Hofmeister Effect in RT-QuIC Seeding Activity of Chronic Wasting Disease Prions.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that causes a fatal neurodegenerative disease in cervids. Cases of CWD are rapidly increasing in North America among wild and farmed cervid populations, and potential for zoonotic transmission is not yet determined. Therefore, in order to manage the disease, it is imperative to devise a system that can detect CWD during its early phases to prevent spread to new captive herds through introduction of CWD-affected animals into otherwise CWD-free herds. Real-time quaking-induced conversion (RT-QuIC) assays have been applied to detect the presence of disease-associated prions from various samples in both animals and humans. In this study, we have tested the use of five Hofmeister anions that range from weakly hydrating to strongly hydrating: Na3citrate, Na2SO4, NaCl, NaI, and NaClO4 in RT-QuIC reactions for CWD seeding activity using different recombinant prion proteins as substrates. This work shows how the ionic environment of the RT-QuIC reaction can enhance or diminish the seeding activity. The use of Na2SO4 or NaI as the sodium salt for RT-QuIC using bank vole recombinant prion substrate for the detection of CWD using brain samples reduces the lag time to detect with reasonable specificity. For detection of the CWD in fecal samples, only NaI showed comparable reduction in lag time relative to NaCl but required reduced temperature to alleviate spontaneous fibril formation in negative control samples. Selection of the proper ion environment and recombinant prion protein substrate will make RT-QuIC a powerful diagnostic tool for early detection of CWD prions, further supporting CWD surveillance in wild and captive cervids.

Real-Time Quaking-Induced Conversion Detection of PrPSc in Fecal Samples From Chronic Wasting Disease Infected White-Tailed Deer Using Bank Vole Substrate.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that is fatal to free-range and captive cervids. CWD has been reported in the United States, Canada, South Korea, Norway, Finland, and Sweden, and the case numbers in both wild and farmed cervids are increasing rapidly. Studies indicate that lateral transmission of cervids likely occurs through the shedding of infectious prions in saliva, feces, urine, and blood into the environment. Therefore, the detection of CWD early in the incubation time is advantageous for disease management. In this study, we adapt real-time quacking-induced conversion (RT-QuIC) assays to detect the seeding activity of CWD prions in feces samples from clinical and preclinical white-tailed deer. By optimizing reaction conditions for temperature as well as the salt and salt concentration, prion seeding activity from both clinical and preclinical animals were detected by RT-QuIC. More specifically, all fecal samples collected from 6 to 30 months post inoculation showed seeding activity under the conditions of study. The combination of a highly sensitive detection tool paired with a sample type that may be collected non-invasively allows a useful tool to support CWD surveillance in wild and captive cervids.

Evaluation of Antemortem Diagnostic Techniques in Goats Naturally Infected With Scrapie.

Scrapie is a naturally occurring transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Sheep and goats can be infected with scrapie as lambs or kids via contact with the placenta or placental fluids, or from ingestion of prions shed in the environment and/or bodily fluids (e.g., saliva, urine, and feces). Like other TSEs, scrapie is generally not diagnosed before extensive and irreversible brain damage has occurred. Therefore, a reliable method to screen animals may facilitate diagnosis. Additionally, while natural scrapie in sheep has been widely described, naturally acquired goat scrapie is less well-characterized. The purpose of this study was to better understand natural goat scrapie in regard to disease phenotype (i.e., incubation period, clinical signs, neuroanatomical deposition patterns of PrPSc, and molecular profile as detected by Western blot) and to evaluate the efficacy of antemortem tests to detect scrapie-positive animals in a herd of goats. Briefly, 28 scrapie-exposed goats were removed from a farm depopulated due to previous diagnoses of scrapie on the premises and observed daily for 30 months. Over the course of the observation period, antemortem biopsies of recto-anal mucosa-associated lymphoid tissue (RAMALT) were taken and tested using immunohistochemistry and real-time quaking-induced conversion (RT-QuIC), and retinal thickness was measured in vivo using optical coherence tomography (OCT). Following the observation period, immunohistochemistry and Western blot were performed to assess neuroanatomical deposition patterns of PrPSc and molecular profile. Our results demonstrate that antemortem rectal biopsy was 77% effective in identifying goats naturally infected with scrapie and that a positive antemortem rectal biopsy was associated with the presence of clinical signs of neurologic disease and a positive dam status. We report that changes in retinal thickness are not detectable over the course of the observation period in goats naturally infected with scrapie. Finally, our results indicate that the accumulation of PrPSc in central nervous system (CNS) and non-CNS tissues is consistent with previous reports of scrapie in sheep and goats.

