Surface Carbohydrate Research Articles

Phage-derived polysaccharide depolymerase potentiates ceftazidime efficacy against Acinetobacter baumannii pneumonia via low-serum-dependent mechanisms

The emergence of multidrug-resistant Acinetobacter baumannii (MDR-AB), which most commonly manifests as pneumonia, has posed significant clinical challenges and called for novel treatment strategies. Phage depolymerases, which degrade bacterial surface carbohydrates, have emerged as potential antimicrobial agents. However, their preclinical application is limited to systemic infections due to their dependency on serum-mediated bacterial killing. To extend the treatment paradigm of depolymerase to low-serum lung infections, we explored the feasibility of applying phage depolymerase to potentiate antibiotic efficacy in controlling MDR-AB pneumonia. Using a model depolymerase, Dpo71, we observed that it could effectively potentiate antibiotic efficacy against MDR-AB2 bacteria in low-serum conditions mimicking lung milieu but showed no adjuvant effect in serum-free conditions. Unprecedentedly, we reported this low-serum-dependent mechanism that polysaccharide-degrading enzyme Dpo71 exposed bacteria to serum-induced membrane permeabilization and oxidative phosphorylation pathway inhibition, leading to a weakened ATP-dependent efflux pump and strengthened ROS-induced membrane permeabilization. These joint effects facilitated antibiotic (ceftazidime, CFZ) binding, ultimately exerting bactericidal effects. Resultantly, the bacterial load in the lungs of the Dpo71-CFZ combination group was significantly reduced compared with the Dpo71-alone and CFZ-alone groups. Overall, this study unravels the low-serum-dependent mechanisms by which depolymerase potentiated antibiotic efficacy, highlighting its potential as a novel strategy to enhance antibiotic activity against severe pneumonia.

N-linked protein glycosylation in Nanobdellati (formerly DPANN) archaea and their hosts.

Members of the kingdom Nanobdellati, previously known as DPANN archaea, are characterized by ultrasmall cell sizes and reduced genomes. They primarily thrive through ectosymbiotic interactions with specific hosts in diverse environments. Recent successful cultivations have emphasized the importance of adhesion to host cells for understanding the ecophysiology of Nanobdellati. Cell adhesion is often mediated by cell surface carbohydrates, and in archaea, this may be facilitated by the glycosylated S-layer protein that typically coats their cell surface. In this study, we conducted glycoproteomic analyses on two co-cultures of Nanobdellati with their host archaea, as well as on pure cultures of both host and non-host archaea. Nanobdellati exhibited various glycoproteins, including archaellins and hypothetical proteins, with glycans that were structurally distinct from those of their hosts. This indicated that Nanobdellati autonomously synthesize their glycans for protein modifications probably using host-derived substrates, despite the high energy cost. Glycan modifications on Nanobdellati proteins consistently occurred on asparagine residues within the N-X-S/T sequon, consistent with patterns observed across archaea, bacteria, and eukaryotes. In both host and non-host archaea, S-layer proteins were commonly modified with hexose, N-acetylhexosamine, and sulfonated deoxyhexose. However, the N-glycan structures of host archaea, characterized by distinct sugars such as deoxyhexose, nonulosonate sugar, and pentose at the nonreducing ends, were implicated in enabling Nanobdellati to differentiate between host and non-host cells. Interestingly, the specific sugar, xylose, was eliminated from the N-glycan in a host archaeon when co-cultured with Nanobdella. These findings enhance our understanding of the role of protein glycosylation in archaeal interactions.IMPORTANCENanobdellati archaea, formerly known as DPANN, are phylogenetically diverse, widely distributed, and obligately ectosymbiotic. The molecular mechanisms by which Nanobdellati recognize and adhere to their specific hosts remain largely unexplored. Protein glycosylation, a fundamental biological mechanism observed across all domains of life, is often crucial for various cell-cell interactions. This study provides the first insights into the glycoproteome of Nanobdellati and their host and non-host archaea. We discovered that Nanobdellati autonomously synthesize glycans for protein modifications, probably utilizing substrates derived from their hosts. Additionally, we identified distinctive glycosylation patterns that suggest mechanisms through which Nanobdellati differentiate between host and non-host cells. This research significantly advances our understanding of the molecular basis of microbial interactions in extreme environments.

