Ly6a disruption or with blocking antibodies reduced transduction of mouse brain endothelial cells by AAV-PHP.eB, while ectopic expression of Ly6a increased AAV-PHP.eB transduction of HEK293T and CHO cells by 30-fold or more. Importantly, we demonstrate that this newly discovered mode of AAV binding and transduction can occur independently of other known AAV receptors. These findings illuminate the previously reported species- and strain-specific tropism characteristics of the AAV-PHP.B vectors and inform ongoing efforts to develop next-generation AAV vehicles for human CNS gene therapy.The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of the AAV-PHP.B vectors. Leveraging CNS tropism differences across 13 mouse strains, we systematically determined a set of genetic variants that segregate with the permissivity phenotype, and rapidly identified LY6A as an essential receptor for the AAV-PHP.B vectors. Interfering with LY6A by CRISPR/Cas9-mediated  AAV vectors are proving to be safe, versatile vehicles for ications –4. Howevvolution –8. Our gvolution  and its volution , to crosvolution ,9,10, involution ,12 and nvolution .A critical question has been how the AAV-PHP.B vectors cross the BBB and whether this mechanism can be translated to other species and, ultimately, humans. The enhanced CNS tropism of AAV-PHP.B and AAV-PHP.eB appears to extend to rats ,15, wherThe dramatically enhanced CNS tropism of AAV-PHP.B present in C57BL/6J but absent in BALB/cJ mice  extended11 vector genomes (vg)/animal of AAV-PHP.eB, which packaged an AAV genome encoding a green fluorescent protein (GFP) with a nuclear localization signal (NLS-GFP). Intravenous administration of AAV-PHP.eB resulted in robust GFP expression in the brains of permissive lines such as C57BL/6J, but not in those of nonpermissive mice such as BALB/cJ; we identified seven permissive and six nonpermissive lines  responsible for the difference in CNS transduction by AAV-PHP.eB. Diverging from conventional diversity outbred or genetic linkage studies requiring the inter-breeding of mouse lines, we leveraged the open-source software Hail  to compive lines .Ly6a and Ly6c1 genes (www.BrainRNAseq.org) [Ly6a and Ly6c1 are expressed in brain endothelial cells (Ly6 locus is linked to susceptibility to mouse adenovirus (MAV1) [LY6 gene family also influences susceptibility to infection by HIV1 [Ly6/LY6 genes and various virus susceptibilities, the mechanisms remain elusive and have not been leveraged in the development of gene therapy vectors.Using the permissivity data to filter the WGS dataset, we reduced the number of high and medium impact genetic variants to missense SNPs in the c1 genes . RNA seqseq.org)  indicateal cells . Intrigus (MAV1) , which ps (MAV1) . The LY6 by HIV1 ,28, Flav by HIV1 ), Influe by HIV1 , and Mar by HIV1 . DespiteLy6a or Ly6c1 is associated with the differential AAV-PHP.eB tropism across mouse lines. Immunohistochemistry (IHC) assays revealed that LY6A is abundant within the CNS endothelium of C57Bl/6J mice but not in BALB/cJ mice  from C57BL/6J mice, which express both genes , and is predicted to reduce the likelihood of GPI-anchor modification [To determine whether AAV-PHP.eB directly binds LY6A and whether either of the missense SNPs (D63G or V106A) in the BALB/cJ y6a gene  affect by6a gene  from HEKfication   cells and derivatives that expose excess galactose (Lec2 cells) or are unable to add galactose to their glycoproteins (Lec8 cells) , which wsduction  to Pro5 c2 cells , potentic8 cells . The finLy6a-expressing cells permissive to AAV-PHP.eB transduction in the absence of AAVR, which is essential for the intracellular trafficking of nearly all AAV capsids including AAV9 [Ly6a, was nearly absent in the brains of AAVR KO mice , identified using CREATE [Ly6a is sufficient to render both human and hamster cells permissive to the enhanced binding and transduction of AAV-PHP.eB. Importantly, these findings demonstrate that AAVs can be engineered to utilize entirely new cell entry/transduction mechanisms rendering the novel capsids less dependent on interactions with the receptors that natural AAV serotypes rely on for transduction.Our finding that Ly6a homolog in primates, other cellular factors that share key properties with LY6A such as abundant luminal surface exposure on brain endothelium, localization within lipid micro-domains through GPI anchoring, or specific recycling/intracellular trafficking capabilities, may be prime molecular targets for gene delivery vectors in mice, NHPs, and humans. Notably, other LY6 proteins with homologs in primates are present within the CNS endothelium and can be explored and potentially harnessed for AAV capsid engineering. Developing capsids and/or other biologicals that target these receptors can open up new therapeutic avenues for treating a wide range of currently intractable neurological diseases.Although there is no direct p that a random mouse line is permissive from a Beta distribution, based on the two known mouse phenotypes. Second, we sampled a subset of additional commercially available lines , providing a distribution over the number of candidate variants at each mouse sample size. This simulation informed our decision regarding the number of mouse lines to order and test in parallel.Access to WGS data for 36 commercially available mouse lines  made it le lines . Third, Ly6 expression vectors were cloned into an AAV-CAG-WPRE-hGH pA backbone obtained from Viviana Gradinaru through Addgene (#99122). GFP, 2A-luciferase, Ly6a, and Ly6c1 (splice variant 1) cDNAs were synthesized as gBlocks (IDT). The NLS-GFP were synthesized using the N-terminal SV40 NLS sequence present in the Addgene plasmid #99130. The CMV-SaCAS9 vector  was obtained from Dr. Feng Zhang through Addgene (#61591). sgRNAs specifically targeting Ly6a or Ly6c1 were cloned after the U6 promoter using a single bridge oligo for each reaction as recommended . The Broad GPP sgRNA tool for SaCAS9 was used to identify suitable SaCAS9 target sites [The AAV-PHP.eB Rep-Cap trans plasmid was generated by gene synthesis (GenScript). AAV9, AAV-PHP.B, AAV-PHP.B2, and AAV-PHP.B3 were generated by replacing the AAV-PHP.eB variant region with that of AAV9, AAV-PHP.B, B2, or B3 using isothermal HiFi DNA Assembly (NEB). The reporter and Ly6a sgRNA cloning:The following primers were used for AATTACCTGCCCCTACCCTGAGTTTTAGTACTCTGGAAACAG,5’-CTTGTGGAAAGGACGAAACACCGCTTTCAATATTAGGAGGGCAGGTTTTAGTACTCTGGAAACAG,5’-CTTGTGGAAAGGACGAAACACCGAATATTGAAAGTATGGAGATCGTTTTAGTACTCTGGAAACAG5’-CTTGTGGAAAGGACGAAACACCGLy6c1 sgRNA cloning:The following primers were used for ACTGCAGTGCTACGAGTGCTAGTTTTAGTACTCTGGAAACAG,5’-CTTGTGGAAAGGACGAAACACCGCAGTTACCTGCCGCGCCTCTGGTTTTAGTACTCTGGAAACAG,5’-CTTGTGGAAAGGACGAAACACCGGATTCTGCATTGCTCAAAACAGTTTTAGTACTCTGGAAACAG5’-CTTGTGGAAAGGACGAAACACCGThe following primers were used for biodistribution assays and/or binding assaysGFP: 5’-TACCCCGACCACATGAAGCAG, 5’-CTTGTAGTTGCCGTCGTCCTTGGcg: 5’-AAGGGACCTTTACCAGTGATGTG, 5’-ACTTACTCTCGCCTTCCTCGGMouse GCG: 5’-ATGCTGAAGGGACCTTTACCAG, 5’-ACTTACTCTCGCCTTCCTCGGHuman Gcg: 5’-ATGCTGAAGGGACCTTTACCAG, 5’-CTCGCCTTCCTCTGCCTTTCHO Ly6a and Ly6c1 were generated and purified to knockout the respective genes in C57BL/6 mouse primary brain microvascular endothelial cells . AAV vectors (1x106 vg/cell) were used to transduce cells every three days for three rounds to achieve higher knockout efficiency. Cells were passaged as necessary.AAV-PHP.eB vectors with sgRNA sequences targeting HEK293T , Pro5 (CRL-1781), Lec2 (CRL-1736), and Lec8 (CRL-1737) were obtained from ATCC. BMVECs were obtained from Cell Biologics (C57-6023) and cultured as directed by the manufacturer.Recombinant AAVs were generated by triple transfection of HEK293T cells using polyethylenimine (PEI) and purified by ultracentrifugation over iodixanol gradients as previously described .TM MP system #1708280.The virus overlay assay was performed as previously reported  with somAll procedures were performed as approved by the Broad Institute IACUC (0213-06-18) or Massachusetts General Hospital IACUC . AKR/J (000648), BALB/cJ (000651), CBA/J (000656), CAST/EiJ (000928), C57Bl/6J (000664), C57BL/J (000668), DBA/2J (000671), FVB/NJ (001800), LP/J (000676), MOLF/EiJ (000550), NOD/ShiLtJ (001976), NZB/B1NJ (000684), and PWK/PhJ (003715) were obtained from the Jackson Laboratory (JAX). AAVR KO mice were a generous gift from Dr. J.E. Carette (Stanford) to Dr. Balazs and have been previously described . RecombiMice were anesthetized with Euthasol (Broad) or ketamine (MGH) and transcardially perfused with phosphate buffered saline (PBS) at room temperature followed by 4% paraformaldehyde (PFA) in PBS. Tissues were post-fixed overnight in 4% PFA in PBS and sectioned by vibratome. IHC was performed on floating sections with antibodies diluted in PBS containing 10% donkey serum, 0.1% Triton X-100, and 0.05% sodium azide. Primary antibodies were incubated at room temperature overnight. The sections were then washed and stained with secondary  for four hours or overnight. Primary antibodies used were mouse anti-AAV capsid , LY6A , LY6C , Glut1 . To expose the internal B1 capsid epitope in intact capsids, tissue sections or cells on coverslips were treated for 15 or 7 minutes, respectively, with 2M HCl at 37°C. The treated samples were then washed extensively with PBS prior to addition of the primary antibody.11 vg of AAV vector packaged into the indicated capsid. One or two hours after injection, the mice were perfused with PBS and tissues were collected and frozen at -80°C. Samples were processed for AAV genome biodistribution analysis by measuring the number of vector genomes (vg) in the sample using absolute qPCR for the GFP sequence. The values are reported as vg/mouse genome after normalizing to the number of copies of host cell genomes using primers recognizing mouse Gcg or human GCG as previously described [Five- to six-week-old C57Bl/6J mice, BALB/cJ mice, AAVR WT FVB/NJ, or AAVR KO FVB/NJ mice were injected intravenously with 10Images were taken on an Axio Imager.Z2 Basis Zeiss 880 laser scanning confocal microscope fitted with the following objectives: PApo 10x/0.45 M27, Plan-Apochromat 20x/0.8 M27, or Plan-APO 40x/1.4 oil DIC (UV) VIS-IR, or on a Zeiss Imager.M2 . In some whole sagittal section images, the 16-bit green channel (GFP) gamma was adjusted to enable visualization of low- and high-expressing cells while minimizing over-saturation. In all cases, changes to contrast or gamma as well as microscope laser settings were kept constant across sets of images that were directly compared.Ly6 family members (0.5 μg/well) were transfected into HEK293T cells (3x105/well) using PEI or into CHO cells (1.5x105/well) with lipofectamine 3000 reagent  in 24-well plates. 48 hours later, the cells were chilled to 4°C and the media was exchanged with fresh cold media containing the indicated recombinant AAV (105 vg/cell). One hour later, cells were washed three times with cold PBS, then fixed with 4% PFA for IHC or lysed for genomic DNA extraction and qPCR analyses. Vector genomes bound were normalized to the number of cell genomes by using primers specific to Gcg or GCG. For BMVECs, 2x104 cells/well were seeded in 12-well plates the day before exposure to virus. The assay was performed as above except that the AAV vectors were added at 106 vg/cell.The cDNA of Ly6 family members (0.1 μg/well) were transfected into the indicated cells  in 96-well plates  in triplicate. 