ABSTRACT The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans . Both fusions display lytic specificity for streptococcal pathogens and S. aureus . The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.
Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention. Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore- and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention. Bacillus anthracis is a Gram-positive, rod-shaped, aerobic soil bacterium that causes anthrax in humans and other mammals (1Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Google Scholar). Like other Bacillus species, B. anthracis forms endospores (or spores) when vegetative cells are deprived of an essential nutrient (2Priest F.G. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Biology. American Society for Microbiology, Washington, D. C.1993: 3-16Google Scholar). The mature spore is dormant and highly resistant to extreme temperatures, radiation, harsh chemicals, desiccation, and physical damage (3Nicholson W.L. Munakata N. Horneck G. Melosh H.J. Setlow P. Microbiol. Mol. Biol. Rev. 2000; 64: 548-572Google Scholar). These properties allow the spore to persist in the soil for many years until encountering a signal to germinate (4Paidhungat M. Setlow P. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Its Closest Relatives: From Genes to Cells. American Society for Microbiology, Washington, D. C.2002: 537-548Google Scholar). Anthrax is typically contracted by contact with spores (1Mock M. Fouet A. Annu. Rev. Microbiol. 2001; 55: 647-671Google Scholar). Because of their ability to cause a potentially fatal disease and to withstand harsh conditions, spores of B. anthracis have been developed into weapons of mass destruction by numerous countries and terrorist groups (5Webb G.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4355-4356Google Scholar). The effectiveness of B. anthracis spores as a biological weapon was demonstrated when letters laden with spores were mailed in the United States in the fall of 2001. In response to the threat of future releases of lethal spores, new research has been undertaken to enhance our knowledge of B. anthracis biology and pathogenesis. A major goal of such studies is to identify components of the B. anthracis spore that can serve either as molecular targets of spore inactivation or as unique markers that allow rapid and accurate spore detection (6Williams D.D. Benedek O. Turnbough Jr., C.L. Appl. Environ. Microbiol. 2003; 69: 6288-6293Google Scholar). Sporulation in the genus Bacillus begins in the starved vegetative cell with an asymmetric septation that produces large and small genome-containing compartments called the mother cell and forespore, respectively (7Errington J. Microbiol. Rev. 1993; 57: 1-33Google Scholar). The mother cell then engulfs the forespore and surrounds it with a layer of modified peptidoglycan called the cortex and a more external proteinaceous layer called the coat. The spore coat, composed of three sublayers and many different proteins, forms the outermost detectable layer for spores of many species (e.g. B. subtilis) (8Lai E.M. Phadke N.D. Kachman M.T. Giorno R. Vazquez S. Vazquez J.A. Maddock J.R. Driks A. J. Bacteriol. 2003; 185: 1443-1454Google Scholar, 9Henriques A.O. Moran Jr., C.P. Methods. 2000; 20: 95-110Google Scholar). For other Bacillus species, such as B. anthracis, the spore coat is surrounded by another prominent layer called the exosporium, which is synthesized by the mother cell concurrently with the cortex and coat (10Gerhardt P. Fed. Proc. 1967; 26: 1504-1517Google Scholar). After a final stage of maturation, during which covalent modifications occur in the outer layers of the spore, the mother cell lyses and releases the spore (9Henriques A.O. Moran Jr., C.P. Methods. 2000; 20: 95-110Google Scholar, 11Roels S. Losick R. J. Bacteriol. 