Nutrition Design Modeling Method Development for Adana Origin Maize Starch Granules by Structural and Elemental Analysis

The granules of Adana origin maize starch were treated with thermal, acidic and amylase enzyme degradation processes and examined using the Field Emission Gun – Scanning Electron Microscope (FEG-SEM) to determine the structure analysis and composition modeling. The morphology deformation of the starch granules in shape, surface and size, and elemental analysis peaks and data were determined upon degradation. The microstructure images resulted the occurrence of stretches and slit cracks on the nanoparticle surface and high carbohydrate carbon elemental compound. The degraded amylopectin layers upon the conversion stages were notably presented with high clarity ever published. Investigation of the treated starch granules presented that the granules presented an explicit swelled, splintered and spherical granules model, and a high carbon carbohydrate nutrition model. The swelling and gelling existence temperature of the starch samples were detected as 69 °C.

Development and Application of a High-Throughput Method for the Purification and Analysis of Surface Carbohydrates from Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium, and a leading cause of neonatal sepsis in low- and middle-income countries, often associated with anti-microbial resistance. Two types of polysaccharides are expressed on the Kp cell surface and have been proposed as key antigens for vaccine design: capsular polysaccharides (known as K-antigens, K-Ags) and O-antigens (O-Ags). Historically, Kp has been classified using capsule serotyping and although 186 distinct genotypes have been predicted so far based on sequence analysis, many structures are still unknown. In contrast, only 11 distinct OAg serotypes have been described. The characterization of emerging strains requires the development of a high-throughput purification method to obtain sufficient K- and O-Ag material to characterize the large collection of serotypes and gain insight on structural features and potential cross-reactivity that could allow vaccine simplification. Here, this was achieved by adapting our established method for the simple purification of O-Ags, using mild acetic acid hydrolysis performed directly on bacterial cells, followed by filtration and precipitation steps. The method was successfully applied to purify the surface carbohydrates from different Kp strains, thereby demonstrating the robustness and general applicability of the purification method developed. Further, antigen characterization showed that the purification method had no impact on the structural integrity of the polysaccharides and preserved labile substituents such as O-acetyl and pyruvyl groups. This method can be further optimized for scaling up and manufacturing to support the development of high-valency saccharide-based vaccines against Kp.

Inhibitory receptors of plasmacytoid dendritic cells as possible targets for checkpoint blockade in cancer.

Plasmacytoid dendritic cells (pDCs) are the major producers of type I interferons (IFNs), which are essential to mount antiviral and antitumoral immune responses. To avoid exaggerated levels of type I IFNs, which pave the way to immune dysregulation and autoimmunity, pDC activation is strictly regulated by a variety of inhibitory receptors (IRs). In tumors, pDCs display an exhausted phenotype and correlate with an unfavorable prognosis, which largely depends on the accumulation of immunosuppressive cytokines and oncometabolites. This review explores the hypothesis that tumor microenvironment may reduce the release of type I IFNs also by a more pDC-specific mechanism, namely the engagement of IRs. Literature shows that many cancer types express de novo, or overexpress, IR ligands (such as BST2, PCNA, CAECAM-1 and modified surface carbohydrates) which often represent a strong predictor of poor outcome and metastasis. In line with this, tumor cells expressing ligands engaging IRs such as BDCA-2, ILT7, TIM3 and CD44 block pDC activation, while this blocking is prevented when IR engagement or signaling is inhibited. Based on this evidence, we propose that the regulation of IFN secretion by IRs may be regarded as an "innate checkpoint", reminiscent of the function of "classical" adaptive immune checkpoints, like PD1 expressed in CD8+ T cells, which restrain autoimmunity and immunopathology but favor chronic infections and tumors. However, we also point out that further work is needed to fully unravel the biology of tumor-associated pDCs, the neat contribution of pDC exhaustion in tumor growth following the engagement of IRs, especially those expressed also by other leukocytes, and their therapeutic potential as targets of combined immune checkpoint blockade in cancer immunotherapy.

