ORCID Profile
0000-0003-2556-8316
Current Organisation
University of Melbourne
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Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Biochemistry and Cell Biology | Biochemistry and Cell Biology not elsewhere classified | Bacteriology |
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in the Agricultural and Veterinary Sciences | Human Biological Preventatives (e.g. Vaccines)
Publisher: Proceedings of the National Academy of Sciences
Date: 24-01-2022
Abstract: Sulfoquinovose, a sulfosugar derivative of glucose, is produced by most photosynthetic organisms and contains up to half of all sulfur in the biosphere. Several pathways for its breakdown are known, though they provide access to only half of the carbon in sulfoquinovose and none of its sulfur. Here, we describe a fundamentally different pathway within the plant pathogen Agrobacterium tumefaciens that features oxidative desulfurization of sulfoquinovose to access all carbon and sulfur within the molecule. Biochemical and structural analyses of the pathway’s key proteins provided insights how the sulfosugar is recognized and degraded. Genes encoding this sulfoquinovose monooxygenase pathway are present in many plant pathogens and symbionts, alluding to a possible role for sulfoquinovose in plant host–bacteria interactions.
Publisher: IOP Publishing
Date: 07-2012
Publisher: Springer Science and Business Media LLC
Date: 11-2021
DOI: 10.1038/S41592-021-01309-X
Abstract: Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometry based glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O -glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved ‘high-coverage’ and ‘high-accuracy’ glycoproteomics search solutions. This study concludes that erse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.
Publisher: Wiley
Date: 06-04-2015
DOI: 10.1111/MMI.12986
Abstract: Multiple species within the Acinetobacter genus are nosocomial opportunistic pathogens of increasing relevance worldwide. Among the virulence factors utilized by these bacteria are the type IV pili and a protein O-glycosylation system. Glycosylation is mediated by O-oligosaccharyltransferases (O-OTases), enzymes that transfer the glycan from a lipid carrier to target proteins. O-oligosaccharyltransferases are difficult to identify due to similarities with the WaaL ligases that catalyze the last step in lipopolysaccharide synthesis. A bioinformatics analysis revealed the presence of two genes encoding putative O-OTases or WaaL ligases in most of the strains within the genus Acinetobacter. Employing A. nosocomialis M2 and A. baylyi ADP1 as model systems, we show that these genes encode two O-OTases, one devoted uniquely to type IV pilin, and the other one responsible for glycosylation of multiple proteins. With the exception of ADP1, the pilin-specific OTases in Acinetobacter resemble the TfpO/PilO O-OTase from Pseudomonas aeruginosa. In ADP1 instead, the two O-OTases are closely related to PglL, the general O-OTase first discovered in Neisseria. However, one of them is exclusively dedicated to the glycosylation of the pilin-like protein ComP. Our data reveal an intricate and remarkable evolutionary pathway for bacterial O-OTases and provide novel tools for glycoengineering.
Publisher: Proceedings of the National Academy of Sciences
Date: 28-08-2023
Abstract: The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow–derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress–induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.
Publisher: Research Square Platform LLC
Date: 16-02-2022
DOI: 10.21203/RS.3.RS-1358538/V1
Abstract: C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 C-mannosyltransferase in the endoplasmic reticulum and that DPY19 -deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. P. falciparum gametogenesis was imaged in its entirety in four dimensions using lattice light-sheet microscopy. This revealed defects in egress and exflagellation for D DPY19 microgametes. While exflagellation was diminished, D DPY19 microgametes still fertilized macrogametes, forming ookinetes but these were abrogated for mosquito infection. The gametogenesis defects corresponded with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum , and the ookinete defect was concordant with defective CTRP secretion on the D DPY19 background. Genetic complementation of DPY19 restored ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 in the early secretory pathway ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.
Publisher: Wiley
Date: 25-11-2009
Abstract: C ylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed C ylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.
Publisher: American Chemical Society (ACS)
Date: 30-10-0027
Publisher: Cold Spring Harbor Laboratory
Date: 20-04-2021
DOI: 10.1101/2021.04.20.440587
Abstract: 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 pglL BC even when pglL BC 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.
Publisher: Springer Science and Business Media LLC
Date: 21-02-2019
DOI: 10.1038/S41467-019-08869-9
Abstract: Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O -linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.
Publisher: American Society for Microbiology
Date: 20-07-2020
DOI: 10.1128/AEM.00750-20
Abstract: Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, Rhizobium leguminosarum SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.
Publisher: Public Library of Science (PLoS)
Date: 09-01-0100
DOI: 10.1371/JOURNAL.PPAT.1009802
Abstract: Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A . baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium’s pathobiology, which remains poorly understood. A . baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A . baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A . baumannii . We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A . baumannii .
Publisher: Springer Science and Business Media LLC
Date: 13-05-2020
DOI: 10.1038/S41467-020-16223-7
Abstract: The mucosal epithelium secretes a host of protective disulfide-rich peptides, including the trefoil factors (TFFs). The TFFs increase the viscoelasticity of the mucosa and promote cell migration, though the molecular mechanisms underlying these functions have remained poorly defined. Here, we demonstrate that all TFFs are alent lectins that recognise the GlcNAc-α-1,4-Gal disaccharide, which terminates some mucin-like O-glycans. Degradation of this disaccharide by a glycoside hydrolase abrogates TFF binding to mucins. Structural, mutagenic and biophysical data provide insights into how the TFFs recognise this disaccharide and rationalise their ability to modulate the physical properties of mucus across different pH ranges. These data reveal that TFF activity is dependent on the glycosylation state of mucosal glycoproteins and alludes to a lectin function for trefoil domains in other human proteins.