1995; 177: 6263-6275Google Scholar). Of particular interest in current studies of the B. anthracis spore is the exosporium, which is the primary permeability barrier of the spore and the source of spore surface antigens (10Gerhardt P. Fed. Proc. 1967; 26: 1504-1517Google Scholar, 12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). As the outermost surface of the spore, the exosporium interacts with the soil environment, detection devices, spore-binding cells in the mammalian host, and host defenses. Thus, it is likely that the exosporium plays an important role in spore survival and/or pathogenesis (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). To demonstrate such a role, it is necessary to characterize individual exosporium components. Early studies revealed that spores of B. anthracis and closely related species (e.g. Bacillus cereus and Bacillus thuringiensis) possess an exosporium composed of a paracrystalline basal layer and an external hairlike nap, which exhibits a strain-specific length up to 600 Å (10Gerhardt P. Fed. Proc. 1967; 26: 1504-1517Google Scholar, 13Gerhardt P. Ribi E. J. Bacteriol. 1964; 88: 1774-1789Google Scholar, 14Hachisuka Y. Kojima K. Sato T. J. Bacteriol. 1966; 91: 2382-2384Google Scholar, 15Beaman T.C. Pankratz H.S. Gerhardt P. J. Bacteriol. 1971; 107: 320-324Google Scholar, 16Kramer M.J. Roth I.L. Can. J. Microbiol. 1968; 14: 1297-1299Google Scholar). The exosporium constitutes about 2% of the mass of the spore and contains approximately 50% protein, 20% lipid, 20% carbohydrate, and 10% other components (17Matz L.L. Beaman T.C. Gerhardt P. J. Bacteriol. 1970; 101: 196-201Google Scholar). A recent proteomic analysis of the exosporium suggested that it contains at least 137 different proteins (18Liu H. Bergman N.H. Thomason B. Shallom S. Hazen A. Crossno J. Rasko D.A. Ravel J. Read T.D. Peterson S.N. Yates J.I. Hanna P.C. J. Bacteriol. 2004; 186: 164-178Google Scholar). However, this analysis was performed with an exosporium fraction prepared from spores that were not purified sufficiently to remove contaminating proteins released into the growth medium by lysed cells (44Williams D.D. Turnbough Jr., C.L. J. Bacteriol. 2004; 186: 566-569Google Scholar). Analyses of the exosporium prepared from highly purified spores indicates that about 20 different protein species are present in or tightly associated with the exosporium (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar, 19Todd S.J. Moir A.J.G. Johnson M.J. Moir A. J. Bacteriol. 2003; 185: 3373-3378Google Scholar, 44Williams D.D. Turnbough Jr., C.L. J. Bacteriol. 2004; 186: 566-569Google Scholar). The first B. anthracis exosporium protein identified, and one of the most interesting, was a glycoprotein called BclA (for Bacillus collagen-like protein of anthracis) (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar, 20Sylvestre P. Couture-Tosi E. Mock M. Mol. Microbiol. 2002; 45: 169-178Google Scholar). BclA is the structural component of the hairlike nap and contains multiple, collagen-like Xaa-Yaa-Gly (or XXG) repeats in its central region (20Sylvestre P. Couture-Tosi E. Mock M. Mol. Microbiol. 2002; 45: 169-178Google Scholar). The number of XXG repeats in BclA varies among strains (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar, 21Sylvestre P. Couture-Tosi E. Mock M. J. Bacteriol. 2003; 185: 1555-1563Google Scholar). This variation is responsible for the different lengths of the hairlike nap found on spores of different B. anthracis strains (21Sylvestre P. Couture-Tosi E. Mock M. J. Bacteriol. 2003; 185: 1555-1563Google Scholar). BclA has also been shown to be the immunodominant protein on the B. anthracis spore surface, because most antibodies raised against spores react with this protein (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). Finally, most of the XXG repeats in the collagen-like region of BclA have the sequence PTG, and nearly all of the XXG repeats contain a threonine residue, which may be a site of attachment of an O-linked oligosaccharide (22Schmidt M.A. Riley L.W. Benz I. Trends Microbiol. 