Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and there is evidence that their interactions negatively affect disease outcome in patients. At the molecular level, interactions between the two bacterial taxa are well-described, with P. aeruginosa usually being the dominant species suppressing S. aureus through a variety of inhibitory molecules. However, in polymicrobial infections, the two species interact over prolonged periods of time, and S. aureus might evolve resistance against inhibitory molecules deployed by P. aeruginosa. Here, we used experimental evolution to test this hypothesis by exposing three different S. aureus strains (Cowan I, 6850, JE2) to the growth-inhibitory supernatant of P. aeruginosa PAO1 over 30 days. We found that all three S. aureus strains rapidly evolved resistance against inhibitory molecules and show that (i) adaptations were strain-specific; (ii) resistance evolution affected the expression of virulence traits; and (iii) mutations in membrane transporters were the most frequent evolutionary targets. Our work indicates that adaptations of S. aureus to co-infecting pathogens could increase virulence and decrease antibiotic susceptibility, because both virulence traits and membrane transporters involved in drug resistance were under selection. Thus, pathogen co-evolution could exacerbate infections and compromise treatment options

Immunohistochemistry for hepatitis E virus capsid protein cross-reacts with cytomegalovirus-infected cells: a potential diagnostic pitfall

Immunohistochemistry for hepatitis E virus (HEV) ORF2 (capsid) protein is a powerful tool for tissue-based diagnosis of hepatitis E, particularly useful in evaluating abnormal liver values in immunocompromised patients. We report here a previously unobserved reactivity of the HEV ORF2 antibody to human cytomegalovirus (CMV) proteins and contrast the staining patterns encountered in HEV and CMV infection, respectively. As part of a routine diagnostic work-up, the liver biopsy of an immunocompromised patient with elevated liver values was examined histologically for infection with viruses including CMV and HEV. Cytopathic changes were found, suggestive of CMV infection, which was confirmed by immunohistochemistry. Surprisingly, reactivity of a portion of CMV-infected cells with a mouse monoclonal antibody (clone 1E6) against HEV ORF2 protein was also detected. This observation prompted a screening of 22 further specimens (including liver, gastrointestinal, lung, brain and placental biopsies) with confirmed CMV infection/reactivation. Immunoreactivity of CMV-infected cells with HEV ORF2 antibody was observed in 18 of 23 specimens. While the HEV ORF2 antibody showed cytoplasmic, nuclear and canalicular positivity in hepatitis E cases, positivity in CMV-infected cells was limited to the nucleus. In conclusion, the HEV ORF2 antibody (clone 1E6) shows unexpected immunoreactivity against CMV proteins. In contrast to the hepatitis E staining pattern with cytoplasmic, nuclear and occasional canalicular positivity, reactivity in CMV-infected cells is restricted to the nucleus. Awareness of this cross-reactivity and knowledge of the differences in staining patterns will prevent pathologists from misinterpreting positive HEV ORF2 immunohistochemistry in liver specimens

Semi-supervised LC/MS alignment for differential proteomics

Motivation: Mass spectrometry (MS) combined with high-performance liquid chromatography (LC) has received considerable attention for high-throughput analysis of proteomes. Isotopic labeling techniques such as ICAT [5,6] have been successfully applied to derive differential quantitative information for two protein samples, however at the price of significantly increased complexity of the experimental setup. To overcome these limitations, we consider a label-free setting where correspondences between elements of two samples have to be established prior to the comparative analysis. The alignment between samples is achieved by nonlinear robust ridge regression. The correspondence estimates are guided in a semi-supervised fashion by prior information which is derived from sequenced tandem mass spectra. Results: The semi-supervised method for finding correspondences was successfully applied to aligning highly complex protein samples, even if they exhibit large variations due to different biological conditions. A large-scale experiment clearly demonstrates that the proposed method bridges the gap between statistical data analysis and label-free quantitative differential proteomics. Availability: The software will be available on the website Contact: [email protected]

Relief food subsistence revealed by microparticle and proteomic analyses of dental calculus from victims of the Great Irish Famine

