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ABSTRACT D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in amurI racDmutant ofVibrio fischerigrown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation ofbsrF, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the samemurI racDmutant from 10¹⁰colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation ingltS, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutantgltSalleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations ingltSenabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutantgltSin the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, thisV. fischerimutant still colonizedEuprymna scolopesand triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. InVibrio fischeri,in transexpression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study. 

Abstract Molecular definition is usually regarded as a prerequisite to achieve protein recognition and functional modulation, particularly for macromolecular interactions. Herein, we report that polymers with specific combinations of monomers arranged into random sequences [random hetero oligomers (RHOs)] can selectively bind to a model protein. Using green fluorescent protein (GFP) as a target, polyacrylates were developed that bound with nanomolar affinity and enhanced fluorescence by >100%. Purification of the polymerization product revealed subpopulations of compositions with distinct affinities and selectivity for GFP over a competing protein. Experimental and computational binding analyses confirmed that there are distinct RHO–GFP interactions, which are influenced by RHO chemical composition. These findings show that sequence‐defined structures are not a prerequisite for selective protein recognition. Synthetic polymers can instead serve as scalable, tunable platforms for molecular recognition—representing a significant leap towards next‐generation sensing, therapeutic, responsive, and catalytic materials in domains previously dominated by biologics or complex peptide scaffolds. 

Abstract Galectin‐3–ligand complexes are characterized by halogen, σ‐hole bonds, hydrogen bonds, cation‐π and CH‐π interactions. Here, we model these non‐covalent interactions with the AMOEBA polarizable force field and conduct an absolute binding free energy analysis on leading galectin‐3 inhibitors. Synthetic drug molecules GB0139, GB1107, and GB1211 were estimated to have binding free energies of −4.3, −6.7, and −9.5 kcal/mol respectively. This compares to −0.3 and 1.4 kcal/mol for the natural ligands, N‐acetyllactosamine type 1 and type 2, respectively. We calculated the electric fields projected along key bonds in each ligand to further rationalize these results. We find that while the hydroxyl groups of the natural ligands interact reasonably well with residues in galectin‐3's binding pocket, structural dynamics weaken the binding pose and favor interactions with water, sometimes yielding to dissociation. In contrast, the more favorable binding energy of GB1211, leading inhibitor in clinical studies, is associated with strong and constant electric fields across the bonds investigated, suggesting a stiffer binding pose with a stabilizing σ‐hole interaction. 

ABSTRACT Corynebacterialesare normal constituents of the human skin microbiota, involved in the metabolism of sweat and sebaceous glands secretions. The resultant compounds are a source of human body malodor. Some species, such asCorynebacterium jeikeium, are also opportunistic pathogens, and can cause systemic infection from colonization of medical devices such as intravascular catheters. Cell lytic enzymes have become effective alternatives to antibiotics and chemical oxidants in eliminating undesirable pathogens. As a baseline for antibacterial enzymatic activity we selected the broadly specific antibacterial enzyme lysozyme. While lysozyme had minimal activity on its own, the activity was significantly potentiated by the addition of a disruptor of the surface lipid layer of the bacterial cell wall, such as the lipase LysB (originating from a mycobacterial bacteriophage) or ethyleneglycol tetraacetic acid. To identify cell lytic enzymes with specific anti‐Corynebacterialactivity, we used a whole enzyme sequence as a probe to interrogate a custom‐builtin silicocDNA database of targetCorynebacterialspecies and their bacteriophages. We also used a consensus enzymatically active domain as a probe. We evaluated the antibacterial activity of the thus discovered enzymes under a panel of application‐relevant conditions, and determined that the newly identified bacterial autolysin NCBI WP_003861765.1 showed activity comparable to that of lysozyme, and the phage endolysin NCBI SLN03091 showed substantially higher activity than lysozyme and was significantly potentiated in the presence of LysB. As a result, we identified a nonchemical, nonantibiotic route to controllingCorynebacterialviability. 

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 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 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 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 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 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.