2003; 11: 554-561Google Scholar, 23Jentoft N. Trends Biochem. Sci. 1990; 15: 291-294Google Scholar). In this report, we describe two O-linked oligosaccharides that are attached to BclA: a 715-Da tetrasaccharide and a 324-Da disaccharide. We show that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. The attachment of each oligosaccharide to BclA may occur through a GalNAc linker, which is lost during oligosaccharide release. Using several analytical techniques, we determine the complete structure of the tetrasaccharide. It contains a unique sugar residue that may be useful for species-specific detection of B. anthracis spores or even serve as a new target for preventing anthrax. Bacterial Strains—The Sterne veterinary vaccine strain of B. anthracis along with B. cereus T and B. thuringiensis ssp. kurstaki were obtained from John Ezzell (U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD). B. subtilis (trpC2) 1A700 (originally designated 168) was obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus, OH). The Sterne strain of B. anthracis is not a human pathogen because it lacks plasmid pXO2, which carries the genes necessary to produce the protective poly-γ-d-glutamic acid capsule of the vegetative cell. Spores of the Sterne strain appear to be essentially identical to spores of virulent strains of B. anthracis (24Read T.D. Peterson S.N. Tourasse N. Baillie L.W. Paulsen I.T. Nelson K.E. Tettelin H. Fouts D.E. Eisen J.A. Gill S.R. Holtzapple E.K. Okstad O.A. Helgason E. Rilstone J. Wu M. Kolonay J.F. Beanan M.J. Dodson R.J. Brinkac L.M. Gwinn M. DeBoy R.T. Madpu R. Daugherty S.C. Durkin A.S. Haft D.H. Nelson W.C. Peterson J.D. Pop M. Khouri H.M. Radune D. Benton J.L. Mahamoud Y. Jiang L. Hance I.R. Weidman J.F. Berry K.J. Plaut R.D. Wolf A.M. Watkins K.L. Nierman W.C. Hazen A. Cline R. Redmond C. Thwaite J.E. White O. Salzberg S.L. Thomason B. Friedlander A.M. Koehler T.M. Hanna P.C. Kolsto A.B. Fraser C.M. Nature. 2003; 423: 81-86Google Scholar). Plasmid and Strain Constructions—Recombinant DNA techniques, preparation of plasmid DNA from Escherichia coli, and transformation of E. coli were carried out by standard procedures (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1989Google Scholar). Electroporation of B. anthracis was performed using unmethylated plasmid DNA isolated from E. coli strain GM1684 (dam-4) (26Koehler T.M. Dai Z. Kaufman-Yarbray M. J. Bacteriol. 1994; 176: 586-595Google Scholar). Mutants of the B. anthracis Sterne strain were constructed by allelic exchange between the chromosome and a mutant locus carried by the shuttle vector pUTE29 as previously described (27Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Google Scholar, 28Dai Z.J.-C. Sirard M. Mock M. Koehler T.M. Mol. Microbiol. 1995; 16: 1171-1181Google Scholar). Without selection with tetracycline, plasmid pUTE29 is not maintained in B. anthracis (26Koehler T.M. Dai Z. Kaufman-Yarbray M. J. Bacteriol. 1994; 176: 586-595Google Scholar). Sitedirected mutagenesis was performed with the QuikChange™ kit from Stratagene. To construct a ΔbclA deletion strain, a DNA segment containing the bclA gene and about 1 kb of flanking sequence on each side was PCR-amplified using genomic B. anthracis Sterne DNA as the template. The PCR product was cloned into plasmid pCR-Blunt II-TOPO (Invitrogen), and a unique BglII restriction site (used below) was introduced 56 bp after the bclA stop codon, in an apparent intergenic region (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar), by site-directed mutagenesis. The unique ApaI restriction site in this plasmid (from the TOPO vector) was also removed by site-directed mutagenesis, and the resultant plasmid was designated pCLT1159. Using this plasmid as the template, outward PCR was used to create a new plasmid in which most of the bclA gene (i.e. codons 27–382 of 400) was removed and replaced with an ApaI site. The region containing the bclA deletion and flanking B. anthracis DNA was then excised and inserted into the multiple cloning site (between PstI and KpnI) of plasmid pUTE29. A kanamycin resistance cassette from plasmid pUC18::Ωkm-2 (29Perez-Casal J. Caparon M.G. Scott J.R. J. Bacteriol. 1991; 173: 2617-2624Google Scholar) was inserted into the unique BglII site of the plasmid. After passage through E. coli strain GM1684 (dam-4), the plasmid was electroporated into B. anthracis. The transformant was grown under conditions that allowed allelic exchange to replace the wild-type bclA locus with the ΔbclA mutation and adjacent kanamycin resistance cassette, while permitting the loss of the recombinant pUTE29 (27Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Google Scholar, 28Dai Z.J.