Exploring disease comorbidity in a module-module interaction network.

Understanding disease comorbidity contributes to improved quality of life in patients who are suffering from multiple diseases. Therefore, to better explore comorbid diseases, the clarification of associations between diseases based on biological functions is essential. In our study, we propose a method for identifying disease comorbidity in a module-based network, named the module-module interaction (MMI) network, which represents how biological functions influence each other. To construct the MMI network, we detected gene modules - sets of genes that have a higher probability of taking part in specific functions - and established a link between these modules. Subsequently, we constructed disease-related networks in the MMI network to understand inherent disease mechanisms and calculated comorbidity scores of disease pairs using Gene Ontology (GO) terms. Our results show that we can obtain further information on disease mechanisms by considering interactions between functional modules instead of between genes. In addition, we verified that predicted comorbid relationships of disease pairs based on the MMI network are more significant than those based on the protein-protein interaction (PPI) network. This study can be useful to elucidate the mechanisms underlying comorbidities for further study, which will provide a broader insight into the pathogenesis of diseases.

Novel Strain of the Chronic Wasting Disease Agent Isolated From Experimentally Inoculated Elk With LL132 Prion Protein.

Chronic wasting disease (CWD) is a fatal, progressive disease that affects cervid species, including Rocky mountain elk (Cervus elaphus nelsoni). There are 2 allelic variants in the elk prion protein gene: L132 (leucine) and M132 (methionine). Following experimental oral challenge with the CWD agent incubation periods are longest in LL132 elk, intermediate in ML132 elk, and shortest in MM132 elk. In order to ascertain whether such CWD-infected elk carry distinct prion strains, groups of Tg12 mice that express M132 elk prion protein were inoculated intracranially with brain homogenate from individual CWD-infected elk of various genotypes (LL132, LM132, or MM132). Brain samples were examined for microscopic changes and assessment of the biochemical properties of disease-associated prion protein (PrPSc). On first passage, mice challenged with LL132 elk inoculum had prolonged incubation periods and greater PrPSc fibril stability compared to mice challenged with MM132 or LM132 inoculum. On second passage, relative incubation periods, western blot profiles, and neuropathology were maintained. These results suggest that the CWD prion isolated from LL132 elk is a novel CWD strain and that M132 PrPC is able to propagate some biophysical properties of the L132 PrPSc conformation.

Role of donor genotype in RT-QuIC seeding activity of chronic wasting disease prions using human and bank vole substrates.

Chronic wasting disease is a transmissible spongiform encephalopathy of cervids. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein (PrPC) to pathogenic conformers (PrPSc), and the pathogenic forms accumulate in the brain and other tissues. Real-time Quaking Induced Conversion (RT-QuIC) can be used for the detection of prions and for prion strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how either PrPSc from cervids of different genotypes or PrPSc from different sources of CWD influence the fibril formation of recombinant bank vole (BV) or human prion proteins using RT-QuIC. We found that reaction mixtures seeded with PrPSc from different genotypes of white-tailed deer or reindeer brains have similar conversion efficiency with both substrates. Also, we observed similar results when assays were seeded with different sources of CWD. Thus, we conclude that the genotypes of all sources of CWD used in this study do not influence the level of conversion of PrPC to PrPSc.

PAD-Beads enrichment enhances detection of PrPSc using real-time quaking-induced conversion.

OBJECTIVE: Scrapie is a transmissible spongiform encephalopathy (TSE) that naturally occurs in sheep and goats. This fatal neurodegenerative disease results from misfolding of the normal cellular prion protein (PrPC) to a pathogenic prion protein form (PrPSc). This pathogenic form, PrPSc, accumulates in the brain and lymphoid tissues. The presence of PrPSc can be detected by an in vitro conversion assay known as real-time quaking induced conversion (RT-QuIC). RT-QuIC has been used to detect PrPSc in a variety of biological tissues from brains to fluids. While this technique is both rapid and sensitive, enhancing the detection of prions would be valuable in the diagnostic laboratories. RESULTS: In this study, we assessed whether PrPSc detection sensitivity of RT-QuIC can be increased by enriching PrPSc in scrapie tissue homogenates using commercially available aggregated protein binding ligands coated magnetic beads (PAD-Beads). Coupling of RT-QuIC to PAD-Beads based cleanup allowed detection of PrPSc rapidly and without dilution of scrapie sheep brain homogenates prior to RT-QuIC. The PAD-Beads sample pretreatment step prior to RT-QuIC is a useful enhancement in the diagnosis of TSEs.