48 hours later, cells were transduced with AAV-CAG-GFP-2A-Luciferase-WPRE packaged into AAV9 or AAV-PHP.eB. Luciferase assays were performed with Britelite plus Reporter Gene Assay System . Luciferase activity was reported as relative light units (RLU) as raw data or normalized to non-transfected control wells transduced with AAV9, or a control transduced without a sgRNA ( a sgRNA .Ly6a or control DNA (pUC19). The cells were incubated with AAV-PHP.eB:CAG-GFP-2A-Luc vectors  at 37°C.BMVECs were seeded at 2,000 cells/well in a 96-well plate. The following day, cells were incubated with LY6C or LY6A rat IgG antibodies at indicated concentration  at 4°C with gentle shaking for one hour. AAV-PHP.eB:CAG-GFP-2A-Luc vectors were added at 13,000 vg/cell at 37°C and the Luciferase reporter assay was performed 48 hours post-transduction. Antibody inhibition assays were performed on HEK293 cells as above with the following changes: HEK293 cells were seeded at 20,000 cells/well in 96-well plate the day before transfection with 11 vg/animal) or AAV-PHP.eB . LY6A IHC in In vitro transduction and binding experiments display the means from three independent experiments. In in vivo assays or distinct samples or transfections in cell culture assays.Microsoft Excel and Prism 8 were used for data analysis. For the comparison between AAV9 and AAV-PHP.eB biodistribution, a group size of six per group  was used based on prior data that indicated a large effect size. No animals or samples were excluded from the analysis. https://github.com/tpoterba/mouse-PHP.eB-simulation.The compiled variant dataset, filtered for variants of predicted high and medium impact, from all mice within the Mouse Genomes Project is provided as a resource to the research community . The codS1 TableThe predicted variant types, their count among all 36 mouse strains in the mouse genome project ,2 databa(PDF)Click here for additional data file.S2 TableLy6a present in permissive and nonpermissive mouse strains encode protein products likely to be modified by a GPI anchor. Within the amino acid sequences, SNPs are highlighted in red and underlined text; predicted GPI-anchor sites (ω-sites) are highlighted in bold green text, and cleaved C-terminal hydrophobic tail sequences are highlighted in magenta text.The predGPI prediction model  was used(PDF)Click here for additional data file.S3 TableThe lines highlighted in bolded text were used in the analysis presented in (PDF)Click here for additional data file.S1 Fig11 vg/mouse AAV-CAG-NLS-GFP packaged into the indicated capsid.Images of GFP fluorescence in whole brain sagittal sections from C57BL/6J (left column) or BALB/cJ (right column) two weeks after intravenous injection of 1x10(TIF)Click here for additional data file.S2 FigSagittal whole brain images show LY6A IHC in several representative permissive and nonpermissive mouse lines.(TIF)Click here for additional data file.S3 Fig(A) The individual sgRNA data used to generate (TIF)Click here for additional data file.S4 FigLy6a isoform 1.The schematic on the left shows the structure of the exons and introns of the  The genetic variants present in at least one of the nonpermissive strains (column headers in blue text) but not the permissive strains (column headers in green) are shown. Variants that segregate between permissive and nonpermissive strains are highlighted in orange text. Variants within exons are highlighted in gray. Variants are shown as homozygous (2), heterozygous (1), absent (0), or as data not available (-).(PDF)Click here for additional data file.S1 FileThe presence of the gene variants is given as 1 for heterozygous, 2 for homozygous, or 0 for absent.(ZIP)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(XLSX)Click here for additional data file.


Hemi-PD rats exhibited an ipsilateral decrease in striatal mAchR densities between 6 and 16%. Moreover, a massive and constant decrease in striatal nAchR density by 57% was found. A second goal of the study was to disclose receptor-related mechanisms for the positive motor effect of intrastriatally injected Botulinum neurotoxin-A (BoNT-A) in hemi-PD rats in the apomorphine rotation test. Therefore, the effect of intrastriatally injected BoNT-A in control and hemi-PD rats on mAchR and nAchR densities was analyzed and compared to control animals or vehicle-injected hemi-PD rats. BoNT-A administration slightly reduced interhemispheric differences of mAchR and nAchR densities in hemi-PD rats. Importantly, the BoNT-A effect on striatal nAchRs significantly correlated with behavioral testing after apomorphine application. This study gives novel insights of 6-OHDA-induced effects on striatal mAchR and nAchR densities, and partly explains the therapeutic effect of BoNT-A in hemi-PD rats on a cellular level.Cholinergic neurotransmission has a pivotal function in the caudate-putamen, and is highly associated with the pathophysiology of Parkinson's disease. Here, we investigated long-term changes in the densities of the muscarinic receptor subtypes M Acetylcholine (Ach) effects are mediated via metabotropic G-protein coupled muscarinic receptors (mAchRs) and ionotropic nicotinic receptors (nAchRs). They are important for cognitive functions such as working, episodic and spatial memory, or attention  as well as mAChRs (M1-M5 subtypes) interact in a complex manner to modulate synaptic plasticity in the striatum. M1 receptors presynaptically located on medium spiny neurons (MSNs) inhibit GABA release  the primary neuropathological characteristic is the dopaminergic denervation of the CPu caused by progressive dopaminergic cell death in the substantia nigra pars compacta (SNpc)   and nAchRs (α4β2 subtype) up to a survival time of 9 months using quantitative in vitro receptor autoradiography. First, we analyzed time-dependent changes in receptor densities in 6-OHDA-induced hemi-PD rats relative to controls. Then the long-term effects of intrastriatal BoNT-A injections in naïve rats were investigated. To our knowledge, this is the first study analyzing long-term effects of 6-OHDA and intrastriatal BoNT-A injections.To explore the role of cholinergic receptors and its changes after DA deprivation and following BoNT-A injection, we comprehensively examined striatal receptor densities of mAchRs  aged 3 months and weighing 250–280 g were obtained from Charles River WIGA . Animals were housed in a temperature-controlled room (22 ± 2°C) under a fixed 12 h light/12 h dark cycle and had free access to food and water 24 h a day. Animal treatment was in line with legal obligations of the animal welfare act and all animal experiments were approved by the state Animal Research Committee of Mecklenburg-Western Pomerania (LALLF M-V/TSD/7221.3-1.1-003/13).Anesthesia was performed via intraperitoneal (i.p.) injection of a mixture of ketamine (50 mg/kg body weight) and xylazine (4 mg/kg body weight). Rats were operated under aseptic conditions at a weight of 285–305 g. Animals were fixed in a rat stereotactic apparatus  and 6-OHDA solution was injected evenly over 4 min via a 26 gauge 5 μl Hamilton syringe into the right MFB. The coordinates of the 6-OHDA injection with reference to bregma were: anterior-posterior = −2.3 mm, lateral = −1.5 mm and ventral = −9.0 mm  and cut in frontal sections (20 μm) on a cryostat . Mounting was performed on gelatin-coated and pre-cooled (−20°C) glass slides followed by a drying procedure . Sections were obtained from 7 predefined levels, which were selected to cover the entire rostro-caudal extent of the CPu. The coordinates with reference to bregma were: level 1: +1.56 mm, level 2: −0.36 mm, level 3: −0.84 mm, level 4: −2.16 mm, level 5: −2.52 mm, level 6: −3.60 mm, and level 7: −5.20 mm , the agonistic binding site of the M2 receptor with [3H]oxotremorine-M, and its antagonistic binding site with [3H]AF-DX 384. The antagonistic ligand [3H]AF-DX 384 binds not only to M2 receptors, but also to M4 receptors in a regional specific manner  and the α4β2 nicotinic receptor with [3H]epibatidine (α4β2 agonist). All ligands were purchased from Perkin Elmer . Autoradiographic processing of the sections was performed in 3 main steps: rehydration and elimination of endogenous ligands, incubation with the respective tritiated ligand in the absence  or presence (nonspecific binding) of a specific non-radioactive ligand as displacer, and a final washing step to remove non-bound ligand and buffer salts. Sections were then dried and co-exposed with different plastic standards (representing different radioactivity concentrations) against ß-sensitive films  for 9 weeks , 12 weeks ([3H]pirenzepine) or 15 weeks . Resulting autoradiographs were developed using a Hyperprocessor  and digitized with a CCD-camera . Details of autoradiographic processing for each receptor are shown in Table Autoradiography was performed according to published protocols . Staining of cell bodies was performed with 0.1% cresyl-violet solution . These sections were used as histological reference during the delineation of the CPu in the autoradiographs.http://www.mcid.co.uk/) was used for measurement of receptor densities. Digitized autoradiographs were loaded and regions of interest (ROIs) were defined manually by comparison with the neighbouting cell-body stained sections. For each ROI, the average and background-corrected gray values were extracted. For densitometric analysis the gray values of all processed sections were transformed into receptor densities (in fmol/mg protein) by means of standard curves derived from the co-exposed plastic standards. In the ensuing linearized autoradiographs, which can be color coded for visualization purposes, the receptor density in fmol/mg protein of each pixel can be calculated according to:The software MCID Analysis v7.0  , B is a constant representing the number of decays per unit of time and radioactivity [Ci/min], Wb the known equivalent protein weight of a standard [mg], Sa the specific activity of the ligand [Ci/mmol], KD the dissociation constant of the ligand [nM], and L the free concentration of the ligand during incubation [nM]. Displayed receptor density values are the area-weighted means of all ROIs of an animal containing the CPu. Since the tritiated ligands do not only bind to a single receptor type, the KD value for each ligand is important to reach the necessary selectivity for demonstration of a single receptor type. Therefore, the ligand concentration in the incubation buffer was set at 50% of the KD value for the dissociation of the ligand-receptor binding. This results in a high specificity of receptor labeling.where post-hoc ANOVA analysis of variance). The dependent variable was “receptor density” and the covariate was the respective treatment group separately for the left and right hemispheres not considering survival time followed by Bonferroni correction with the factor group . Correlations between relative receptor density and apomorphine-induced rotations as well as time-dependent correlations of right-left differences were analyzed with linear regression followed by a two-sided Pearson correlation test. Possible significant differences in body weight between 2 groups were analyzed using factorial ANOVA. p < 0.05 was considered statistically significant.For all statistical observations, IBM SPSS Statistics version 20.0 was used. Gaussian distribution of receptor density values was tested with the Kolmogorov-Smirnov test. Then, data was analyzed with a between-subject Univariate General Linear Model  and nAchRs (α4β2) longitudinally between 3 weeks and 9 months post lesion in 5 experimental groups  significantly abolished contralateral rotations for up to 3 months. Vehicle injection in hemi-PD rats (group LV) did not affect the rotational behavior, as previously shown  demonstrated a contralateral net rotation of about 8 min1 receptors showed a mean concentration of 5,092 ± 2,331 fmol/mg (mean ± SD) without significant interhemispheric differences   , M Figures . 