-C. Sirard M. Mock M. Koehler T.M. Mol. Microbiol. 1995; 16: 1171-1181Google Scholar). The mutant locus was confirmed by PCR amplification of the bclA region and sequencing the DNA product, and the mutant strain was designated CLT292. To construct mutant strains carrying a reduced number of XXG repeats in the bclA gene, we first performed outward PCR with primers 5′-ACTGGGCCCACTGGTGCTACCGGACTG and 5′-AGTGGGCCCAGTTGGTCCAGTAGTACC and plasmid pCLT1159 (bclA+) as the template. One primer hybridizes to a unique site, whereas the other hybridizes to multiple sites within the central region of bclA. The resulting PCR products were digested with ApaI and circularized to produce plasmids carrying a partial deletion (not all the same) of the bclA repeat region. After cloning and propagating the plasmids in E. coli, the deletions were defined by sequence analysis. For each deletion plasmid, a fragment containing the mutant bclA gene and flanking sequences was excised and inserted into plasmid pUTE29 as above. A spectinomycin-resistance cassette from plasmid pJRS312 (Ω-sp) (27Saile E. Koehler T.M. J. Bacteriol. 2002; 184: 370-380Google Scholar) was inserted into the unique BglII site downstream of bclA. The resulting plasmid was used for allelic replacement of the bclA locus using strain CLT292 (ΔbclA kan) as the recipient. We confirmed that each mutant strain was kanamycin-sensitive and spectinomycin-resistant, and carried the expected deletion. To construct a strain unable to synthesize l-rhamnose, we generated a derivative of plasmid pUTE29 that contained a spectinomycin-resistance cassette (i.e. Ω-sp) flanked by sequences upstream and downstream of the rmlD gene of B. anthracis. This plasmid was used for allelic replacement of the rmlD locus of the Sterne strain as described above. The mutant strain, designated CLT274, contains a deletion that removes codons 11–264 of the 284 rmlD codons, and the deleted codons are replaced by the Ω-sp cassette. Preparation of Spores and Purified Exosporium—Spores were prepared from cultures grown at 37 °C for 48–72 h on liquid or solid Difco sporulation medium, extensively washed in cold (4 °C) distilled water, sedimented through 50% Renografin to remove vegetative cells and debris, and washed again in cold water (30Knurr J. Benedek O. Heslop J. Vinson R.B. Boydston J.A. McAndrew J. Kearney J.F. Turnbough Jr., C.L. Appl. Environ. Microbiol. 2003; 69: 6841-6847Google Scholar, 31Nicholson W.L. Setlow P. Harwood C.R. Cutting S.M. Molecular Biological Methods for Bacillus. John Wiley & Sons, Ltd., West Sussex, UK1990: 391-450Google Scholar). Spores were stored in water at 4 °C (protected from light) and quantitated microscopically using a Petroff-Hausser counting chamber. The exosporium was removed from spores by passage through a French press and then highly purified by differential centrifugation as previously described (12Steichen C. Chen P. Kearney J.F. Turnbough Jr., C.L. J. Bacteriol. 2003; 185: 1903-1910Google Scholar). Monosaccharide Analysis by Gas Chromatography—The monosaccharide compositions of spores, exosporium, and other samples were determined by gas chromatographic analysis of the trimethylsilyl derivatives of the sugar methyl glycosides. Samples were dried in a vacuum centrifuge, resuspended in 400 μl of 1.45 n methanolic HCl, and heated at 80 °C overnight. The methanolic HCl was removed by vacuum centrifugation, and the sample was resuspended in 200 μl of methanol, followed by the addition of 20 μl of acetic anhydride and 20 μl of pyridine. This mixture was allowed to react for 30 min at room temperature and then evaporated to dryness. The samples were then trimethylsilylated using 50 μl of Tri-Sil (Pierce), and the vials were sealed under argon. The trimethylsilylated glycosides were separated and quantitated on an HP 5890 gas chromatograph equipped with a 30-m HP-1 wide bore fused silica column coated with a 0.88-μm layer of cross-linked methyl silicone gum. Samples were applied to the column with an automatic injector, and sugars were detected by flame ionization. Determination of the Absolute Configuration