Biology subjects

Scott, N. E.

Publications and source records attributed to Scott, N. E..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have previously been confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a sensitive and streamlined approach for proteome and N-terminome analyses in a single analytical method without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves neo-N- termini identification by >2.5 fold, resulting in identification of >2,882 unique neo-N-termini from limited sample amounts. Within murine spleens, this approach identifies 6,366 proteins and 2,528 unique neo-N-termini, with 235 cleavage events enriched in wild-type compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrates the utility of FAIMS as an accessible method to improve depth and quality of N- terminomics studies.

biochemistry↗

bioRxiv2023

Human gut bacteria tailor extracellular vesicle cargo for the breakdown of diet- and host-derived glycans

Extracellular vesicles (EV) are produced in all three domains of life, and their biogenesis have common ancient origins in eukaryotes and archaea. Although bacterial vesicles were discovered several decades ago and multiple roles have been attributed to them, no mechanism has been established for vesicles biogenesis in bacteria. For this reason, there is a significant level of skepticism about the biological relevance of bacterial vesicles. In Bacteroides thetaiotaomicron (Bt), a prominent member of the human intestinal microbiota, outer membrane vesicles (OMVs) have been proposed to play key physiological roles. By employing outer membrane- and OMV-specific markers fused to fluorescent proteins we visualized OMV biogenesis in live-cells. We performed comparative proteomic analyses to demonstrate that Bt actively tailors its vesicle cargo to optimize the breakdown of diet- and host-derived complex glycans. Surprisingly, our data suggests that OMV are not employed for mucin degradation. We also show that, in Bt, a negatively-charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. We conclude that OMVs are the result of an exquisitely orchestrated mechanism. This work lays the foundation for further investigations into the physiological relevance of OMVs and their roles in gut homeostasis. Furthermore, our work constitutes a roadmap to guide EV biogenesis research in other bacteria.

microbiology↗

bioRxiv2023

Identification of levoglucosan degradation pathways in bacteria and sequence similarity network analysis

Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated from soil by metabolic enrichment using levoglucosan as the sole carbon source. Genome sequencing and proteomics analysis revealed the expression of a series of genes encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan {beta}-eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and an associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident, while the expressed genes contained a range of putative sugar phosphate isomerases/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbours of LgdA revealed that homologues of LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One group of sugar phosphate isomerase/xylose isomerase homologues (named LgdB3) was identified with limited distribution that is mutually exclusive with LgdB2, and we propose that they may fulfill a similar function. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds, suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight diversity within the LGDH pathway, through which bacteria utilize levoglucosan as a nutrient source.

microbiology↗

bioRxiv2022

Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen. Significance StatementCurrently there are no licensed vaccines targeting K. pneumoniae, but several are in development. Two prominent K. pneumoniae surface polysaccharides (capsule and O-antigen) represent attractive vaccine targets; however, the relative efficacy of these potential vaccines against K. pneumoniae strains has not been directly compared. To inform future vaccine development, we evaluate two bioconjugate vaccines (targeting either capsule or O-antigen) demonstrating that each are immunogenic in murine models. However, we find that K. pneumoniae capsule largely inhibits recognition by antibodies raised against O-antigen. Further, we demonstrate that a capsule-based vaccine outperforms an O-antigen vaccine against both cKp and hvKp in murine models of pneumonia and bacteremia, suggesting that capsule-based vaccines offer superior protection from some K. pneumoniae infections.

microbiology↗

bioRxiv2022

An expanded proteomic survey of the human parasite Leishmania major focusing on changes in null mutants of the Golgi GDP-Mannose/Fucose/Arabinopyranose transporter LPG2 or the mitochondrial fucosyltransferase FUT1