6-OHDA  Figures . Notably) Figure .1 receptor density of −7.0% 2 weeks after BoNT-A. This reduction significantly decreased  with longer post injection survival , and changed into an increase   did not result in interhemispheric differences when densities were averaged over all analyzed survival times Figure . Conside) Figure .1 receptors when densities were averaged over all survival times   did not significantly affect the right-left difference of Ms Figure . Notably) Figure . Vehicle Figures  as compa1 receptor density with apomorphine-induced rotations for groups LB and LV and did not find a significant correlation .To analyze a possible relation between interhemispheric differences and apomorphine-induced rotations and the effect of BoNT-A and Sham injection in this context, we correlated relative M2 receptor density in the CPu of control rats (group C) exhibited a mean concentration of 949 ± 95 fmol/mg (mean ± SD) showing no significant side differences : −0.7% (L3W), 2.0% (L6W), −13.0% (L6W1M), −6.1% (L6W3M), −6.7% (L6W6M), −7.3% (L6W9M)  Figure .2 agonist binding of rats treated only with BoNT-A (group B) showed no significant interhemispheric differences when the mean was calculated over all post-injection time points  depending on post-injection times of BoNT-A starting with an ipsilateral decrease at 2 weeks and ending with an increase at 9 months: −8% (B2W), −13.8% (B1M), −0.4% (B3M), 2.7% (B6M), 13.3% (B9M)  Figure .2 agonist binding was not significantly altered by post-treatment of hemi-PD rats with BoNT-A (group LB) or with vehicle (group LV) when the mean effect was calculated over all survival times :   Figure .R2 = 0.2559, p = 0.0544) between the interhemispheric difference of these M2 receptor binding sites for groups LB and LV and the apomorphine-induced rotations.We found no significant correlation (3H]-AFDX-384) binding of 16% when densities were averaged over all examined time points , a mean density of 3,876 ± 357 fmol/mg (mean ± SD) was measured. No significant interhemispheric differences were found Figures . Injecti Figures . Further Figures .R2 = 0.68926, p = 0.0001) depending on post BoNT-A injection-time, starting with an ipsilateral decrease at 2 weeks and ending with a slight increase at 9 months: −16% (B2W), −10.9% (B1M), −9.4% (B3M), −0.6% (B6M), 3.5% (B9M)  resulted in a non-significant interhemispheric decrease (−8%) in antagonistic receptor binding when densities were averaged over all examined time points Figure . However) Figure .R2 = 0.317, p = 0.0001):  with motor behavior in the apomorphine rotation test.Right-left differences for groups LB and LV did not correlate (3 receptors in control rats (group C)  compared to the drug naïve rats (group C)  density of 7,321 ± 733 fmol/mg was found for M Figures . The dif Figures . However) Figure .R2 = 0.309, p = 0.001) time dependence of the BoNT-A effect (group B) on the M3 receptor densities was observed: −3% (B2W), –.3% (B1M), −2.0% (B3M), 4.4% (B6M), 7.8% (9M)  post-injection time dependency    Figure . In the  Figures .R2 = 0.1433, p = 0.1643) correlation between the right-left difference of M3 receptor density in the CPu of groups LB and LV and the apomorphine-induced rotational behavior.We observed no significant (4β2 receptors exhibited a density of 387 ± 29 fmol/mg (mean ± SD) in the CPu of control rats (group C), and no significant interhemispheric differences were observed  from values well above control levels at 2 weeks to 3 months after BoNT-A to control values at 6 and 9 months post-injection   did not result in significant interhemispheric differences when densities were averaged over all post-injection time points Figure , althoug) Figure .2 = 0.174, p = 0.014) negative correlation between interhemispheric differences and increasing post-injection times was found   Figure . In vehi) Figure .R2 = 0.5493, p = 0.0016) of the interhemispheric difference of nicotinic receptors in the groups LB and LV and apomorphine-induced rotations was found  show control values 3 weeks after 6-OHDA application followed by a decrease of ~10% over the subsequent 9 months  and the M3 receptor are also decreased by ~10% compared to control value, but do not show any significant recovery over time  3 weeks after 6-OHDA injection, but no sign of any recovery during the following period  leads to a 10% decrease of Mr Figure . The M2/s Figure . The M2  Figures , 4D. Thed Figure .1 and M3 receptor densities of about 5–10% followed by a steady recovery, reaching 5–10% over the control value 9 months later  show an initial decrease of slightly more than 10%, followed by a steep increase resulting in a density of ~10% over control values 9 months after BoNT-A treatment  leads to an initial (2 weeks after injection) decrease in M Figures , 4D. Alst Figure . The antr Figure . The nicd Figure .1 receptor density which is well comparable to that of the untreated (group L) and vehicle injected (group LV) hemi-PD rats over all time points  and M3 receptor densities caused by 6-OHDA application is compensated by BoNT-A, since changes over time in the densities of these receptors in the LB group result in values comparable to those of controls 9 months after BoNT-A injection  reaches nearly the values of the untreated hemi-PD (L group) rats  leads to an Ms Figure . The dec Figures , 4D. Thes Figure . The lar4β2 receptor densities and post-injection time in drug naïve (group B) and BoNT-A treated (group LB) hemi-PD rats, but results in a positive correlation between all examined muscarinic cholinergic binding site  and M3) densities. Apparently, the nicotinic and muscarinic receptors react differentially to BoNT-A treatment.Most notable is the fact that BoNT-A injection causes a negative correlation between nicotinic αin vitro receptor autoradiography to analyze time-dependent changes in the densities of mAchRs  and nAchRs (α4β2) in hemi-PD rats with or without unilateral BoNT-A injection into the striatum. Receptor densities were measured in the striatum ipsi- and contralateral to the 6-OHDA injection side, and expressed as relative values by calculating the difference between ipsilateral and contralateral receptor densities normalized by the contralateral density. BoNT-A was injected ipsilaterally.Progressive dopaminergic cell death and the consequent DA deficit in the CPu can only partly explain PD's symptomatology, hence various compensatory changes in dopaminergic and other neurotransmitter systems have been described Barone, . Here, w2/D3 receptor density  , whereas the other half of the MSNs express enkephalin as well as dopamine D2 receptors and mainly project to the globus pallidus externus (EGP)  were both described to mediate DA release from dopaminergic terminals. However, studies report conflicting results on the DA inhibiting or facilitating effects. The role of different receptor subtypes, especially of mAchRs located on MSNs and corticostriatal or thalamostriatal afferents , the DA release of dopaminergic terminals is modified indirectly by mAchRs via nAchRs  results in decreased Ach release from cholinergic interneurons, and by this in a reduced activation of nAchR on dopaminergic axons. Consequently, inhibition of DA release is observed after activation of mAchRs for single-spike modes of DA neurons, activation is found for burst firing mode  in the CPu was significantly reduced by 19%   and M3 receptors, whereas differences of M1, and M2 (agonist binding) receptors were not significantly changed  and the drug naïve hemi-PD rats (group L). Thus, BoNT-A induced an increase of nAchR density in groups B and LB.Seemingly, BoNT-A induced an increase of nAchRs on non-dopaminergic structures, e.g., cortical afferents or striatal interneurons, since dopaminergic terminals no longer exist in hemi-PD. An initially increased nAchR density compared to controls and vehicle-injected rats was found after BoNT-A injection which may be explained by the reduction of striatal Ach content and a concomitant upregulation of nAchRs. This effect, however, disappears after 6–9 months after BoNT-A injection. Interestingly, the opposite effect of BoNT-A was seen in mAchR densities; here the reduction in striatal Ach content was followed by a downregulation of mAchR densities.This study expands our understanding of 6-OHDA- and BoNT-A-induced changes in striatal mAchR and nAchR expression. It provides hypotheses for the positive BoNT-A effect on motor performance in hemi-PD rats based on changes in cholinergic receptor densities. The differential outcome of striatal BoNT-A application on Ach receptor densities can be interpreted as a further hint to the fact, that the densities of mAchRs and nAchRs are regulated differently by the availability of its transmitter (Sivam et al., The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.AW and KZ designed and planned the project. AW performed 6-OHDA lesioning and BoNT-A injections. OS and AW provided the analyzing strategy, analyzed data, and designed concepts for figures. AH performed apomorphine-induced rotation tests and MC the receptor binding experiments. FK and TM analyzed data. TM, AW, NP-G, and KZ interpreted data and wrote the manuscript. All authors have approved the final article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry we identified two proteins, named “LukS-I” and “LukF-I”, encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermedius LukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections.Bacterial infections from  S. pseudintermedius is the primary cause of pyoderma (skin infection), the most common canine dermatologic disease, and is also associated with urinary tract infections, wound and surgical site infections, external ear otitis, abscess formation, mastitis and endocarditis [S. pseudintermedius isolates tested in our University of Tennessee College of Veterinary Medicine Bacteriology Laboratory from patients are methicillin-resistant (MRSP) and high levels of resistance occurs in other regions of the United States [carditis –3. Approd States .Alternative approaches to control staphylococcal infections, such as vaccines, have been difficult to develop. This is likely rooted in the ability of the bacteria to neutralize and/or destroy important components of their host defenses. Some staphylococcal toxins impact the innate immune