Cristina De Castro’s research while affiliated with Parthenope University of Naples and other places

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Publications (206)


The N-glycan structures attached to the chlorovirus glycoproteins used in this study
The left panel reports the three different viruses along with the class they belong to (upper part of each individual panel). On the right, the N-glycan structures of PBCV-1 antigenic variants are reported along with their antigenic classification (upper part of each individual panel). Note that PBCV-1 glycoprotein has 4 N-linked sites, none in a typical consensus sequon13,16,18,20. Dotted linkages in PBCV-1 and P91 N-glycans indicate that certain monosaccharides are non-stoichiometric substituents. The red box in the PBCV-1 N-glycan encloses the four monosaccharide residues conserved in all the chloroviruses studied to date. In ATCV-1, the methyl groups are enclosed in brackets because they are nonstoichiometric substituents.
Binding of viral glycoproteins to human and plant lectins
A Human C-type lectins ELISA results: wells were coated with viral glycoproteins and the binding to DC-SIGN, Langerin and MGL was evaluated in a calcium-containing buffer. B Plant lectins ELISA results: wells were coated with viral glycoproteins and the binding to VVL, HPA, SBA and RCA was evaluated. The experiments were performed in duplicate and data were normalized over signal from BSA-coated wells. Error bars indicate standard deviations. OD Optical density, VVL Vicia villosa lectin, HPA Helix pomatia agglutinin, SBA Soybean agglutinin, RCA Ricinus communis agglutinin.
IL-6 and IL-10 ELISA detection from moDC
IL-6 (A) and IL-10 (B) secretion from human monocytes-derived dendritic cells (n = 4) stimulated with the viral glycoproteins at 20 μg/mL, detected via ELISA in the supernatants after 20 h stimulation. Mean values from four different experiments (four different human donors) are reported. Error bars indicate standard deviations. Experimental data were analysed by ANOVA multiple comparison (Tukey’s multiple comparison test), with a statistical significance level of alpha=0.05. Significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.
IL-6 and IL-10 ELISA detection from RAW264.7 cells
Concentration of IL-6 (A) and IL-10 (B) secreted from RAW264.7 Lucia macrophage cell line challenged with 20 µg/mL of the MCP glycans of PBCV-1, ATCV-1, OSy-NE5, P91, EIL-1, and PIL6. Accumulated IL-6 and IL-10 concentrations in 20 h supernatants were measured with ELISA. Data are means ± standard error (n = 4). Experimental data were analysed by ANOVA multiple comparison (Tukey’s multiple comparison test), with a statistical significance level of alpha=0.05. Significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.
Healthy donors (n = 12) IgG binding to the viral glycoproteins
A IgG ELISA results: wells were coated with viral glycoproteins and human IgG was specifically detected. Single OD normalized values are reported from 12 different donors, as well as the mean value from each group. Data were normalized over signal from BSA-coated wells. B Heat map from the IgG ELISA results. OD: Optical density. Experimental data were analysed by ANOVA multiple comparison (Tukey’s multiple comparison test), with a statistical significance level of alpha=0.05. Significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.
Carbohydrate-mediated interactions between chloroviruses and the immune system
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December 2024

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62 Reads

Communications Biology

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Sven Bruijns

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Understanding the molecular mechanisms which drive and modulate host-pathogen interactions are essential when designing effective therapeutic and diagnostic approaches aimed at controlling infectious diseases. Certain large and giant viruses have recently been discovered as components of the human virome, yet little is known about their interactions with the host immune system. We have dissected the role of viral N-linked glycans during the interaction between the glycoproteins from six chloroviruses (belonging to three chlorovirus classes: NC64A, SAG, and Osy viruses) and the representative carbohydrate-binding receptors of the innate immune system. Using solid-phase assays we have identified the binding of viral glycoproteins to different C-type lectins in a carbohydrate-dependent manner. These experiments verified the importance of D-rhamnose in modulating their binding to C-type lectins DC-SIGN and Langerin. In vitro assays further determined the ability of the chlorovirus glycoproteins to trigger secretion of cytokines Interleukins 6 and 10 (IL-6 and IL-10) in human monocyte-derived dendritic cells and mouse macrophages. Additionally, IgG from healthy human controls recognized certain chlorovirus glycoproteins, indicating the significance of human environmental viral exposures. Collectively, these results demonstrate the ability of the innate and adaptive immune systems to recognize chlorovirus glycoproteins, a process dependent on their specific N-glycan structures.