The trypanosomatid protozoan parasite Leishmania has a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes in Leishmania, encoding the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporter LPG2, and the mitochondrial fucosyltransferase FUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally known Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both{Delta} lpg2- and{Delta} fut1s mutants with FUT1-dependent changes linked to marked alterations within mitochondrial associated proteins while LPG2-dependent changes impacted many pathways including the secretory pathway. While the FUT1 enzyme has been shown to fucosylate peptides in vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans within Leishmania glycopeptides isolated using HILIC enrichment. Combined this work provides a critical resource for the community on the observable Leishmania proteome as well as highlights phenotypic changes associated with LPG2 or FUT1 ablation which may guide the development of future therapeutics. ImportanceLeishmania is a widespread trypanosomatid protozoan parasite of humans with [~]12 million cases ranging from mild to fatal, and hundreds of millions asymptomatically infected. This work advances knowledge of the experimental proteome by nearly 2 fold, to more than 6500 proteins a great resource to investigators seeking to decode how this parasite is transmitted and causes disease, and new targets for therapeutic intervention. The ultradeep proteomics approach identified potential proteins underlying the persistence without pathology phenotype of deletion mutants of the Golgi nucleotide transporter LPG2, showing many alterations and several candidates. Studies of a rare deletion mutant of the mitochondrial fucosyltransferase FUT1 revealed changes underlying its strong mitochondrial dysfunction, but did not reveal examples of fucosylation of either peptides or N-glycans. This suggests this vital proteins elusive target(s) may be more complex than the methods used could detect, or may not be a protein, perhaps another glycoconjugate or glycolipid.

microbiology↗

bioRxiv2022

InvL, an invasin-like adhesin, is a type II secretion system substrate required for Acinetobacter baumannii uropathogenesis.

Acinetobacter baumannii is an opportunistic pathogen of growing concern, as isolates are commonly multidrug resistant. While A. baumannii is most frequently associated with pulmonary infections, a significant proportion of clinical isolates come from urinary sources, highlighting its uropathogenic potential. The type II secretion system (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in urinary tract infection (UTI) remains unknown. Herein, we used a catheter-associated UTI (CAUTI) model to demonstrate that a modern urinary isolate, UPAB1, requires the T2SS for full virulence. A proteomic screen to identify putative UPAB1 T2SS effectors revealed an uncharacterized lipoprotein with structural similarity to the intimin-invasin family, which serve as type V secretion system (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the {beta}-barrel domain associated with T5SSs, but was confirmed to require the T2SS for both surface localization and secretion. This makes InvL the first identified T2SS effector belonging to the intimin-invasin family. InvL was confirmed to be an adhesin, as the protein bound to extracellular matrix components and mediated adhesion to urinary tract cell lines in vitro. Additionally, the invL mutant was attenuated in the CAUTI model, indicating a role in Acinetobacter uropathogenesis. Finally, bioinformatic analyses revealed that InvL is present in nearly all clinical isolates belonging to international clone 2, a lineage of significant clinical importance. In all, we conclude that the T2SS substrate InvL is an adhesin required for A. baumannii uropathogenesis. IMPORTANCEWhile pathogenic Acinetobacter can cause various infections, we recently found that 20% of clinical isolates come from urinary sources. Despite the clinical relevance of Acinetobacter as a uropathogen, few virulence factors involved in urinary tract colonization have been defined. Herein, we identify a novel type II secretion system effector, InvL, which is required for full uropathogenesis by a modern urinary isolate. Though InvL has predicted structural similarity to the intimin-invasin family of autotransporter adhesins, InvL is predicted to be anchored to the membrane as a lipoprotein. Similar to other invasin homologs however, we demonstrate that InvL is a bona fide adhesin capable of binding extracellular matrix components and mediating adhesion to urinary tract cell lines. In all, this work establishes InvL as an adhesin important for Acinetobacters urinary tract virulence, and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.

microbiology↗

bioRxiv2021

Getting more out of co-immunoprecipitation mass spectrometry experiments by reducing interference using FAIMS.

Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of protein interaction networks. In instances where a suitable antibody is not available, it is common to graft synthetic tags onto target protein sequences and allowing the use of commercially available antibodies for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the selective elution of protein complexes, competitive displacement using a large molar excess of the tag peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis can be applied to FLAG-Tag co-immunoprecipitation experiment to increase the depth of protein coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of interacting proteins and as a result, deeper biological insights, without the need for additional sample handling or altering sample preparation protocols.

biochemistry↗

bioRxiv2021

Burkholderia PglL enzymes are serine preferring oligosaccharidetransferases which target conserved proteins across the Burkholderia genus

Glycosylation is increasingly recognised as a common protein modification within bacterial proteomes. While great strides have been made in identifying species that contain glycosylation systems, our understanding of the proteins and sites targeted by these enzymes is far more limited. Within this work we explore the conservation of glycoproteins and O-linked glycosylation sites across the pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach we generate high-confidence glycoproteomes and associated glycosylation sites in two widely utilized B. cenocepacia strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively at serine residues and that glycoproteins/glycosylation sites are highly conserved across 294 publicly available B. cenocepacia genomes. Consistent with this we demonstrate that the substitution of Serine for Threonine residues in a model protein results in a dramatic decrease in glycosylation efficiency by the oligosaccharidetransferase pglLBC even when pglLBC is overexpressed. This preference for glycosylation at Serine residues is observed across at least 9 Burkholderia glycoproteomes supporting that Serine is the dominant residue targeted by pglL-mediated glycosylation across the Burkholderia genus. Using population genomics we observe that pglL targeted glycosylated proteins are common across Burkholderia species. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharidetransferases that target conserved and shared protein substrates across the Burkholderia genus.

microbiology↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

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 for N- and O-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.

biochemistry↗

bioRxiv2020

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for diagnosis of glanders infections

We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that mainly infects horses, mules, donkeys and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100 % specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections. IMPORTANCEGlanders is a severe zoonotic disease caused by the Gram-negative bacterium Burkholderia mallei, which affects horses, mules and donkeys, as well as humans. B. mallei is also considered a category B biothreat agent. Due to insufficient pathognomonic symptoms in the early stages of glanders, diagnosis can be difficult. Complement fixation is the most accurate and reliable serological test prescribed by the World Organization for Animal Health; however, this test has a considerable number of false-positive results. We have recently described a conserved protein O-glycosylation pathway present in all species of the Burkholderia genus; we also demonstrated that Burkholderia-infected humans develop anti-glycan antibodies. Here, we exploited this system to construct and evaluate a synthetic glycoengineered protein antigen as a diagnostic tool for glanders. Our results show 100 % specificity in the detection of antibodies from infected horses, indicating that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

microbiology↗

bioRxiv2020

What are we missing by using hydrophilic enrichment? Improving bacterial glycoproteome coverage using total proteome and FAIMS analysis.

Hydrophilic Interaction Liquid Chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterisation of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of short comings including requiring large amounts of starting material, potentially introducing chemical artefacts such as formylation, and biasing/under-sampling specific classes of glycopeptides. Here we investigate HILIC enrichment independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (B. cenocepacia, B. dolosa and B. ubonensis) we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments yet are readily identified in whole proteome samples. Using Field Asymmetric Waveform IMS (FAIMS) fractionation we show that at low compensation voltages (CVs) short aliphatic glycopeptides can be enriched from complex samples providing an alternative means to identify glycopeptides recalcitrant to hydrophilic based enrichment. Combining whole proteome and FAIMS analysis we show that the observable glycoproteome of these Burkholderia species is at least 30% larger than initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also enrichable at low FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.

microbiology↗

bioRxiv2020

The glycoprotease CpaA secreted by medically relevant Acinetobacter species targets multiple O-linked host glycoproteins

