Proteomic analysis of ceftazidime and meropenem-exposed Pseudomonas aeruginosa ATCC 9027.

BACKGROUND: Pseudomonas aeruginosa is well known for its intrinsic ability to resist a wide range of antibiotics, thus complicates treatment. Thus, understanding the response of the pathogen to antibiotics is important for developing new therapies. In this study, proteomic response of P. aeruginosa to the commonly used anti-pseudomonas antibiotics, ceftazidime (Caz) and meropenem (Mem) was investigated. METHODS: P. aeruginosa ATCC 9027, an antibiotic-susceptible strain, was exposed to sub-MIC values of antibiotics either Caz or Mem for 14 days to obtain E1 strains and then cultured in antibiotic-free environments for 10 days to obtain E2 strains. Proteomes of the initial and E1, E2 strains were identified and comparatively analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) in cooperation with nano LC-MS/MS. Noted up and down-regulated proteins were confirmed with quantitative reverse transcriptase PCR (qRT-PCR). RESULTS: Overall, 1039 and 1041 proteins were identified in Caz and Mem-exposed strains, respectively. Upon antibiotic exposure, there were 7-10% up-regulated (Caz: 71, Mem: 85) and down-regulated (Caz: 106, Mem: 69) proteins (1.5-fold change cut-off). For both Caz and Mem, the DEPs were primarily the ones involved in metabolic process, membrane, virulence, protein synthesis, and antibiotic resistance in which proteins involved in antibiotics resistance tended to be up-regulated while proteins involved in protein synthesis and metabolic process were down-regulated. Noted proteins included beta-lactamase AmpC which was up-regulated and OprD which was down-regulated in both the antibiotic-exposed strains. Besides, biofilm formation related proteins TssC1 and Hcp1 in Caz- exposed strains and the membrane/ periplasmic proteins Azu and PagL in Mem-exposed strains were found significantly down-regulated. qRT-PCR results confirmed the expression change of AmpC, Hcp1 and OprD proteins. CONCLUSION: Exposure of Pseudomonas aeruginosa to sub-MIC values of Caz and Mem resulted in around 10% change in its proteome. Not only proteins with confirmed roles in antibiotic resistance mechanisms changed their expression but also virulence- associated proteins. Both Caz and Mem response involved up-regulation of AmpC and down-regulation of OprD. While TssC1 and Hcp1 were responsible for Caz response, Azu and PagL were more likely involved in Mem response.

Proteomics Analysis of Early Developmental Stages of Zebrafish Embryos.

Zebrafish is a well-recognized organism for investigating vertebrate development and human diseases. However, the data on zebrafish proteome are scarce, particularly during embryogenesis. This is mostly due to the overwhelming abundance of egg yolk proteins, which tend to mask the detectable presence of less abundant proteins. We developed an efficient procedure to reduce the amount of yolk in zebrafish early embryos to improve the Liquid chromatography-tandem mass spectrometry (LC-MS)-based shotgun proteomics analysis. We demonstrated that the deyolking procedure resulted in a greater number of proteins being identified. This protocol resulted in approximately 2-fold increase in the number of proteins identified in deyolked samples at cleavage stages, and the number of identified proteins increased greatly by 3-4 times compared to non-deyolked samples in both oblong and bud stages. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a high number of functional proteins differentially accumulated in the deyolked versus non-deyolked samples. The most prominent enrichments after the deyolking procedure included processes, functions, and components related to cellular organization, cell cycle, control of replication and translation, and mitochondrial functions. This deyolking procedure improves both qualitative and quantitative proteome analyses and provides an innovative tool in molecular embryogenesis of polylecithal animals, such as fish, amphibians, reptiles, or birds.

Snapshot of proteomic changes in Aspergillus oryzae during various stages of fermentative processing of pea protein isolate.

Pea (Pisum sativum) is one of the most abundant and sustainable alternate source of protein. Although pea proteins have good quantities of most of the essential amino acids, they have a limited supply of tryptophan, methionine and cysteine. Moreover, pea proteins have poor techno-functional properties compared to proteins from animal sources, limiting their use in certain food applications. Bioprocessing techniques like solid-state fermentation (SSF) and enzymatic processing have been explored to improve the nutrient profile and functionality of pea proteins. However, there is a lack of information about proteomic changes in the food matrix during fermentation of the pea substrate. In this research, samples during SSF of pea protein isolate with Aspergillus oryzae were used for shotgun mass spectrometry (LC-MS/MS) analysis to identify the underlying functional pathways which play direct or indirect roles in enabling the colonization of the substrate leading to potential improvement of functional and nutritional value of pea protein. Results revealed the identity of A. oryzae proteins involved in different metabolic pathways that differed during various stages of SSF. Among them, methionine synthase was identified as an abundant protein, which catalyzes methionine biosynthesis. This might suggest how fermentation processes could be used to improve the presence of sulfur containing amino acids to rebalance the essential amino acid profile and improve the nutritional quality of pea proteins.

iTRAQ-based quantitative proteomics suggests mitophagy involvement after Rice black-streaked dwarf virus acquisition in insect vector small brown planthopper Laodelphax striatellus Fallén.