system, the first line of defense against this pathogen –8. AntibLeukocidins are a family of potent toxins contributing to the pathogenicity of staphylococci . LeukociStaphylococcus aureus including HlgAB, LukMF, HlgCB, LukAB/HG, LukED, Panton-Valentine leukocidins (LukSF-PV/PVL), and LukPQ, some of which are host and cell specific [hlgAB and hlgCB and lukAB/HG), prophage associated  or reside on a pathogenicity island (LukED) [A total of seven different bicomponent pore-forming toxins (BCPFTs) have been identified and characterized in specific . The enc (LukED) –8, 11–13Staphylococcus intermedius was identified and characterized previously [S. pseudintermedius strains including (S. pseudintermedius type strain CCUG49543T) isolated from dogs without characterization of the actual protein function.A bi-component toxin (LukS-I + LukF-I) from eviously . Descloueviously  has repoS. aureus LukS-PV and LukF-PV subunits [S. aureus USA300 sepsis and this effect could also be achieved by passive transfer of rabbit anti-LukS-mut9 antisera [In a previous study Karauzum et al. rationally designed mutants of subunits . They tesubunits . Rabbit subunits . LukS-muantisera .S. pseudintermedius by mass spectrometry (MS) to determine the abundance of secreted Luk-I, characterize the cytotoxic effect of recombinant S. pseudintermedius Luk-I on canine PMNs and develop attenuated, nontoxic LukS-I and LukF-I. Attenuated LukS-I and LukF-I were tested for their antigenicity and PMN killing. Furthermore, antibody raised against S. pseudintermedius attenuated Luk-I in clinically healthy dogs was evaluated for its ability to neutralize wild-type LukS-I and LukF-I. The results from this study suggest that S. pseudintermedius LukS-I and LukF-I may serve as key components in a vaccine or as part of an immunotherapeutic approach.The purpose of the present study was to analyze the secretome of Experimental protocols were reviewed and approved by the University of Tennessee Institutional Animal Care and Use Committee (IACUC) including obtaining blood samples of dog (2474–0716) and injecting dogs with recombinant protein for producing antibodies (2572–1217).S. pseudintermedius strains used in this study, representing the most common multilocus sequence types (ST) previously reported in the United States [The d States , 4, 20, d States , 4, 20. LukS-I-M and pMA- attenuated LukF-I-M, each containing a mutated, synthetic S. pseudintermedius gene (designed as described below) with BamHI/NotI cloning sites, was obtained commercially .Plasmid construct pMA- attenuated 600 = 0.4–0.6 was reached.Bacterial colonies grown on blood agar plates were inoculated into 5ml of sterile trypticase soy broth (TSB)  and incubated overnight at 37°C with shaking at 225 rpm . Fifty microliters of overnight culture were inoculated into 5ml of fresh, sterile TSB to initiate log-phase bacterial cultures. Bacteria were grown at 37°C with shaking at 225 rpm until an optical density of OD S. pseudintermedius supernatant/ secretome were prepared for shotgun LC-MS/MS analysis as previously described [Log-phase bacterial cultures of 06–3228, 08–1661 and NA45 were centrifuged at 10,000 x g for 30 minutes at 4°C  and the supernatant was collected and passed through a 0.45μm filter . The filtrate was concentrated using a centrifugal filter  and stored at -20°C until further analysis. Samples interrogating escribed .Peptides were separated and analyzed with a 2-step MudPIT LC-MS/MS protocol  over a 4-hr period then measured with a hybrid LTQ XL-Orbitrap  mass spectrometer (MS) at Oak Ridge National Laboratories, Oak Ridge, Tennessee, USA. PeptideS. pseudintermedius 06–3228 strain with corresponding units in other leukotoxins ( was performed and a rooted phylogenetic tree (UPGMA (unweighted pair group method with arithmetic mean)) of Luk-I was generated with Geneious version 11.0.3 [S. pseudintermedius protein A serving as an outgroup.Multiple sequence alignment (MSA) of mature LukS-I and LukF-I subunits of n 11.0.3  with S. S. pseudintermedius Luk-I that may shape the protein function and specificity, a multiple alignment of amino acid sequences of the DR4 region in the rim domain (an important region for S-component receptor binding) of S. aureus LukE, HlgA, LukM and LukP, S. pseudintermedius and S. intermedius LukS-I was performed.In order to identify the unique residues in S-component of http://www.psort.org/psortb/)[The bacterial localization prediction tool, PSORTb version 3.0.2 (/psortb/) was usedS. pseudintermedius LukS-I and LukF-I modeling and binding site prediction was performed using Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) (http://www.sbg.bio.ic.ac.uk/phyre2) [http://www.sbg.bio.ic.ac.uk/3dligandsite/)[S. aureus LukSF-PV, LukED and LukPQ as a basis to predict the critical amino acids for protein function. PHAST (http://phast.wishartlab.com/index.html) and PHASTER [http://phaster.ca/) were used for prophage detection in a total of 22 S. pseudintermedius isolates./phyre2)  and the andsite/) using S. PHASTER , 29 (httS. pseudintermedius strain 06–3228 obtained from blood agar plates were grown in TSB at 37°C with 225 rpm shaking. DNA was extracted using a MO BIO DNA Isolation Kit (QIAGEN Inc. Cat No.12224-50) according to the manufacturer’s instructions. Oligonucleotide primers  ( were designed using a PrimerQuest Tool (https://www.idtdna.com/Primerquest/Home/Index) based on the genomic sequence of S. pseudintermedius strain 06–3228 (20). The native LukS-I and LukF-I open reading frames (ORF)  without the regions encoding the predicted N-terminal signal peptide were amplified from S. pseudintermedius 06–3228 genomic DNA and the ORF of mutant LukS-I and LukF-I were amplified from pMA-attenuated LukS-I-M and pMA-attenuated LukF-I-M plasmids , respectively  and the following cycling conditions were performed: initial denaturation at 95°C for 90 seconds, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute followed by a final extension at 72°C for 5 minutes. All ORFs were amplified without a histidine tag because pETBlue-2 allowed T7lac promoter-based expression of target genes with C-terminal histidine tagged sequences. PCR products were Sanger sequenced at The University of Tennessee Genomics Core facility.Bacteria from a single colony of S. pseudintermedius native and mutant LukS-I and mutant LukF-I, their PCR products were digested with NotI and BamHI, then ligated into pETBlue-2  and transformed into DH5-alpha E. coli chemically-competent cells  by heat shock. The DH5-alpha E. coli were plated on LB agar plates with 100 μg/mL ampicillin. The plasmid constructs were transformed into DE3 pLacI E. coli chemically-competent cells  by heat shock and the pLacI E. coli were plated on LB agar containing 50μg/ml ampicillin and 20μg/ml chloramphenicol.To clone S. pseudintermedius native and mutant LukS-I and mutant LukF-I, a single colony of DE3 pLacI E. coli was inoculated into LB broth containing 50μg/ml ampicillin and 20μg/ml chloramphenicol and bacteria grown overnight at 37°C with 225 rpm shaking. LB broth containing 50μg/ml ampicillin and 20μg/ml chloramphenicol was inoculated with a 1:100 dilution of overnight culture and grown at 37°C with 225 rpm shaking until a 600 nm optical density of between 0.4 and 0.6 was reached. Protein expression was induced by addition of 1 mM Isopropyl β-D-1-thiogalatopyranoside (IPTG)  and bacteria were grown for 4 hr at 30°C with shaking at 225 rpm. Bacterial cultures were centrifuged at 12,000 x g for 5 min in 5 ml of protein extraction reagent  and 20 μl of 100X protease inhibitor  and incubated for 30 min at 37°C in a shaking incubator at 225 rpm. Bacteria were pelleted by centrifugation at 12,000 x g for 45 min at 4°C. Recombinant proteins were purified using affinity purification .To express recombinant Kluyveromyces lactis . Recombinant protein was purified from K. lactis supernatant using affinity purification . Protein concentrations were determined using a bicinchoninic acid (BCA) assay .Recombinant native LukF-I was cloned using an integrative expression vector (pKLAC2) and expressed in Protein samples were resolved by SDS-PAGE in 4–12% polyacrylamide gels  and electrophoretically transferred onto nitrocellulose membranes . The blots were blocked overnight in 5% (wt/vol) nonfat dried milk powder in 0.05% polyethylene glycol sorbitan monolaurate (Tween 20) containing phosphate buffered saline (PBS-T) at 4°C. The blocked membranes were incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated anti-6xhis tag monoclonal antibody  in 0.05% PBS-T for 1 h with 225 rpm shaking at room temperature. After five washes with 0.05% PBS-T bound antibodies were detected using chloronaphthol substrate solution .E.coli were purified using affinity chromatography (as above) and endotoxin concentrations were measured using a chromogenic LAL Endotoxin Assay Kit . Recombinant attenuated LukS-I and LukF-I at 20 μg each / 0.5 cc in phosphate buffered saline (PBS) (pH 7.2) were injected in the lateral thorax by the subcutaneous route, into three clinically normal dogs. Injections were given once every 7 days for a total of three injections with a control dog receiving PBS (pH 7.2) only. Blood (6 cc) was collected from a jugular vein 4 times, on days -7, 8, 15 and 29. The collected blood was left undisturbed at room temperature for 30 min followed by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge.Recombinant attenuated LukS-I and LukF-I produced in For measurement of recombinant protein antigenicity, attenuated LukS-I and LukF-I were coated separately onto ELISA plates  at 2μg/ml in PBS (pH 7.2). The plates were washed with 0.05% PBS-T and incubated with two-fold serial diluted serum from dogs (injected with recombinant proteins) for 1 h at 37°C, then bound IgG was detected using HRP-conjugated goat anti-dog IgG heavy and light chain . ELISA assays plates were washed three times with PBS-T between all incubations, bound antibodies were detected using TMB substrate , reactions were stopped with 0.18 M sulphuric acid and optical density read at 450 nm on a plate reader . The experiment was repeated a minimum of three times and a p-value of <0.05 was considered significant for all the experiments unless otherwise stated.pseudintermedius 06–3228 supernatant) in a volume of 500 μl in RPMI medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator for 30 min. Supernatant of S. pseudintermedius 06–3228 was harvested at log phase to test the toxic effect of secreted LukS-IR. PMNs were stained with 1 μl of Sytox Green  for 30 min, washed with PBS (pH 7.2) twice and analyzed using a flow cytometer (Attune acoustic focusing cytometer). In order to measure the protective effect of anti-LukS-IR on canine PMNs, recombinant LukS-I and LukF-I were incubated with canine anti-attenuated LukS-I LukF-I at a dilution of 1:100 for 30 min at 37°C, then tested with the cell permeability assay as previously described.Canine blood was collected from healthy dogs using a sterile blood collection system with EDTA anti-coagulant (BD Vacutainer). Then, 600 μl of dog blood was added to 1 ml of red blood cell lysing buffer  for 30 min at 37°C in 15 ml sterile plastic tube, centrifuged and re-suspended in 1ml RPMI medium supplemented with 