Analysis of Bacterial Phosphorylcholine-Related Genes Reveals an Association between Type-Specific Biosynthesis Pathways and Biomolecules Targeted for Phosphorylcholine Modification.

Many bacterial surface proteins and carbohydrates are modified with phosphorylcholine (ChoP), which contributes to host mimicry and can also promote colonization and survival in the host. However, the ChoP biosynthetic pathways that are used in bacterial species that express ChoP have not been systematically studied. For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as Neisseria meningitidis and Neisseria gonorrhoeae. This raises a question as to the origin of the ChoP used for macromolecule biosynthesis in these species. In the current study, we used in silico analyses to identify the potential pathways involved in ChoP biosynthesis in genomes of the 26 bacterial species reported to express a ChoP-modified biomolecule. We used the four known ChoP biosynthetic pathways and a ChoP transferase as search terms to probe for their presence in these genomes. We found that the Lic-1 pathway is primarily associated with organisms producing ChoP-modified carbohydrates, such as lipooligosaccharide. Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins. Additionally, ChoP biosynthesis pathways, such as phospholipid N-methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP-modified proteins. Thus, a major finding of this study is the association of a particular ChoP biosynthetic pathway with a cognate, target ChoP-modified surface factor; i.e., protein versus carbohydrate. This survey failed to identify a known biosynthetic pathway for some species that express ChoP, indicating that a novel ChoP biosynthetic pathway(s) may remain to be identified. IMPORTANCE The modification of bacterial surface virulence factors with phosphorylcholine (ChoP) plays an important role in bacterial virulence and pathogenesis. However, the ChoP biosynthetic pathways in bacteria have not been fully understood. In this study, we used in silico analysis to identify potential ChoP biosynthetic pathways in bacteria that express ChoP-modified biomolecules and found the association between a specific ChoP biosynthesis pathway and the cognate target ChoP-modified surface factor.

Tet-Dependent Gene Expression in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.

Fluorescent nanoprobes based on quantum dots conjugated to Cramoll to assess surface carbohydrates of Aeromonas spp.

The association of quantum dots (QDs) to carbohydrate-binding proteins – lectins – has revealed novel biotechnological strategies for glycobiology studies. Herein, carboxyl-coated QDs were conjugated by adsorption to Cramoll, a glucose/mannose lectin obtained from Cratylia mollis seeds. Then, the conjugates were optically characterized and used to evaluate the surface carbohydrate profiles of four Aeromonas species isolated from the tambaqui fish (Colossoma macropomum). All the Aeromonas cells were labeled by the conjugate. Inhibition assays with methyl-α-D-mannopyranoside and mannan were performed to confirm the labeling specificity. Cramoll-QDs conjugates presented high brightness and showed similar absorption and emission profiles compared to bare QDs. According to the labeling pattern of Aeromonas spp. by the conjugate, results suggested that A. jandaei and A. dhakensis strains may harbor a higher content of more complex glucose/mannose surface glycans, with more available sites for Cramoll-QDs interaction, than A. hydrophila and A. caviae. Noteworthy, the Cramoll-QDs conjugates demonstrated to be potential tools for bacterial characterization based on superficial carbohydrate detection.

Sialyl Lewis X mediates interleukin-1 beta-induced trophoblast adhesion to endometrial cells during human embryo implantation.

