Broad Protection against Invasive Fungal Disease from a Nanobody Targeting the Active Site of Fungal ß-1,3-Glucanosyltransferases.

Invasive fungal disease accounts for about 3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insight about the role of ß-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

Water Sculpts the Distinctive Shapes and Dynamics of the Tumor-Associated Carbohydrate Tn Antigens: Implications for Their Molecular Recognition.

The tumor-associated carbohydrate Tn antigens include two variants, αGalNAc- O-Thr and αGalNAc- O-Ser. In solution, they exhibit dissimilar shapes and dynamics and bind differently to the same protein receptor. Here, we demonstrate experimentally and theoretically that their conformational preferences in the gas phase are highly similar, revealing the essential role of water. We propose that water molecules prompt the rotation around the glycosidic linkage in the threonine derivative, shielding its hydrophobic methyl group and allowing an optimal solvation of the polar region of the antigen. The unusual arrangement of αGalNAc- O-Thr features a water molecule bound into a "pocket" between the sugar and the threonine. This mechanism is supported by trapping, for the first time, such localized water in the crystal structures of an antibody bound to two glycopeptides that comprise fluorinated Tn antigens in their structure. According to several reported X-ray structures, installing oxygenated amino acids in specific regions of the receptor capable of displacing the bridging water molecule to the bulk-solvent may facilitate the molecular recognition of the Tn antigen with threonine. Overall, our data also explain how water fine-tunes the 3D structure features of similar molecules, which in turn are behind their distinct biological activities.

Cyclooxygenase-2 immunoexpression in intestinal epithelium and lamina propria of cats with inflammatory bowel disease and low grade alimentary lymphoma.

BACKGROUND: Cyclooxygenase 2 (COX-2) is an inducible isoform by cellular activation, proinflammatory cytokines and growth factors. The aims of the current study were to evaluate COX-2 immunoexpression in epithelial and lamina propria (LP) of cats with inflammatory bowel disease (IBD) and low grade alimentary lymphoma (LGAL), as well as to correlate them with clinical signs and histopathological scoring. Cats diagnosed with IBD and LGAL (2007-2013) were included in the current study. Feline chronic enteropathy activity index (FCEAI) was calculated for all cases. Control group was composed by 3 healthy indoor cats and 5 sick cats died or were euthanized (non-gastrointestinal illness). Diagnosis and classification of IBD and LGAL was established according to the WSAVA gastrointestinal standardization group template and the National Cancer Institute formulation, respectively. Furthermore, a modified WSAVA template was applied for LGAL evaluation. Immunolabelling for COX-2 (polyclonal rabbit anti-murine antibody) was performed on biopsy samples. Epithelial and LP (inflammatory or neoplastic cells) COX-2 immunolabelling was calculated according to the grade and intensity. The most representative segment scored by the WSAVA and the modified WSAVA were used for statistical analysis. RESULTS: Significant difference was found regarding COX-2 intensity overexpression in the epithelial cells of IBD and LGAL groups when compared to control cats, but not between the groups of sick cats, whereas no differences were found regarding the grade of immunoreactivity between groups. No difference was found for COX-2 immunoexpression at the LP between all groups. However, 3 cats from LGAL group showed COX-2 expression in neoplastic cells at the LP. There were no correlations between epithelial or LP COX-2 expression and FCEAI and histological alterations. CONCLUSIONS: Increased COX-2 intensity at the epithelial cells observed in cats with IBD and LGAL may be secondary to the inflammatory response or a protective function in the intestinal reparation. COX-2 expression at the LP was presented in 33% of LGAL. This result provides a reason for further investigation concerning the role of COX-2 expression in feline alimentary lymphoma.

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.

A Trapped Covalent Intermediate of a Glycoside Hydrolase on the Pathway to Transglycosylation. Insights from Experiments and Quantum Mechanics/Molecular Mechanics Simulations.

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

Design of α-S-Neoglycopeptides Derived from MUC1 with a Flexible and Solvent-Exposed Sugar Moiety.

The use of vaccines based on MUC1 glycopeptides is a promising approach to treat cancer. We present herein several sulfa-Tn antigens incorporated in MUC1 sequences that possess a variable linker between the carbohydrate (GalNAc) and the peptide backbone. The main conformations of these molecules in solution have been evaluated by combining NMR experiments and molecular dynamics simulations. The linker plays a key role in the modulation of the conformation of these compounds at different levels, blocking a direct contact between the sugar moiety and the backbone, promoting a helix-like conformation for the glycosylated residue and favoring the proper presentation of the sugar unit for molecular recognition events. The feasibility of these novel compounds as mimics of MUC1 antigens has been validated by the X-ray diffraction structure of one of these unnatural derivatives complexed to an anti-MUC1 monoclonal antibody. These features, together with potential lack of immune suppression, render these unnatural glycopeptides promising candidates for designing alternative therapeutic vaccines against cancer.