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Unveiling the GT114 family: Structural characterization of A075L, a glycosyltransferase from Paramecium bursaria chlorella virus‐1 (PBCV‐1)

November 2024

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70 Reads

Protein A075L is a β‐xylosyltransferase that participates in producing the core of the N‐glycans found in VP54, the major viral capsid protein of Paramecium bursaria chlorella virus‐1 (PBCV‐1). In this study, we present an X‐ray crystallographic analysis of the apo form of A075L, along with its complexes with the sugar donor and with a trisaccharide acceptor. The protein structure shows a typical GT‐B folding, with two Rossmann‐like fold domains, in which the acceptor substrate binds to the N‐terminal region, and the nucleotide‐sugar donor binds to the C‐terminal region. We propose that the catalytic mechanism follows a direct displacement SN2‐like reaction, where Asp73 serves as a catalytic base that deprotonates the incoming nucleophile of the acceptor, facilitating direct displacement of the UDP with the inversion of the anomeric configuration of the acceptor without metal ion dependence, while the interactions with side chains of Arg158 and Arg208 stabilize the developing negative charge. Using isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, high‐performance liquid chromatography, and molecular dynamics simulations, the catalytic activity and specificity of this enzyme have been unraveled.



A. muciniphila LOS chemical structure
A The glycan components of the LOS from A. muciniphila sketched according to the symbology of monosaccharide residues (in the inset); the structure of the core region has been assigned by HR MS and NMR analyzes of the acetic acid and full deacylated products; B, C, D multiplicity edited HSQC NMR spectra of (from left to right) A. muciniphilaOS (full deacylated LOS, 1.2 GHz), OS1 and OS2 (from acetic acid hydrolysis, at 950 MHz): zoom of the anomeric region. Letters refers to the spin systems and are as reported in Supplementary Tables S1, S2 and S3. The glycosidic linkages of Zt and K residues are putative and can be interchanged.
Mass spectrometry of LOS and lipid A
A HR and sub-ppm mass measurement of Nona- and Tetradeca- oligosaccharides from the deacylated LOS performed by ESI-FT-ICR MS (mass spectrum deconvoluted to [M + H]⁺). BA. muciniphilalipid A. Negative ion MALDI-TOF mass spectrum of the lipid A from A. muciniphila. The families of lipid A species that differ in the degree of acetylation are indicated as “Hexa”, “Penta”, and “Tetra” Lipid A (P: phosphate group). The proposed interpretation of relevant ion peaks is reported in Table S3. The structure of the main bis-phosphorylated lipid A species for each family (Tetra, Penta and Hexa) has been sketched in each colored panel. The C17:0 moiety has been depicted in its linear form for description purposes only.
Injection of A. muciniphila LOS in mice triggers mRNA expression in the liver
A Outline of the experimental setup. Two groups of mice were injected interperitoneally with 230 µl of LOS solution (generating a final concentration of 300 µg/kg body weight; LOS group, n = 8) or control group (CT; 50 mM Tris-NaCl buffer pH 7; CT group, n = 7). B Expression of relevant genes such as Toll like receptor 2 and 4 (TLR2, TLR4), monocyte chemoattractant protein 1 (MCP1), Lipopolysaccharides binding protein (LBP), interleukin 1beta and 10 (IL1β and IL10) were determined in the liver by qPCR, RPL19 (ribosomal protein L19) was chosen as housekeeping genes. All the data were compared to the CT group as fold changed (CT group set at 1). Data are expressed as mean ± SEM and compared using two-tailed Mann Whitney test (*p-value < 0.05; ***p-value < 0.0005). Source data are provided as a Source Data file.
Activation of TLR4 and TLR2 by A. muciniphila LOS and its structural components
The signaling capacity of A. muciniphila LOS, OS and lipid A was evaluated by incubating them in HEK-Blue reporter cell lines (A), expressing hTLR4 (B) and hTLR2 (C). A comparative analysis of LOS signaling to the different hTLR2-1, hTLR2-6, and hTLR2 KO1/6 cell lines was made, as the hTLR2 reporter cell line expresses both TLR1 and TLR6, allowing hetero- and homodimerization of TLR2 with TLR1, TLR6 and TLR2 (D). All experiments were performed at least in three biological replicates and representative experiments of three technical replicates are shown. The dose-response curves depict normalized mean TLR responses of three replicates +- SD, and log of the agonist in ng/mL. (Abbreviations used: LOS = lipooligosaccharide, OS = oligosaccharide, hTLR = human Toll-Like Receptor, TLR4 = TLR4 expressing cell line, TLR2 = TLR2 expressing cell line also harboring TLR1 and TLR6, TLR2-1 = cell line only harboring TLR2 and TLR1 heterodimers, TLR2-6 = cell line in which TLR2 and TLR6 are expressed and can form heterodimers, TLR KO = cell line in which all TLR receptors are knocked out, except TLR2). Source data are provided as a Source Data file, and more details are recorded in the “Methods” section.
The lipooligosaccharide of the gut symbiont Akkermansia muciniphila exhibits a remarkable structure and TLR signaling capacity

