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ABSTRACT Pseudomonas aeruginosais considered one of the most challenging, drug-resistant, opportunistic pathogens partly due to its ability to synthesize robust biofilms. Biofilm is a mixture of extracellular polymeric substances (EPS) that encapsulates microbial cells, leading to immune evasion, antibiotic resistance, and thus higher risk of infection. In the cystic fibrosis lung environment,P. aeruginosaundergoes a mucoid transition, defined by overproduction of the exopolysaccharide alginate. Alginate encapsulation results in bacterial resistance to antibiotics and the host immune system. Given its role in airway inflammation and chronic infection, alginate is an obvious target to improve treatment forP. aeruginosainfection. Previously, we demonstrated polysaccharide lyase Smlt1473 fromStenotrophomonas maltophiliastrain k279a can catalyze the degradation of multiple polyuronidesin vitro, including D-mannuronic acid (poly-ManA). Poly-ManA is a major constituent ofP. aeruginosaalginate, suggesting that Smlt1473 could have potential application against multidrug-resistantP. aeruginosaand perhaps other microbes with related biofilm composition. In this study, we demonstrate that Smlt1473 can inhibit and degrade alginate fromP. aeruginosa. Additionally, we show that testedP. aeruginosastrains are dominant in acetylated alginate and that all but one have similar M-to-G ratios. These results indicate that variation in enzyme efficacy among the isolates is not primarily due to differences in total EPS or alginate chemical composition. Overall, these results demonstrate Smlt1473 can inhibit and degradeP. aeruginosaalginate and suggest that other factors including rate of EPS production, alginate sequence/chain length, or non-EPS components may explain differences in enzyme efficacy. IMPORTANCEPseudomonas aeruginosais a major opportunistic human pathogen in part due to its ability to synthesize biofilms that confer antibiotic resistance. Biofilm is a mixture of polysaccharides, DNA, and proteins that encapsulate cells, protecting them from antibiotics, disinfectants, and other cleaning agents. Due to its ability to increase antibiotic and immune resistance, the exopolysaccharide alginate plays a large role in airway inflammation and chronicP. aeruginosainfection. As a result, colonization withP. aeruginosais the leading cause of morbidity and mortality in CF patients. Thus, it is an obvious target to improve the treatment regimen forP. aeruginosainfection. In this study, we demonstrate that polysaccharide lyase, Smlt1473, inhibits alginate secretion and degrades established alginate from a variety of mucoidP. aeruginosaclinical isolates. Additionally, Smlt1473 differs from other alginate lyases in that it is active against acetylated alginate, which is secreted during chronic lung infection. These results suggest that Smlt1473 may be useful in treating infections associated with alginate-producingP. aeruginosa, as well as have the potential to reduceP. aeruginosaEPS in non-clinical settings. 

Abstract People living with HIV (PLWH) experience increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors associated with this vulnerability remain uncertain. In the general population, alterations in theN-glycans on IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgGN-glycans in cross-sectional and longitudinal samples from 1214 women and men, living with and without HIV. PLWH exhibit an accelerated accumulation of pro-aging-associated glycan alterations and heightened expression of senescence-associated glycan-degrading enzymes compared to controls. These alterations correlate with elevated markers of inflammation and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit a reduced ability to elicit anti-HIV Fc-mediated immune activities. These findings hold potential for the development of biomarkers and tools to identify and prevent premature aging and comorbidities in PLWH. 

ABSTRACT Whole genome sequencing has revealed that the genome ofStaphylococcus aureuspossesses an uncharacterized 5-gene operon (SAOUHSC_00088–00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus assscfor staphylococcal surface carbohydrate. We found that thesscgenes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed inEscherichia coliand extracted by heat treatment. Ssc was also conjugated to AcrA fromCampylobacter jejuniinE. coliusing protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting ofN-acetylgalactosamine. We further demonstrated that the expression of thesscgenes inS. aureusaffected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCESurface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens.Staphylococcus aureusproduces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide inS. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide. 

Abstract Polymeric donors of gasotransmitters, gaseous signaling molecules such as hydrogen sulfide, nitric oxide, and carbon monoxide, hold potential for localized and extended delivery of these reactive gases. Examples of gasotransmitter donors based on polysaccharides are limited despite the availability and generally low toxicity of this broad class of polymers. In this work, we sought to create a polysaccharide H₂S donor by covalently attachingN‐thiocarboxyanhydrides (NTAs) to amylopectin, the major component of starch. To accomplish this, we added an allyl group to an NTA, which can spontaneously hydrolyze to release carbonyl sulfide and ultimately H₂S via the ubiquitous enzyme carbonic anhydrase, and then coupled it to thiol‐functionalized amylopectin of three different molecular weights (MWs) through thiol‐ene “click” photochemistry. We also varied the degree of substitution (DS) of the NTA along the amylopectin backbone. H₂S release studies on the six samples, termed amyl‐NTAs, with variable MWs (three) and DS values (two), revealed that lower MW and higher DS led to faster release. Finally, dynamic light scattering experiments suggested that aggregation increased with MW, which may also have affected H₂S release rates. Collectively, these studies present a new synthetic method to produce polysaccharide H₂S donors for applications in the biomedical field. 

Abstract Despite the recent progress on the solution-phase enzymatic synthesis of heparan sulfate (HS) and chondroitin sulfate (CS), solid-phase enzymatic synthesis has not been fully investigated. Here, we describe the solid-phase enzymatic synthesis of HS and CS backbone oligosaccharides using specialized linkers. We demonstrate the use of immobilized HS linker to synthesize CS, and the use of immobilized CS linker to synthesize HS. The linkers were then digested with chondroitin ABCase and heparin lyases, respectively, to obtain the products. Our findings uncover a potential approach for accelerating the synthesis of structurally homogeneous HS and CS oligosaccharides. 

