Search

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project – Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance forN- andO-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved “high coverage” and “high accuracy” glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251±70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

Beer is perhaps the oldest biotechnology and continues to this day to be one of the most popular beverages worldwide. Brewing is and always has been dependent on agricultural food sources, primarily barley (Hordeum vulgare). Not only is the brewing process reliant on the agricultural production of grains, it also relies on and encompasses many connected bio-processes: hop farming, germination, malting, yeast propagation, mashing, boiling, fermentation, and maturation. Proteins are key drivers of these bio-processes, facilitating many biochemical changes: allowing germination to begin, breaking down sugars and proteins in the mash, and affecting mouth-feel and flavor in the finished beer, to name a few. Mass spectrometry proteomics allows detailed insights into the identity, abundance, location, modifications, and interactions of proteins across complex proteomes. This chapter will provide an overview of how proteomics has been applied to brewing and its related bio-processes.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analyzed the glycoproteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyze sequential window acquisition of all theoretical mass spectra data-independent acquisition mass spectrometry data based on glycopeptides identified by Byonic (Protein Metrics; version 2.13.17). We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the liqueur de tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared with 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. To our knowledge, this work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic sequential window acquisition of all theoretical mass spectra/data-independent acquisition workflow that is broadly applicable to other sample types., Graphical Abstract, Highlights • Development of an automated glycoproteomic sequential window acquisition of all theoretical mass spectra workflow. • Application to three pairs of commercial-scale experimental sparkling wines. • Decreased protein abundance in cuvée during the aging process. • Different yeast strains produce varying levels of yeast proteins., In Brief Glycoproteins in sparkling wine are especially important for its sensory properties. Here, we developed a novel quantitative glycoproteomic approach to investigate the glycoproteomes of sparkling wine. The protein- and glycopeptide-centric approaches enabled robust quantification from small volumes of sparkling wine and highlighted key changes that may contribute to sparkling wine quality.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins., The pathogenesis of Streptococcus pyogenes (GAS) causing scarlet fever has been associated with the presence of prophages, such as ΦHKU.vir, and their products. Here, the authors characterize the exotoxins SpeC and Spd1 of ΦHKU.vir and show these to act synergistically to facilitate nasopharyngeal colonization in mice.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses., Zacchi et al. attempt to bridge the gap between understanding the impact of process variables and determination of critical post-translational modifications on a highly complex glycoprotein, factor IX. They implement an LC-MS/MS approach based on data independent acquisition which allows measurement of all detectable peptides, even with heterogeneous modifications.

The Endoplasmic Reticulum (ER) is responsible for the folding and post-translational modification of secretory proteins, as well as for triaging misfolded proteins. During folding, there is a complex yet only partially understood interplay between disulfide bond formation, which is an enzyme catalyzed event in the oxidizing environment of the ER, along with other post-translational modifications (PTMs) and chaperone-supported protein folding. Here, we used the glycoprotein torsinA as a model substrate to explore the impact of ER redox homeostasis on PTMs and protein biogenesis. TorsinA is a AAA+ ATPase with unusual oligomeric properties and controversial functions. The deletion of a C-terminal glutamic acid residue (∆E) is associated with the development of Early-Onset Torsion Dystonia, a severe movement disorder. TorsinA differs from other AAA+ ATPases since it is an ER resident, and as a result of its entry into the ER torsinA contains two N-linked glycans and at least one disulfide bond. The role of these PTMs on torsinA biogenesis and function and the identity of the enzymes that catalyze them are poorly defined. Using a yeast torsinA expression system, we demonstrate that a specific protein disulfide isomerase, Pdi1, affects the folding and N-linked glycosylation of torsinA and torsinA∆E in a redox-dependent manner, suggesting that the acquisition of early torsinA folding intermediates is sensitive to perturbed interactions between Cys residues and the quality control machinery. We also highlight the role of specific Cys residues during torsinA biogenesis and demonstrate that torsinA∆E is more sensitive than torsinA when these Cys residues are mutated.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analysed the glyco/proteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyse Sequential Window Acquisition of all THeoretical mass spectra (SWATH) Data Independent Acquisition (DIA) mass spectrometry data based on glycopeptides identified by Byonic. We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the Liqueur de Tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared to 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. This work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic SWATH/DIA workflow that is broadly applicable to other sample types.

Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX post-translational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.

Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used Sequential Window Acquisition of all THeoretical ions Mass Spectrometry (SWATH-MS) proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We verified our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here we report the comprehensive molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for optimal growth in human blood, confers resistance to neutrophil killing, and degrades neutrophil extracellular traps (NETs). Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the etiology and pathogenesis of scarlet fever-causing GAS mediated by phage ΦHKU.vir exotoxins.

Proteomic analysis of bioreactor supernatants can inform on cellular metabolic status, viability, and productivity, as well as product quality, which can in turn help optimize bioreactor operation. Incubating mammalian cells in bioreactors requires the addition of polymeric surfactants such as Pluronic F68, which reduce the sheer stress caused by agitation. However, these surfactants are incompatible with mass spectrometry proteomics and must be eliminated during sample preparation. Here, we compared four different sample preparation methods to eliminate polymeric surfactants from filtered bioreactor supernatant samples: organic solvent precipitation; filter-assisted sample preparation (FASP); S-Trap; and single-pot, solid-phase, sample preparation (SP3). We found that SP3 and S-Trap substantially reduced or eliminated the polymer(s), but S-Trap provided the most robust clean-up and highest quality data. Additionally, we observed that SP3 sample preparation of our samples and in other published datasets was associated with partial alkylation of cysteines, which could impact the confidence and robustness of protein identification and quantification. Finally, we observed that several commercial mammalian cell culture media and media supplements also contained polymers with similar mass spectrometry profiles, and we suggest that proteomic analyses in these media will also benefit from the use of S-Trap sample preparation.

Granular protein is an important structural feature in determining starch digestibility. High-amylose wheat starch (HAWS) with80% amylose content contains more granular protein than wild-type starch. As analyzed by mass spectrometry-based proteomics, granular-bound starch synthase (GBSS) is the major granular protein in isolated starch materials. GBSS content increases with amylose content (Spearman's correlation, p 0.05), whereas the abundance relative to other proteins is similar among starches. Multiple amylase inhibitors were also identified. From Michaelis-Menten analysis, HAWS has a similar K

The Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. Thereby, RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.

Neisseria meningitidis, a common cause of sepsis and bacterial meningitis, infects the meninges and central nervous system (CNS), primarily via paracellular traversal across the blood-brain barrier (BBB) or blood-cerebrospinal fluid barrier. N. meningitidis is often present asymptomatically in the nasopharynx, and the nerves extending between the nasal cavity and the brain constitute an alternative route by which the meningococci may reach the CNS. To date, the cellular mechanisms involved in nerve infection are not fully understood., Neisseria meningitidis, a common cause of sepsis and bacterial meningitis, infects the meninges and central nervous system (CNS), primarily via paracellular traversal across the blood-brain barrier (BBB) or blood-cerebrospinal fluid barrier. N. meningitidis is often present asymptomatically in the nasopharynx, and the nerves extending between the nasal cavity and the brain constitute an alternative route by which the meningococci may reach the CNS. To date, the cellular mechanisms involved in nerve infection are not fully understood. Peripheral nerve glial cells are phagocytic and are capable of eliminating microorganisms, but some pathogens may be able to overcome this protection mechanism and instead infect the glia, causing cell death or pathology. Here, we show that N. meningitidis readily infects trigeminal Schwann cells (the glial cells of the trigeminal nerve) in vitro in both two-dimensional and three-dimensional cell cultures. Infection of trigeminal Schwann cells may be one mechanism by which N. meningitidis is able to invade the CNS. Infection of the cells led to multinucleation and the appearance of atypical nuclei, with the presence of horseshoe nuclei and the budding of nuclei increasing over time. Using sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics followed by bioinformatics pathway analysis, we showed that N. meningitidis induced protein alterations in the glia that were associated with altered intercellular signaling, cell-cell interactions, and cellular movement. The analysis also suggested that the alterations in protein levels were consistent with changes occurring in cancer. Thus, infection of the trigeminal nerve by N. meningitidis may have ongoing adverse effects on the biology of Schwann cells, which may lead to pathology.

Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric assay. Both methods resulted in comparable kinetic parameters. From the colorimetric assay the kcat and KM values are 131 s−1 and 0.25 mM, respectively, and from the ITC assay the corresponding parameters are 30 s−1 and 0.45 mM, respectively. The continuous ITC-based assay will facilitate functional studies for an enzyme that is an emerging target for the development of addiction treatments.

