Transcriptome responses of Arabidopsis to necrotrophic fungus Alternaria brassicae reveal pathways and candidate genes associated with resistance.

Alternaria leaf blight (ALB), caused by a necrotrophic fungus Alternaria brassicae is a serious disease of oleiferous Brassicas resulting in significant yield losses worldwide. No robust resistance against A. brassicae has been identified in the Brassicas. Natural accessions of Arabidopsis show a spectrum of responses to A. brassicae ranging from high susceptibility to complete resistance. To understand the molecular mechanisms of resistance/ susceptibility, we analysed the comparative changes in the transcriptome profile of Arabidopsis accessions with contrasting responses- at different time points post-infection. Differential gene expression, GO enrichment, pathway enrichment, and weighted gene co-expression network analysis (WGCNA) revealed reprogramming of phenylpropanoid biosynthetic pathway involving lignin, hydroxycinnamic acids, scopoletin, anthocyanin genes to be highly associated with resistance against A. brassicae. T-DNA insertion mutants deficient in the biosynthesis of coumarin scopoletin exhibited enhanced susceptibility to A. brassicae. The supplementation of scopoletin to medium or exogenous application resulted in a significant reduction in the A. brassicae growth. Our study provides new insights into the transcriptome dynamics in A. brassicae-challenged Arabidopsis and demonstrates the involvement of coumarins in plant immunity against the Brassica pathogen A. brassicae.

Genomic analysis of phylogroup D Escherichia coli strains using novel de-novo reference-based guided assembly.

Escherichia coli are highly diverse bacteria with different pathogenic types, serotypes and phylogenetic types/phylotypes. In recent years, infections with E. coli have increased worldwide and so has the emergence of antibiotic resistant strains. In the present study we have assembled, annotated and analysed genome sequences of three strains of the phylogroup D of E. coli. These strains were isolated from the river Yamuna, a prominent anthropogenic urban river of northern India. These strains showed varied antibiotic susceptibilities, one was susceptible to all the antibiotics tested except ampicillin while of the other two, one was multi-ß-lactam resistant and the other was multi-drug resistant (resistant to multiple ß-lactams, fluoroquinolones and kanamycin). The short-sequence reads were assembled into contigs using the de-novo approach and further, scaffolding of contigs was performed by using the best reference genome for a particular isolate which resulted in a significant increase in the N50 value of each assembly. The bioinformatics assembly approach used in this study could be easily applied to study other bacterial genomes.

Comprehensive genomic analysis of hypocholesterolemic probiotic Enterococcus faecium LR13 reveals unique proteins involved in cholesterol-assimilation.

Hypercholesterolemia is a major risk factor for cardiovascular diseases (CVDs). Chemotherapeutic agents for CVDs exhibit several side effects. Specific probiotics with hypocholesterolemic effects can be safe and effective alternatives to chemotherapeutics. Here, we have analyzed and compared the genome of a novel rhizospheric Enterococcus faecium LR13 cholesterol-assimilating probiotic with other probiotic/pathogenic E. faecium strains to discern genetic factors underlying probiotic efficacy and cholesterol-assimilation. Genomic analyses of E. faecium probiotic strains revealed that LR13 and WEFA23 (cholesterol-assimilating probiotics) harbored 21 unique proteins absent in non-cholesterol-assimilating probiotics. Of these, 14 proteins could directly help in cholesterol-assimilation by producing short chain fatty acids, lipid (sterol) transport and membrane stabilization, and bile salt hydrolase activity. This suggests that cholesterol-assimilation is an intrinsic, strain-specific trait exhibited by probiotics with a specific genetic constitution. Moreover, the unique proteins identified in this study can serve as biomarkers for discerning/characterizing cholesterol-assimilating probiotics as novel biotherapeutics against CVDs.

Protein expression profiling, in silico classification and pathway analysis of cariogenic bacteria Streptococcus mutans under bacitracin stress conditions.

Introduction. Streptococcus mutans is a cariogenic bacterium that causes dental caries as well as being implicated in other dental pathologies and infective endocarditis. Bacitracin is a bactericidal antibiotic that induces cell wall stress in Gram-positive bacteria.Gap Statement. S. mutans is among the most characterized Gram-positive bacteria. However, the transcriptome and proteome of S. mutans have received less attention, and they are actually key in understanding the pathogenesis of any bacteria. In this study, we extracted the whole proteome of S. mutans grown under bacitracin stress. Such a proteome is anticipated to offer deep insights related to physiological dynamic fluctuations and, consequently, it may provide 'proteomic signatures' to be identified as potential targets.Aim. The aim of the study is to explore the general stress response that S. mutans exhibits at the proteome level when cell wall stress is imposed on it.Methodology. A sub-MIC concentration of bacitracin was added to the growth media of S. mutans followed by whole-cell protein extraction. The proteome was then subjected to high-throughput proteomics analysis, i.e. liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins obtained through LC-MS/MS underwent analyses such as gene ontology, KEGG (Kyoto Encyclopaedia of Genes and Genomes) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis, and STRING for functional annotation, pathway enrichment and protein-protein interaction (PPI) networks, respectively. These proteins were also categorized into functional classes using the PANTHER (Protein Annotation Through Evolutionary Relationship) classification system.Result. LC-MS/MS produced data from 321 identified proteins. From these, 41 and 30 were found to be significantly over- (≥2 fold change) and underexpressed (≤0.4 fold change), respectively. In the upregulated proteins we mostly observed sortases and proteins involved in the EPS biosynthesis pathway, whereas among the downregulated proteins the majority related to glycolysis.Conclusion. The sortase family of proteins appear to be potential targets because they regulate various virulence factors and therefore can be targeted to inhibit multiple virulence pathways simultaneously. This study offers an understanding of proteomic fluctuations in response to cell wall stress and can thus help in identifying key players mediating virulence.