Abstract Stereoselective reactions have played a vital role in the emergence of life, evolution, human biology, and medicine. However, for a long time, most industrial and academic efforts followed a trial-and-error approach for asymmetric synthesis in stereoselective reactions. In addition, most previous studies have been qualitatively focused on the influence of steric and electronic effects on stereoselective reactions. Therefore, quantitatively understanding the stereoselectivity of a given chemical reaction is extremely difficult. As proof of principle, this paper develops a novel composite machine learning method for quantitatively predicting the enantioselectivity representing the degree to which one enantiomer is preferentially produced from the reactions. Specifically, machine learning methods that are widely used in data analytics, including Random Forest, Support Vector Regression, and LASSO, are utilized. In addition, the Bayesian optimization and permutation importance tests are provided for an in-depth understanding of reactions and accurate prediction. Finally, the proposed composite method approximates the key features of the available reactions by using Gaussian mixture models, which provide suitable machine learning methods for new reactions. The case studies using the real stereoselective reactions show that the proposed method is effective and provides a solid foundation for further application to other chemical reactions. 

ABSTRACT Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacteriumAnabaenasp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment ofAnabaena, the analysis of the extracellular metabolites and proteins of afurC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction ofAnabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome offurC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins andamiCgene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, includingamiC1andamiC2,could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in thefurC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments infurC-overexpressing cells, namely 1,6-anhydro-N-acetyl-β-D-muramic acid (anhydroMurNAc) and its associated disaccharide (β-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.IMPORTANCECyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions.Anabaenasp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator inAnabaena, which modulates the response to several stresses. Here, we show thatfurC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer. 

Abstract Spectroscopic techniques generate one-dimensional spectra with distinct peaks and specific widths in the frequency domain. These features act as unique identities for material characteristics. Deep neural networks (DNNs) has recently been considered a powerful tool for automatically categorizing experimental spectra data by supervised classification to evaluate material characteristics. However, most existing work assumes balanced spectral data among various classes in the training data, contrary to actual experiments, where the spectral data is usually imbalanced. The imbalanced training data deteriorates the supervised classification performance, hindering understanding of the phase behavior, specifically, sol-gel transition (gelation) of soft materials and glycomaterials. To address this issue, this paper applies a novel data augmentation method based on a generative adversarial network (GAN) proposed by the authors in their prior work. To demonstrate the effectiveness of the proposed method, the actual imbalanced spectral data from Pluronic F-127 hydrogel and Alpha-Cyclodextrin hydrogel are used to classify the phases of data. Specifically, our approach improves 8.8%, 6.4%, and 6.2% of the performance of the existing data augmentation methods regarding the classifier’s F-score, Precision, and Recall on average, respectively. Specifically, our method consists of three DNNs: the generator, discriminator, and classifier. The method generates samples that are not only authentic but emphasize the differentiation between material characteristics to provide balanced training data, improving the classification results. Based on these validated results, we expect the method’s broader applications in addressing imbalanced measurement data across diverse domains in materials science and chemical engineering. 

Abstract SARS-CoV-2 infection causes spike-dependent fusion of infected cells with ACE2 positive neighboring cells, generating multi-nuclear syncytia that are often associated with severe COVID. To better elucidate the mechanism of spike-induced syncytium formation, we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical stimulator for spike-induced cell-cell fusion. We show that HS binds spike and promotes spike-induced ACE2 clustering, forming synapse-like cell-cell contacts that facilitate fusion pore formation between ACE2-expresing and spike-transfected human cells. Chemical or genetic inhibition of HS mitigates ACE2 clustering, and thus, syncytium formation, whereas in a cell-free system comprising purified HS and lipid-anchored ACE2, HS stimulates ACE2 clustering directly in the presence of spike. Furthermore, HS-stimulated syncytium formation and receptor clustering require a conserved ACE2 linker distal from the spike-binding site. Importantly, the cell fusion-boosting function of HS can be targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice. Thus, HS, as a host factor exploited by SARS-CoV-2 to facilitate receptor clustering and a stimulator of infection-associated syncytium formation, may be a promising therapeutic target for severe COVID. 

Abstract A mouse monoclonal antibody (mAb FL100A) previously prepared againstFlavobacterium psychrophilum(Fp) CSF259‐93 has now been examined for binding to lipopolysaccharides (LPS) of this strain andFp950106‐1/1. The corresponding O‐polysaccharides (O‐PS) of these strains are formed by identical trisaccharide repeats composed ofl‐Rhamnose (l‐Rha), 2‐acetamido‐2‐deoxy‐l‐fucose (l‐FucNAc) and 2‐acetamido‐4‐R₁‐2,4‐dideoxy‐d‐quinovose (d‐Qui2NAc4NR₁) where R₁represents a dihydroxyhexanamido moiety. The O‐PS loci of these strains are also identical except for the gene (wzy1orwzy2) that encodes the polysaccharide polymerase. Accordingly, adjacent O‐PS repeats are joined throughd‐Qui2NAc4NR₁andl‐Rha bywzy2‐dependent α(1–2) linkages inFpCSF259‐93 versuswzy1‐dependent β(1–3) linkages inFp950106‐1/1. mAb FL100A reacted strongly withFpCSF259‐93 O‐PS and LPS but weakly or not at all withFp950106‐1/1 LPS and O‐PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5‐times stronger to homologousFpCSF259‐93 LPS than to LPS from a strain with a different R‐group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O‐PS. Thus,FpCSF259‐93 O‐PS formed a stable well‐defined compact helix in which the R₁groups were displayed in a regular pattern on the helix exterior while unreactiveFp950106‐1/1 O‐PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2‐linkedF. psychrophilumO‐PS and LPS.