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Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein–protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.

Abstract Background Host–pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection. Results We find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status. Conclusions We show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the host’s dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance of diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted.

Tapasin is part of the peptide loading complex necessary for presenting antigenic peptides on MHC-I for the induction of adaptive immunity. Here the authors show that tapasin interacts with MHC-I in both conserved and allele-specific regions to promote antigen presentation, with tapasin L18 and K16 residues both implicated in this molecular interaction.

Species-specific diversities are particular features of mammalian chloride channel regulator, calcium activated (CLCA) genes. In contrast to four complex gene clusters in mammals, only two CLCA genes appear to exist in chickens. CLCA2 is conserved in both, while only the galline CLCA1 (gCLCA1) displays close genetic distance to mammalian clusters 1, 3 and 4. In this study, sequence analyses and biochemical characterizations revealed that gCLCA1 as a putative avian prototype shares common protein domains and processing features with all mammalian CLCA homologues. It has a transmembrane (TM) domain in the carboxy terminal region and its mRNA and protein were detected in the alimentary canal, where the protein was localized in the apical membrane of enterocytes, similar to CLCA4. Both mammals and birds seem to have at least one TM domain containing CLCA protein with complex glycosylation in the apical membrane of enterocytes. However, some characteristic features of mammalian CLCA1 and 3 including entire protein secretion and expression in cell types other than enterocytes seem to be dispensable for chicken. Phylogenetic analyses including twelve bird species revealed that avian CLCA1 and mammalian CLCA3 form clades separate from a major branch containing mammalian CLCA1 and 4. Overall, our data suggest that gCLCA1 and mammalian CLCA clusters 1, 3 and 4 stem from a common ancestor which underwent complex gene diversification in mammals but not in birds.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Polynucleobacter asymbioticus strain QLW-P1DMWA-1T represents a group of highly successful heterotrophic ultramicrobacteria that is frequently very abundant (up to 70% of total bacterioplankton) in freshwater habitats across all seven continents. This strain was originally isolated from a shallow Alpine pond characterized by rapid changes in water temperature and elevated UV radiation due to its location at an altitude of 1300 m. To elucidate the strain’s adjustment to fluctuating environmental conditions, we recorded changes occurring in its transcriptomic and proteomic profiles under contrasting experimental conditions by simulating thermal conditions in winter and summer as well as high UV irradiation. To analyze the potential connection between gene expression and regulation via methyl group modification of the genome, we also analyzed its methylome. The methylation pattern differed between the three treatments, pointing to its potential role in differential gene expression. An adaptive process due to evolutionary pressure in the genus was deduced by calculating the ratios of non-synonymous to synonymous substitution rates for 20 Polynucleobacter spp. genomes obtained from geographically diverse isolates. The results indicate purifying selection.

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that Staphylococcus aureus is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.

Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are converted into electro-chemical signals by mechanically activated ion channels. We describe here the presence of mechanically activated currents in melanoma cells that are dependent on TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 exhibit defective mechanoelectrical transduction, decreased motility and increased dissociation from organotypic spheroids. By analysing cell adhesion properties, we demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular adhesions and regulates melanoma cell migration and cell-cell interactions.

Abstract Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.

Here, we provide the dataset associated with our research article ‘label-free quantitative proteomic analysis of harmless and pathogenic strains of infectious microalgae, Prototheca spp.’ (Murugaiyan et al., 2017) [1]. This dataset describes liquid chromatography–mass spectrometry (LC–MS)-based protein identification and quantification of a non-infectious strain, Prototheca zopfii genotype 1 and two strains associated with severe and mild infections, respectively, P. zopfii genotype 2 and Prototheca blaschkeae. Protein identification and label-free quantification was carried out by analysing MS raw data using the MaxQuant-Andromeda software suit. The expressional level differences of the identified proteins among the strains were computed using Perseus software and the results were presented in [1]. This DiB provides the MaxQuant output file and raw data deposited in the PRIDE repository with the dataset identifier PXD005305.

Feae’s viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300–1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.

Here, we provide the dataset associated with our research article on comprehensive screening of Brucella immunoreactive proteins using sera of naturally infected hosts published in Biochemical and Biophysical Research Communications Wareth et al., 2015 [1]. Whole-cell protein extracts were prepared from Brucella abortus and Brucella melitensis, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently western blotting was carried out using sera from bovines (cows and buffaloes) and small ruminants (goats and sheep). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [2] with the dataset identifiers PXD001270 and DOI:10.6019/PXD001270.

The extracellular membranes of the inner ear are essential constituents to maintain sensory functions, the cupula for sensing torsional movements of the head, the otoconial membrane for sensing linear movements and accelerations like gravity, and the tectorial membrane in the cochlea for hearing. So far a number of structural proteins have been described, but for the gelatinous cupula precise data are missing. Here, we describe for the first time a major proteinogenic component of the cupula structure with an apparent molecular mass of 45 kDa from salmon. Analyses of respective peptides revealed highly conserved amino-acid sequences with identity to zona pellucida-like domain proteins. Immunohistochemistry studies localized the protein in the ampulla of the inner ear from salmon and according to its anatomical appearance we identified this glycoprotein as Cupulin. Future research on structure and function of zona pellucida-like domain proteins will enhance our knowledge of inner ear diseases, like sudden loss of vestibular function and other disturbances.

In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment.

Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P