of Rhamnose Residues—To distinguish between the d- and l-forms of rhamnose residues, the (+)-2-butyl glycosides were prepared and analyzed by gas chromatography as previously described (32Leontein K. Lindberg B. Lonngren J. Carbohydr. Res. 1978; 62: 359-362Google Scholar). However, HCl rather than trifluoroacetic acid was used as the catalyst, and rather than using acetate derivatives, as in the original procedure, trimethylsilyl derivatives were prepared as described above. The retention time of the uncommon d-rhamnose, for which a standard was not available, was determined by chromatography of the (–)-2-butyl glycoside of l-rhamnose. Hydrazinolysis of Glycoproteins—Selective hydrazinolysis was used to release O-linked oligosaccharides from spore glycoproteins. B. anthracis spores (1010) or exosporium samples were dried in a vacuum centrifuge and desiccated overnight over P2O5 under vacuum. Anhydrous hydrazine (1 ml) was added to each sample in a glass ampoule, which was flushed with argon and flame-sealed. The samples were heated at 60 °C for 5 h to specifically release O-linked oligosaccharides (33Patel T. Bruce J. Merry A. Bigge C. Wormald M. Jaques A. Parekh R. Biochemistry. 1993; 32: 679-693Google Scholar). The hydrazine was evaporated under vacuum and the residue was resuspended in 3 ml of water. The mixture was centrifuged at 14,000 × g for 10 min, and the supernatant containing oligosaccharides was collected. Gel Filtration Chromatography and Assay of Rhamnose-containing Oligosaccharides—The supernatant obtained from the hydrazinolysis procedure was loaded onto a 170 × 2.2 cm Bio-Gel P4 (fine; Bio-Rad) column, and the oligosaccharides were eluted with 0.1 m acetic acid. Three-ml fractions were collected, and a 250-μl sample of each fraction was assayed for 6-deoxy sugars (e.g. rhamnose) using the Dische-Shettles protocol (34Dische Z. Shettles L.B. J. Biol. Chem. 1948; 175: 595-603Google Scholar). Mass Spectrometry—Mass spectrometry was performed with a Micromass Q-TOF 2 mass spectrometer. Samples were introduced by flow injection into a stream of 50% acetonitrile containing 0.1% formic acid delivered by a Harvard model 22 syringe pump and were ionized by the electrospray mode. NMR Spectroscopy—Approximately 750 μg of the purified tetrasaccharide was lyophilized, dissolved in 450 μl of Me2SO-d6 (99.99% deuterium), and transferred to a 5-mm NMR tube. NMR data were collected on a Bruker DRX-500 NMR spectrometer using a 5 mm TXI probe equipped with x, y, z gradients at a probe temperature of 25 °C. A few measurements were repeated at 600 MHz on an Avance DRX-600 NMR spectrometer. NMR experiments were performed on samples stored for 3–4 days at 4 °C. Standard Bruker pulse sequences were used, except for the 13C-coupled HSQC, where the program was modified to remove the 13C decoupling during acquisition. Proton and carbon chemical shifts were referenced to an internal Me2SO peak (2.490 ppm for proton and 39.5 ppm for 13C). In addition to standard 1H and 13C one-dimensional NMR spectra, a series of homo- and heteronuclear two-dimensional NMR data sets were obtained. DQF-COSY 1The abbreviations used are: DQF-COSY, double quantum filtered-correlated spectroscopy; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; HMBC, heteronuclear multiple bond correlation; ESI-Q-TOF, electrospray ionization-quadrupole-time of flight; Ant, anthrose. was collected with 4096 data points and 0.409 s acquisition time in the F2 dimension with 800 increments in the indirect dimension. The data matrix was zero-filled in the F1 dimension to give a matrix of 4096 × 2048 points. The two-dimensional TOCSY experiments were performed with various spin lock times of 20, 40, 50, and 70 ms. The two-dimensional NOESY experiments were performed using 400- and 800-ms mixing times. The heteronuclear two-dimensional experiments HSQC, HSQC-TOCSY, and HMBC (with and without bilinear rotation decoupling filter) were performed using pulse field gradient programs. Data processing and plotting were performed using the Bruker Xwinplot program. Analysis of the Exosporium Monosaccharide Composition—As the first step in the identification of BclA oligosaccharides, we