Preparation of lyophilized recombinant prion protein for TSE diagnosis by RT-QuIC.

OBJECTIVE: Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases, often referred as prion diseases. TSEs result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain and lymphatic tissue. Amplification based assays such as real-time quaking induced conversion allow us to assess the conversion of PrPC to PrPSc. Real-time quaking induced conversion (RT-QuIC) can be used for the detection of PrPSc in a variety of biological tissues from humans and animals. However, RT-QuIC requires a continuous supply of freshly purified prion protein and this necessity is not sustainable in a diagnostic laboratory setting. RESULTS: In this study, we developed a method to dry and preserve the prion protein for long term storage allowing for production of the protein and storage for extended time prior to use and room temperature shipping to appropriate diagnostic laboratory destinations facilitating widespread use of RT-QuIC as a diagnostic method.

Source genotype influence on cross species transmission of transmissible spongiform encephalopathies evaluated by RT-QuIC.

Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein to pathogenic ß-rich conformers (PrPSc) that accumulate in higher order structures of the brain and other tissues. This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions and for strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how PrPSc isolated from sheep of different genotypes after inoculation with the scrapie agent influence the fibril formation in vitro using RT-QuIC. We found that reaction mixtures seeded with PrPSc from genotype VRQ/VRQ sheep brains have better conversion efficiency with 132M elk substrate compared to reactions seeded with PrPSc from the brains of sheep with the ARQ/ARQ genotype no matter which strain of scrapie was used to seed the reactions. We also inoculated transgenic mice expressing 132M elk PRNP (Tg12) with the scrapie agent from different genotypes of sheep to compare with our RT-QuIC results. The bioassays support the data showing a significantly shorter incubation period for inoculum from VRQ/VRQ sheep when compared to inoculum from ARQ/ARQ sheep. Thus, we conclude that the genotype of both source and recipient can strongly influence transmission.

PISTON: Predicting drug indications and side effects using topic modeling and natural language processing.

The process of discovering novel drugs to treat diseases requires a long time and high cost. It is important to understand side effects of drugs as well as their therapeutic effects, because these can seriously damage the patients due to unexpected actions of the derived candidate drugs. In order to overcome these limitations, computational methods for predicting the therapeutic effects and side effects have been proposed. In particular, text mining is a widely used technique in the field of systems biology, because it can discover hidden relationships between drugs, genes and diseases from a large amount of literature data. Compared with in vivo/in vitro experiments, text mining derives meaningful results with less time and cost. In this study, we propose an algorithm for predicting novel drug-phenotype associations and drug-side effect associations using topic modeling and natural language processing (NLP). We extract sentences in which drugs and genes co-occur from the abstracts of the literature and identify words that describe the relationship between them using NLP. Considering the characteristics of the identified words, we determine if the drug has an up-regulation effect or a down-regulation effect on the gene. Based on genes that affect drugs and their regulatory relationships, we group the frequently occurring genes and regulatory relationships into topics, and build a drug-topic probability matrix by calculating the score that the drug will have a topic using topic modeling. Using the matrix, a classifier is constructed for predicting the novel indications and side effects of drugs considering the characteristics of known drug-phenotype associations or drug-side effect associations. The proposed method predicts both indications and side effects with a single algorithm, and it can exclude drugs with serious side effects or side effects that patients do not want to experience from among the candidate drugs provided for the treatment of the phenotype. Furthermore, lists of novel candidate drugs for phenotypes and side effects can be continuously updated with our algorithm every time a document is added. More than a thousand documents are produced per day, and it is possible for our algorithm to efficiently derive candidate drugs because it requires less cost than the existing drug repositioning methods. The resource of PISTON is available at databio.gachon.ac.kr/tools/PISTON.

