Peptidoglycan-associated lipoprotein (Pal) is a potential vaccine candidate from Haemophilus influenzae that highly conserved in Gram-negative bacteria and anchored to the outer membrane through an N-terminal lipid attachment. Pal stabilizes by providing noncovalent link peptidoglycan (PG) layer periplasmic domain. Using NMR spectroscopy, we determined three-dimensional structure of complex between domain biosynthetic precursor (PG-P), UDP-N-acetylmuramyl-L-Ala-alpha-d-Glu-m-Dap-D-Ala-d-Ala...

Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the highly conserved across viral genotypes, suggesting an role this modification life cycle. Here, we analyzed N-glycosylation receptor-binding domain (RBD) strain M41 and assessed receptor binding. Ten single Asn–to–Ala substitutions at predicted M41–RBD were evaluated...

The influenza virus surface glycoprotein hemagglutinin (HA) is the major target of host neutralizing antibodies. oligosaccharides HA can contribute to HA's antigenic characteristics. After a leap humans from zoonotic host, gain N-glycosylation sequons over time as part its fitness strategy. This glycosylation expansion has not been studied at structural level. Here we examine H3N2 strains that have engineered closely mimic sites gained between 1968 through 2002 starting with pandemic A/Hong...

Infectious bronchitis virus is the causative agent of infectious in chickens. Upon infection chicken flocks, poultry industry faces substantial economic losses by diminished egg quality and increased morbidity mortality infected animals. While all IBV strains infect respiratory tract via ciliated epithelial layer trachea, some can also replicate kidneys, dividing into following two pathotypes: nonnephropathogenic (example, IBV-M41) nephropathogenic viruses (including IBV-QX). Here, we set...

In the present study, glycosylation patterns of 2014-2015 influenza vaccine season standard antigens A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012 were revealed, sensitivity single radial immunodiffusion (SRID) potency test to was tested. Differences in hemagglutinin site composition heterogeneity seen produced different cell substrates suggest differences processing downstream immune responses. The SRID used for release is not sensitive under use conditions....

The SARS-CoV-2 spike protein is heavily glycosylated, having 22 predicted N-glycosylation sites per monomer. It also O-glycosylated, although the number of O-glycosites less defined. Recent studies show that glycans play critical roles in viral entry and infection. monomer has two subdomains, S1 S2, a receptor-binding domain (RBD) within domain. In this study, we have characterized site-specific glycosylation patterns HEK293 recombinant RBD domains as well intact derived from whole virus...

Phenazines, including pyocyanin and iodonin, are biologically active compounds that believed to confer producing organisms with a competitive growth advantage, also thought be virulence factors in certain diseases cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized series of poorly characterized steps by enzymes encoded seven-gene cistron Pseudomonas other organisms. Despite the biological importance these compounds, our understanding their mode action, biochemistry...

HI0719 belongs to a large family of highly conserved proteins with no definitive molecular function and is found in organisms ranging from bacteria humans. We describe the NMR structure HI0719, first solution for member this family. The overall fold similar crystal structures two homologues, YabJ Bacillus subtilis YjgF Escherichia coli, all three are that chorismate mutase, although there little sequence homology apparent functional connection. homotrimer distinct cavity located at subunit...

Caenorabditis elegans bus-4 glycosyltransferase mutants are resistant to infection by Microbacterium nematophilum, Yersinia pestis and pseudotuberculosis have altered susceptibility two Leucobacter species Verde1 Verde2. Our objective in this study was define the glycosylation changes leading phenotype better understand how these lead pathogen resistance. We performed MALDI-TOF MS, tandem MS GC/MS experiments reveal fine structural detail for N- O-glycan pools. observed dramatic O-glycans...

O-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active the release O-glycans. Moreover, chemical releasing methods, such as β-elimination/Michael addition, are not specific O-glycan and can also eliminate phosphoryl substitutions. Both these events leave behind deaminated serine threonine thus lead ambiguous structural conclusions. Recently, O-protease OpeRATOR, derived from intestinal bacteria expressed Escherichia coli, become...

TolR is a part of the Pal/Tol system which forms five-member, membrane-spanning, multiprotein complex that conserved in Gram-negative bacteria. The helps to maintain integrity outer membrane and has been proposed be involved several other cellular processes including cell division. Obtaining structure interest not only help explain many functions but also gain an understanding homologues ExbD MotB provide more targets for antibacterial treatments. In addition, may insights into how colicins...