Glycogen is a highly branched glucose polymer which plays an important role in glucose storage and the maintenance of blood sugar homeostasis. The dimeric protein glycogenin can self-glucosylate to act as a primer for glycogen synthesis, eventually resulting in small (∼20 nm diameter) glycogen β particles with a dimer of glycogenin at their core. In the liver, glycogen is also found in the form of α particles: large bound composites of many β particles. Here, we provide evidence using qualitative and quantitative proteomics and size-exclusion chromatography from healthy rat, mouse, and human liver glycogen that glycogenin is the binding agent linking β particles together into α particles.

Interspecies electron transfer (IET) occurs in many microbial communities, enabling extracellular electron exchange for syntrophic utilization of mixed resources. Various mechanisms of IET have been characterized including direct IET (DIET) and hydrogen IET (HIT) but their evolution throughout syntrophic adaptation has not been investigated through an omics approach. A syntrophic coculture of Geobacter sulfurreducens and Pseudomonas aeruginosa was established and evolved in restricted medium. The medium required cooperative metabolism due to preferential utilization of formate and fumarate by P. aeruginosa and G. sulfurreducens respectively. Pure cultures did not yield significant growth while substantial growth was observed in cocultures. The syntrophy was not reliant on phenazine, since Δphz mutant strain cocultures grew, however appeared to rely on cytochromes as evidenced from the stunted growth G. sulfurreducens ΔomcZ and ΔomcS mutant cocultures. SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) proteomic analysis of initial cocultures revealed upregulation in DIET-associated cytochromes, whereas adapted cocultures revealed upregulation in HybA, a G. sulfurreducens uptake hydrogenase critical to HIT. This suggests DIET plays a critical role in the establishment of syntrophy between G. sulfurreducens and P. aeruginosa but is later consolidated with HIT as the cocultures adapt. This is the first instance to show a temporal distribution of DIET and HIT within the same coculture.

Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment., Author summary The first step in the biogenesis of secretory proteins is the targeting and translocation into the endoplasmic reticulum (ER). Secretory proteins enter the ER via a gated channel in the ER membrane called the translocon, a protein complex composed of Sec61p/Sec61α, Sbh1p/Sec61β and Sss1p/Sec61γ. As a protein conducting channel the translocon must be sealed in a regulated manner to prevent the free flow of ions and small molecules between the ER and cytosol. We have discovered that mutations in Sec61γ that arise in cancer affect this seal but not the ability of this protein complex to translocate secretory proteins into the ER. We hypothesise that altered translocon gating contributes to malignancy by influencing factors such as migration, autophagy and chemotherapy resistance.

Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germination of the seeds before harvest from the field. This can result in inefficiencies in industrial malting. We used Sequential Window Acquisition of all THeoretical ions Mass Spectrometry (SWATH-MS) proteomics to measure changes in the barley seed proteome throughout germination. We found a large number of proteins involved in desiccation tolerance and germination inhibition rapidly decreased in abundance after imbibition. This was followed by a decrease in proteins involved in lipid, protein and nutrient reservoir storage, consistent with induction and activation of systems for nutrient mobilisation to provide nutrients to the growing embryo. Dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, with differences in sulfur metabolic enzymes, endogenous alpha-amylase/trypsin inhibitors, and histone proteins. We verified our findings with analysis of germinating barley seeds from two commercial malting facilities, demonstrating that key features of the dynamic proteome of germinating barley seeds were conserved between laboratory and industrial scales. The results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy. SIGNIFICANCE: Germination is critical to the reproduction and propagation of flowering plants, and in industrial malting. Dormancy, where seeds fail to germinate upon steeping, can result in inefficiencies in industrial malting. Our DIA/SWATH-MS proteomics analyses identified key changes during germination, including an initial loss of proteins involved in desiccation tolerance and germination inhibition, followed by decreases in lipid, protein and nutrient reservoir storage. These changes were consistent between laboratory and industrial malting scales, and therefore demonstrate the utility of laboratory-scale barley germination as a model system for industrial malt house processes. We also showed that dormant seeds that failed to germinate showed substantial biochemical activity distinct from that of seeds undergoing germination, consistent with dormancy being an actively regulated state. Our results provide a more detailed understanding of the changes in the barley proteome during germination and give possible target proteins for testing or to inform selective breeding to enhance germination or control dormancy.