Proteomic Analysis of the Colistin-resistant E. coli Clinical Isolate: Explorations of the Resistome.

BACKGROUND: Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. OBJECTIVE: The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. METHODS: In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. RESULTS: LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. CONCLUSION: We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

Identification of Binding Partners of CsaA - An Archaeal Chaperonic Protein of Picrophilus torridus.

BACKGROUND: CsaA is among the few chaperones which are present in both bacteria and archaea, but absent in eukaryotes. There are no reports on interactome analysis of CsaA from archaea, till date. Identification of binding partners of CsaA might be helpful in understanding CsaA-associated processes in Picrophilus torridus an extreme thermoacidophilic euryarchaeon. OBJECTIVES: The present study was conducted to identify the binding partners of CsaA of P. torridus (PtCsaA). METHODS: The binding partners of PtCsaA were isolated and identified using a pull down assay and liquid chromatography-mass spectrometry (LC-MS). RESULTS: The results revealed twelve potential binding partners of CsaA. These were thermosome subunits (Q6KZS2 and Q6L132), nascent polypeptide-associated complex protein (Q6L1N3), elongation factor 1-alpha (Q6L202), uncharacterized protein (Q6L0Y6), citrate synthase (Q6L0M8), asparaginyl- tRNA synthetase (Q6L0M5), succinyl-CoA synthetase beta chain (Q6L0B4), pyruvate ferredoxin oxidoreductase alpha and beta chain proteins (Q6KZA7 and Q6KZA6, respectively), malate dehydrogenase (Q6L0C3) and reversed fumarylacetoacetase (Q6KZ97). Functional categorization revealed that of these, six proteins were involved in energy metabolic pathways, three were archaeal chaperones, two were involved in translation and one might be a transcription regulator. STRING-based analysis of the protein-protein interactions of the experimental interactome revealed strong interactions among them. CONCLUSION: PtCsaA might be a multifaceted protein which besides translation might also play important role in metabolic processes of P. torridus. However, further experiments investigating the binding partners of CsaA in other archaea are required for a better understanding of CsaA-associated processes in archaea.

Comparative in-silico proteomic analysis discerns potential granuloma proteins of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is one of the three pathogenic species of the genus Yersinia. Most studies regarding pathogenesis of Y. pseudotuberculosis are based on the proteins related to Type III secretion system, which is a well-known primary virulence factor in pathogenic Gram-negative bacteria, including Y. pseudotuberculosis. Information related to the factors involved in Y. pseudotuberculosis granuloma formation is scarce. In the present study we have used a computational approach to identify proteins that might be potentially involved in formation of Y. pseudotuberculosis granuloma. A comparative proteome analysis and conserved orthologous protein identification was performed between two different genera of bacteria - Mycobacterium and Yersinia, their only common pathogenic trait being ability to form necrotizing granuloma. Comprehensive analysis of orthologous proteins was performed in proteomes of seven bacterial species. This included M. tuberculosis, M. bovis and M. avium paratuberculosis - the known granuloma forming Mycobacterium species, Y. pestis and Y. frederiksenii - the non-granuloma forming Yersinia species and, Y. enterocolitica - that forms micro-granuloma and, Y. pseudotuberculosis - a prominent granuloma forming Yersinia species. In silico proteome analysis indicated that seven proteins (UniProt id A0A0U1QT64, A0A0U1QTE0, A0A0U1QWK3, A0A0U1R1R0, A0A0U1R1Z2, A0A0U1R2S7, A7FMD4) might play some role in Y. pseudotuberculosis granuloma. Validation of the probable involvement of the seven proposed Y. pseudotuberculosis granuloma proteins was done using transcriptome data analysis and, by mapping on a composite protein-protein interaction map of experimentally proved M. tuberculosis granuloma proteins (RD1 locus proteins, ESAT-6 secretion system proteins and intra-macrophage secreted proteins). Though, additional experiments involving knocking out of each of these seven proteins are required to confirm their role in Y. pseudotuberculosis granuloma our study can serve as a basis for further studies on Y. pseudotuberculosis granuloma.