Thermodynamic characterization for the denatured state of bovine prion protein and the BSE Associated variant E211K.

Propagation of transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. The most common hereditary prion disease is a genetic form of Creutzfeldt-Jakob disease in humans, in which a mutation in the prion gene results in a glutamic acid to lysine substitution at position 200 (E200K) in PrP. In cattle, the analogous amino acid substitution is found at residue 211 (E211K) and has been associated with a case of bovine spongiform encephalopathy. Here, we have compared the secondary structure of E211K to that of wild type using circular dichroism and completed a thermodynamic analysis of the folding of recombinant wild type and E211K variants of the bovine prion protein. The secondary structure of the E211K variant was essentially indistinguishable from that of wild type. The thermodynamic stability of E211K substitution showed a slight destabilization relative to the wild type consistent with results reported for recombinant human prion protein and its mutant E200K. In addition, the E211K variant exhibits a similarly compact denatured state to that of wild type based upon similar m-value and change in heat capacity of unfolding for the proteins. Together these results indicate that residual structure in the denatured state of bPrP is present in both the wild type protein and BSE associated variant E211K. Given this observation, as well as folding similarities reported for other disease associated variants of PrP it is worth consideration that functional aspects of PrP conformation may play a role in the misfolding process.

Pathologic and biochemical characterization of PrPSc from elk with PRNP polymorphisms at codon 132 after experimental infection with the chronic wasting disease agent.

BACKGROUND: The Rocky Mountain elk (Cervus elaphus nelsoni) prion protein gene (PRNP) is polymorphic at codon 132, with leucine (L132) and methionine (M132) allelic variants present in the population. In elk experimentally inoculated with the chronic wasting disease (CWD) agent, different incubation periods are associated with PRNP genotype: LL132 elk survive the longest, LM132 elk are intermediate, and MM132 elk the shortest. The purpose of this study was to investigate potential mechanisms underlying variations in incubation period in elk of different prion protein genotypes. Elk calves of three PRNP genotypes (n = 2 MM132, n = 2 LM132, n = 4 LL132) were orally inoculated with brain homogenate from elk clinically affected with CWD. RESULTS: Elk with longer incubation periods accumulated relatively less PrPSc in the brain than elk with shorter incubation periods. PrPSc accumulation in LM132 and MM132 elk was primarily neuropil-associated while glial-associated immunoreactivity was prominent in LL132 elk. The fibril stability of PrPSc from MM132 and LM132 elk were similar to each other and less stable than that from LL132 elk. Real-time quaking induced conversion assays (RT-QuIC) revealed differences in the ability of PrPSc seed from elk of different genotypes to convert recombinant 132 M or 132 L substrate. CONCLUSIONS: This study provides further evidence of the importance of PRNP genotype in the pathogenesis of CWD of elk. The longer incubation periods observed in LL132 elk are associated with PrPSc that is more stable and relatively less abundant at the time of clinical disease. The biochemical properties of PrPSc from MM132 and LM132 elk are similar to each other and different to PrPSc from LL132 elk. The shorter incubation periods in MM132 compared to LM132 elk may be the result of genotype-dependent differences in the efficiency of propagation of PrPSc moieties present in the inoculum. A better understanding of the mechanisms by which the polymorphisms at codon 132 in elk PRNP influence disease pathogenesis will help to improve control of CWD in captive and free-ranging elk populations.

Use of bovine recombinant prion protein and real-time quaking-induced conversion to detect cattle transmissible mink encephalopathy prions and discriminate classical and atypical L- and H-Type bovine spongiform encephalopathy.

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving conversion from the normal cellular prion protein to the pathogenic misfolded conformation (PrPSc). This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. Extensive work has been done to demonstrate that RT-QuIC is a rapid, specific, and highly sensitive prion detection assay. RT-QuIC uses recombinant prion protein to detect minute amounts of PrPSc. RT-QuIC has been successfully used to detect PrPSc from different prion diseases with a variety of substrates including hamster, human, sheep, bank vole, bovine and chimeric forms of prion protein. However, recombinant bovine prion protein has not been used to detect transmissible mink encephalopathy (TME) or to differentiate types of bovine spongiform encephalopathy (BSE) in samples from cattle. We evaluated whether PrPSc from TME and BSE infected cattle can be detected with RT-QuIC using recombinant bovine prion proteins, and optimized the reaction conditions to specifically detect cattle TME and to discriminate between classical and atypical BSE by conversion efficiency. We also found that substrate composed of the disease associated E211K mutant protein can be effective for the detection of TME in cattle and that wild type prion protein appears to be a practical substrate to discriminate between the different types of BSEs.