Glycans decorate proteins and affect their biological function, including protection against proteolytic degradation. However, pathogenic, and commensal bacteria have evolved specific glycoproteases that overcome the steric impediment posed by carbohydrates, cleaving glycoproteins precisely at their glycosylation site(s). Medically relevant Acinetobacter strains employ their type II secretion system (T2SS) to secrete the glycoprotease CpaA, which contributes to virulence. Previously, CpaA was shown to cleave two O-linked glycoproteins, factors V and XII, leading to reduced blood coagulation. In this work, we show that CpaA cleaves a broader range of O-linked human glycoproteins, including several glycoproteins involved in complement activation, such as CD55 and CD46. However, only CD55 was removed from the cell surface, while CD46 remained unaltered during the A. nosocomialis infection assay. We show that CpaA has a unique consensus target sequence that consists of a glycosylated serine or threonine residue after a proline residue (P-S/T), and its activity is not affected by sialic acids. Molecular modeling and mutagenesis analysis of CpaA suggest that the indole ring of Trp493 and the ring of the Pro residue in the substrate form a key interaction that contributes to CpaA sequence selectivity. Similar bacterial glycoproteases have recently gained attention as tools for proteomic analysis of human glycoproteins, and CpaA appears to be a robust and attractive new component of the glycoproteomics toolbox. Combined, our work provides insight into the function and possible application of CpaA, a member of a widespread class of broad-spectrum bacterial glycoproteases involved in host-pathogen interactions. IMPORTANCECpaA is a glycoprotease expressed by members of the Acinetobacter baumannii-calcoaceticus complex and it is the first bona fide secreted virulence factor identified in these species. Here, we show that CpaA cleaves multiple targets precisely at O-glycosylation sites preceded by a Pro residue. This feature, together with the observation that sialic acid does not impact CpaA activity, makes of this enzyme an attractive tool for the analysis of O-linked human protein for biotechnical and diagnostic purposes. Previous work identified proteins involved in blood coagulation as targets of CpaA. Our work broadens the set of targets of CpaA, pointing towards additional roles in bacteria-host interactions. We propose that CpaA belongs to an expanding class of functionally-defined glycoproteases that targets multiple O-linked host glycoproteins.

microbiology↗

bioRxiv2020

Proteomic identification of Coxiella burnetii effector proteins targeted to the host cell mitochondria during infection

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver a cohort of bacterial proteins, termed effector proteins into the host cell during infection by sophisticated protein translocation systems which manipulate cellular processes and functions. Despite the importance of these proteins during infection the functional contribution of individual effectors is poorly characterised, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here we present development of a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify 4 novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localisation of ectopically expressed proteins in epithelial cells confirmed the mitochondrial localisation, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein is imported into mitochondria and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to the study of intracellular host-pathogen interactions occurring during infection, providing a robust strategy to examine the sub-cellular localisation of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

cell biology↗

bioRxiv2020

Open database searching enables the identification and comparison of glycoproteomes without defining glycan compositions prior to searching

Mass spectrometry has become an indispensable tool for the characterisation of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. While glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses prior to database searching. Utilising this approach, we demonstrate how wide tolerance searching can be used to compare glycan utilisation across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.

microbiology↗

bioRxiv2019

Trefoil factors share a lectin activity that defines their role in mucus

The trefoil factors (TFFs) are disulfide-rich mucosal peptides that protect the epithelium by promoting cell migration and increasing the viscoelasticity of the mucosa. Here we show that all TFFs are divalent lectins that recognise the GlcNAc--1,4-Gal disaccharide, which terminates type-III mucin-like O-glycans. Structural, mutagenic and biophysical data support a model of mucus viscoelasticity that features non-covalent cross-linking of glycoproteins by TFFs.

biochemistry↗

bioRxiv2019

Loss of O-linked protein glycosylation in Burkholderia cenocepacia impairs biofilm formation and siderophore production via alteration of quorum sensing regulation