This study provides direct evidence of the dependency on relief food in Ireland around the time of the Great Famine (1845 to 1852) through dental calculus analysis of archaeological human remains. The findings show a dominance of corn (maize) and milk from the identified foodstuffs and corroborate the contemporaneous historical accounts of diet and subsistence. It shows that microparticle and proteomic analyses, even when based on small archaeological samples, can provide a valid snapshot of dietary patterns and food consumption. The occurrence of egg protein, generally only included in the diet for the better-off social classes, also highlights how these analytical techniques can provide unanticipated insights into the variability of diet in historical populations.Food and diet were class markers in 19th-century Ireland, which became evident as nearly 1 million people, primarily the poor and destitute, died as a consequence of the notorious Great Famine of 1845 to 1852. Famine took hold after a blight (Phytophthora infestans) destroyed virtually the only means of subsistence—}the potato crop{—}for a significant proportion of the population. This study seeks to elucidate the variability of diet in mid{–}19th-century Ireland through microparticle and proteomic analysis of human dental calculus samples (n = 42) from victims of the famine. The samples derive from remains of people who died between August 1847 and March 1851 while receiving poor relief as inmates in the union workhouse in the city of Kilkenny (52{°}39' N, -7{°}15' W). The results corroborate the historical accounts of food provisions before and during the famine, with evidence of corn (maize), potato, and cereal starch granules from the microparticle analysis and milk protein from the proteomic analysis. Unexpectedly, there is also evidence of egg protein{—}a food source generally reserved only for export and the better-off social classes{—which highlights the variability of the prefamine experience for those who died. Through historical contextualization, this study shows how the notoriously monotonous potato diet of the poor was opportunistically supplemented by other foodstuffs. While the Great Irish Famine was one of the worst subsistence crises in history, it was foremost a social disaster induced by the lack of access to food and not the lack of food availability

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3’-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein–protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species

Grape ASR-Silencing Sways Nuclear Proteome, Histone Marks and Interplay of Intrinsically Disordered Proteins

In order to unravel the functions of ASR (Abscisic acid, Stress, Ripening-induced) proteins in the nucleus, we created a new model of genetically transformed grape embryogenic cells by RNAi-knockdown of grape ASR (VvMSA). Nuclear proteomes of wild-type and VvMSA-RNAi grape cell lines were analyzed by quantitative isobaric tagging (iTRAQ 8-plex). The most significantly up- or down-regulated nuclear proteins were involved in epigenetic regulation, DNA replication/repair, transcription, mRNA splicing/stability/editing, rRNA processing/biogenesis, metabolism, cell division/differentiation and stress responses. The spectacular up-regulation in VvMSA-silenced cells was that of the stress response protein VvLEA D-29 (Late Embryogenesis Abundant). Both VvMSA and VvLEA D-29 genes displayed strong and contrasted responsiveness to auxin depletion, repression of VvMSA and induction of VvLEA D-29. In silico analysis of VvMSA and VvLEA D-29 proteins highlighted their intrinsically disordered nature and possible compensatory relationship. Semi-quantitative evaluation by medium-throughput immunoblotting of eighteen post-translational modifications of histones H3 and H4 in VvMSA-knockdown cells showed significant enrichment/depletion of the histone marks H3K4me1, H3K4me3, H3K9me1, H3K9me2, H3K36me2, H3K36me3 and H4K16ac. We demonstrate that grape ASR repression differentially affects members of complex nucleoprotein structures and may not only act as molecular chaperone/transcription factor, but also participates in plant responses to developmental and environmental cues through epigenetic mechanisms

Proteome and Interactome Linked to Metabolism, Genetic Information Processing, and Abiotic Stress in Gametophytes of Two Woodferns

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades, together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution

PepSplice: cache-efficient search algorithms for comprehensive identification of tandem mass spectra