Plant viruses trigger numerous responses in their insect vectors. Using iTRAQ-based quantitative proteomics analysis, early responses of the insect vector, the small brown planthopper (Laodelphax striatellus Fallén, SBPH), after acquiring Rice black-streaked dwarf virus (RBSDV) at 3 days and 5 days post first access to diseased plants (padp) were revealed. A total of 582 differentially abundant proteins (DAPs) in SBPH with a fold change >1.500 or <0.667 (p-value < 0.05) were identified. The proteomic analysis in SBPH at 3 days padp revealed 106 highly abundant proteins and 193 of low abundance, while 5 days padp revealed 214 highly abundant proteins and 182 of low abundance. Among them, 51 highly abundant proteins and 42 of low abundance were shown consistently at both 3 days and 5 days padp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapping and Gene Ontology (GO) term classification suggested impairment of mitochondria in SBPH after RBSDV acquisition, and the 77 out of 582 differentially abundant SBPH proteins analyzed by the STRING program revealed the interaction network of the mitochondrial DAPs, showing an overall down-regulation of mitochondrial proteins including the electron transport chain proteins and mitochondrial ribosome proteins. The high abundance of Parkin at 5 days padp suggests that activation of mitophagy induced degradation of mitochondria occurred. Further verification of autophagy/mitophagy-related genes by reverse-transcription quantitative RT-PCR (RT-qPCR) in SBPH after RBSDV acquisition showed up-regulation of the autophagy receptors Optineurin (OPTN), Sequestosome-1 (SQSTM1, also known as p62) and Tax1-binding protein 1 (TAX1BP1) which targets ubiquitinated damaged mitochondria during mitophagy. The phosphorylation of the three autophagy receptors may be up-regulated through an increase of transcription level TRAF-associated NFκB activator (TANK)-binding kinase 1 (TBK1). As a result, an overall reduction in the abundance of mitochondrial proteins was observed and the selective autophagic degradation was up-regulated through increased transcription level of OPTN, p62/SQSTM1, TAX1BP1 and TBK1. Therefore, acquisition of RBSDV associated with up-regulated autophagy and selective mitochondrial degradation in SBPH suggest prevention of mitochondrial-mediated apoptosis and extension of the vector life span. BIOLOGICAL SIGNIFICANCE: RBSDV causes severe yield loss in rice plants. RBSDV is transmitted efficiently only through SBPH. It is important to understand how RBSDV infects SBPH in a persistent, circulative and propagative manner. However, there has been no study on the interaction between RBSDV and SBPH at the early acquisition stage using a proteomics approach. In this study, we combined iTRAQ technique and LC-MS/MS to analyze the vector proteomics at both the initial and latent infection stages after RBSDV acquisition and verified the results by RT-qPCR. Our results revealed that significantly low DAPs were involved in various pathways, including biosynthesis of secondary metabolites, ribosomes, carbon metabolism, biosynthesis of amino acids and TCA cycle. Further clustering of the DAPs revealed significant changes in SBPH mitochondria, including decreased proteins in mitochondrial ribosomes and electron transport chain complex I, II and V. On the other hand, there was a high abundance of Parkin, suggesting the occurrence of mitochondria damage and subsequent Parkin-mediated mitophagy for clearance of impaired mitochondria. Moreover, the decreased level of PMPCB in terms of gene expression and protein abundance suggested decreased PINK1 turnover, promoting Parkin/PINK1-mediated mitophagy. Further analysis on autophagy/mitophagy-related gene transcription level indicated up-regulation of OPTN, p62/SQSTM1, TAX1BP1 and TBK1, promoting selective autophagy in SBPH after RBSDV acquisition. These findings provided new insights into the effects of RBSDV on SBPH after early acquisition by selective degradation of mitochondria, especially on reprogramming of energy metabolism and decreased mitochondria biogenesis, to prevent apoptosis and prolong the life span of SBPH post virus acquisition.

Directive Effect of Chain Length in Modulating Peptide Nano-assemblies.

