A network-based approach to identify substrate classes of bacterial glycosyltransferases

Background

Bacterial interactions with the environment- and/or host largely depend on the bacterial glycome. The specificities of a bacterial glycome are largely determined by glycosyltransferases (GTs), the enzymes involved in transferring sugar moieties from an activated donor to a specific substrate. Of these GTs their coding regions, but mainly also their substrate specificity are still largely unannotated as most sequence-based annotation flows suffer from the lack of characterized sequence motifs that can aid in the prediction of the substrate specificity.

Results

In this work, we developed an analysis flow that uses sequence-based strategies to predict novel GTs, but also exploits a network-based approach to infer the putative substrate classes of these predicted GTs. Our analysis flow was benchmarked with the well-documented GT-repertoire of Campylobacter jejuni NCTC 11168 and applied to the probiotic model Lactobacillus rhamnosus GG to expand our insights in the glycosylation potential of this bacterium. In L. rhamnosus GG we could predict 48 GTs of which eight were not previously reported. For at least 20 of these GTs a substrate relation was inferred.

Conclusions

We confirmed through experimental validation our prediction of WelI acting upstream of WelE in the biosynthesis of exopolysaccharides. We further hypothesize to have identified in L. rhamnosus GG the yet undiscovered genes involved in the biosynthesis of glucose-rich glycans and novel GTs involved in the glycosylation of proteins. Interestingly, we also predict GTs with well-known functions in peptidoglycan synthesis to also play a role in protein glycosylation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-349) contains supplementary material, which is available to authorized users.     

The Recent Recombinant Evolution of a Major Crop Pathogen,Potato virus Y

Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVY^NTN occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs to be minimised to prevent further such occurrences.     

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.Equine grass sickness (EGS, or equine dysautonomia) is a predominantly fatal, rapid multiple system neuropathy of grazing horses with reported incidence rates of 2.1–2.3% (reviewed by (1, 2)). An apparently identical disease occurs in cats, dogs, hares, rabbits, llamas, and possibly sheep (3–9). EGS is associated with chromatolysis of sympathetic and parasympathetic postsynaptic neurons, particularly in the enteric nervous system, as well as autonomic presynaptic and somatic lower motor neurons in the brainstem and spinal cord (10). EGS is subdivided into acute, subacute, and chronic forms according to the severity of clinical signs that largely reflect enteric and autonomic neurodegeneration, including dysphagia, generalized ileus, sweating, salivation, ptosis, rhinitis sicca, and tachycardia. While the etiology of EGS remains unknown, some evidence supports it being a toxic infection with Clostridium botulinum type C or D (11, 12). Ultrastructural studies suggest that the lesion in EGS primarily involves the glycoprotein biosynthetic pathway of specific neurons since the rough endoplasmic reticulum and Golgi complexes are consistently affected, while other organelles, including mitochondria, appear relatively normal (13). However, while the ultrastructural and cellular appearance of affected neurons has been studied extensively, little is known about the molecular mechanisms that contribute to neurodegeneration.The overarching aim of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS using a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. The cranial (superior) cervical ganglion (CCG), which supplies sympathetic innervation to the head and neck, was selected because chromatolysis of a high proportion of CCG neurons is a consistent feature of EGS (Fig. 1 and Supplemental Fig. 1 (14)). Here, proteomic analysis was carried out using isobaric tag for relative and absolute quantitation (iTRAQ) tools, which are now well established in small animal models of human neurodegenerative conditions but which are not routinely utilized in large animal models or large animal intrinsic conditions. This proteomic analysis was coupled with quantitative fluorescent Western blotting (QFWB), immunohistochemistry (IHC), and in silico based techniques in an attempt to identify the molecular pathways and processes that may be contributing to neurodegeneration in EGS. Here, we report widespread changes in the CCG of EGS horses, including significant disruption to a broad range of functional pathways clustering around candidates commonly associated with protein misfolding/aggregation responses in human neurodegenerative conditions.Open in a separate windowFig. 1.Equine grass sickness is a predominantly fatal, acute multiple system neuropathy of grazing horses. (A) Example photograph of a horse exhibiting typical appearance associated with chronic EGS. There is ptosis, and generalized muscle weakness as evidenced by the base narrow stance, low head and neck carriage, and leaning against a wall for support. Generalized muscle atrophy and reduced abdominal volume are also evident. (B) Example BIII-tubulin-stained sections from cranial cervical ganglia (CCG), which are known to exhibit neuronal perturbations in this disease. The visible puncta are BIII positive neurons. (C) High power micrographs stained CCG sections from B showing BIII positive neuronal profiles. (D) Quantification of BIII positive neurons demonstrates that there is still equivalent neuronal density in ganglia at terminal stages of the disease (control 6.25 ± 0.12, EGS 6.10 ± 0.20 cells per 100 μm², mean ± Standard Error (S.E) n = 4 cases per condition, n = 116 grids measured. See Materials and Methods for more information). (E) Example H and E stained sections from control and EGS-affected CCG demonstrates that while the neuronal density may be similar, many of the neurons exhibit chromatolysis. Scale bar = 0.75ft (A), 0.5 cm (B), 35 μm (C), 100 μm (E).This study therefore represents the first application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease). It is also the first to demonstrate correlation and conservation spanning the degenerative molecular cascades from an apparently unrelated condition of large animals to small animal models with altered neuronal vulnerability and a range of human neurological conditions from childhood neurodegenerative conditions such as spinal muscular atrophy through to diseases associated with advancing age such as Alzheimer''s. Finally, this study highlights the feasibility and benefits of applying differential proteomics techniques to the investigation of the neurodegenerative processes in diseases of large animals.     

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice

Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4 % w/w lard) or a high-fat [HF; 21 % w/w lard and cholesterol (0/15 % w/w)] diet with different B vitamin compositions for 16 weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P < 0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P < 0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0446-y) contains supplementary material, which is available to authorized users.     

A rapid and inexpensive method for the direct PCR amplification of DNA from plants

? Premise of the study: We present a rapid and inexpensive alternative to DNA isolation for polymerase chain reaction (PCR) amplification from plants. ? Methods and Results: The method involves direct PCR amplification from material macerated in one buffer, followed by dilution and incubation in a second buffer. We describe the procedure and demonstrate its application for nuclear and plastid DNA amplification across a broad range of vascular plants. ? Conclusions: The method is fast, easy to perform, cost-effective, and consequently ideal for large sample numbers. It represents a considerable simplification of present approaches requiring DNA isolation prior to PCR amplification and will be useful in plant systematics and biotechnology, including applications such as DNA barcoding.