Comparative sub-cellular proteome analyses reveals metabolic differentiation and production of effector-like molecules in the dieback phytopathogen Phytophthora cinnamomi.

Phytopathogenic oomycetes pose a significant threat to global biodiversity and food security. The proteomes of these oomycetes likely contain important factors that contribute to their pathogenic success, making their discovery crucial for elucidating pathogenicity. Phytophthora cinnamomi is a root pathogen that causes dieback in a wide variety of crops and native vegetation world-wide. Virulence proteins produced by P. cinnamomi are not well defined and a large-scale approach to understand the biochemistry of this pathogen has not been documented. Soluble mycelial, zoospore and secreted proteomes were obtained and label-free quantitative proteomics was used to compare the composition of the three sub-proteomes. A total of 4635 proteins were identified, validating 17.7% of the predicted gene set. The mycelia were abundant in transporters for nutrient acquisition, metabolism and cellular proliferation. The zoospores had less metabolic related ontologies but were abundant in energy generating, motility and signalling associated proteins. Virulence-associated proteins were identified in the secretome such as candidate effector and effector-like proteins, which interfere with the host immune system. These include hydrolases, cell wall degrading enzymes, putative necrosis-inducing proteins and elicitins. The secretome elicited a hypersensitive response on the roots of a model host and thus suggests evidence of effector activity

Myristoylated protein kinase C beta II peptide inhibitor exerts dose-dependent inhibition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced leukocyte superoxide release

Protein kinase C (PKC) phosphorylation of leukocyte NADPH oxidase is essential to generatesuperoxide (SO) release. Inhibition of leukocyte SO release attenuates inflammation mediated vascular injury. However, the role of PKC isoforms mediating this response has not been fully elucidated. We hypothesize that PKC beta II (βII) isoform positively regulates leukocyte NADPH oxidase, and that a cell-permeable (myr)-PKC βII peptide inhibitor (N-myr-SLNPEWNET) would dose-dependently attenuate fMLP induced leukocyte SO release. fMLP is a leukocyte chemoattractant cell membrane receptor agonist. We isolated leukocytes by peritoneal lavage from male Sprague-Dawley rats using standard methods. fMLP (1 M)-induced leukocyte SO release was measured for 120 sec spectrophotometrically by reduction of ferricytochrome c in the presence/absence of myr-PKC βII peptide inhibitor (0.2 to 20 M) in 5 x 106 leukocytes. After each assay, cell viability was determined by 0.3% trypan blue exclusion. fMLP-induced leukocyte SO release increased peak absorbance to 0.18±0.03 in controls (n=20). This response was dose-dependently inhibited by myr-PKC βII peptide inhibitor at 0.17±0.05 (0.2 M; n=9), 0.14±0.05 (0.5 M; n=11), 0.1±0.05 (1 M; n=9), 0.05±0.03 (5 M; n=9), 0.04±0.03 (10 M; n=8) and 0.05±0.03 (20 µM; n=7) and was significantly attenuated in the 5 to 20 M range compared to controls (p\u3c0.05). Moreover, cell viability was \u3e 94±1% in all study groups. These results suggest that myr-PKC βII peptide inhibitor dose-dependently inhibits fMLP-induced leukocyte SO release in the 0.2 to 5 M dose-range and these effects are attributed to inhibition of PKC βII isoform

Proteomic analysis revealed that the oomyceticide phosphite exhibits multi-modal action in an oomycete pathosystem