Cell surface carbohydrate antigens sialyl Lewis X (sLeX) and Lewis Y (LeY) are paramount glycoconjugates and are abundantly expressed in the receptive endometrium. Furthermore, among the important biological functions of both antigens is their role in leukocytes adhesion and extravasation. Interleukin-1 beta (IL-1β) is involved in the process of human embryo implantation and placenta development. Here, we used an in vitro model to investigate whether sLeX and LeY are playing a role in the embryo implantation process mediated by IL-1β. Our results are showing that the expression of cell surface sLeX was enhanced in endometrial RL95-2 cells after exposure to IL-1β. RT-qPCR detection indicated that the transcript level of glycosyltransferase gene fucosyltransferase 3 (FUT3) was significantly elevated and that of FUT4/7 and ST3 beta-galactoside alpha-2,3-sialyltransferase 3/4 (ST3GAL3/4) were decreased by treatment with IL-1β. Modulatory role of glycosyltransferase FUT3 on sLeX biosynthesis was determined by FUT3 siRNA transfection in RL95-2 cells. Results showed that the expression level of sLeX was suppressed, but no change was observed in regard to LeY. Moreover, IL-1β promoted the HTR-8/SVneo trophoblast spheroids attachment to the RL95-2 endometrial monolayer, which was partially blocked by anti-sLeX antibody and FUT3 knockdown. Gene expression analysis of the RNA-seq transcriptome data from human secretory endometrium demonstrated a significantly higher level of FUT3 in the mid-secretory phase compared to the early secretory phase, which was correlated with the expression of IL1B. In summary, the inflammatory microenvironment at the fetomaternal interface can regulate the glycosylation pattern of endometrial cells at the time of implantation. SLeX can be significantly induced by IL-1β via increasing FUT3 expression, which facilitates the trophoblast adhesion during embryo implantation.

Effects of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Infection on the Surface Glycoprofiling of Porcine Pulmonary Microvascular Endothelial Cells.

Previously, our study has demonstrated that porcine pulmonary microvascular endothelial cells (PPMVECs) were susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and produced a significant non-specific immune response to it. The significance of microvascular endothelial glycocalyx is increasingly attracting attention, and its rich carbohydrate components are not only important signaling molecules, but also remarkably influence the signaling of most proteins. Comprehending changes in the carbohydrate chains contributes to understanding cell functions. This study aimed to reveal the effects of HP-PRRSV infection on the surface carbohydrate chains of PPMVECs. PPMVECs were isolated and cultured in vitro and infected with HP-PRRSV HN and JXA1 strains. Scanning electron microscopy analysis indicated that at 48 h post-infection, some broken holes were in their cell membranes, and that the surface fibrous glycocalyx was obviously reduced or even disappeared. Lectin microarray analysis indicated that the fluorescence intensities of 8 and 7 lectin sites were significantly changed by the HP-PRRSV HN and JXA1 strains, respectively, among which there were 6 common lectin sites. The up-regulation of common lectins (RCA-I, LEL, and STL) and the down-regulation of common lectins (LCA, DSA, and PHA-E) were confirmed by lectin fluorescence staining and lectin flow cytometry, respectively. Together, the results show that the HP-PRRSV infection can induce the glycocalyx disruption of PPMVECs and their surface glycoprofiling changes, and that the poly-N-acetyllactosamine and complex N-glycan are the main up-regulated and down-regulated carbohydrate chains, respectively. Our findings may provide insights into revealing the pathogenesis of HP-PRRSV from the perspective of glycobiology.

β-1,3-Glucan/chitin unmasking in the Saccharomyces cerevisiae mutant, Δmnn9, promotes immune response and resistance of the Pacific oyster (Crassostrea gigas) to Vibrio coralliilyticus infection

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component β-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, β-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of β-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for β-glucan-binding protein (CgβGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of β-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to β-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgβGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked β-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding.

An Efficacious Transgenic Strategy for Triple Knockout of Xeno-Reactive Antigen Genes GGTA1, CMAH, and B4GALNT2 from Jeju Native Pigs.

Pigs are promising donors of biological materials for xenotransplantation; however, cell surface carbohydrate antigens, including galactose-alpha-1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd blood group antigens, play a significant role in porcine xenograft rejection. Inactivating swine endogenous genes, including GGTA1, CMAH, and B4GALNT2, decreases the binding ratio of human IgG/IgM in peripheral blood mononuclear cells and erythrocytes and impedes the effectiveness of α-Gal, Neu5Gc, and Sd, thereby successfully preventing hyperacute rejection. Therefore, in this study, an effective transgenic system was developed to target GGTA1, CMAH, and B4GALNT2 using CRISPR-CAS9 and develop triple-knockout pigs. The findings revealed that all three antigens (α-Gal, Neu5Gc, and Sd) were not expressed in the heart, lungs, or liver of the triple-knockout Jeju Native Pigs (JNPs), and poor expression of α-Gal and Neu5G was confirmed in the kidneys. Compared with the kidney, heart, and lung tissues from wild-type JNPs, those from GGTA1/CMAH/ B4GALNT2 knockout-recipient JNPs exhibited reduced human IgM and IgG binding and expression of each immunological rejection component. Hence, reducing the expression of swine xenogeneic antigens identifiable by human immunoglobulins can lessen the immunological rejection against xenotransplantation. The findings support the possibility of employing knockout JNP organs for xenogeneic transplantation to minimize or completely eradicate rejection using multiple gene-editing methods.