BACKGROUND: RADA-4 (Ac-RADARADARADARADA-NH2) is the most extensively studied and marketed self-assembling peptide, forming hydrogel, used to create defined threedimensional microenvironments for cell culture applications. OBJECTIVES: In this work, we use various biophysical techniques to investigate the length dependency of RADA aggregation and assembly. METHODS: We synthesized a series of RADA-N peptides, N ranging from 1 to 4, resulting in four peptides having 4, 8, 12, and 16 amino acids in their sequence. Through a combination of various biophysical methods including thioflavin T fluorescence assay, static right angle light scattering assay, Dynamic Light Scattering (DLS), electron microscopy, CD, and IR spectroscopy, we have examined the role of chain-length on the self-assembly of RADA peptide. RESULTS: Our observations show that the aggregation of ionic, charge-complementary RADA motifcontaining peptides is length-dependent, with N less than 3 are not forming spontaneous selfassemblies. CONCLUSION: The six biophysical experiments discussed in this paper validate the significance of chain-length on the epitaxial growth of RADA peptide self-assembly.

iTRAQ-based protein analysis provides insight into heterologous superinfection exclusion with TMV-43A against CMV in tobacco (Nicotiana benthamiana) plants.

Heterologous superinfection exclusion (HSE) is a phenomenon of an initial virus infection which prevents reinfection by a distantly related or unrelated challenger virus strain in the same host. Here, we demonstrate that a mild strain mutant of Tobacco mosaic virus (TMV-43A) can protect Nicotiana benthamiana plants against infection by a challenger Cucumber mosaic virus (CMV)-Fny strain. The isobaric tags for relative and absolute quantification (iTRAQ) technique was used to investigate proteome of N. benthamiana plant during HSE. Our results indicated that in superinfected plants, the PSI and PSII proteins in the photosynthetic pathway increased in abundance, providing sufficient energy to plants for survival. The fatty acid synthesis-related proteins acetyl-CoA carboxylase 1-like and fatty acid synthase were decreased in abundance, affecting the formation of virus replication complex, which in turn reduced CMV replication and lessen hijacking of basic building blocks of RNA transcription and protein synthesis required for normal host functions. This is the first analyses of host proteins that are correlated to HSE between two unrelated plant viruses TMV-43A and CMV in N. benthamiana plants. BIOLOGICAL SIGNIFICANCE: CMV is one of the most studied host-virus interaction models in plants. It infects both monocot and dicot crop plants, causing significant economic losses. Superinfection exclusion (also known as cross protection) is one of the methods to combat virus infection. However, there is lack of proteome information of heterologous superinfection exclusion between two taxonomically unrelated plant viruses (such as between CMV and TMV). An iTRAQ-based quantitative approach was used to study proteomics of superinfection, where TMV-43A acts as a protector of N. benthamiana plants against its challenger CMV. Results showed that TMV-43A protects host plants and prevents plant death from CMV infection. This study provided insights into host responses involving multiple host pathways: photosynthesis, plant defence, carbon metabolism, translation and protein processing, fatty acid metabolism and amino acid biosynthesis. The findings provide a reference database for other viruses and increase our knowledge in host proteins that are correlated to superinfection.

Comparative proteomics of Tobacco mosaic virus-infected Nicotiana tabacum plants identified major host proteins involved in photosystems and plant defence.

Tobacco mosaic virus (TMV) is a positive single-stranded RNA virus. Its 5' end ORF codes for the replicase proteins, namely 126 kDa and 183 kDa, respectively. These proteins interact with many host proteins to form a virus replication complex (VRC). This study aims to dissect the proteome profile of TMV-infected Nicotiana tabacum in host cellular and molecular pathways. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyse the differential global proteomic profile of TMV infected and mock infected plants. Out of 1897 total proteins, we identified 407 differentially abundant proteins and grouped them into three functional categories, namely metabolism, cellular processes and signalling processing. Our results showed that photosynthesis, carbon metabolism, plant defence, protein synthesis, and protein processing in the endoplasmic reticulum were significantly altered. Carbon metabolism and photosynthesis were present in very low abundance, whereas accumulation of reactive oxygen species and misfolded proteins lead to the accumulation of thioredoxin H-type 1. In conclusion, we identified several key host proteins that are involved in TMV infection/replication in N. tabacum plants. SIGNIFICANCE OF THE STUDY: TMV is one of the most widely studied plant virus. It is used as a tool to study host-virus interaction. There are several host proteins reported that facilitate VRC formation and replication of TMV. However, there is limited knowledge in the expression regulation of these host proteins upon TMV infection. This study is the first report that investigates the response of host protein expression involved in TMV infection through a quantitative proteomics technique iTRAQ, combined with LC-MS/MS analysis. We used TMV-infected Nicotiana tabacum plants to investigate the effects of TMV infection on host proteins. Our results revealed differential abundance of proteins involving various pathways in protein translation, protein processing, photosynthesis and plant defence. There was a high abundance of thioredoxin H-type 1, a protein that counters oxidative stress and accelerated regulation of fatty acid synthesis to provide additional lipid molecules for VRC formation. There was a significant reduction in abundance of psaA and psbB proteins in the photosynthetic pathways. Our results identified key candidate host proteins involved in TMV-infected N. tabacum for functional studies in future.

iTRAQ-based analysis of leaf proteome identifies important proteins in secondary metabolite biosynthesis and defence pathways crucial to cross-protection against TMV.