Background: Over 26 million people suffer from heart failure (HF) globally. Current diagnosis of HF relies on clinical evaluation, blood assays and imaging techniques. Our aim is to develop a diagnostic assay to detect HF in at risk individuals within the community using human saliva as a medium, potentially leading to a simple, safe early warning system. Methods: Saliva samples were collected from healthy controls (n=36) and HF patients (n=75). Salivary proteome profiles were analysed by Sequential Window Acquisition of All Theoretical fragment ion spectra – Mass Spectrometry (SWATH-MS). A total of 738 proteins were quantified and 177 proteins demonstrated significant differences between HF patients and healthy controls. Candidate biomarkers were chosen based on their abundance and difference between the two cohorts. A multi-protein panel was developed using logistic regression analysis. The diagnostic performance of the multi-protein panel was assessed using receiver operative characteristic curves. The candidate proteins were further confirmed, using western blot analysis, and validated technically, using an independent biological cohort. Results: A group of six proteins were chosen in the discovery phase as potential candidates based on their differences in the abundance between the two cohorts. During the validation phase, two of the proteins were not detected with western blotting and as such were removed. The final panel consists of four proteins with sensitivity of 83.3%, specificity of 62.5% with an area under ROC curve of 0.78 in discriminating healthy controls from NYHA class I/II HF patients, and was validated in a second independent cohort study. Conclusion: Analysis of salivary proteome using SWATH-MS revealed novel HF-specific protein candidates yielding high diagnostic performance. A multi-centre longitudinal clinical trial will be the next step before clinical implementation of this panel.

Nontypeable

Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings.Graphical Abstract

Glycosylation is a complex posttranslational modification that is critical for regulating the functions of diverse proteins. Analysis of protein glycosylation is made challenging by the high degree of heterogeneity in both glycan occupancy and structure. Here, we describe methods for data-independent acquisition (SWATH) mass spectrometry analysis of structure and occupancy of N-glycans from yeast cell wall glycoproteins.

Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. Crystal structures of this enzyme in complex with ATP analogues (to 2.20 and 2.60 A resolution, respectively) complement the recent structure of rkbPAP with a bound ADP analogue (ChemBioChem 20 (2019) 1536). Together these complexes provide the first structural insight of a PAP in complex with molecules that mimic biologically relevant substrates. Homology modeling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and thale cress (AtPAP26) that are efficient in hydrolyzing phytate and PEP as preferred substrates, respectively. The combining of crystallographic data, substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus) resulted in the identification of several active site residues that are important in defining the substrate specificities of plant PAPs; of particular relevance is the identification of a motif (“REKA”) that is characteristic for plant PAPs that possess phytase activity. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus acquisition and use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production.

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively. GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component. Postprint

Non-typeableHaemophilus influenzae(NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonisation of host cells are crucial steps in bacterial pathogenesis, and in many strains of NTHi interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins areN-glycoproteins which are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters ofhmw1Aandhmw2Adirectly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters ofhmw1Candhmw2Cin diverse NTHi isolates. In anex vivoassay, we systematically altered substrate and glycosyltransferase expression and showed that both of these factors affected the site-specific efficiency of glycosylation on HMW-A. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Site-directed mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss–of–function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg(2+) transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg(2+) levels and that reduced Mg(2+) impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.

The accumulation of advanced glycation end products is implicated in the development and progression of diabetic kidney disease. No study has examined whether stimulating advanced glycation clearance via receptor manipulation is reno-protective in diabetes. Podocytes, which are early contributors to diabetic kidney disease and could be a target for reno-protection. To examine the effects of increased podocyte oligosaccharyltransferase-48 on kidney function, glomerular sclerosis, tubulointerstitial fibrosis and proteome (PXD011434), we generated a mouse with increased oligosaccharyltransferase-48kDa subunit abundance in podocytes driven by the podocin promoter. Despite increased urinary clearance of advanced glycation end products, we observed a decline in renal function, significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane thickening and foot process effacement and tubulointerstitial fibrosis. Analysis of isolated glomeruli identified enrichment in proteins associated with collagen deposition, endoplasmic reticulum stress and oxidative stress. Ultra-resolution microscopy of podocytes revealed denudation of foot processes where there was co-localization of oligosaccharyltransferase-48kDa subunit and advanced glycation end-products. These studies indicate that increased podocyte expression of oligosaccharyltransferase-48kDa subunit results in glomerular endoplasmic reticulum stress and a decline in kidney function.