10% fetal bovine serum. PMNs were incubated with recombinant proteins  were incubated with 50μl of 10mM EZ-Link Sulfo-NHS-LC-Biotin reagent   for 30 min at room temperature. Excess biotin was removed using an Amicon Ultra-0.5 Centrifugal Filter Unit with a 30 kDa molecular weight cut-off . The biotin-labeled proteins were stored at -20°C until further use.Purified recombinant To test the binding of wild type and attenuated LukS-I and LukF-I to canine PMNs, biotin labelled recombinant proteins were incubated with PMNs from a clinical healthy dog for 30 minutes at room temperature, the cells were washed, then PMNs were incubated with 1:500 dilution of avidin-FITC conjugate  at room temperature for 30 minutes in the dark. Unbound conjugate was removed by washing and the amount of binding was determined using a flow cytometer (Attune acoustic focusing cytometer).Data was analyzed using ANOVA tests and followed by post hoc multiple comparisons with Tukey’s adjust. Diagnostic analysis was performed on residuals for checking normality and equal variance assumptions. Rank data transformation was applied when assumptions were violated. Statistical significance was identified at the level of 0.05. Analyses were conducted in JMP pro 14 for Windows .Secreted proteins, were isolated and digested to peptides with trypsin and analyzed by LC-MS/MS. The high mass accuracy of intact peptides and their fragmentation profiles were searched against genome-derived amino acid sequences for each protein in the sequenced genome of each strain. Identified peptides were then mapped back to their respective proteins and proteins quantified by both sequence coverage as well as area-under-the-curve abundance. Using this semi-quantitative data, along with categorizing each protein by their predetermined cellular location (pSORT), we were able to identify both LukS-I and LukF-I proteins in supernatant at relatively high levels, i.e. rank 5 (LukF-I) and rank 7 (LukS-I) out of 92 total extracellular proteins identified , putting both in the top 10% of the extracellular dataset. These MS identification confirmed the extracellular existence of these proteins in all virulent strains and that they are indeed expressed at relatively high-levels, thus serving as candidates for the further analysis (presented here and on-going).The percent coverage of LukS-I and LukF-I were calculated by dividing the number of amino acids in all found peptides by the total number of amino acids in the mature protein sequence S. pseudintermedius strains including 06–3221, 08–1661 and NA45 with amino acid identities over 99.4% between strains.Multiple sequence alignment (MSA) analysis showed that Luk-I is conserved among S. pseudintermedius 06–3228. A BLAST search of Φ Staphy_96_NC_007057 using complete genome sequences of S. pseudintermedius strains available in the GenBank database and others sequenced in our lab but not yet published, revealed that approximately 7 Kb of the phage are present in all of S. pseudintermedius isolates examined . They also contain the coding DNA sequences (CDS) of ascorbate-specific PTS system EII A, B and C components, probable L-ascorbate-6-phosphate lactonase UlaG (L-ascorbate utilization protein G), phosphoglycerate mutase family 2 and hypothetical protein.A 14.9 kb incomplete prophage (similar to Φ Staphy_96_NC_007057) was identified in the genome of S. pseudintermedius LukS-I is closely related to S. intermedius LukS-I, S. aureus LukE and LukP. However, S. pseudintermedius LukF-I is closely related to S. intermedius LukF-I and S. aureus gamma hemolysin subunit B  analysis showed that Luk-I in 1 server .2 web portal, shows that LukF-I consist of β-strands organized as four antiparallel β-sheets and three very short α-helices  71, Tryptophan (W) 176, Arginine (R) 179, W256, Phenylalanine (F) 259 and histidine (H) 260 as a critical residues located in the rim domain and identified as important for phospholipid binding on the membranes of canine PMNs while Serine (S) 33 and Asparagine (N) 39 located in the β-sandwich (cap) domain as essential amino acids to interact with side residues of LukS-I which stimulate oligomerization. These residues are conserved among S. intermedius LukF-I and S. aureus LukD  region in the rim domain (an important region for S-component receptor binding) of ifferent .We designed attenuated LukS-I and LukF-I with mutations away from the rim region because we need to disrupt the ability of LukS-I and LukF-I to interact side by side and form oligomers, a function critically required for cytolysis.S. pseudintermedius LukS-I and S. aureus LukS-PV share the critical residues T28, K99 and S211 for interaction and oligomerization with the corresponding F-component [2, and MSA of amino acid sequences of S. pseudintermedius LukS-I and S. aureus LukS-PV  59 and Asparagine (N) 65 in the mature LukF-I protein as essential residues for side-by-side interactions with LukS-I to form oligomers. To test that we designed attenuated LukF-I with the following substitutions S59D and N65A.E.coli and LukF-I was secreted in the culture supernatant of K. lactis using an integrative expression vector (pKLAC2). Recombinant proteins were purified using HisPur Ni-NTA resin under native conditions and eluted using an imidazole gradient. The molecular weights of LukS-I, LukS-I-M, LukF-I and LukF-I-M determined in western blots were of the expected sizes  (Recombinant polyhistidine tagged wild type and mutant LukS-I and LukF-I were generated in ctively) . The endS. pseudintermedius wild type and attenuated LukS-I and LukF-I were detected using an indirect ELISA, with sera collected on days -7, 8, 15 and 29, after the second injection of attenuated Luk-I (on day 15) and was higher on day 29 (P < 0.0001) compared to pre-injection control sera (Specific antibodies against rol sera  Tables.S. pseudintermedius 06–3228 supernatant dilution (P <0.0001) (Canine PMNs were highly susceptible to Luk-I with lysis induced within 30 minutes at a concentration of 200 ng of each leukototxin component (P <0.0001) and a 1:2 <0.0001)  Tables. <0.0001) .To test the effect of critical amino acid substitutions on attenuated LukS-I and LukF-I binding to canine PMNs, biotin labelled recombinant wild type and attenuated LukS-I and LukF-I were incubated with canine PMNs and their binding detected using FITC conjugated avidin. No significant difference in MFI of attenuated or wild type recombinant LukS-I and LukF-I was found (P >0.05) .Dog anti-Luk-I at a dilution of 1:100 preincubated with Luk-I showed a significant reduction in mean fluorescent intensity (MFI) compared with Luk-I treatment alone .S. pseudintermedius and kill canine PMNs by pores formation and cell lysis. Luk-I, a member of the leukocidin family, is highly conserved among S. pseudintermedius isolates. Luk-I is associated with an incomplete prophage that occurs in degenerate form across all S. pseudintermedius isolates for which genomic sequence is available.We identified a bicomponent leukotoxin, Luk-I, composed of LukS-I and LukF-I proteins that secreted by S. pseudintermedius Luk-I in the dog, in line with the assumed function of other phage-encoded leukocidins that similarly have a host-specific function and distribution. For example, LukMF is highly toxic to ruminant neutrophils and LukPQ preferentially kills horse neutrophils [In accordance with its host distribution in a canine opportunistic pathogen, LukS-IR is cytotoxic against dog PMNs. This highlights the immune-evasive attribute of trophils , 7, 31.S. aureus γ-hemolysin LukF component and S. pseudintermedius LukS-I and LukF-I model, we designed attenuated LukS-I and LukF-I with mutations away from the rim region. The attenuated LukS-I and LukF-I bind to canine leukocytes without causing any significant killing which indicates that mutations disrupt the ability of attenuated LukS-I and LukF-I to oligomerize, an essential function required for cytolysis. This observation is in agreement with the findings of Karauzum et al [Based on the crystal structure of β-sandwich (cap) domain of um et al  with LukE. coli suggests that endotoxin did not play a role in neutrophil killing.The low concentration of endotoxin in the recombinant proteins and the low toxicity exhibited by the attenuated protein produced in E.coli was not successful and for unknown reasons this protein appeared to be lethal to these bacteria; therefore, a yeast protein expression system (K. lactis) was used for this protein. K. lactis is considered the best alternative to bacterial expression systems. It efficiently produces recombinant proteins in culture supernatant that are also secreted by their native host [K. lactis protein expression is driven by a variant of the strong PLAC4 promoter (PLAC2) [E.coli [Expression of LukF-I in ive host –34. K. l (PLAC2)  that lac [E.coli .The high titer of Luk-I-specific, neutralizing antibodies produced in a clinically healthy dog injected with with attenuated LukS-I and LukF-I is similar to the antibody production that Karauzum et al  and AdhiS. aureus. Taking the same factors into account, a potent and effective vaccine against S. pseudintermedius would involve identifying antigenic targets conserved among a wide variety of strains and sequence types. Understanding the nature of extracellular proteins and their role in virulence and pathogenesis is critical for vaccine development against S. pseudintermedius infection.With widespread antimicrobial resistance, it is critical to find alternative approaches, such as vaccines, to control staphylococcal infections. Previous studies –38 conclS. pseudintermedius isolates 06–3221, 08–1661 and NA45, representing the three clonal complexes that predominate in the United States [Using mass spectrometry and genomic information, it was possible to identify a unique leucocidin present in S. pseudintermedius. To our knowledge this is the only leucocidin from a species other than S. aureus characterized to date. Mutant versions of the proteins had a reduced cytotoxic effect on dog PMNs and anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of wild type canine leukotoxin. Therefore, these mutants may serve as important components of a multivalent vaccine for prophylaxis or control of S. pseudintermedius infections. Such a vaccine may neutralize extracellular toxins responsible for host tissue destruction and immunosuppression and may help the host immune system control infections.In conclusion, we describe a unique pore-forming toxin from S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property.The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles  In. InN-met+, PS2.M forms an antiparallel G-quadruplex initially at optical concentrations, but with additional time or at higher initial strand concentrations an increasing amount of a multistranded parallel G-quadruplexes appear .,41.