O-linked protein glycosylation is a conserved feature of the Burkholderia genus. For Burkholderia cenocepacia, the addition of the trisaccharide {beta}-Gal-(1,3)--GalNAc-(1,3)-{beta}-GalNAc to membrane exported proteins is required for virulence and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. This study demonstrates that the global B. cenocepacia proteome undergoes dramatic changes consistent with alterations in global transcriptional regulation in the absence of glycosylation. Using luciferase reporter assays and DNA cross-linking analysis, we confirm the repression of the master quorum sensing regulon CepR/I in response to the loss of glycosylation, which leads to the abolition of biofilm formation, defects in siderophore production, and reduced virulence. The abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants except for BCAL1086 and BCAL2974, which were found in reduced amount, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, as well as for reduced virulence and siderophore production. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins.\n\nIMPORTANCEProtein glycosylation is increasingly recognised as a common protein modification in bacterial species. Despite this commonality our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that virulence attenuation observed within glycosylation-null B. cenocepacia strains are consistent with alteration of the master virulence regulator CepR. The repression of CepR transcription and its associated phenotypes support a model in which the virulence defects observed in glycosylation-null strains are at least in part due to transcriptional alteration and not the direct result of the loss of glycosylation per-se. This research unravels the pleotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

microbiology↗

bioRxiv2018

A dual motif mediates outer-membrane translocation and packing of glycosidases into Bacteroides Outer Membrane Vesicles.

Outer membrane vesicles (OMV) are spherical structures derived from the outer membrane (OM) of Gram-negative bacteria. Bacteroides spp. are prominent components of the human gut microbiota, and OMV produced with these species are proposed to play key roles in gut homeostasis. OMV biogenesis in Bacteroides is a poorly understood process. Here, we revisited the protein composition of B. theta OMVs by mass spectrometry. We confirmed that OMVs produced by this organism contain large quantities of glycosidases and proteases, with most of them being lipoproteins. We found that most of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as the sus operon. We examined the subcellular localization of the components of the Sus system, and found that the alpha-amylase SusG is highly enriched in OMVs while the oligosaccharide importer SusC remains mostly in the OM. We show that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES) that mediates translocation of SusG from the periplasmic face of the OM towards the extracellular milieu and is required for SusG to localize preferentially to OMVs. We also show that surface-exposed SusG in OMVs is active and can rescue growth of bacterial cells incapable of growing on starch as only carbon source. Our results support the role of OMVs as \"public goods\" that can be utilized by other organisms with different metabolic capabilities.\n\nIMPORTANCESpecies from the Bacteroides genus are predominant members of the human gut microbiota. OMVs in Bacteroides have been shown to be important for the homeostasis of complex host-commensal relationships, mainly involving immune tolerance and protection from disease. OMVs carry many enzymatic activities involved in the cleavage of complex polysaccharides and have been proposed as public goods that can provide growth to other bacterial species by release of polysaccharide breakdown products into the gut lumen. Nevertheless, the mechanistic nature of OMV biogenesis is unclear for Bacteroides spp. This works shows the presence of a negatively-charged rich amino acid dual motif that is required for efficient packing of the surface-exposed alpha-amylase SusG into OMVs. Discovery of this motif (LES) is the first step in the generation of tailor made probiotic interventions that can exploit LES-related sequences to generate Bacteroides strains displaying proteins of interest in OMVs.

microbiology↗

bioRxiv2018

An atlas of protein-protein interactions across mammalian tissues

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the complete network of biologically relevant protein-protein interactions, the interactome, has therefore been a central objective of high-throughput biology. Yet, because widely used methods for high-throughput interaction discovery rely on heterologous expression or genetically manipulated cell lines, the dynamics of protein interactions across physiological contexts are poorly understood. Here, we use a quantitative proteomic approach combining protein correlation profiling with stable isotope labelling of mammals (PCP SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide the first proteome-scale survey of interactome dynamics across mammalian tissues, revealing over 27,000 unique interactions with an accuracy comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewiring of protein interactions across tissues is widespread, and is poorly predicted by gene expression or coexpression. Rewired proteins are tightly regulated by multiple cellular mechanisms and implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

systems biology↗