Motivation: Tandem mass spectrometry allows for high-throughput identification of complex protein samples. Searching tandem mass spectra against sequence databases is the main analysis method nowadays. Since many peptide variations are possible, including them in the search space seems only logical. However, the search space usually grows exponentially with the number of independent variations and may therefore overwhelm computational resources. Results: We provide fast, cache-efficient search algorithms to screen large peptide search spaces including non-tryptic peptides, whole genomes, dozens of posttranslational modifications, unannotated point mutations and even unannotated splice sites. All these search spaces can be screened simultaneously. By optimizing the cache usage, we achieve a calculation speed that closely approaches the limits of the hardware. At the same time, we control the size of the overall search space by limiting the combinations of variations that can co-occur on the same peptide. Using a hypergeometric scoring scheme, we applied these algorithms to a dataset of 1 420 632 spectra. We were able to identify a considerable number of peptide variations within a modest amount of computing time on standard desktop computers. Availability: PepSplice is available as a C++ application for Linux, Windows and OSX at www.ti.inf.ethz.ch/pw/software/pepsplice/. It is open source under the revised BSD license. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Tuning heterologous glucan biosynthesis in yeast to understand and exploit plant starch diversity

Background: Starch, a vital plant-derived polysaccharide comprised of branched glucans, is essential in nutrition and many industrial applications. Starch is often modified post-extraction to alter its structure and enhance its functionality. Targeted metabolic engineering of crops to produce valuable and versatile starches requires knowledge of the relationships between starch biosynthesis, structure, and properties, but systematic studies to obtain this knowledge are difficult to conduct in plants. Here we used Saccharomyces cerevisiae as a testbed to dissect the functions of plant starch biosynthetic enzymes and create diverse starch-like polymers. Results: We explored yeast promoters and terminators to tune the expression levels of the starch-biosynthesis machinery from Arabidopsis thaliana. We systematically modulated the expression of each starch synthase (SS) together with a branching enzyme (BE) in yeast. Protein quantification by parallel reaction monitoring (targeted proteomics) revealed unexpected effects of glucan biosynthesis on protein abundances but showed that the anticipated broad range of SS/BE enzyme ratios was maintained during the biosynthetic process. The different SS/BE ratios clearly influenced glucan structure and solubility: The higher the SS/BE ratio, the longer the glucan chains and the more glucans were partitioned into the insoluble fraction. This effect was irrespective of the SS isoform, demonstrating that the elongation/branching ratio controls glucan properties separate from enzyme specificity. Conclusions: Our results provide a quantitative framework for the in silico design of improved starch biosynthetic processes in plants. Our study also exemplifies a workflow for the rational tuning of a complex pathway in yeast, starting from the selection and evaluation of expression modules to multi-gene assembly and targeted protein monitoring during the biosynthetic process. Keywords: Amylopectin structure; Arabidopsis thaliana; Heterologous expression in yeast; Parallel reaction monitoring, Proteomics; Starch biosynthesis; YFP reporter

Amnion Cells in Tailored Hydrogels Deposit Human Amnion Native Extracellular Matrix

Fetal therapies regularly result in iatrogenic preterm premature rupture of the fetal membranes (iPPROM), which is associated with preterm birth. Biomaterials that promote the healing of traumatized fetal membranes (FMs) may prevent iPPROM-associated preterm births, addressing this unmet clinical need. Here, a fully defined synthetic poly(ethylene glycol) (PEG) hydrogel is developed to study the healing functions of human amnion-derived mesenchymal stromal cells (hAMCs) in 3D cultures. A pipeline to analyze extracellular matrix (ECM) proteins deposited by hAMCs in PEG hydrogels is established involving label-free quantification of mass-spectrometry data. Owing to the contaminant-free PEG hydrogels and a short fetal bovine serum (FBS)-free culture period, 128 ECM proteins, of which 97 are present in the native amnion, are identified. Upon stimulation with platelet-derived growth factor BB (PDGF-BB), a cell proliferation and migration inducing factor, hAMCs remodel their surroundings and deposit ECM proteins pericellularly. Among the most abundantly deposited amnion proteins, transforming growth factor β-induced protein ig-h3 (TGFβi), a very distinctive amnion protein that is involved in the wound healing cascade, is identified. These data support the potential of PDGF-BB to promote the repair of traumatized FMs and encourage its use for the engineering of biomaterials for FM healing, to ultimately prevent iPPROM