Markers of endothelial glycocalyx dysfunction in Clarkson disease

BackgroundClarkson disease (monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome, ISCLS) is a rare idiopathic condition marked by transient, relapsing-remitting episodes of systemic microvascular hyper-permeability, which liberates plasma fluid and macromolecules into the peripheral tissues. This pathology manifests clinically as the abrupt onset of hypotensive shock, hemoconcentration, and hypoalbuminemia.MethodsWe analysed endothelial glycocalyx (eGCX)-related markers in plasma from patients with ISCLS during acute disease flares and convalescence by ELISA and comprehensive proteomic profiling. We evaluated eGCX-related components and gene expression in cultured endothelial cells using RNA-sequencing, real-time PCR, and fluorescence staining.ResultsSerum levels of eGCX-related core components including hyaluronic acid (HA) and the core proteoglycan soluble syndecan-1 (sCD138) were elevated at baseline and during acute ISCLS flares. Serial measurements demonstrated that sCD138 levels peaked during the recovery (post-leak) phase of the illness. Proteomic analysis of matched acute and convalescent ISCLS plasma revealed increased abundance of eGCX-related proteins, including glypicans, thrombospondin-1 (TSP-1), and eGCX-degrading enzymes in acute compared to remission plasma. Abundance of endothelial cell damage markers did not differ in acute and baseline plasma. Expression of several eGCX-related genes and surface carbohydrate content in endothelial cells from patients with ISCLS did not differ significantly from that observed in healthy control cells.ConclusionseGCX dysfunction, but not endothelial injury, may contribute to clinical symptoms of acute ISCLS.Serum levels of of eGCX components including sCD138 may be measured during acute episodes of ISCLS to monitor clinical status and therapeutic responses.

Rotavirus C Replication in Porcine Intestinal Enteroids Reveals Roles for Cellular Cholesterol and Sialic Acids.

Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs. Group C rotavirus (RVC) is an emerging pathogen increasingly reported in pigs and humans worldwide, and is currently recognized as the major cause of gastroenteritis in neonatal piglets that results in substantial economic losses to the pork industry. However, little is known about RVC pathogenesis due to the lack of a robust cell culture system, with the exception of the RVC Cowden strain. Here, we evaluated the permissiveness of porcine crypt-derived 3D and 2D intestinal enteroid (PIE) culture systems for RVC infection. Differentiated 3D and 2D PIEs were infected with porcine RVC (PRVC) Cowden G1P[1], PRVC104 G3P[18], and PRVC143 G6P[5] virulent strains, and the virus replication was measured by qRT-PCR. Our results demonstrated that all RVC strains replicated in 2D-PIEs poorly, while 3D-PIEs supported a higher level of replication, suggesting that RVC selectively infects terminally differentiated enterocytes, which were less abundant in the 2D vs. 3D PIE cultures. While cellular receptors for RVC are unknown, target cell surface carbohydrates, including histo-blood-group antigens (HBGAs) and sialic acids (SAs), are believed to play a role in cell attachment/entry. The evaluation of the selective binding of RVCs to different HBGAs revealed that PRVC Cowden G1P[1] replicated to the highest titers in the HBGA-A PIEs, while PRVC104 or PRVC143 achieved the highest titers in the HBGA-H PIEs. Further, contrasting outcomes were observed following sialidase treatment (resulting in terminal SA removal), which significantly enhanced Cowden and RVC143 replication, but inhibited the growth of PRVC104. These observations suggest that different RVC strains may recognize terminal (PRVC104) as well as internal (Cowden and RVC143) SAs on gangliosides. Finally, several cell culture additives, such as diethylaminoethyl (DEAE)-dextran, cholesterol, and bile extract, were tested to establish if they could enhance RVC replication. We observed that only DEAE-dextran significantly enhanced RVC attachment, but it had no effect on RVC replication. Additionally, the depletion of cellular cholesterol by MβCD inhibited Cowden replication, while the restoration of the cellular cholesterol partially reversed the MβCD effects. These results suggest that cellular cholesterol plays an important role in the replication of the PRVC strain tested. Overall, our study has established a novel robust and physiologically relevant system to investigate RVC pathogenesis. We also generated novel, experimentally derived evidence regarding the role of host glycans, DEAE, and cholesterol in RVC replication, which is critical for the development of control strategies.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