The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic kidney disease (DKD). There has been interest in investigating the potential of AGE clearance receptors, such as oligosaccharyltransferase-48kDa subunit (OST48) to prevent the detrimental effects of excess AGE accumulation seen in the diabetic kidney. Here the objective of the study was to increase the expression of OST48 to examine if this slowed the development of DKD by facilitating the clearance of AGEs. Groups of 8-week-old heterozygous knock-in male mice (n=9-12/group) over-expressing the gene encoding for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST+/-) and litter mate controls were randomised to either (i) no diabetes or (ii) diabetes induced via multiple low-dose streptozotocin and followed for 24 weeks. By the study end, global over expression of OST48 increased glomerular OST48. This facilitated greater renal excretion of AGEs but did not affect circulating or renal AGE concentrations. Diabetes resulted in kidney damage including lower glomerular filtration rate, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. In diabetic mice, tubulointerstitial fibrosis was further exacerbated by global increases in OST48. There was significantly insulin effectiveness, increased acute insulin secretion, fasting insulin concentrations and AUCinsulin observed during glucose tolerance testing in diabetic mice with global elevations in OST48 when compared to diabetic wild-type littermates. Overall, this study suggested that despite facilitating urinaryrenal AGE clearance, there were no benefits observed on kidney functional and structural parameters in diabetes afforded by globally increasing OST48 expression. However, the improvements in insulin secretion seen in diabetic mice with global over-expression of OST48 and their dissociation from effects on kidney function warrant future investigation.

Mashing is a key step in beer brewing in which starch and proteins are solubilized from malted barley in a hot water extraction and digested to oligomaltose and free amino nitrogen. We used SWATH-MS to measure the abundance and site-specific modifications of proteins throughout a small-scale pale ale mash. Proteins extracted from the malt at low temperatures early in the mash decreased precipitously in abundance at higher temperatures late in the mash due to temperature/time-induced unfolding and aggregation. We validated these observations using experimental manipulation of time and temperature parameters in a micro-scale pale ale mash. Correlation analysis of temperature/time-dependent abundance showed that sequence and structure were the main features that controlled protein abundance profiles. Partial proteolysis by barley proteases was common early in the mash. The resulting proteolytically clipped proteins were particularly sensitive and were preferentially lost at high temperatures late in the mash, while intact proteins remained soluble. The beer brewing proteome is therefore driven by the interplay between protein solubilisation and proteolysis, which are in turn determined by barley variety, growth conditions, and brewing process parameters.

Mass spectrometry (MS) proteomics allows systematic identification, characterization, and relative quantification of the full suite of proteins in a biological sample, and is a powerful analytical approach for investigation of many aspects of the biology of Neisseria meningitidis. Here, we describe methods for robust and efficient sample preparation of the proteome of N. meningitidis suitable for diverse MS proteomics workflows.

Glycosylation is a complex posttranslational modification that is critical for regulating the functions of diverse proteins. Analysis of protein glycosylation is made challenging by the high degree of heterogeneity in both glycan occupancy and structure. Here, we describe methods for data-independent acquisition (SWATH) mass spectrometry analysis of structure and occupancy of N-glycans from yeast cell wall glycoproteins.

Mass spectrometry (MS) proteomics allows systematic identification, characterization, and relative quantification of the full suite of proteins in a biological sample, and is a powerful analytical approach for investigation of many aspects of the biology of Neisseria meningitidis. Here, we describe methods for robust and efficient sample preparation of the proteome of N. meningitidis suitable for diverse MS proteomics workflows.

Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we sampled tissue and uterine fluid from six pre- and six postpubertal Brahman heifers. Post-pubertal heifers were sampled in the luteal phase. Gene expression of the uterine tissue was investigated with RNA-sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P-value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 9 10 À6 ). We also identified 79 proteins (out of 230 proteins) that were DE (P < 1 9 10 À5 ) in the uterine fluid. When we compared proteomics and transcriptome results, four DE proteins were identified as being encoded by DE genes: OVGP1, GRP, CAP1 and HBA. Except for CAP1, the other three had lower expression post-puberty. The function of these four genes hypothetically related to preparation of the uterus for a potential pregnancy is discussed in the context of puberty. All DE genes and proteins were also used in pathway and ontology enrichment analyses to investigate overall function. The DE genes were enriched for terms related to ribosomal activity. Transcription factors that were deemed key regulators of DE genes are also reported. Transcription factors ZNF567, ZNF775, RELA, PIAS2, LHX4, SOX2, MEF2C, ZNF354C, HMG20A, TCF7L2, ZNF420, HIC1, GTF3A and two novel genes had the highest regulatory impact factor scores. These data can help to understand how puberty influences uterine function.