The glycosylation patterns of four recombinant H5 hemagglutinins (HAs) derived from A/Mallard/Denmark/64650/03 (H5N7) have been characterized. proteins were expressed in (i) HEK293T cells to produce complex glycoforms, (ii) treated with Vibrio cholera neuraminidase provide asialo-complex (iii) HEK293S GnTI(-) predominantly the canonical Man5GlcNAc2 glycoform, and (iv) Drosophila S2 insect producing primarily paucimannose glycoforms. Previously, these HAs used investigate effect different...

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from viral envelope to bind angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor mediate host-cell entry. Currently, main recombinant S production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a truncated at transmembrane domain engineered express C-terminal trimerization motif...

Here we present a Caenorhabditis elegans N-glycan shotgun array. This nematode serves as model organism for many areas of biology including but not limited to tissue development, host-pathogen interactions, innate immunity, and genetics. N-glycans contain structural motifs that are also found in other nematodes well trematodes lepidopteran species. Glycan binding toxins interact with C. glycoconjugates do so some agriculturally relevant species, such Haemonchus contortus, Ascaris suum,...

For several decades, it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve efficacy. However, some key questions for manufacturing stable and immunogenic rNAs remain answered.

People with disorders such as cancer, autoimmune disease, diabetes, or obesity often have metabolic dysregulation of cellular glycosylation and also more severe influenza a reduced immune response to the virus, vaccine efficacy. Since viruses that infect people do not show consistent genomic variations, it is generally assumed altered biology mainly related host factors.

Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA of low-pathogenicity avian virus (LPAIV) subtypes. These pose a pandemic threat through reassortment and emergence in human populations. Since presence high-mannose glycosites dictates SP-D activity, ability to predict these glycosite glycan subtypes may value. Here, we investigate activities two recombinant forms...

Abstract The solution structure of the acidic protein HI1450 from Haemophilus influenzae has been determined by NMR spectroscopy. homologues in ten other bacterial species including Escherichia coli , Vibrio cholerae and Yersinia pestis but there are no functional assignments for any members family. Thirty‐one amino acids this 107‐residue aspartates or glutamates, contributing to an unusually low pI 3.72. secondary elements arranged α‐α‐β‐β‐β‐β order with two alpha helices packed against...

Recently, the solution structure of hypothetical protein HI1450 from Haemophilus influenzae was solved as part a structure-based effort to understand function. The distribution its many negatively charged residues and weak sequence homology uracil DNA glycosylase inhibitor (Ugi) suggested that may act double-stranded (dsDNA) mimic. We present supporting evidence here show interacts with dsDNA-binding HU-alpha. interaction between HU-alpha H. is characterized using calorimetry NMR...

A novel bacterial ribosome binding protein, protein Y (also known as YfiA), was recently shown to reside at the 30S/50S subunit interface and stabilize ribosomal 70S complex against dissociation low magnesium ion concentrations. We report here three-dimensional NMR structure in solution of a homologue from Haemophilus influenzae, HI0257, that has 64% sequence identity Y. The 107 residue β-α-β-β-β-α folding topology with two parallel α-helices packed same side four-stranded β-sheet. closest...

The solution structure of the 154-residue conserved hypothetical protein HI0004 has been determined using multidimensional heteronuclear NMR spectroscopy. sequence homologs in many organisms ranging from bacteria to humans and is believed be essential Haemophilus influenzae, although an exact function yet defined. It a alpha-beta-alpha sandwich architecture consisting central four-stranded beta-sheet with alpha2-helix packed against one side four alpha-helices (alpha1, alpha3, alpha4,...

We recently reported that members of group 1 influenza A virus (IAV) containing H2, H5, H6, and H11 hemagglutinins (HAs) are resistant to lung surfactant protein D (SP-D). H3 viruses, 2 IAV, have high affinity for SP-D, which depends on the presence high-mannose glycans at glycosite N165 head HA. The low SP-D viruses is due complex an analogous HA, replacement with glycan this site evoked strong interaction SP-D. Thus, if IAV were make zoonotic leap humans, pathogenicity such strains could...