ABSTRACTDespite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition.IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as “O antigen.” This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

Exploiting phage-derived carbohydrate depolymerases for combating infectious diseases.

Bacteria are protected against the immune system of their human hosts, as well as against predators such as phages, by expressing diverse surface carbohydrates. Some phages produce specialized depolymerases which can degrade those carbohydrates. Here, we discuss the biological role of depolymerases and how they can be exploited to develop new therapeutic strategies against pathogens.

Utility of mean platelet volume in differentiating intrahepatic cholangiocarcinoma from hepatocellular carcinoma

BackgroundIntrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are the most prevalent histologic types of primary liver cancer. HCC and ICC differ in treatment and prognosis, warranting an effective differential diagnosis between them. This study aimed to explore the clinical value of mean platelet volume (MPV) to discriminate between HCC and ICC.Material/methodsWe performed a retrospective analysis of ICC and HCC patients who were from the Harbin Medical University Cancer Hospital, China. Logistic regression analysis was used to identify the independent factors for the differentiation of HCC and ICC. A receiver operating characteristic curve was built to evaluate the diagnostic performance of the potential model. An independent validation study was performed to validate the diagnostic ability.ResultsICC patients were detected in 146 out of 348 patients in the primary cohort. MPV levels were decreased in ICC patients compared with those in HCC patients. Logistic regression analysis revealed that MPV was an independent factor in distinguishing HCC from ICC. A combination of sex, hepatitis B surface antigen, MPV, alpha-fetoprotein, and carbohydrate antigen 19–9 demonstrated a good capability to differentiate HCC from ICC. Similar results were achieved in the validation cohort.ConclusionsMPV may be a new marker to help distinguish ICC from HCC. Further validation studies are required.

Structural Characterization and Evaluation of an Epitope at the Tip of the A-Band Rhamnan Polysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Density of Compatible Ligands on the Surface of Food Particles Modulates Sorting Efficiency in the Blue Mussel Mytilus edulis

The adhesion between food particles and mucus is a fundamental process in particle sorting in suspension-feeding bivalves that requires specific recognition. Interactions between carbohydrate-binding proteins (lectins) expressed on the feeding organs and carbohydrates present on microbial cell surface can provide this specificity. Microalga cell surface carbohydrates (MCSC) represent unique patterns that can be considered as species-specific fingerprints. In this study, sorting efficiencies in blue mussels Mytilus edulis fed with microalgae having modified MCSC and engineered microspheres coated with target carbohydrates was measured. The nature and quantities of surface carbohydrates required to trigger sorting in mussels was evaluated and the relationship between ligand quantities and sorting efficiency (SE) was determined. Mussels fed with Chlamydomonas which MCSC were blocked with ConA or PEA lectins (affinity to mannose and glucose) led to a significant decrease of the sorting efficiencies, not observed when the lectin UEA (affinity to fucose) was used. The ability of commercial lectins to inhibit sorting was not linear and a threshold was noted between 30 and 45 ug lectins per million algae cells. Further, mussels were fed with microspheres coated with neoglycoproteins. Results showed that glucose-BSA, but not fucose-BSA, has an effect on particle sorting in mussels, and 1.08 x 109 molecules of glucose per microspheres, corresponding to a density of 6.99 x 106 molecules of glucose per µm2, triggers particle selection. These findings support that selection of food particles by mussels rely on the strength of the bond between suspended particle and the mucosal layer that mediate sorting, and that these bonds depend on the quantity of compatible ligands on each particle.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)–associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano–liquid chromatography coupled with electrospray ionization–mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.