus luteus gen. nov., sp. nov., an LL-diaminopimelic acid-containing coccoid actinomycete from soil.

A gram-positive, aerobic actinomycete was isolated from soil. Spherical cells of this organism occur singly or form packets, which may cluster. The diagnostic diamino acid of the cell wall peptidoglycan is LL-diaminopimelic acid. The predominate menaquinone is MK-8 (H4), and the main fatty acids are 13-methyl tetradecanoic acid and 12-methyl tetradecanoic acid. The diagnostic polar lipids are phosphatidylethanolamine and phosphatidylinositol. The DNA base composition is 73 mol% G + C. Comparison of 16S ribosomal DNA sequences showed that this isolate is a phylogenetic neighbor of Terrabacter tumescens and Intrasporangium calvum. Genotypic, chemotaxonomic, morphological, and physiological characteristics are used to describe a new genus and species, Terracoccus luteus gen. nov., sp. nov. The type strain is strain IMET 7848 (= DSM 44267).

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family.

Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.

Fatty acid characterization of rapidly growing pathogenic aerobic actinomycetes as a means of identification.

The fatty acid compositions of 39 type strains and 529 clinical or reference strains of pathogenic aerobic actinomycetes were analyzed after standardized culture by using the Microbial Identification System (MIS). Library entries for each type strain were created by using the MIS Library Generation Software, and the fatty acid profiles of clinical and reference strains were compared to these library entries. The bacteria separated into two large groups based upon major amounts of branched-chain or of saturated or monounsaturated straight-chain fatty acids. Identification of isolates was possible by using only the type strains for comparison, but fatty acid heterogeneity occurred within most species.

Isolation and structure of two novel muscarinic receptor antagonists.

The structures of two novel muscarinic receptor antagonists, 1 and 2, were determined by their spectral data and high-resolution mass measurements of their degradation products. Both are aliphatic long-chain compounds and contain amide and keto functionalities. The major microbial metabolite [1] contains three terminal guanidino groups and the minor compound [2] has two terminal guanidino groups.