Phytopathogenic oomycetes constitute some of the most devastating plant pathogens and cause significant crop and horticultural yield and economic losses. The phytopathogen Phytophthora cinnamomi causes dieback disease in native vegetation and several crops. The most commonly used chemical to control P. cinnamomi is the oomyceticide phosphite. Despite its widespread use, the mode of action of phosphite is not well understood and it is unclear whether it targets the pathogen, the host, or both. Resistance to phosphite is emerging in P. cinnamomi isolates and other oomycete phytopathogens. The mode of action of phosphite on phosphite-sensitive and resistant isolates of the pathogen and through a model host was investigated using label-free quantitative proteomics. In vitro treatment of sensitive P. cinnamomi isolates with phosphite hinders growth by interfering with metabolism, signalling and gene expression; traits that are not observed in the resistant isolate. When the model host Lupinus angustifolius was treated with phosphite, proteins associated with photosynthesis, carbon fixation and lipid metabolism in the host were enriched. Increased production of defence-related proteins was also observed in the plant. We hypothesise the multi-modal action of phosphite and present two models constructed using comparative proteomics that demonstrate mechanisms of pathogen and host responses to phosphite. Significance: Phytophthora cinnamomi is a significant phytopathogenic oomycete that causes root rot (dieback) in a number of horticultural crops and a vast range of native vegetation. Historically, areas infected with phosphite have been treated with the oomyceticide phosphite despite its unknown mode of action. Additionally, overuse of phosphite has driven the emergence of phosphite-resistant isolates of the pathogen. We conducted a comparative proteomic study of a sensitive and resistant isolate of P. cinnamomi in response to treatment with phosphite, and the response of a model host, Lupinus angustifolius, to phosphite and its implications on infection. The present study has allowed for a deeper understanding of the bimodal action of phosphite, suggested potential biochemical factors contributing to chemical resistance in P. cinnamomi, and unveiled possible drivers of phosphite-induced host plant immunity to the pathogen

Gene validation and remodelling using proteogenomics of Phytophthora cinnamomi, the causal agent of Dieback

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen

Comparison of the effects of myristrolated and transactivating peptide (TAT) conjugated mitochondrial fission peptide inhibitor (P110) in myocardial ischemia/reperfusion (I/R) injury

During myocardial I/R, cardiac mitochondrial fission-fusiondynamics are altered towards mitochondrial fission which during I/R is associated with a shortening of mitochondria, decreased ATP production, and increased reactive oxygen species, factors known to promote cardiomyocyte death. Therefore, inhibiting mitochondrial fission may be a strategy to salvage damaged cardiac myocytes during I/R and limit infarct size. Given that cell membrane permeability of peptides is crucial for efficacy, we compared theeffects of a novel mitochondrial fission peptide inhibitor, P110 (DLLPRGT) that was conjugated to either a TAT carrier peptide YGRKKRRQRRR-GG-DLLPRGT (MW=2427 g/mol) or myristic acid myr-DLLPRGT (MW=981 g/mol) to determine which of these peptide formulations would be more potent to attenuate cardiac contractile dysfunction and infarct size in isolated perfused rat hearts subjected to I (30 min)/R (90 min). We found that myr-P110 (1 M; n=6) given for 10 min before ischemia and for 20 min post-reperfusion, significantly restored the maximal rate of left ventricular developed pressure (dP/dtmax) to 49 ± 7% compared to TAT-conjugated P110 (1 M; n=6) and untreated controls (n=9), which only recovered to 26 ± 5% and 28 ± 4% of baseline values at 90 min post-reperfusion, respectively (p\u3c0.05). Myr-P110 also significantly reduced infarct size to 28± 2% compared to controls which had an infarct size of 46±3% (p\u3c0.01). Whereas, TAT-conjugated P110 had an infarct size of 35 ± 3% and was not statistically different from controls using ANOVA analysis. Preliminary results suggest myr-P110 would be a more effective formulation to salvage heart tissue after myocardial infarction

The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Protein Kinase Beta II (PKC-βІI) inhibitor exerts cardioprotective effects in Myocardial Ischemia/Reperfusion Injury (MI/R)

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to the post-reperfused cardiac injury and contractile dysfunction. Activation of Protein Kinase C beta II (PKC βII) has been associated with increased ROS release from myocardial I/R tissue, decreased endothelial-derived nitric oxide, and increased infarct size. We tested the hypothesis that using a cell permeable PKC βII peptide inhibitor (PKC βII-) (N-myr-SLNPEWNET, MW=1300 g/mol, 10μM or 20μM) will attenuate infarct size and improve post-reperfused cardiac function compared to untreated controls in isolated perfused rat hearts subjected to I(30min)/R(90 min). PKC βII- treated hearts (both 10 and 20 μM) significantly improved postreperfused cardiac function (e.g. left ventricular developed pressure [LVDP], and dP/dt max) compared to controls (all

Protein kinase C beta II (PKC ßII) peptide inhibitor exerts cardioprotective effects in myocardial ischemia/reperfusion injury