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody.

The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment.

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

Pilot study: duodenal MDR1 and COX2 gene expression in cats with inflammatory bowel disease and low-grade alimentary lymphoma.

Objectives Multidrug resistance 1 (MDR1) encodes a protein called P-glycoprotein (P-gp), which serves as an efflux pump membrane protein implicated in intestinal homeostasis and drug resistance. Cyclooxygenase-2 (COX2) is a key enzyme in the synthesis of proinflammatory prostaglandins, tumourigenesis and in mucosal defence. Despite the importance of MDR1 and COX2, changes in their mRNA levels have not been studied in cats with inflammatory bowel disease (IBD) and low-grade alimentary lymphoma (LGAL). The present study aimed to determine the mRNA levels of MDR1 and COX2 in cats with IBD and LGAL, and to evaluate their correlation with clinical signs, histological severity and between genes. Methods Cats diagnosed with IBD (n = 20) and LGAL (n = 9) between 2008 and 2015 were included in the current study. Three healthy animals composed the healthy control cats group in which endoscopy was performed immediately before the ovariohysterectomy. All duodenal biopsy samples were obtained by endoscopy. Feline chronic enteropathy activity index was calculated for all cases. IBD histopathology was classified according to severity. MDR1 and COX2 mRNA levels were determined by absolute reverse transcriptase-quantitative real-time PCR. Results Statistically significant differences were observed for MDR1 and COX2 mRNA levels between the IBD and LGAL groups. No correlations were observed between molecular gene expression, feline chronic enteropathy activity index and histological grading for IBD, and between MDR1 and COX2 genes. However, a positive statistically significant correlation was observed between MDR1 and COX2 expression in the duodenum of cats. Conclusions and relevance MDR1 and COX2 gene expression is increased in cats with LGAL compared with cats with IBD. The control group tended to have lower values than both diseased groups. These results suggest that these genes may be involved in the pathogenesis of IBD or LGAL in cats.

Inhibitors against Fungal Cell Wall Remodeling Enzymes.

Fungal ß-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cation-π and face-to-face π-π interactions. The lead compound is capable of inhibiting AfGel4 with an IC50 value of 42 µm.

Prevalence of selected infectious disease agents in stray cats in Catalonia, Spain.

OBJECTIVES: The objective of the current study was to investigate the prevalence rates of the following infectious agents in 116 stray cats in the Barcelona area of Spain: Anaplasma phagocytophilum, Bartonella species, Borrelia burgdorferi, Chlamydia felis, Dirofilaria immitis, Ehrlichia species, feline calicivirus (FCV), feline herpesvirus-1 (FHV-1), feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), haemoplasmas, Mycoplasma species and Rickettsia species. METHODS: Serum antibodies were used to estimate the prevalence of exposure to A phagocytophilum, Bartonella species, B burgdorferi, Ehrlichia species and FIV; serum antigens were used to assess for infection by D immitis and FeLV; and molecular assays were used to amplify nucleic acids of Anaplasma species, Bartonella species, C felis, D immitis, Ehrlichia species, FCV, FHV-1, haemoplasmas, Mycoplasma species and Rickettsia species from blood and nasal or oral swabs. RESULTS: Of the 116 cats, 63 (54.3%) had evidence of infection by Bartonella species, FeLV, FIV or a haemoplasma. Anaplasma species, Ehrlichia species or Rickettsia species DNA was not amplified from these cats. A total of 18/116 cats (15.5%) were positive for FCV RNA (six cats), Mycoplasma species DNA (six cats), FHV-1 DNA (three cats) or C felis DNA (three cats). CONCLUSIONS AND RELEVANCE: This study documents that shelter cats in Catalonia are exposed to many infectious agents with clinical and zoonotic significance, and that flea control is indicated for cats in the region.

Detection of tumor-associated glycopeptides by lectins: the peptide context modulates carbohydrate recognition.

Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide-lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.

Pyogranulomatous mural folliculitis in a cat treated with methimazole.

An 11-year-old spayed female domestic shorthair cat was presented for polydipsia, hyperactivity and bilateral thyroid gland enlargement. Total T4 (TT4) was in the upper interval range; therefore, an early hyperthyroidism was suspected. A treatment trial with methimazole was started, as the owner refused further tests. Six months later the owner stopped the treatment. One year later, clinical signs persisted and TT4 was still in the upper interval range. Methimazole was re-introduced but 48 h later the cat presented non-pruritic alopecia with erythema, scales and perilesional yellowish crusts. Pyogranulomatous mural folliculitis was diagnosed by histopatological examination of the skin biopsies. Methimazole was withdrawn and macroscopic lesions healed and disappeared histologically in 15 days. An idiosyncratic drug reaction to methimazole was suspected. To the best of our knowledge, this is the first report of feline pyogranulomatous mural folliculitis likely secondary to an adverse drug reaction to methimazole administration.