Analysis of Powassan Virus Genome Sequences from Human Cases Reveals Substantial Genetic Diversity with Implications for Molecular Assay Development.

Powassan virus (POWV) is an emerging tick-borne virus that causes severe meningoencephalitis in the United States, Canada, and Russia. Serology is generally the preferred diagnostic modality, but PCR on cerebrospinal fluid, blood, or urine has an important role, particularly in immunocompromised patients who are unable to mount a serologic response. Although the perceived poor sensitivity of PCR in the general population may be due to the biology of infection and health-seeking behavior (with short viremic periods that end before hospital presentation), limitations in assay design may also contribute.

Clinical utility of EBV DNA testing of blood in immunocompetent and immunocompromised patients: A single-center experience.

Objectives: Epstein-Barr virus (EBV) causes different clinical presentations in immunocompetent and immunocompromised persons, and thus indications for testing vary between these populations. We reviewed our institution's EBV DNA testing across these populations to understand its clinical utility and appropriateness.

Methods: We conducted a retrospective chart review of adult patients with positive EBV nucleic acid amplification (NAAT) testing from November 2022 to 2023.

Evaluation of Lyme serologic quantitative test indexes: High first-tier test index values predict positive second-tier result in standard and modified 2-tier Lyme testing algorithms.

Objectives: In this study, we evaluated the potential utility of reporting a quantitative Lyme serologic test index to improve the utility of results from first-tier Lyme assays.

Methods: Serum from consecutive samples sent to our laboratory for Lyme testing were tested on 2 commercial first-tier Lyme assays and evaluated to determine the probability of second-tier confirmation based on the serologic index value.

Results: For both assays, we identified an index value above which 100% of samples confirmed on second-tier testing using both standard and modified 2-tier testing algorithms.

Laboratory Testing for Powassan Virus: Past, Present, and Future.

Powassan virus is a tick-borne flavivirus that can cause severe neuroinvasive disease, with areas of endemicity in the Northeast and Midwest United States, Canada, and Russia. Diagnosis is challenging and relies on a high index of suspicion and choosing the right test based on duration of infection and the patient's immune status. This review covers laboratory testing for Powassan virus, including historical considerations, modern options, and methods being developed in the research space.

Cerebrospinal fluid metagenomics has greatest added value as a test for Powassan virus among patients in New England with suspected central nervous system infection.

Cerebrospinal fluid (CSF) metagenomic next generation sequencing (mNGS) can detect diverse pathogens in patients with central nervous system infection. Due to its high cost and unclear clinical utility, it is typically reserved for patients with unrevealing routine workups. A multi-center retrospective analysis of real-world CSF mNGS was performed involving orders between 2017 and 2022 at a large New England healthcare system.

The structure of NAD consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD hydrolysis and map the conformational effects of its interaction with NAD using hydrogen-deuterium exchange-mass spectrometry.

Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysis by Endo-β-N-Acetylglucosaminidases.

Glycosylation is a common posttranslational modification of proteins and refers to the covalent addition of glycans, chains of polysaccharides, onto proteins producing glycoproteins. The glycans influence the structure, function, and stability of proteins. They also play an integral role in the immune system, and aberrantly glycosylated proteins have wide ranging effects, including leading to diseases such as autoimmune conditions and cancer.

Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases.

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component.

Variant With Multiple Resistance Mutations Detected in an Immunocompromised Patient Receiving Atovaquone Prophylaxis.

We report genomic sequences with multiple mutations in the atovaquone-target region of cytochrome b, including a newly identified Y272S mutation, plus 1 mutation of undetermined significance in the azithromycin-associated ribosomal protein L4. The parasite was sequenced from an immunocompromised patient on prophylactic atovaquone for pneumonia before diagnosis of babesiosis.

Chop-Chop: The Future of Bacterial Enzymes in Transfusion Medicine.

The discovery of bacterial enzymes with specificity for IgG antibodies has led to breakthroughs in several autoantibody-mediated diseases. Two such enzymes, IdeS and EndoS, degrade IgG by different mechanisms, and have separately shown promise in numerous animal models of autoimmune diseases. Recently, imlifidase (the international nonproprietary name for IdeS) has advanced to clinical trials, where it has performed remarkably well in desensitizing patients to enable kidney transplantation, and in anti-glomerular basement membrane disease.

Molecular Basis of Selective Cytokine Signaling Inhibition by Antibodies Targeting a Shared Receptor.

Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy.

Sculpting therapeutic monoclonal antibody N-glycans using endoglycosidases.

Immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders. The chemical composition of the N-glycan on the fragment crystallizable (Fc) region determines the effector functions through interaction with the Fc gamma receptors and complement proteins. The chemoenzymatic synthesis using endo-β-N-acetylglucosaminidases (ENGases) emerged as a strategy to obtain antibodies with customized glycoforms that modulate their therapeutic activity.

GH18 endo-β-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases.

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2.

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity.

Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation.

Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation.

Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides.

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-β-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine.

Structural insights into the mechanisms and specificities of IgG-active endoglycosidases.

The conserved N-glycan on Asn297 of immunoglobulin G (IgG) has significant impacts on antibody effector functions, and is a frequent target for antibody engineering. Chemoenzymatic synthesis has emerged as a strategy for producing antibodies with homogenous glycosylation and improved effector functions. Central to this strategy is the use of enzymes with activity on the Asn297 glycan.

Molecular Basis of Broad Spectrum -Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2.

Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. secretes a unique endo-β--acetylglucosaminidase, EndoS2, which deglycosylates the conserved -linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions.

Small-Molecule Inhibitor of FosA Expands Fosfomycin Activity to Multidrug-Resistant Gram-Negative Pathogens.

The spread of multidrug or extensively drug-resistant Gram-negative bacteria is a serious public health issue. There are too few new antibiotics in development to combat the threat of multidrug-resistant infections, and consequently the rate of increasing antibiotic resistance is outpacing the drug development process. This fundamentally threatens our ability to treat common infectious diseases.

Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S.

Endoglycosidase S (EndoS) is a bacterial endo-β-N-acetylglucosaminidase that specifically catalyzes the hydrolysis of the β-1,4 linkage between the first two N-acetylglucosamine residues of the biantennary complex-type N-linked glycans of IgG Fc regions. It is used for the chemoenzymatic synthesis of homogeneously glycosylated antibodies with improved therapeutic properties, but the molecular basis for its substrate specificity is unknown. Here, we report the crystal structure of the full-length EndoS in complex with its oligosaccharide G2 product.

Structure and Dynamics of FosA-Mediated Fosfomycin Resistance in Klebsiella pneumoniae and Escherichia coli.

Fosfomycin exhibits broad-spectrum antibacterial activity and is being reevaluated for the treatment of extensively drug-resistant pathogens. Its activity in Gram-negative organisms, however, can be compromised by expression of FosA, a metal-dependent transferase that catalyzes the conjugation of glutathione to fosfomycin, rendering the antibiotic inactive. In this study, we solved the crystal structures of two of the most clinically relevant FosA enzymes: plasmid-encoded FosA3 from and chromosomally encoded FosA from (FosA).

Getting oriented with antibodies.

Neisseria meningitidis is a Gram-negative bacterium capable of causing deadly invasive disease. Two recently developed vaccines against N. meningitidis serogroup B include recombinant factor H binding protein (fHbp), a surface protein that meningococci use to evade the host immune system.