The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

Recognition of Dimeric Lewis X by Anti-Dimeric Lex Antibody SH2.

The carbohydrate antigen dimeric Lewis X (DimLex), which accumulates in colonic and liver adenocarcinomas, is a valuable target to develop anti-cancer therapeutics. Using the native DimLex antigen as a vaccine would elicit an autoimmune response against the Lex antigen found on normal, healthy cells. Thus, we aim to study the immunogenic potential of DimLex and search internal epitopes displayed by DimLex that remain to be recognized by anti-DimLex monoclonal antibodies (mAbs) but no longer possess epitopes recognized by anti-Lex mAbs. In this context, we attempted to map the epitope recognized by anti-DimLex mAb SH2 by titrations and competitive inhibition experiments using oligosaccharide fragments of DimLex as well as Lex analogues. We compare our results with that reported for anti-Lex mAb SH1 and anti-polymeric Lex mAbs 1G5F6 and 291-2G3-A. While SH1 recognizes an epitope localized to the non-reducing end Lex trisaccharide, SH2, 1G5F6, and 291-2G3-A have greater affinity for DimLex conjugates than for Lex conjugates. We show, however, that the Lex trisaccharide is still an important recognition element for SH2, which (like 1G5F6 and 291-2G3-A) makes contacts with all three sugar units of Lex. In contrast to mAb SH1, anti-polymeric Lex mAbs make contact with the GlcNAc acetamido group, suggesting that epitopes extend further from the non-reducing end Lex. Results with SH2 show that this epitope is only recognized when DimLex is presented by glycoconjugates. We have reported that DimLex adopts two conformations around the ß-d-GlcNAc-(1→3)-d-Gal bond connecting the Lex trisaccharides. We propose that only one of these conformations is recognized by SH2 and that this conformation is favored when the hexasaccharide is presented as part of a glycoconjugate such as DimLex-bovine serum albumin (DimLex-BSA). Proper presentation of the oligosaccharide candidate via conjugation to a protein or lipid is essential for the design of an anti-cancer vaccine or immunotherapeutic based on DimLex.

Synthesis of defined mono-de-N-acetylated ß-(1→6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity.

Many clinically-relevant biofilm-forming bacterial strains produce partially de-N-acetylated poly-ß-(1→6)-N-acetyl-d-glucosamine (dPNAG) as an exopolysaccharide. In Gram-negative bacteria, the periplasmic protein PgaB is responsible for partial de-N-acetylation of PNAG prior to its export to the extracellular space. In addition to de-N-acetylase activity found in the N-terminal domain, PgaB contains a C-terminal hydrolase domain that can disrupt dPNAG-dependent biofilms and hydrolyzes dPNAG but not fully acetylated PNAG. The role of this C-terminal domain in biofilm formation has yet to be determined in vivo. Further characterization of the enzyme's hydrolase activity has been hampered by a lack of specific dPNAG oligosaccharides. Here, we report the synthesis of a defined mono de-N-acetylated dPNAG penta- and hepta-saccharide. Using mass spectrometry analysis and a fluorescence-based thin-layer chromatography (TLC) assay, we found that our defined dPNAG oligosaccharides are hydrolase substrates. In addition to the expected cleavage site, two residues to the reducing side of the de-N-acetylated residue, minor cleavage products on the non-reducing side of the de-N-acetylation site were observed. These findings provide quantitative data to support how PNAG is processed in Gram-negative bacteria.

Ega3 from the fungal pathogen Aspergillus fumigatus is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4-linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (ß/α)8-fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the -2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.

Convergent synthesis of tetra- and penta-saccharide fragments of dimeric Lewis X.

The convergent synthesis of tetra- and penta-saccharide fragments of the TACA dimeric Lex is described. The synthetic strategy relied on the preparation of a protected GlcNTCA-(1,3)-Gal-(1,4)-GlcNAc trisaccharide diol free at O-3 of both glucosamine residues. Key steps in the preparation of this diol involved glycosylation at O-4 of N-acetylglucosamine using activation of a trichloroacetimidate with BF3·Et2O at 40 °C, removal of the non-reducing end O-3' chloroacetate with thiourea, and glycosylation with a N-trichloroacetamido glucosamine trichloroacetimidate donor. After conversion to the diol acceptor, the trisaccharide was selectively fucosylated at the nonreducing end under NIS/TMSOTf activation, or di-fucosylated under CuBr2/Bu4NBr activation. The protected tetra- and pentasaccharides were then efficiently deprotected under dissolving metal conditions and the nonreducing end glucosamine residues were N-acetylated during the reaction work up. The deprotected compounds will be used as soluble competitors to characterize the epitopes recognized by anti-polymeric Lex antibodies.

The effect of methylation on the intrinsic photophysical properties of simple rhodamines.