Cross-protection is a phenomenon in which infection with a mild virus strain protects host plants against subsequent infection with a closely related severe virus strain. This study showed that a mild strain mutant virus, Tobacco mosaic virus (TMV)-43A could cross protect Nicotiana benthamiana plants against wild-type TMV. Furthermore, we investigated the host responses at the proteome level to identify important host proteins involved in cross-protection. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyze the proteome profiles of TMV, TMV-43A and cross-protected plants at different time-points. Our results showed that TMV-43A can cross-protect N. benthamiana plants from TMV. In cross-protected plants, photosynthetic activities were augmented, as supported by the increased accumulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) enzymes, which are crucial for chlorophyll biosynthesis. The increased abundance of ROS scavenging enzymes like thioredoxins and L-ascorbate peroxidase would prevent oxidative damage in cross-protected plants. Interestingly, the abundance of defence-related proteins (14-3-3 and NbSGT1) decreased, along with a reduction in virus accumulation during cross-protection. In conclusion, we have identified several important host proteins that are crucial in cross-protection to counter TMV infection in N. benthamiana plants. BIOLOGICAL SIGNIFICANCE: TMV is the most studied model for host-virus interaction in plants. It can infect wide varieties of plant species, causing significant economic losses. Cross protection is one of the methods to combat virus infection. A few cross-protection mechanisms have been proposed, including replicase/coat protein-mediated resistance, RNA silencing, and exclusion/spatial separation between virus strains. However, knowledge on host responses at the proteome level during cross protection is limited. To address this knowledge gap, we have leveraged on a global proteomics analysis approach to study cross protection. We discovered that TMV-43A (protector) protects N. benthamiana plants from TMV (challenger) infection through multiple host pathways: secondary metabolite biosynthesis, photosynthesis, defence, carbon metabolism, protein translation and processing and amino acid biosynthesis. In the secondary metabolite biosynthesis pathway, enzymes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) play crucial roles in chlorophyll biosynthesis during cross protection. In addition, accumulation of ROS scavenging enzymes was also found in cross-protected plants, providing rescues from excessive oxidative damage. Reduced abundance of plant defence proteins is correlated to reduced virus accumulation in host plants. These findings have increased our knowledge in host responses during cross-protection.

In planta proximity-dependent biotin identification (BioID) identifies a TMV replication co-chaperone NbSGT1 in the vicinity of 126 kDa replicase.

Tobacco mosaic virus (TMV) is a positive, single-stranded RNA virus. It encodes two replicases (126 kDa and 183 kDa), a movement protein and a coat protein. These proteins interact with host proteins for successful infection. Some host proteins such as eEF1α, Tm-1, TOM1, 14-3-3 proteins directly interact with Tobamovirus replication proteins. There are host proteins in the virus replication complex which do not interact with viral replicases directly, such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. We have used Proximity-dependent biotin identification (BioID) technique to screen for transient or weak protein interactions of host proteins and viral replicase in vivo. We transiently expressed BirA* tagged TMV 126 kDa replicase in TMV infected Nicotiana benthamiana plants. Among 18 host proteins, we identified NbSGT1 as a potential target for further characterization. Silencing of NbSGT1 in N. benthamiana plants increased its susceptibility to TMV infection, and overexpression of NbSGT1 increased resistance to TMV infection. There were weak interactions between NbSGT1 and TMV replicases but no interaction between them was found in Y2H assay. It suggests that the interaction might be transient or indirect. Therefore, the BioID technique is a valuable method to identify weak or transient/indirect interaction(s) between pathogen proteins and host proteins in plants. BIOLOGICAL SIGNIFICANCE: TMV is a well characterized positive-strand RNA virus model for study of virus-plant host interactions. It infects >350 plant species and is one of the significant pathogens of crop loss globally. Many host proteins are involved in TMV replication complex formation. To date there are few techniques available for identifying interacting host proteins to viral proteins. There is limited knowledge on transient or non-interacting host proteins during virus infection/replication. In this study, we used agroinfiltration-mediated in planta BioID technique to identify transiently or non-interacting host proteins to viral proteins in TMV-infected N. benthamiana plants. This technique allowed us to identify potential candidate proteins in the vicinity of TMV 126 kDa replicase. We have selected NbSGT1 and its overexpression suppresses TMV replication and increase plant resistance. NbSGT1 is believed to interact transiently or indirectly with TMV replicases in the presence of Hsp90/Hsp70. BioID is a novel and powerful technique to identify transiently or indirectly interacting proteins in the study of pathogen-host interactions.