September 2024

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188 Reads

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2 Citations

The cell-envelope of Gram-negative bacteria contains endotoxic lipopolysaccharides (LPS) that are recognized by the innate immune system via Toll-Like Receptors (TLRs). The intestinal mucosal symbiont Akkermansia muciniphila is known to confer beneficial effects on the host and has a Gram-negative architecture. Here we show that A. muciniphila LPS lacks the O-polysaccharide repeating unit, with the resulting lipooligosaccharide (LOS) having unprecedented structural and signaling properties. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each containing three 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residues. The lipid A moiety appears as a mixture of differently phosphorylated and acylated species and carries either linear or branched acyl moieties. Peritoneal injection of the LOS in mice increased higher gene expression of liver TLR2 than TLR4 (100-fold) and induced high IL-10 gene expression. A. muciniphila LOS was found to signal both through TLR4 and TLR2, whereas lipid A only induced TLR2 in a human cell line. We propose that the unique structure of the A. muciniphila LOS allows interaction with TLR2, thus generating an anti-inflammatory response as to compensate for the canonical inflammatory signaling associated with LOS and TLR4, rationalizing its beneficial host interaction.


Fig. 3. Comparison of biofilm architecture and COMSTAT analysis of CLSM images of M. haemolytica wild-type and mutant E09. Five-day-old biofilms of the wild-type (A and B) and mutant E09 (C and D) were imaged by CLSM under the orthogonal (A and C) and topographical (B and D) view after FISH using the
Fig. 5. Polysaccharide composition of M. haemolytica biofilms in comparison to CPS by GC-MS. Carbohydrate composition of capsular polysaccharide (CPS) from planktonic M. haemolytica wild-type (A), biofilm polysaccharide from the wild-type (B), and biofilm polysaccharide from mutant E09 (C). The profiles of polysaccharides detected in the biofilm of the wild-type and mutant E09 were similar to the profile of CPS from the wild-type. Glc, Gal, and Man stand for glucose, galactose and mannose, respectively. Hep, HexNA, and HexN indicate the presence of a heptose, a hexosaminuronic acid, and a hexosamine residue, respectively, of unknown stereochemistry due to the lack of the appropriate standards. "i" indicates impurity, C16:0 and C18:0 are two long chain fatty acids that occur as contaminants. The signals eluted before 13.5 min are impurities and are not labeled.
Fig. 8. CPS staining and quantification for M. haemolytica strains. The CPS (clear halo) of M. haemolytica wild-type (A), mutant E09 (B), and wecB-complemented strain (C) was visualized by Maneval's staining (100X magnification). Enhanced magnification of the wecB-complemented strain shows elongated cells are multiple, individual cells (arrows). Inset is a further magnified view of three cells (arrowhead) (D). Released carbohydrate content from the cell surface after heat treatment was determined by Anthrone's assay (E). Bars represent the means and the standard deviations of samples tested in triplicate. Significance was determined by twotailed t-tests; *, P < 0.05.
Fig. 11. FISH of polymicrobial biofilms by H. somni strain 2336ΔIbpA9 and M. haemolytica on bovine cells. Five-day-old polymicrobial biofilms were formed by either co-inoculating H. somni 2336ΔIbpA9 and M. haemolytica on day one (A) or sequentially inoculating H. somni 2336ΔIbpA9 on day one and M. haemolytica on day 3 (B) on bovine turbinate cells. H. somni was stained using an H. somni oligonucleotide 16 S rRNA probe labeled with Texas Red. M. haemolytica was stained using an M. haemolytica oligonucleotide 16 S rRNA probe labeled with 6-FAM. Scale bars: 50 μm. COMSTAT analysis of biofilm coverage by each species after coinoculation or sequential inoculation is shown on right. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Relationship between capsule production and biofilm formation by Mannheimia haemolytica, and establishment of a poly-species biofilm with other Pasteurellaceae

