Dixon plot analyses used to determine the inhibition constant of NGT (show the chemical structures of the inhibitors. features give StrH specificity for the mannose arms of the aglycon) making the enzyme very specific for terminal GlcNAc residues in complex conversation is an example of an expanding paradigm of how this bacterium attacks and utilizes host glycans to inhabit and invade human tissues (9, 17). is usually a commensal bacterium inhabiting the human upper respiratory tract of healthy individuals, but in some circumstances it can progress beyond the nasopharynx to other normally sterile sites and cause diseases ranging from comparatively minor infections, like otitis media, to lethal infections such as pneumonia, bacteremia, and meningitis (18). The occurrence of infections remains high in both developed and developing countries with high-risk groups, including children, the elderly, and immuno-compromised individuals. Approximately 15 million cases of invasive infections are reported annually with 1.6 million of these resulting in death (19). Given that the primary route of access and/or colonization niche is the human nasopharynx, encounters an environment that is rich in the complex carbohydrates displayed by the host tissues. In keeping with this, large level testing has suggested that a significant number of Cloxacillin sodium genes encoding putative and known glycoside hydrolases, including StrH, may participate in virulence (20,C22). These are progressively being validated as important components of the host-bacterium conversation, such as nutritional support, enhanced adherence, and immune system modulation (9, 17, 23,C28). It was from your perspective of the importance of carbohydrate-processing enzymes to virulence and the role of StrH in this that we noted the presence in the genome of a second gene encoding a family 20 glycoside hydrolase. This gene appears to be present in all sequenced strains of the bacterium. The putative product of this gene, referred to as GH20C, displays 70% amino acid sequence identity with the structurally characterized GcnA, which displays both enzymes. Experimental Procedures Materials Oligosaccharides were obtained from V-labs (Covington, LA). PUGNAc and NGT synthesized in-house were kind gifts from Dr. David Vocadlo. GalPUGNAc and GalNGT synthesized in-house were kind gifts from Dr. Keith Stubbs. All other reagents, including GlcNAc and GalNAc, were purchased from Sigma unless normally specified. Cloning The gene fragment encoding GH20C (amino acids 1C626) was PCR-amplified from TIGR4 genomic DNA using the oligonucleotide primers GH20C-Fw (5-CATATCGCTAGCATGGTAAGATTTACAG) and GH20C-Rv (5-GGTGGTCTCGAGTTAAGTCGTATAAATC). The amplified DNA fragment was cloned using standard procedures into pET 28a (Novagen) using 5 NheI and 3 XhoI restriction sites, respectively. The producing plasmid, pGH20C, encoded the desired polypeptide fused to an N-terminal hexa-histidine tag by a thrombin protease cleavage site. Standard PCR site-directed mutagenesis procedures were used to expose the E223Q substitution into pGH20C using the oligonucleotide primers GH20CE223Q-Fw (5-ATCGGGATGGACCAGGCCCACTTGGTT) and GH20CE223Q-Rv (5-AACCAAGTGGGCCTGGTCCATCCCGAT) (52). The DNA sequence fidelity of all constructs was verified by bidirectional sequencing. Protein Production and Purification All constructs were transformed into the expression strain, BL21 Star (DE3) strain. Six liters of yeast/tryptone broth, made up of 50 g/ml kanamycin, was inoculated with the transformed cells and Cloxacillin sodium incubated at 37 C. Once an optical density of 1 1 at 595 nm was reached, protein production was induced Cloxacillin sodium by the addition of isopropyl 1-thio–d-galactopyranoside to a final concentration of 0.5 mm. Incubation of the cultures was continued overnight with shaking FUT3 at 16 C. Cells were harvested by centrifugation at 5000 for 10 min and ruptured by chemical lysis. The cleared supernatant of the cell lysate was loaded onto a Ni2+-nitrilotriacetic acid-immobilized metal affinity chromatography column. Polypeptide was eluted with binding buffer (20 mm Tris, pH 8.0) containing increasing concentrations of imidazole (0C500 mm). GH20C was concentrated and buffer-exchanged into 20 mm Tris, pH 8.0, in a stirred ultrafiltration unit (Amicon). Purified protein was further purified by size exclusion chromatography using a Sephacryl S-200 column (GE Healthcare) and concentrated using a stirred cell.