+ sho+. These findings are important for the biosensing application utilizing hemin-G-quadruplex peroxidase because conformational switching between the catalytically inactive and the active forms of the G-quadruplex is crucially important for this application. In the next section we will discuss representative applications, mainly DNA detection, utilizing the conformation switch.These studies with various G-quadruplexes strongly suggest that the peroxidase-catalytically active form is the parallel conformation induced by KHemin-G-quadruplex peroxidases have been widely applied to biosensing for various target molecules and ions. In particular, hemin-G-quadruplex peroxidases have been widely applied for sequence-specific DNA detection.2O2, to chemiluminescence. Based on this scheme, the detection of the target oligonucleotide was possible to a detection limit of 0.6 μM  to the target DNA and a G-quadruplex forming sequence (III) A 43]. I. I43]. If 0.6 μM . The samf 0.6 μM B 44]. T. T2O2, tf 0.6 μM B. The hy2 system .2O2 system after 30 PCR cycles  using primers with a 5' tail sequence, which includes a G-quadruplex forming sequence (I) tethered to the cytosine-rich (C-rich) complementary sequence (II) via an oxyethyleneglycol bridge (III) A 45]. B. B45]. BR cycles . The detR cycles B 46]. U. U2O2 syof 10 fM  [46]. Inof 10 fM C 47]. A. A2O2 sy2 system . 3O4-Au core shell nanoparticle (2O2 system (Novel DNA detection techniques utilizing a probe DNA tethered to nanoparticles have been developed. Zhang’s group immobilized both ends of a stem-loop type probe DNA onto a Feparticle A 48]. T. T3O4-Au2 system .2O2 system is 100 pM (Chemiluminescence resonance energy transfer (CRET) of the chemiluminescence from oxidized luminol to quantum dots (QDs) was further combined for DNA detection B 49]. T. T49]. Ts 100 pM .2O2 using the pyrolytic graphite electrode coated with hemin-G-quadruplex-cetyltrimethylammonium bromide (CTAB) film [2O2 can be observed. The currents depend on the target DNA concentration, and the detection limit of the target DNA is 1 pM  film . After tAB) film . They im is 1 pM . Electro+ in body fluids may cause a potentially fatal condition known as hypokalemia; in contrast, excess of K+ may increase the risk of high blood pressure and stroke [+, Cd2+, Hg2+ and Pb2+ usually act as highly toxic environmental pollutants and have severe adverse effects on human health [+ is one of the most studied coordination metal ions in G-quadruplexes. K+ enhances the peroxidase-like activity of various G-quadruplexes by changing and stabilizing the structures [+ enables the measurement of K+ concentrations  base is usually utilized in the detection of Hg2+ (2+ and hemin (2+-T base pairs (Form II) by the addition of Hg2+ and hemin. Peroxidase-like activity of the G-quadruplex with hemin enables the selective detection of Hg2+ over other metal ions. The binding of Ag+ to DNA bases can also be exploited for the detection of Ag+ [+ to G base disrupts the G-quadruplex structure of CatG4  separation. In order to overcome these drawbacks, some protein detection techniques using hemin-G-quadruplex peroxidase have been developed to date. A DNA aptamer for thrombin, which plays a role in blood coagulation by catalyzing the conversion of fibrinogen to fibrin in blood coagulation, forms a G-quadruplex structure ,74,75. T+ [2O2 system porphine (TMPyP4), which is a cationic porphyrin derivative composed of four modified pyrrol units and four cationic functional groups [Since the discovery that the human telomere G-quadruplex inhibits telomerase activity, development of G-quadruplex-ligands that inhibit telomerase activity via induction or stabilization of the G-quadruplex has become an area of great interest B 19,20,,24,25,26l groups ,24,25,26l groups ,89,90,91l groups ,23,25. Tl groups . Actualll groups ,92,93. N3-3') with a dissociation constant (Kd) of 42 μM in the presence of 100 mM KCl. On the contrary, little or no absorbance change was observed for ssDNA (5'-T21-3') or dsDNA (5'-AGAAGAGAAAGA-3'/5'-TCTTTCTCTTCT-3') (Kd of Cu(II)APC for the G-quadruplex in the presence of excess decoy dsDNA, lambda DNA (a condition intended to mimic conditions in cell nuclei) was almost the same as that in the absence of lambda DNA APC, TTCT-3') . These rmbda DNA . Furthermbda DNA . Ni(II)Adentical . Similar to hemin, the selective binding property of Cu(II)APC and Ni(II)APC is attributable to the electrostatic repulsion between anionic groups of anionic phthalocyanines and anionic phosphate groups of dsDNA. In contrast, lambda DNA prevented TMPyP4 from binding to the G-quadruplex, which is thought to be due to nonspecific binding of TMPyP4 to dsDNA .Its highly selective binding property suggests that Cu(II)APC can inhibit telomerase activity with or without excess dsDNA. Hence, the telomerase inhibitory effect was studied using a modified telomere repeat amplification protocol (TRAP), which comprises two reactions: a telomerase reaction in the presence of 63 mM KCl at 30 °C for 30 min, and a polymerase chain reaction (PCR) using a 50-fold diluted telomerase product as template  25,26,9,95. The results from the TRAP assay were in accordance with the results of the binding studies; Cu(II)APC inhibited telomerase activity with an IC50 value of 1.2 μM in both the absence and presence of lambda DNA . Furtherin vitro and in vivo applications related to G-quadruplexes because ssDNA and/or dsDNA coexist and could prevent the interaction between ligand and G-quadruplex in most applications. On the other hand, anionic porphyrins, hemin and NMM, and anionic phthalocyanines bind to G-quadruplexes with high selectivity [in vivo application of hemin-G-quadruplex peroxidase. For example, the reactive oxygen species (ROS) scavenging system using peroxidases is crucial for cell resistance because ROS plays a role in various severe diseases including cancer [Most G-quadruplex ligands are π-planar compounds with cationic groups for forming π-π stacking interactions with G-quartets and electrostatic interactions with anionic phosphate groups of G-quadruplexes ,24,25,26ectivity ,92,93. Sectivity ,97,98. Sectivity ,102,103.g cancer ,105. Thuin vivo applications of the anionic ligand, the specificity of anionic phthalocyanines for the human telomere G-quadruplex over ssDNA and dsDNA is also essential for efficient telomerase inhibition in cell nuclei. Furthermore, recent bioinformatic studies have shown that many putative G-quadruplex-forming sequences in the genome are enriched in the promoter regions of oncogenes including C-MYC, C-KIT, H-RAS and K-RAS [For nd K-RAS ,106,107.nd K-RAS ,22,23,24Finally, phthalocyanines also represent promising molecules in the area of organic electronics because of their physical properties. In particular, dye-sensitized solar cells using phthalocyanines have been attracting increasing attention ,109. How


Wheat streak mosaic virus  is an economically important virus infecting wheat in the Great Plains region of the USA. Previously, we reported that the P1 protein of WSMV acts as a viral suppressor of RNA silencing. In this study, we delineated the minimal region of WSMV P1 and examined its mechanisms in suppression of RNA silencing. We found that the 25 N-terminal amino acids are dispensable, while deletion of a single amino acid at the C-terminal region completely abolished the RNA silencing suppression activity of P1. Electrophoretic mobility shift assays with in vitro expressed P1 revealed that the P1 protein formed complexes with green fluorescent protein-derived 180-nt dsRNA and 21 and 24-nt ds-siRNAs, and WSMV coat protein-specific 600-nt dsRNA. These data suggest that the P1 protein of WSMV binds to dsRNAs in a size- and sequence-independent manner. Additionally, in vitro dicing assay with human Dicer revealed that the P1 protein efficiently protects dsRNAs from processing by Dicer into siRNAs, by forming complexes with dsRNA. Sequence comparison of P1-like proteins from select potyvirid species revealed that WSMV P1 harbors a glycine-tryptophan (GW) motif at the C-terminal region. Disruption of GW motif in WSMV P1 through W303A mutation resulted in loss of silencing suppression function and pathogenicity enhancement, and abolished WSMV viability. These data suggest that the mechanisms of suppression of RNA silencing of P1 proteins of potyvirid species appear to be broadly conserved in the family Potyviridae. RNA silencing or post-transcriptional gene silencing (PTGS) is one of the most conserved eukaryotic regulatory pathways of many biological processes such as development, genome stability, and responses to abiotic and antiviral defenses ,2,3. RNATurnip crinkle virus or NSs protein of Tomato spotted wilt virus bind to long dsRNAs to protect them from the activity of DCL [Rice yellow mottle virus directly block DCL4 to hamper the production of siRNAs [Cymbidium ring spot virus or 2b of Cucumber mosaic virus indirectly impede the formation of RISC by sequestering PTGS-generated short dsRNAs (ds-siRNAs), which also depletes mobile inducers of systemic silencing [Beet western yellows virus and the P25 protein of Potato virus X (PVX) were mediated ubiquitination of AGO1 for proteolytic degradation [Sweet potato mild mottle virus (SPMMV) interacts with AGO1 of assembled RISC to block target hydrolysis [Potato virus A (PVA), interact with SGS3 protein cofactor of RDR6 [Rice yellow stunt virus directly blocks RDR6 to prevent its ability to generate long dsRNAs for secondary siRNAs production [VSRs of different groups of viruses are highly divergent in sequence; however, their RNA silencing pathway targets are functionally conserved. A few silencing suppressors such as coat protein (CP) of y of DCL ,13. Somef siRNAs . VSRs liilencing ,16. Howeradation ,18. Convdrolysis . In orde of RDR6 , which ioduction . The ubiPotyviridae consists of the largest number of plant RNA viruses that are characterized by a mono- or bi-partite positive-sense single-stranded RNA genome [Potyvirus (eg. Tobacco etch virus) [Rymovirus (eg. Agropyron mosaic virus) [Potyvirus, PVA [Ipomovirus employ P1 as the suppressor of RNA silencing. Ipomoviral species assign their P1 as a VSR, irrespective of the presence or absence of HC-Pro in their genomes. Cassava brown streak virus and Cucurbit vein yellowing virus (CVYV), which lack HC-Pro, assign their P1 and P1b as VSRs, respectively [Tritimovirus [Poacevirus [The family A genome . The genA genome . Among th virus) ,25 and Rc virus) . Interesrus, PVA . In contectively ,28. Howeectively . Furtherimovirus  and Poacacevirus  were alsacevirus ,30. Furtacevirus ,32.Wheat streak mosaic virus (WSMV), the type species of the genus Tritimovirus of the family Potyviridae, is the most economically important virus infecting wheat in the Great Plains region of the USA [Aceria tosichella Keifer) in a persistent manner [ the USA . WSMV ist manner ,35. The t manner . The gent manner .In this study, the counter defense mechanisms employed by WSMV P1 to suppress plant-induced RNA silencing were examined. We found that WSMV P1 interacts with dsRNAs in a size and sequence independent manner and protects long dsRNAs from the hydrolytic activity of recombinant Dicer. Additionally, we found that a GW dipeptide, a putative AGO-binding linear peptide motif, in WSMV P1 plays an important role in suppression of RNA silencing.Agrobacterium tumefaciens strain EHA105. pWSMV-GFP-P1-W303A was created by introducing W303A point mutation in pSP6-WSMV-GFP-6K1/CI [ClaI and AscI restriction sites. For protein expression in the bacterial system, P1 or P1-W303A cistrons were cloned between the NcoI and BamHI restriction