September 2024

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36 Reads

Biofilm

Mannheimia haemolytica is one of the bacterial agents responsible for bovine respiratory disease (BRD). The capability of M. haemolytica to form a biofilm may contribute to the development of chronic BRD infection by making the bacteria more resistant to host innate immunity and antibiotics. To improve therapy and prevent BRD, a greater understanding of the association between M. haemolytica surface components and biofilm formation is needed. M. haemolytica strain 619 (wild-type) made a poorly adherent, low-biomass biofilm. To examine the relationship between capsule and biofilm formation, a capsule-deficient mutant of wild-type M. haemolytica was obtained following mutagenesis with ethyl methanesulfonate to obtain mutant E09. Loss of capsular polysaccharide (CPS) in mutant E09 was supported by transmission electron microscopy and Maneval's staining. Mutant E09 attached to polyvinyl chloride plates more effectively, and produced a significantly denser and more uniform biofilm than the wild-type, as determined by crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy with COMSTAT analysis. The biofilm matrix of E09 contained predominately protein and significantly more eDNA than the wild-type, but not a distinct exopolysaccharide. Furthermore, treatment with DNase I significantly reduced the biofilm content of both the wild-type and E09 mutant. DNA sequencing of E09 showed that a point mutation occurred in the capsule biosynthesis gene wecB. The complementation of wecB in trans in mutant E09 successfully restored CPS production and reduced bacterial attachment/biofilm to levels similar to that of the wild-type. Fluorescence in-situ hybridization microscopy showed that M. haemolytica formed a poly-microbial biofilm with Histophilus somni and Pasteurella multocida. Overall, CPS production by M. haemolytica was inversely correlated with biofilm formation, the integrity of which required eDNA. A poly-microbial biofilm was readily formed between M. haemolytica, H. somni, and P. multocida, suggesting a mutualistic or synergistic interaction that may benefit bacterial colonization of the bovine respiratory tract.




A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron

April 2024

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413 Reads

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6 Citations

Nature Immunology

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8⁺ T cell response, and triggers macrophage ‘nutritional immunity’ to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into ‘oncobiotics’ for a multi-layered approach to cancer therapy.



Citations (75)


... However, the finding that at higher concentrations A. muciniphila LPS can elicit similar effects on DCs as E. coli LPS and can induce tolerance toward E. coli LPS would claim of a role of TLR4 in its recognition. In line with this hypothesis, Garcia-Vello and colleagues recently demonstrated that A. muciniphila LPS exhibits an unprecedented chemical structure being devoid of the O-antigen and carrying a unique mono-phosphorylated lipid A consisting of a balanced mixture of tetra-, penta-and hexa-acylated forms [62]. This unique structure of A. muciniphila LPS preferentially activated the TLR2 pathway in human TLR-transfected Hek293 cells and exhibited a low proinflammatory potential in animal studies [62]. ...