Real-Time Quaking-Induced Conversion Detection of Bovine Spongiform Encephalopathy Prions in a Subclinical Steer.

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal prion diseases that result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain. In vitro assays such as serial protein misfolding amplification and real-time quaking-induced conversion (RT-QuIC) allow assessment of the conversion of PrPC to PrPSc. RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. However, there is no such comparison of RT-QuIC data between BSE positive and presymptomatic cattle. Further, the current study assesses prion distribution in multiple brain regions of clinically ill or subclinical animals. Here, we compare RT-QuIC reactions seeded with brain samples collected from experimentally inoculated cattle that were clinically ill or subclinically affected with BSE. The results demonstrate RT-QuIC seeding in various brain regions of an animal with subclinical BSE despite being determined negative by immunohistochemistry. Bioassay of the subclinical animal and RT-QuIC of brainstem from inoculated knockout (PRNP-/-) cattle were used to confirm infectivity in the subclinical animal and determine that RT-QuIC reactions were not the result of residual inoculum, respectively. These results confirm that RT-QuIC is a highly sensitive prion detection assay that can detect prions in a steer prior to the onset of clinical signs of BSE.

Discovery of a Unique Extracellular Polysaccharide in Members of the Pathogenic Bacillus That Can Co-form with Spores.

An exopolysaccharide, produced during the late stage of stationary growth phase, was discovered and purified from the culture medium of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis when strains were grown in a defined nutrient medium that induces biofilm. Two-dimensional NMR structural characterization of the polysaccharide, named pzX, revealed that it is composed of an unusual three amino-sugar sequence repeat of [-3)XylNAc4OAc(α1-3)GlcNAcA4OAc(α1-3)XylNAc(α1-]n The sugar residue XylNAc had never been described previously in any glycan structure. The XNAC operon that contains the genes for the assembly of pzX is also unique and so far has been identified only in members of the Bacillus cereus sensu lato group. Microscopic and biochemical analyses indicate that pzX co-forms during sporulation, so that upon the release of the spore to the extracellular milieu it becomes surrounded by pzX. The relative amounts of pzX produced can be manipulated by specific nutrients in the medium, but rich medium appears to suppress pzX formation. pzX has the following unique characteristics: a surfactant property that lowers surface tension, a cell/spore antiaggregant, and an adherence property that increases spores binding to surfaces. pzX in Bacillus could represent a trait shared by many spore-producing microorganisms. It suggests pzX is an active player in spore physiology and may provide new insights to the successful survival of the B. cereus species in natural environments or in the hosts.

The Biosynthesis of UDP-D-QuiNAc in Bacillus cereus ATCC 14579.

N-acetylquinovosamine (2-acetamido-2,6-di-deoxy-D-glucose, QuiNAc) is a relatively rare amino sugar residue found in glycans of few pathogenic gram-negative bacteria where it can play a role in infection. However, little is known about QuiNAc-related polysaccharides in gram-positive bacteria. In a routine screen for bacillus glycan grown at defined medium, it was surprising to identify a QuiNAc residue in polysaccharides isolated from this gram-positive bacterium. To gain insight into the biosynthesis of these glycans, we report the identification of an operon in Bacillus cereus ATCC 14579 that contains two genes encoding activities not previously described in gram-positive bacteria. One gene encodes a UDP-N-acetylglucosamine C4,6-dehydratase, (abbreviated Pdeg) that converts UDP-GlcNAc to UDP-4-keto-4,6-D-deoxy-GlcNAc (UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose); and the second encodes a UDP-4-reductase (abbr. Preq) that converts UDP-4-keto-4,6-D-deoxy-GlcNAc to UDP-N-acetyl-quinovosamine in the presence of NADPH. Biochemical studies established that the sequential Pdeg and Preq reaction product is UDP-D-QuiNAc as determined by mass spectrometry and one- and two-dimensional NMR experiments. Also, unambiguous evidence for the conversions of the dehydratase product, UDP-α-D-4-keto-4,6-deoxy-GlcNAc, to UDP-α-D-QuiNAc was obtained using real-time 1H-NMR spectroscopy and mass spectrometry. The two genes overlap by 4 nucleotides and similar operon organization and identical gene sequences were also identified in a few other Bacillus species suggesting they may have similar roles in the lifecycle of this class of bacteria important to human health. Our results provide new information about the ability of Bacilli to form UDP-QuiNAc and will provide insight to evaluate their role in the biology of Bacillus.