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.

The glycoproteome remains severely understudied because of significant analytical challenges associated with glycoproteomics, the system-wide analysis of intact glycopeptides. This review introduces important structural aspects of protein N-glycosylation and summarizes the latest technological developments and applications in LC-MS/MS-based qualitative and quantitative N-glycoproteomics. These maturing technologies provide unique structural insights into the N-glycoproteome and its synthesis and regulation by complementing existing methods in glycoscience. Modern glycoproteomics is now sufficiently mature to initiate efforts to capture the molecular complexity displayed by the N-glycoproteome, opening exciting opportunities to increase our understanding of the functional roles of protein N-glycosylation in human health and disease.

Changes in brain metabolism are a hallmark of Alcohol Use Disorder (AUD). Determining how AUD changes the brain proteome is critical for understanding the effects of alcohol consumption on biochemical processes in the brain. We used data-independent acquisition mass spectrometry proteomics to study differences in the abundance of proteins associated with AUD in pre-frontal lobe and motor cortex from autopsy brain. AUD had a substantial effect on the overall brain proteome exceeding the inherent differences between brain regions. Proteins associated with glycolysis, trafficking, the cytoskeleton, and excitotoxicity were altered in abundance in AUD. We observed extensive changes in the abundance of key metabolic enzymes, consistent with a switch from glucose to acetate utilization in the AUD brain. We propose that metabolic adaptations allowing efficient acetate utilization contribute to ethanol dependence in AUD.

Barley is an important cereal grain used for beer brewing, animal feed, and human food consumption. Fungal disease can impact barley production, as it causes substantial yield loss and lowers seed quality. We used sequential window acquisition of all theoretical ions mass spectrometry (SWATH-MS) to measure and quantify the relative abundance of proteins within seeds of different barley varieties under various fungal pathogen burdens (ProteomeXchange Datasets PXD011303 and PXD014093). Fungal burden in the leaves and stems of barley resulted in changes to the seed proteome. However, these changes were minimal and showed substantial variation among barley samples infected with different pathogens. The limited effect of intrinsic disease resistance on the seed proteome is consistent with the main mediators of disease resistance being present in the leaves and stems of the plant. The seeds of barley varieties accredited for use as malt had higher levels of proteins associated with starch synthesis and beer quality. The proteomic workflows developed and implemented here have potential application in quality control, breeding and processing of barley, and other agricultural products.

Carbohydrate analyses are often challenging due to the structural complexity of these molecules, as well as the lack of suitable analytical tools for distinguishing the vast number of possible isomers. The coupled technique, ion mobility-mass spectrometry (IM-MS), has been in use for two decades for the analysis of complex biomolecules, and in recent years it has emerged as a powerful technique for the analysis of carbohydrates. For carbohydrates, most studies have focused on the separation and characterization of isomers in biological samples. IM-MS is capable of separating isomeric ions by drift time, and further characterizing them by mass analysis. Applications of IM-MS in carbohydrate analysis are extremely useful and important for understanding many biological mechanisms and for the determination of disease states, although efforts are still needed for higher sensitivity and resolution.

Flocculation is one of the most important characteristics of brewing yeast as it allows for the easy and cheap removal of cells after fermentation. The genes responsible for both the Flo1 and NewFlo flocculation phenotypes are well characterized. However, the relationship between Flo protein abundance and flocculation efficiency is poorly understood. In this present study, we used mass spectrometry proteomics to compare the cell wall and whole cell proteomes of commercial yeast strains with diverse flocculation behaviors. We found that the relative abundance of Flo1/5 or Flo10 in the cell wall was correlated with the ability of these yeast strains to flocculate. Analysis of whole cell proteomes identified differences in the proteomes of yeast strains and identified the potential for high metabolic diversity. Characterization of the cell wall and whole cell proteomes during fermentation showed high levels of Flo10 in cells that settled early during fermentation. Our data reveal the diversity of the cell wall and global proteomes of brewing yeast, highlighting the potential biochemical diversity present in yeast that can be utilized in the production of fermented beverages.