Reference:

Akkermansia muciniphila- and Pathogenic Bacteria-Derived Endotoxins Differently Regulate Human Dendritic Cell Generation and γδ T Lymphocyte Activation
The lipooligosaccharide of the gut symbiont Akkermansia muciniphila exhibits a remarkable structure and TLR signaling capacity

... Further research should also focus on the interactions between CARMN and other components of the tumor microenvironment, such as its relationship with inflammatory factors and cytokine expression. CARMN's potential role in regulating inflammation-related signaling pathways, like NF-κB or STAT3, should be explored [92,93]. If CARMN is involved in modulating inflammatory responses, it could help shape the tumor immune environment, either promoting or suppressing immune evasion. ...

The human gut symbiont Ruminococcus gnavus displays strain-specific exopolysaccharides modulating the host immune response
  • Citing Article
  • September 2024

Carbohydrate Polymers

... Glycosylation is one of the most frequent post-translational modifications in proteins from eukaryotic cells (Hu, Guo, & Li, 2006). The glycans that modify glycoproteins have been associated with functions such as contributing to folding, stability, and solubility, mediating the reactivity of the immune system, cell adhesion and differentiation (Lembo, Molinaro, De Castro, Berti, & Biagini, 2024). The development of methodologies to elucidate the contribution of oligosaccharides to the functional properties of glycoproteins is of major importance for research groups working in protein characterization. ...

Impact of glycosylation on viral vaccines
  • Citing Article
  • June 2024

Carbohydrate Polymers

... Advances in targeted sequencing and metagenomics have provided deep insights into microbial ecosystems of foods, elucidating their impact on flavor, preservation, safety, and ultimately human health [2,3]. For example, the health benefits of fermented food are increasingly recognized [4][5][6][7][8][9]. The characterization of microbiota during key fermentation stages is crucial for consistent food production and, therefore, extensively studied [10,11]. ...

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron

Nature Immunology

... L. decastes, an edible mushroom of significant nutritional value, is abundant in protein, various amino acids, vitamins, and polysaccharides. It possesses numerous physiological properties, including antioxidant, anti-aging, anti-tumor, anti-inflammatory, hypoglycemic, hypolipidemic, anti-thrombotic, radiation protection, blood sugar-and lipid-reducing, anti-obesity, and immune system-enhancing capabilities [4,[7][8][9][10][11]. ...

Identification of a capsular polysaccharide from Enterococcus faecium U0317 using a targeted approach to discover immunogenic carbohydrates for vaccine development
  • Citing Article
  • December 2023

Carbohydrate Polymers

... The plasmids were amplified and enriched in E. coli DH5α and then transformed into P. pastoris GS115 according to the user manual (Invitrogen, Pichia Expression version G). As previously described in [19], the P. pastoris transformants were selected with MD medium, and the positive clones were cultivated and induced to produce enzymes with BMGY medium and BMMY medium in sequence. ...

N-glycosylation in Archaea: Unusual sugars and unique modifications
  • Citing Article
  • October 2023

Carbohydrate Research

... What tradeoff might remain that provides an advantage for vraFG loss? Cell envelope modifications often affect phage susceptibility, including multiple WTA-modifications recently characterized in Staphylococci 41,42 . We applied phage-containing supernatant obtained in our mechanism screen (from isolate 32.1; Methods) and quantified plaque-forming-units (PFU). ...

Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis

... In view of these properties, rhamnolipids have the potential to act as a natural flocculant for the aggregation of MPs and enable their subsequent removal by sedimentation or flotation. In addition, taxonomic and metabolic pathway analyses have shown that Pseudomonas is much more abundant on MPs than in the environment (Esposito et al., 2023;Wu et al., 2020). Inspired by the change in surface tension, Jiang et al. (2023) used anionic surfactants to increase the hydrophobicity and flotation efficiency of MPs. ...

Rhamnolipid Self-Aggregation in Aqueous Media: A Long Journey toward the Definition of Structure–Property Relationships

International Journal of Molecular Sciences

... At the same time, the 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose) found in the same N-linked glycan has not been previously described and is only one of two known monosaccharides presenting a direct C-S bond [39]. The glycan attached to selected asparagine residues of membrane proteins in Thermococcus kodakarensis comprises five sugars and a myo-inositol phosphate [59]. This represents the first example of inositol as a component of an N-linked glycan. ...

Structural Determination and Chemical Synthesis of the N‐Glycan from the Hyperthermophilic Archaeon Thermococcus kodakarensis