The rational design of rhodamines and other fluorescent probes for different functions would benefit from an improved understanding of their photophysics. Key photophysical properties, including fluorescence, depend on the outcome of competing pathways for intra- and intermolecular energy flow within and from excited state molecules. In the work reported here, we simplify this complex landscape by eliminating solvent interactions, revealing intrinsic photophysical effects of systematic structural changes. Selected-ion laser-induced fluorescence (SILIF) is used to examine the effects of stepwise N-methylation on a rhodamine scaffold, starting with the simple rhodamine 123, in the gas phase. Fluorescence excitation and emission spectra together with fluorescence lifetime measurements are reported and discussed. While the systematic decrease in gas-phase 0-0 transition energy by 500 cm-1 per methylation is in line with expectations from solution studies, other trends are observed that are not apparent in solution studies. These include a notable narrowing of spectral profiles, three-fold decrease in Stokes shift and an ∼three-fold increase in brightness as the number of N-methylations rises from zero to four. Most surprising, while rhodamine 123 displays the expected textbook mirror-image symmetry between excitation and emission spectra, the emission spectrum of its tetra N-methylated derivative is ∼30% broader than the excitation spectrum. The likelihood that this difference reflects emission prior to complete vibrational redistribution of energy within the excited state of the larger rhodamines is discussed. This suggestion goes against conventional wisdom about the timescale of energy redistribution within molecules of this size, an understanding which was developed from solution studies. Overall, this study furthers our understanding of energy flow within an important class of fluorophores, highlights the consequences of energy flow between fluorophores and surrounding solvent, and provides benchmark experimental data for solvent-free chromophores to assist and calibrate computational work.

The Association of Streptococcus gallolyticus Subspecies pasteurianus Bacteremia with the Detection of Premalignant and Malignant Colonic Lesions.

Streptococcus gallolyticus subspecies (subsp.) gallolyticus (formerly bovis biotype I) bacteremia has been associated with colonic adenocarcinoma. The bovis species underwent reclassification in 2003. Subtypes of gallolyticus are associated with colonic malignancy but are less frequent, resulting in less awareness. A 71-year-old male admitted with worsening lower back pain and fevers. Initial vital signs and laboratory data were within normal limits. MRI revealed lumbosacral osteomyelitis and antibiotics were initiated. Blood cultures showed Streptococcus species, prompting a transesophageal echocardiogram (TEE) revealing vegetations on the mitral and aortic valves. The etiology for his endocarditis was unclear. A colonoscopy was suggested, but his clinical instability made such a procedure intolerable. Final cultures revealed Streptococcus gallolyticus subsp. pasteurianus (previously bovis biotype II). After antibiotic completion he underwent aortic grafting with valve replacements. Later, he was readmitted for Streptococcus bacteremia. After a negative TEE, colonoscopy revealed a 2.5 × 3 cm cecal tubulovillous adenoma with high-grade dysplasia suspicious for his origin of infection. Clinicians understand the link between Streptococcus gallolyticus subsp. gallolyticus (bovis type I) and malignancy, but the new speciation may be unfamiliar. There are no guidelines for managing S. gallolyticus subsp. pasteurianus bacteremia; therefore a colonoscopy should be considered when no source is identified.

Orthoesters formation leading to mismatched Helferich glycosylations at O-3 of N-trichloroacetylated glucosamine residues.

Using trisaccharide diol acceptors displaying two glucosamine residues free at O-3, we observed that α-l-fucosylation with α armed donor proceeded smoothly at the most accessible N-trichloroacetyl nonreducing end glucosamine residue. In contrast, glycosylations with peracetylated glycosyl bromide donors activated under Helferich conditions seemed to proceed preferentially or exclusively at the more sterically hindered N-acetylated reducing end unit. Thus, we concluded that disarmed donors were mismatched at O-3 of the N-trichloroacetylated glucosamine residue regardless of α or ß configuration of the glycosidic bond formed and d or l configuration of the donor. Interestingly orthoester formation occurred in some cases at this position while they were not observed at the reducing end unit. Conversion of the nonreducing end trichloroacetamido to an acetamido allowed the Helferich catalyzed galactosylation to occur at both positions and revealed the impact of the N-trichloroacetamido on the mismatched glycosylations. Changing the activation conditions from the mild Lewis acid Hg(CN)2 to the stronger acid AgOTf revealed that in fact ß-d-galactosylation at the less hindered N-trichloroacetylated residue was kinetically favored over that at the reducing end residue. Isolation of equal amounts of orthoester at this position suggested that it was formed first but that the strong AgOTf Lewis acid was able to promote rearrangement to the ß-d-galactosidic bond. These results shed additional light on the apparent mismatch of disarmed glycosyl donors with hydroxyl groups deemed more accessible. Depending on electronic factors imposed by the acceptor and activation conditions, transient unstable orthoester formation may explain in some cases why these donors appear mismatched with the most accessible hydroxyl groups which are otherwise glycosylated by armed donors.

Multi-arm PEG/silica hydrogel for sustained ocular drug delivery.

In the present study, a series of sustained drug delivery multiarm poly(ethylene glycol) (PEG)/silica hydrogels were prepared and characterized. The hydrogels were formed by hydrolysis and condensation of poly(4-arm PEG silicate) using the sol-gel method. The relationships between water content in the PEG/silica hydrogel and stability as well as rheological properties were evaluated. Scanning electron microscopy analysis of the PEG/silica hydrogels revealed water content-dependent changes in microstructure. An increase in water content resulted in larger pores within the hydrogel, longer gelation time and higher viscosity. The PEG/silica hydrogels were loaded with dexamethasone (DMS) or dexamethasone sodium phosphate (DMSP), drugs that are hydrophobic and hydrophilic in nature, respectively. Evaluation of in vitro release revealed a zero-order release profile for DMS over the first 6 days, suggesting that degradation of the silica hydrogel matrix was the primary mechanism of drug release. It was also found that the drug-release profile could be tailored by varying the water content used during hydrogel preparation. In contrast, more than 90% of DMSP was released within 1 h, suggesting that DMSP release was only controlled by diffusion. Overall, results from this study indicate that PEG/silica hydrogels may be promising drug-eluting depot materials for the sustained delivery of hydrophobic, ophthalmic drugs.