Biofilms play a critical role in the colonization, persistence, and pathogenesis of many human pathogens. Multiple mucosa-associated pathogens have evolved a mechanism of rapid adaptation, termed the phasevarion, which facilitates a coordinated regulation of numerous genes throughout the bacterial genome. This epigenetic regulation occurs via phase variation of a DNA methyltransferase, Mod. The phasevarion of nontypeable Haemophilus influenzae (NTHI) significantly affects the severity of experimental otitis media and regulates several disease-related processes. However, the role of the NTHI phasevarion in biofilm formation is unclear. The present study shows that the phasevarions of multiple NTHI clinical isolates regulate in vitro biofilm formation under disease-specific microenvironmental conditions. The impact of phasevarion regulation was greatest under alkaline conditions that mimic those known to occur in the middle ear during disease. Under alkaline conditions, NTHI strains that express the ModA2 methyltransferase formed biofilms with significantly greater biomass and less distinct architecture than those formed by a ModA2-deficient population. The biofilms formed by NTHI strains that express ModA2 also contained less extracellular DNA (eDNA) and significantly less extracellular HU, a DNABII DNA-binding protein critical for biofilm structural stability. Stable biofilm structure is critical for bacterial pathogenesis and persistence in multiple experimental models of disease. These results identify a role for the phasevarion in regulation of biofilm formation, a process integral to the chronic nature of many infections. Understanding the role of the phasevarion in biofilm formation is critical to the development of prevention and treatment strategies for these chronic diseases. IMPORTANCE Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phasevariable regulon, to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.

The adipose tissue has been recognized as an active endocrine organ which can modulate numerous physiological processes such as metabolism, appetite, immunity, and reproduction. The aim of this study was to look for differentially abundant proteins and their biological functions in the abdominal adipose tissue between pre- and postpubertal Brahman heifers. Twelve Brahman heifers were divided into 2 groups and paired on slaughter day. Prepubertal heifers had never ovulated and postpubertal heifers were slaughtered on the luteal phase of their second estrous cycle. After ensuring the occurrence of puberty in postpubertal heifers, abdominal adipose tissue samples were collected. Mass spectrometry proteomic analysis identified 646 proteins and revealed that 171 proteins showed differential abundance in adipose tissue between the pre- and postpuberty groups (adjusted P-value < 0.05). Data are available via ProteomeXchange with identifier PXD009452. Using a list of 51 highly differentially abundant proteins as the target (adjusted P-value < 10(−5)), we found 14 enriched pathways. The results indicated that gluconeogenesis was enhanced when puberty approached. The metabolism of glucose, lipids, and AA in the adipose tissue mainly participated in oxidation and energy supply for heifers when puberty occurred. Our study also revealed the differentially abundant proteins were enriched for estrogen signaling and PI3K-Akt signaling pathways, which are known integrators of metabolism and reproduction. These results suggest new candidate proteins that may contribute to a better understanding of the signaling mechanisms that relate adipose tissue function to puberty. Protein–protein interaction network analysis identified 4 hub proteins that had the highest degrees of connection: PGK1, ALDH5A1, EEF2, and LDHB. Highly connected proteins are likely to influence the functions of all differentially abundant proteins identified, directly or indirectly.

© 2018 American Chemical Society. Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.

We describe an update to the Herschel-SPIRE Fourier-Transform Spectrometer (FTS) calibration for extended sources, which incorporates a correction for the frequency-dependent far-field feedhorn efficiency, $\eta_\mathrm{FF}$. This significant correction affects all FTS extended-source calibrated spectra in sparse or mapping mode, regardless of the spectral resolution. Line fluxes and continuum levels are underestimated by factors of 1.3-2 in the Spectrometer Long-Wavelength band (SLW, 447-1018 GHz; 671-294 $\mu$m) and 1.4-1.5 in the Spectrometer Short-Wavelength band (SSW, 944-1568 GHz; 318-191 $\mu$m). The correction was implemented in the FTS pipeline version 14.1 and has also been described in the SPIRE Handbook since Feb 2017. Studies based on extended-source calibrated spectra produced prior to this pipeline version should be critically reconsidered using the current products available in the Herschel Science Archive. Once the extended-source calibrated spectra are corrected for $\eta_\mathrm{FF}$, the synthetic photometry and the broadband intensities from SPIRE photometer maps agree within 2-4% -- similar levels to the comparison of point-source calibrated spectra and photometry from point-source calibrated maps. The two calibration schemes for the FTS are now self-consistent: the conversion between the corrected extended-source and point-source calibrated spectra can be achieved with the beam solid angle and a gain correction that accounts for the diffraction loss., Comment: 9 pages, 6 figures, 1 table, MNRAS in press

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.