analyzed the monosaccharide composition of exosporium preparations purified from spores of wild-type and ΔbclA strains of B. anthracis (Sterne). Equal amounts of each exosporium sample were subjected to methanolysis, and trimethylsilyl derivatives of the resulting methyl glycosides were separated by gas chromatography. Methanolysis gives rise to several isomeric methyl glycosides in defined ratios for a particular sugar (35Bhatti T. Chambers R.E. Clamp J.R. Biochim. Biophys. Acta. 1970; 222: 339-347Google Scholar). The results showed that the wild-type exosporium contained at least four major monosaccharides that were absent in the ΔbclA exosporium (Fig. 1). Based on the retention times and isomeric ratios of sugar standards, we identified the most abundant monosaccharide residue as rhamnose and a second residue as GalNAc. Because bacteria are known to make both d- and l-rhamnose, we determined the absolute configuration of the rhamnose in the exosporium. The chiral 2-butyl glycosides of rhamnose were prepared, and the trimethylsilyl derivatives were analyzed by gas chromatography (32Leontein K. Lindberg B. Lonngren J. Carbohydr. Res. 1978; 62: 359-362Google Scholar). Only l-rhamnose was present (data not shown). The other two major monosaccharide residues that appear to be associated with BclA are labeled A and B in Fig. 1A. Component A is a unique monosaccharide described in detail below, and component B is most likely 3-O-methyl l-rhamnose based on a preliminary characterization (data not shown). The latter assignment is supported by the previous identification of 3-O-methyl rhamnose as a component of B. anthracis spores (36Fox A. Black G.E. Fox K. Rostovtseva S. J. Clin. Microbiol. 1993; 31: 887-894Google Scholar). The gas chromatogram of the ΔbclA exosporium contained a minor peak of a sugar with a retention time similar to that of the major methyl glycoside isomer of rhamnose (Fig. 1B). However, this sugar does not appear to be rhamnose. The same minor peak was present in a chromatogram of a ΔrmlD derivative of the Sterne strain that is unable to synthesize l-rhamnose (data not shown). Based on retention times and isomeric ratios of sugar standards, the minor sugar was tentatively identified as ribose, which was previously reported to be present in low levels in the exosporium of B. cereus T (17Matz L.L. Beaman T.C. Gerhardt P. J. Bacteriol. 1970; 101: 196-201Google Scholar) and in spores of B. anthracis (36Fox A. Black G.E. Fox K. Rostovtseva S. J. Clin. Microbiol. 1993; 31: 887-894Google Scholar). The only BclA-associated peak present in the chromatogram of the ΔrmlD strain was that of GalNAc. Although the four BclA-associated monosaccharides described above were major components of the exosporium carbohydrate, they were relatively minor components of the carbohydrate present in intact spores (data not shown). Thus, these monosaccharides appear to be components of glycoconjugates that are primarily or uniquely found in the exosporium. Isolation of Rhamnose-containing Oligosaccharides Associated with BclA—The numerous threonine residues in the collagen-like region of BclA and the monosaccharide composition of the exosporium indicated that one or more rhamnose-containing oligosaccharides were O-linked to BclA. To isolate these oligosaccharides, 1010 purified spores of the Sterne strain of B. anthracis were treated with anhydrous hydrazine under conditions that released only O-linked oligosaccharides. The free oligosaccharides were separated on a Bio-Gel P4 column (2.2 × 170 cm), and column fractions were assayed for rhamnose. Two oligosaccharide peaks were detected (Fig. 2). Based on the elution times of oligosaccharide standards, the larger peak corresponded to a tetrasaccharide, whereas the smaller peak corresponded to a disaccharide. Analysis of individual column fractions by ESI-Q-TOF mass spectrometry indicated that each oligosaccharide peak was essentially homogeneous and that the tetrasaccharide and disaccharide had masses of 715 and 324 Da, respectively. Both oligosaccharides were also isolated from purified exosporium of the Sterne strain following hydrazinolysis and gel filtration chromatography as described