Pen and Pal are nucleotide-sugar dehydratases that convert UDP-GlcNAc to UDP-6-deoxy-D-GlcNAc-5,6-ene and then to UDP-4-keto-6-deoxy-L-AltNAc for CMP-pseudaminic acid synthesis in Bacillus thuringiensis.

CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP(+) and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD(+)-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.

The biosynthesis of UDP-d-FucNAc-4N-(2)-oxoglutarate (UDP-Yelosamine) in Bacillus cereus ATCC 14579: Pat and Pyl, an aminotransferase and an ATP-dependent Grasp protein that ligates 2-oxoglutarate to UDP-4-amino-sugars.

Surface glycan switching is often observed when micro-organisms transition between different biotic and abiotic niches, including biofilms, although the advantages of this switching to the organism are not well understood. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(ß1→, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. The molecular mechanism involved in attaching 2-hydroxyglutarate to 4-amino-FucNAc has not been determined. Here, we show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4″-aminotransferase (herein referred to as Pat) that, in the presence of pyridoxal phosphate, transfers the primary amino group of l-Glu to C-4″ of UDP-4-keto-6-deoxy-d-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate. Pat also converts 4-keto-xylose, 4-keto-glucose, and 4-keto-2-acetamido-altrose to their corresponding UDP-4-amino-sugars. Bc5272 encodes a carboxylate-amine ligase (herein referred as Pyl) that, in the presence of ATP and Mg(II), adds 2-oxoglutarate to the 4-amino moiety of UDP-4-amino-FucNAc to form UDP-Yelosamine and ADP. Pyl is also able to ligate 2-oxoglutarate to other 4-amino-sugar derivatives to form UDP-Yelose, UDP-Solosamine, and UDP-Aravonose. Characterizing the metabolic pathways involved in the formation of modified nucleotide sugars provides a basis for understanding some of the mechanisms used by bacteria to modify or alter their cell surface polysaccharides in response to changing growth and environmental challenges.

Folding of a tryptophan zipper peptide investigated on the basis of the nuclear Overhauser effect and thermal denaturation.

Short, secondary-structure-containing peptides are suitable models for the study of protein folding due to their relative simplicity. Here, we investigate thermal denaturation of the tryptophan zipper peptide, trpzip4, a peptide that forms a ß-hairpin in solution. In order to monitor the thermal denaturation of peptides or small proteins, chemical shift values of H(α) or H(N) may be used. However, various factors other than secondary structure can influence chemical shift values, such as side-chain orientation of nearby aromatic residues. Nuclear Overhauser effect (NOE) intensity from backbone interproton cross peaks is an alternative way to study thermal denaturation, as long as various factors that give rise to a change in NOE intensity upon changing the temperature are considered. As a relative indicator for denaturation, we define a cutoff temperature, where half of the initial NOE intensity is lost for each backbone interproton cross peak. For trpzip4, this cutoff temperature is highest for residues in the central part of the structure and lowest for residues near the termini. These observations support the notion that the structure of the trpzip4 peptide is stabilized by a hydrophobic cluster formed by tryptophan residues located in the central region of the ß-hairpin.

Methanol strengthens hydrogen bonds and weakens hydrophobic interactions in proteins--a combined molecular dynamics and NMR study.

A combined simulation and experimental study was performed to investigate how methanol affects the structure of a model peptide BBA5. BBA5 forms a stable ß-hairpin-α-helix structure in aqueous solutions. Molecular dynamics simulations were performed in water and methanol/water solutions using all-atom explicit models. NMR experiments were used to test the calculated results. The combined theoretical and experimental studies suggest that methanol strengthens the interactions between the polar backbone of the peptide and thus enhances the secondary structure formation; at the same time methanol weakens the hydrophobic interactions and results in an expansion of the hydrophobic core and an increase in gyration.