sites of pMAL-c5X and then chemically transformed into a NEB express strain of Escherichia coli . Herculase II Fusion DNA polymerase  was used for all PCR reactions. The presence of introduced deletions or mutations in all constructs was verified by sequencing on Applied Biosystems 3730xl DNA Analyzer at the University of Florida ICBR Core DNA sequencing facility.WSMV infectious cDNA clone (pSP6-WSMV) generated from isolate Sidney 81  was usedP-6K1/CI . pPVX-WSP-6K1/CI  between 2 and 100 µM Acetosyringone to the optical density of 1.0 at 600 nm. This suspension was incubated at room temperature for ~3 h and mixed with 1.0 OD600 agrobacterial suspension of pPZP212-35S:GFP (35S:ssGFP) [N. benthamiana at the 6–8 leaf stage. Plants were maintained in a growth chamber at 24–26 °C with a 14 h photoperiod. Agroinfiltrated leaves were observed for green fluorescence under long range UV light at 3 and 6 days post-agroinfiltration (dpa) and photographed with a Nikon D70 DSLR camera using an orange filter.Agrobacteria harboring P1 cistron or its mutants were grown overnight and resuspended in 10 mM MES, pH 5.45 containing 10 mM MgClS:ssGFP)  and infiN. benthamiana leaf patches using the TriPure isolation reagent . Two µg of total RNA per sample was electrophoresed in a 1% agarose-formaldehyde gel or 15% urea-polyacrylamide gel, followed by blotting onto a positively charged nylon membrane for detecting GFP mRNA or siRNAs, respectively, using GFP-specific digoxigenin (DIG)-labelled antisense riboprobes as described in Tatineni et al. [Total RNA was extracted from 400 mg of agroinfiltrated i et al. .Multiple sequence alignment of select P1 sequences of potyvirid species was performed using deep substitution scoring matrix, BLOSUM62 . GenBankN. benthamiana leaves at the six-leaf stage and incubated in a growth chamber at 25 °C with a 16 h photoperiod. In vitro transcripts were synthesized from linearized pWSMV-GFP (pSP6-WSMV-GFP-6K1/CI) , pWSMV-GN. benthamiana plants inoculated with PVX, PVX-WSMV-P1, or PVX-WSMV-P1-W303A at 10 and 15 dpi using TriPure isolation reagent, followed by reverse transcription with random primers and PCR with primers Tr-206 and Tr-207 [Total nucleic acids were isolated from wheat leaves inoculated with WSMV-GFP, WSMV-GFP-P1-W303A, or WSMV-GFP-ΔP1 by the glycine-phenol method as described in McNeil et al. . One micd Tr-207 . N. benthamiana actin as an internal reference gene for normalization. Non-specific expression from buffer-inoculated plants was subtracted from relative expression values from PVX-infected samples.Real-time RT-PCR (RT-qPCR) was performed on total RNA extracted from PVX-infected upper symptomatic leaves collected at 10 and 15 dpi. Relative expression of PVX was determined on two biological replicates which were tested in duplicates by RT-qPCR with primers G-35 and G-36  using SsTotal proteins were extracted from inoculated and upper non-inoculated wheat leaves by homogenizing 0.4 g leaf pieces in 2 mL of TPE buffer  containing 1 Complete Mini Protease Inhibitor Cocktail tablet  per 10 mL of TPE buffer as described in Tatineni et al. . The tisE. coli through pMAL-c5X and purified by affinity chromatography with amylose resin . Affinity purified proteins were quantified by the Bradford assay using Quick Start Bradford kit (Bio-Rad). PTGS-like ds-siRNAs were prepared from chemically synthesized 21- or 24-nt 5’P-ssRNA with 2-nt 3’ overhangs  from GFP sequence. These ds-siRNAs were diluted to 1 µM concentration in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA and 50 mM NaCl (TEN buffer). Long dsRNA was prepared by annealing in vitro transcribed positive- and negative-sense ssRNAs derived from GFP (180-nt) or WSMV CP (600-nt) sequences in TEN-T buffer (TEN buffer containing 0.02% Tween 20). Maltose binding protein (MBP)-tagged proteins of WSMV P1 or P1-W303A were expressed in Synthetic ds-siRNAs or ss/dsRNA (100 ng) was incubated with MBP-tagged protein (0.3 nmol) in 30 µL of TEN buffer for 30 min at room temperature. Following incubation, the reaction mixture was analyzed through 4–12% non-denaturing polyacrylamide gel electrophoresis at 4 °C and visualized with SYBR Green stain  as described in Samuel et al. . ImageJ 2 and 1 mM ATP. The Dicer reaction mixture was incubated overnight at 37 °C. Following incubation, the reaction mix was quenched with 10 mM EDTA, resolved on a 4–12% non-denaturing polyacrylamide gel at 4 °C, and visualized by staining with SYBR Green. In the dicing reaction, MBP was used as a negative control for MBP tags and for non-dsRNA binding protein.In vitro dicing reaction was performed by incubating 0.3 nmol of affinity purified MBP-tagged WSMV P1 or MBP and 1.0 µg of 600-nt dsRNA with 0.5 U of recombinant human Dicer enzyme  in a 30 µL reaction mixture of 250 mM NaCl, 30 mM HEPES (pH 8.0), 0.05 mM EDTA, 2.5 mM MgClAgrobacterium-harboring pPZP212 with P1 that contained nine and ten progressive deletions from the N- and C-terminal regions, respectively, were co-infiltrated with 35S:ssGFP reporter into the leaves of N. benthamiana at the 6–8 leaf stage (To examine the WSMV P1 sequence (352 amino acids) harboring functional domains for VSR function, progressive deletions were introduced from the N- and C-terminal regions of P1, followed by agroinfiltration-based GFP reporter assay. af stage A.N. benthamiana leaves infiltrated with N-terminal deletion of 25 amino acids or less. In contrast, none of the C-terminal deletion mutants facilitated prolonged expression of GFP  dipeptide motif toward the C-terminal region, along with few adjacent amino acids, that showed high sequence homology with few other P1-like proteins of potyvirid species A. AdditiN. benthamiana leaves at the 6–8 leaf stage revealed no expression of GFP at 3 and 6 dpa , was generated (nd 6 dpa B. Northend 6 dpa C. These E. coli (The above experiments indicated that the GW motif in WSMV P1 is required for VSR function. To determine whether the loss of silencing suppression function of P1-W303A mutant was due to loss of dsRNA binding activity, we performed in vitro dsRNA binding assays with MBP::P1-W303A. MBP-tagged P1-W303A protein was expressed in E. coli A, lane 3N. benthamiana [N. benthamiana plants inoculated with in vitro transcripts of PVX-P1-W303A showed inward curling of upper leaves with mild mosaic symptoms similar to those elicited by PVX-WSMV-P1 and PVX-WT at 8 dpi. At 15 dpi, plants inoculated with PVX-WSMV-P1 elicited severe stunting and apical necrosis (N. benthamiana plants was confirmed by RT-PCR on total RNA extracted from symptomatic leaves at 15 dpi with primers flanking the site of insertion, and found that the inserted sequences were stably maintained in PVX (Viral suppressors of RNA silencing are known to be inducers of pathogenicity in host plants ,52,53,54thamiana . Here, tthamiana . N. bentnecrosis A. In connecrosis A. Stabild in PVX B. N. benthamiana plants at 10 and 15 dpi by RT-qPCR. Upon normalization with expression of Nb-Actin as a reference gene, accumulation of PVX-WSMV-P1-W303A was 25- and 60-fold less at 10 and 15 dpi, respectively, compared with PVX-WSMV-P1. Moreover, relative expression of PVX-WSMV-P1-W303A was similar to that of PVX-WT (We next examined the accumulation of PVX from PVX-, PVX-WSMV-P1-, or PVX-WSMV-P1-W303A-infected f PVX-WT C. These To test whether disruption of GW motif in P1 cistron affects the viability of WSMV in terms of cell-to-cell or long-distance movement, W303A site-directed mutation was introduced in P1 cistron in WSMV-GFP to generate WSMV-GFP-P1-W303A A. CappedExamining wheat leaves under the fluorescent microscope revealed formation of local foci at 10 dpi only in wheat seedlings inoculated with WSMV-GFP but not from WSMV lacking the P1 or P1 with W303A mutation B. At 21 Due to their obligate parasitic nature, viruses face several challenges in not only utilizing the host cellular resources but also overwhelming host biology to establish systemic infection. In the evolutionary tug-of-war between host and virus, viruses acquired suppressors of RNA silencing to overcome the host-induced defensive RNA silencing ,24. In tIn order to examine the mechanisms of WSMV P1 suppression of RNA silencing, the P1 protein was expressed in vitro as MBP-fusion protein to enhance solubility . ComputaBinding of WSMV P1 to dsRNAs in a size-independent manner is considered as its role in silencing suppression at two different stages: i. Protection of dsRNAs from dicing, which would dramatically reduce siRNA generation, and ii. Sequestration of ds-siRNAs from the host defense system would potentially create an impediment for RISC formation . WSMV P1Sequence analysis of the C-terminal region of WSMV P1 and other potyvirid P1-like sequences revealed high conservation, suggesting functional conservation among P1-like sequences with suppression of RNA silencing function. Loss of VSR function upon deletion of a single amino acid at the C-terminus emphasized the presence of a highly conserved C-terminal domain in WSMV P1 that could function in suppression of RNA silencing. Sequence analyses showed the presence of a conserved GW dipeptide at the C-terminal region. The GW182 family of proteins is known to interact with AGO-PIWI of miRISC to aid in target hydrolysis . The GW N. benthamiana leaves was due to loss of ability to suppress RNA silencing. Chimeric expression of a VSR has been shown to increase pathogenicity of a heterologous virus through its pathogenicity enhancement property [N. benthamiana [N. benthamiana plants by PVX-WSMV-P1-W303A elicited symptoms similar to wild-type PVX. Further evidence demonstrating the significance of GW motif of P1 in the virus life-cycle was the inability of mutant WSMV-GFP-P1-W303A to form viral foci or establish systemic infection in wheat. Suppression of host defensive RNA silencing is imperative for systemic infection of plants, which is possible only in the presence of a bona fide VSR. However, we cannot exclude mutation of W303A might have affected the cleavage of P1. The mutant P1-W303A protein did not lose its ability to bind to dsRNAs but lost the VSR function in a GFP reporter assay. This could be because interaction of P1 with other host-encoded RNA silencing proteins is indispensable for suppression of RNA silencing irrespective of P1’s ability to bind dsRNA for protection from Dicer and siRNA sequestration.Mutagenesis of W303A to disrupt the GW motif of WSMV P1 abolished silencing suppression function, pathogenicity enhancement of a heterologous virus, and replication and movement of WSMV. Loss of GFP expression in the presence of P1-W303A plus a GFP reporter construct in agroinfiltrated property ,44,65, wthamiana . HoweverViral suppressors of RNA silencing play a central role in combating the host defense systems. Studying suppressors of RNA silencing and unveiling their molecular mechanisms are important not only to understand their diversity, regulation, and evolution, but also to develop and fine-tune approaches for disease management. Conclusively, WSMV P1 suppresses host RNA silencing at multiple levels by protecting dsRNA from dicing, sequestering ds-siRNAs, and potentially interacting with Argonaute through GW motif to interfere with RISC formation and target hydrolysis.