above. In addition, two minor peaks corresponding to a pentasaccharide and a trisaccharide were observed with masses of 918 and 527 Da, respectively (data not shown). These masses are equal to those of the tetrasaccharide and disaccharide, respectively, with the addition of a GalNAc residue. The significance of the minor oligosaccharides detected in the exosporium is discussed below. To determine whether the two major oligosaccharides were present in spores lacking BclA, we subjected 1010 spores of a ΔbclA derivative of the Sterne strain to hydrazinolysis and assayed for oligosaccharides as described above. No oligosaccharides were detected in the column fractions (Fig. 2). This result indicated that the tetrasaccharide and disaccharide found in wild-type spores were attached to or at least associated with BclA. Each purified oligosaccharide from wild-type sp
A group B streptococcal (GBS) bacteriophage lysin gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other beta-haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including Streptococcus mutans. The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.
ABSTRACT Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-β- l -rhamnose α-1,3- l -rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD -encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit β-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N -acyltransferase activity.
Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.
Objective
To obtain and anlysis the diffuse idiopathic skeletal hyperostosis(DISH)related miRNAs under 3-D adhesion for cell culture.
Methods
From January 2012 to January 2014, 4 ossific ligamenta flava tissues were obtained from DISH patients and 4 normal ligamenta flava tissues were obtained from trauma patients surgically. Fibroblasts were separated by using collagenase technique and then cultured on human acellular amniotic membrane(HAAM). Each sample was identified by immunofluorescence before harvested. Total RNA was extracted and then quantified by microfluidics analysis. The small RNAs(< 300 nt)were isolated by using a YM-100 Microcon centrifugal filter. μParaflo™ MiRNA microarray assay was performed using a service provider to identify miRNAs whose expression was significantly different between the two groups. Part of differential expression miRNAs were verified by qRT-PCR. Targets of miRNAs were obtained using PicTar 2005, miRanda v5, TargetScan 5.1, their function were analyzed by using Gene Ontology. Functional pathway analysis of miRNAs was performed using KEGG Path-way Analysis. TRANSFAC 7.0 public and Patser were used to get the distribution of transcription factor binding sites.
Results
When grown on HAAM, fibroblasts kept their morphology, distributed in the way of cluster, lived in multi-level of HAAM, and established linkage. Collagen I and III were tested positive in normal group cells. Collagen I, II, III and Osteocalcin were tested positive in DISH group cells by immunefluorescence. In total 15 miRNAs showed differential expression, 12 were up-regulated and 3 were down-regulated. The result of qRT-PCR was consistent with MiRNA microarray assay. Totally 67 target genes were predicted which participated in cell differentiation, cell adhesion, mineralization et al, and had function in regulating MAPK, Wnt, TGF-β, Focal adhesion signal pathway et al. In total 10 transcription factors were predicted in differentially expressed miRNAs.
Conclusion
HAAM can provide fibroblasts with 3D adhesion growth, Some differentially expressed miRNAs may participate in the pathogenesis of DISH.
Key words:
MicroRNAs; Ligamentum flavum; Ossification, heterotopic; Microarray analysis
LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.
ABSTRACT Synthetic peptides corresponding to portions of group B streptococcal peptidoglycan were used to show that the endopeptidase activity of bacteriophage B30 lysin cleaves between d -Ala in the stem peptide and l -Ala in the cross bridge and that the minimal peptide sequence cleaved is dl -γ-Glu-Lys- d -Ala-Ala-Ala. The only glycosidase activity present is that of N -acetyl-β- d -muramidase.
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