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to the post-reperfused cardiac injury and contractile dysfunction. Activation of Protein Kinase C beta II (PKC βII) has been associated with increased ROS release from myocardial I/R tissue, decreased endothelial-derived nitric oxide, and increased infarct size. We tested the hypothesis that using a cell permeable PKC βII peptide inhibitor (PKC βII-) (N-myr-SLNPEWNET, MW=1300 g/mol, 10µM) will attenuate infarct size and improve post-reperfused cardiac function compared to untreated controls in isolated perfused rat hearts subjected to I(30min)/R(45 or 90 min). The 90 min reperfusion group (n=9) showed significantly less recovery to the initial baselines in left ventricular developed pressure (LVDP) (38±6%) and maximal rate of LVDP (+dP/dtmax ) (28±4%), both p˂0.01. The 45 min reperfusion group (n=9) also showed significantly compromised LVDP (46±6%) and +dP/dtmax (35±4%) compared to initial baseline but to a lesser extent than the 90 min group. Conversely, PKC βII- treated hearts significantlyimproved cardiac function compared to controls (all p\u3c0.05). Similarly, 90 min reperfusion (n=7) showed a reduced recovery in LVDP (57±7%) and +dP/dtmax (48±5%) compared to 45 min reperfusion (LVDP: 70±6%; +dP/dtmax: 55±6%; n=7). Furthermore, PKC βII- treated hearts showed significant reduction in infarct size (24±3% and 29±3% for 45 and 90 min reperfusion, respectively) compared to controls (43±2% and 46±3% for 45 and 90 min reperfusion, respectively; [p˂0.01]). The results suggest that PKC βII- is effective in improving cardiac function and reducing infarct size and aids in clinical myocardial infarction/organ transplantation patient recovery

Protein Kinase C Beta II (PKC ßII) Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury

Coronary heart disease is the leading cause of death worldwide, and is primarily attributable to the detrimental effects of tissue infarct after an ischemic insult, The most effective therapeutic intervention for reducing infarct size associated with myocardial ischemia injury is timely and effective reperfusion of blood flow back to the ischemic heart tissue. However, the reperfusion of blood itself can induce additional cardiomyocyte death that can account for up to 50% of the final infarction size. Currently, there are no effective clinical pharmacologic treatments to limit myocardial ischemia/reperfusion (MI/R) injury in heart attack patients. Reperfusion injury is initiated by decreased endothelial derived nitric oxide (NO) which occurs within 5 min of reperfusion, and may in part be explained by PKC ßII mediated activation of NADPH oxidase, which occurs upon cytokine release during MI/. PKC ßII activity is increased in animal models of MI/R and known to exacerbate tissue injury. PKC ßII is known to increase NADPH oxidase activity in leukocytes, endothelial cells and cardiac myocytes via phox47 phosphorylation, and decrease endothelial NO synthase (eNOS) activity via phosphorylation of Thr 495. NADPH oxidase produces superoxide (SO) and quenches endothelial derived NO in cardiac endothelial cells. Moreover, PKC ßII phosphorylation of p66Shc at Ser 36 leads to increased mitochondrial reactive active oxygen species (ROS) production, opening of the mitochondrial permeability transition pore (MPTP), and pro-apoptotic factors leading to cell death and increased infarct size. Therefore, using a pharmacologic agent that inhibits the rapid release of PKC ßII mediated ROS, would attenuate endothelial dysfunction and downstream pro apoptotic pathways when given during reperfusion and should be an ideal candidate to attenuate MI/R injury. PKC ßII peptide inhibitor mechanism of action is to inhibit PKC ßII translocation to cellular substrates such as eNOS, NADPH oxidase, and mitochondrial p66Shc protein that increase ROS leading to opening of the MPTP which in turn leads to consequent release of proapoptotic factors into the cytosol. We\u27ve previously shown that PKC ßII peptide inhibitor restored post-reperfused cardiac function and reduced polymorphornuclear leukocyte (PMN) infiltration in isolated rat hearts subjected to MI(20min)/R(45min) reperfused with PMNs. In addition, the use of PKC ßII peptide inhibitor (10-20 µM) correlated with the inhibition of SO release from isolated leukocytes suggesting that this dose range maybe effective in attenuating ROS production. We extended our research in the current study by using a MI (30min)/R (90min) isolated perfused rat heart model. A cell permeable PKC ßII peptide inhibitor (10-20 µM) was given at the beginning of reperfusion for five minutes. Post-reperfused cardiac function and infarct size were measured and compared to untreated control MI/R hearts