However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA PolyI:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using PolyI:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited PolyI:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of PolyI:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection  The annual influenza vaccines incorporate variants of influenza A(H1N1), A(H3N2), B/Yamagata and B/Victoria, which WHO predicts will dominate the following season. However, the match between predicted and circulating strains can vary, leading to variable efficacy of the vaccine. In addition, influenza viruses pose a constant threat of human pandemics due to the risk of transmission of new variants from animals for which we currently lack appropriate vaccines (According to new estimates by the United States Centers for Disease Control and Prevention (US-CDC), the World Health Organization and global health partners , AH3N, B/YamagNN1, AH3Nvaccines . Due to Influenza strains infect respiratory epithelial cells, which may lead to cytopathic viral effects . Attachm−/− mice, which survived longer than wild-type mice following lethal influenza infection despite sustained high viral load in the lungs . Cells were then treated or not with the following molecules: synthetic, endotoxin-free, completely phosphorothioate-modified oligonucleotides named ssON , or an oligonucleotide with the naturally occurring phosphodiester backbone (ssON PO), high molecular weight PolyI:C  or the combination of both, referred as ssON/PolyI:C. The sequence of 35 bases long ssON is: 5′-G*A*A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G*G*T*G-3′, the sequence of the 30-mer is: 5′-A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G-3′, the 25-mer: 5′-T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G-3′, the 20-mer: 5′-T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*A*T*T*G*G-3′ and the 15-mer: 5′- G*G*T*T*T*T*G*A*A*G*T*T*G*T*T-3′, wherein the phosphorothioate modifications are indicated by *.Stock of pandemic H1N1 virus strain A/Cal/07/2009 was kindly provided by Bertin-Pharma, France. MoDC were mock-exposed or exposed to IAV or heat inactivated IAV  at a multiplicity of infection (MOI) of 0.02, 0.2, 1, or 2 for 4 h at 37°C 5%CO5 cells/mL and after 3 days of differentiation, 50% of the medium was replaced and new cytokines added. Staining for flow cytometry was done before and at indicated time points post viral infection. MoDC were incubated with LIVE/DEAD® Fixable near-IR Dead Cell Stain Kit (Life Technologies) followed by staining with CD14-PE-Cy7 (MφP9), CD1a-BV510 (HI149), CD80-PE (L307.4), and CD86-APC (2331 FUN-1) from BD Biosciences. The mouse anti-IAV NP mAb  was detected with a secondary Ab coupled to Alexa Fluor 488 fluorochrome with the Zenon® Kit (Invitrogen). Acquisition was done on a Fortessa flow cytometer (BD Biosciences) and analysis was performed with FlowJo software .Monocytes were isolated from buffy coats using Ficoll centrifugation  after negative selection using the RosetteSep Monocyte Enrichment Kit (StemCell Technologies). Monocyte-derived DC (MoDC) were obtained after 6 days of differentiation in complete RPMI medium  complemented with GM-CSF  and rIL-4 . Cells were seeded at a density of 5 × 10MoDC were exposed to PolyI:C-Alexa488, with or without addition of ssON, on ice in complete 10% RPMI media (or serum free media for PO ON uptake studies), and then transferred to 37°C for 45 min, as previously described , supernatants or MoDC were harvested and kept at −80°C until RNA purification was performed. RNeasy® Plus Mini kit (QIAGEN) was used to extract cellular RNA while the QIAamp® Viral RNA mini kit (QIAGEN) was used for viral RNA purification from supernatants, following manufacturer' spin protocol. Viral load was determined by qRT-PCR  using pandemic H1N1 virus strain A/Cal/07/2009 as internal standard. The following primer sequences 5′-3′ specific for IAV Cal07/09 dist function in R. In the DESeq2 pipeline, the design formulae was set to “~ donor + control” i.e., to analyse the effect of condition while controlling for the donor specific differences. For each comparison, filtering was performed prior to analysis by removing all genes that had zero counts across samples. Shrinkage of log2 fold changes was performed according to the DESeq2 pipeline. Genes with adjusted p-value below 0.05 after Benjamini-Hochberg adjustment, were labeled as differently expressed.Purified RNA molecules were submitted to the National Genomics Infrastructure Sweden Stockholm (NGI) for sequencing. The RNA sequencing was performed with the TruSeq RiboZero kit from Illumina, 25 M reads per sample and 2 × 125 bp. Fastq files were obtained from NGI and the read quality were assessed using FastQC (Version 0.11.5) Trim Galore (Version 0.3.6) was used for adapter removal and quality trimming with a quality threshold of 20 on the Phred scale. Obtained high-quality reads were mapped to Homo sapiens UCSC hg38 (GRCh38.77) reference genome using STAR aligner (version 2.5) with default values and the parameter out Reads Unmapped set to FastX in order to extract the unmapped reads. After STAR alignment, the count data for the aligned reads were generated with HTSeq-count (version 0.6.1). The-m parameter was set to union. Next, the count data sets were imported into the statistical software R (version 3.5.1). The R/Bioconductor package DESeq2 (version 1.22.1) was used to conduct differential expression (DE) analysis according to the workflow outlined in the vignette. Hierarchical clustering was performed on a sample-to-sample distance matrix that was calculated on regularized logarithm transformed counts with the p-value below 0.05.Ingenuity Pathway Analysis (IPA) software  (Ingenuity Systems) was used to identify canonical signaling pathways. To calculate significance of enrichment , the reference molecule set was Ingenuity Knowledge Base (Genes only). Results tables were exported from R after DESeq2 and prior to IPA analysis filtered by adjusted Supernatants were collected from cells at given time points post infection and secretion of cytokines/IFN was measured by standard ELISA according to manufacturer's instructions , IL-12(p70), IL-29; Mabtech, IFN-α; PBL). Protein amount was monitored by 3,3′,5,5′-Tetramethylbenzidine (TMB) absorbance at 450 nm.4 influenza virus or sterile PBS 100 μl.H1N1, A/England/195/2009 was isolated by Public Health England (UK) in SIAT-MDCK cells . Prior tg for 5 min. Supernatants were removed and the cell pellet treated with red blood cell lysis buffer  before centrifugation at 200 × g for 5 min. The remaining cells were resuspended in RPMI 1,640 medium with 10% fetal calf serum, and viable cell numbers determined by trypan blue exclusion. Viral load in vivo was assessed by Trizol extraction of RNA from frozen lung tissue disrupted in a TissueLyzer . RNA was converted into cDNA and quantitative RT-PCR was carried out using bulk viral RNA, for the influenza M gene and mRNA using 0.1 μM forward primer (5′-AAGACAAGACCAATYCTGTCACCTCT-3′), 0.1 μM reverse primer (5′-TCTACGYTGCAGTCCYCGCT-3′), and 0.2 μM probe (5′-FAM-TYACGCTCACCGTGCCCAGTG-TAMRA-3′) on a Stratagene Mx3005p . M-specific RNA copy number was determined using an influenza M gene standard plasmid  and tissues collected as previously described . Blood w plasmid .*P < 0.05, **P < 0.01, ***P < 0.001.All data were analyzed using Prism software . Unless stated otherwise, the non-parametric Kruskal-Wallis unpaired test followed by Dunn's post-test was used to compare against the control (represented with a line), while different treatment groups were compared using non-parametric Mann-Whitney unpaired test (represented with a bar). ns: not significant, in vitro IAV infection, at different time points, as we wanted to avoid release of dsRNA due to cell death, which may affect innate immune responses , but increased to 16.7% ± 3.7 with 0.2 MOI. MoDC incubated with heat-inactivated (HI) IAV did not show any detectable NP+ cells or any cell death 24 h or 48 h post-exposure, as expected  MoDCs were able to up-regulate the surface expression of both CD80 and CD86.Several influenza strains, including pandemic H1N1, have previously been shown to impair the maturation of MoDCs . Howeverependent . Notablye donors . For come donors . The expAV+ MoDC . These dper se in the neighboring non-infected (NP-) MoDCs  MoDCs , which w in “Activation of IRF by Cytosolic Pattern Recognition Receptors” and “Dendritic Cell Maturation,” which are expected after triggering of innate immune responses . FurtherTo get unbiased analyses of transcriptional changes occurring upon TLR3 activation during IAV infection of MoDC, MA plots were generated displaying some of the significantly up- and down-regulated genes in the presence of either PolyI:C or ssON . The IAVThe IAV cultures treated with ssON revealed an up-regulation of ISGs , includiTo get insight to the differential gene expression occurring in the PolyI:C treated IAV cultures, we made MA-plots and volcano plots comparing IAV infected cultures vs. IAV infected cultures treated with PolyI:C IAV 0.02 . It is cAltogether, these data showed induction of IFN-signaling pathways in the IAV cultures regardless of whether cells were treated with PolyI:C or ssON. These data also demonstrated that IAV-infected cultures treated with PolyI:C resulted in further up-regulation of many genes, while ssON treatment did not have any major effect on the baseline gene expression induced by the IAV infection.per se, but significantly inhibited the augmented effect by PolyI:C (TLR3 activation results in expression of both pro-inflammatory cytokines and IFNs . To furt PolyI:C .per se provoke significant IL-12/23(p40) or IL-12(p70) secretions . Inper se.Altogether, these data demonstrate PolyI:C-mediated induction of several pro-inflammatory cytokines and IFNs in the IAV cultures, confirming the transcriptomic analyses. Likewise, the data shows that ssON was able to effectively inhibit PolyI:C-mediated pro-inflammatory cytokine and IFN production but preserved cytokine and IFN responses induced by the IAV infection per se interfere with the qRT-PCR and indicating inhibition at the entry level. To get further insight into ssONs capacity to inhibit IAV infection, we investigated whether the stabilization conferred by the chemical PS modification was required, which was found to be the case as the natural PO backbone did not show any antiviral effect (Extracellular ssON was recently shown to inhibit uptake of ligands taken up by CME and Caveolin-Dependent Endocytosis (CDE) , which al effect . This fil effect . We prevl effect . Therefol effect  and refll effect . MoDCs wl effect . The flul effect . Accordil effect . Altogetin vivo, mice were infected with IAV by intranasal inoculation, as previously described  showed reduced serum cholesterol in both the LDL and HDL fractions . Thus, we performed the in vitro experiments with MoDC, which are susceptible to influenza A without the addition of trypsin.There are limitations of using MoDC ividuals . The reacultures . The siacultures , 42. Subcultures . In vivo surface , 42, whiin vitro experiments . We further provide evidence of reduced viral replication in vivo in mice treated with ssON, supporting further studies investigating ssONs capacity to limit viral infections.Altogether, we here show that treatment with a 35 bases long PS-ssON reduced IAV infection of human primary MoDC www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7803.RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI  at Imperial College London.CP, AD, JB, SP, CA and DB performed the experimental studies and analyzed data. PJ, A-LS, and JA provided the technical input and data analysis. VC and RL provided and grew the influenza strains. JL, JT, and A-LS designed the studies. CP, AD, JB, and A-LS wrote the paper.PJ, AD, and A-LS are authors of patent applications related to the work and are shareholders of TIRmed Pharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.