Publisher: Springer Science and Business Media LLC
Date: 29-07-2022
DOI: 10.1038/S41467-022-32076-8
Abstract: Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19 -deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in Δ DPY19 gametocyte egress and exflagellation. While egress is diminished, Δ DPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum , and the ookinete defect is concordant with defective CTRP secretion on the Δ DPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2550-6_25
Abstract: Large-scale analysis of protein N- and O-linked glycosylation by mass spectrometry has traditionally been performed in eukaryotes by parallel approaches aimed at elucidating glycan structures (glycomics) and their formerly glycosylated peptides (glycoproteomics) without reference to their intact state. Such analyses depend heavily on commercial glycosidases (e.g. protein N-glycosidase F) that can remove glycans from the peptide backbone for separate analyses. Bacterial glycosylation has only recently been identified as a widespread phenomenon. In many cases however, unique bacterial sugars preclude enzymatic removal, therefore ultimately requiring a site-specific approach for intact glycopeptide analysis. Here, we describe protocols for the enrichment of bacterial glycopeptides using zwitterionic-hydrophilic interaction liquid chromatography (ZIC-HILIC) and their analysis using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) with collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) fragmentation for glycan structure elucidation and glycopeptide identification.
Publisher: Cold Spring Harbor Laboratory
Date: 15-06-2021
DOI: 10.1101/2021.06.15.448610
Abstract: 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 s le handling or altering s le preparation protocols.
Publisher: Elsevier BV
Date: 04-2019
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-02-2021
Abstract: A new technology, SUGAR-seq, enables simultaneous detection of surface glycans, epitopes, transcripts, and TCR repertoire.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 05-2020
Abstract: CDK12 and CDK13 regulate POLII elongation rate and processivity and influence the selection of transcription termination sites.
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.MOLMED.2017.10.003
Abstract: Bacterial effector proteins are a specialized class of secreted proteins that are translocated directly into the host cytoplasm by bacterial pathogens. Effector proteins have erse activities and targets, and many mediate post-translational modifications of host proteins. Effector proteins offer potential in novel biotechnological and medical applications as enzymes that may modify human proteins. Here, we discuss the mechanisms used by effectors to subvert the human host through blocking, blunting, or subverting immune mechanisms. This capacity allows bacteria to control host cell function to support pathogen survival, replication and dissemination to other hosts. In addition, we highlight that knowledge of effector protein activity may be used to develop chemical inhibitors as a new approach to treat bacterial infections.
Publisher: American Society for Microbiology
Date: 27-10-2021
Abstract: Tannerella forsythia is an oral pathogen associated with severe forms of periodontal disease characterized by destruction of the tooth’s supporting tissues, including the bone. The bacterium releases a variety of proteins associated with virulence on the surface of outer membrane vesicles.
Publisher: Cold Spring Harbor Laboratory
Date: 28-05-2020
DOI: 10.1101/2020.05.28.121236
Abstract: 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 in idual 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.
Publisher: Portland Press Ltd.
Date: 07-08-2017
DOI: 10.1042/BCJ20160797
Abstract: Many secreted bacterial effector proteins play a critical role in host–pathogen interactions by mediating a variety of post-translational modifications, some of which do not occur natively within the eukaryotic proteome. The characterization of bacterial effector protein activity remains an important step to understanding the subversion of host cell biology during pathogen infection and although molecular biology and immunochemistry remain critical tools for gaining insights into bacterial effector functions, increasingly mass spectrometry (MS) and proteomic approaches are also playing an indispensable role. The focus of this editorial is to highlight the strengths of specific MS approaches and their utility for the characterization of bacterial effector activity. With the capability of new generation MS instrumentation, MS-based technologies can provide information that is inaccessible using traditional molecular or immunochemical approaches.
Publisher: American Chemical Society (ACS)
Date: 09-10-2013
DOI: 10.1021/PR4007365
Abstract: Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.
Publisher: Cold Spring Harbor Laboratory
Date: 19-07-2023
DOI: 10.1101/2023.07.18.549248
Abstract: 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 .5 fold, resulting in identification of ,882 unique neo-N-termini from limited s le 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.
Publisher: Elsevier BV
Date: 06-2017
Publisher: Cold Spring Harbor Laboratory
Date: 03-04-2023
DOI: 10.1101/2023.04.03.535451
Abstract: 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.
Publisher: Informa UK Limited
Date: 02-2009
Abstract: C ylobacter species are a major cause of disease in mammalian systems. The most common human etiological agent within this genus is C ylobacter jejuni - the leading cause of bacterial gastroenteritis in the developed world. While this organism has been extensively studied at the cellular level, and the genome sequences of several strains have now been elucidated, little is known regarding the role of in idual proteins in virulence processes, such as adhesion, colonization and toxicity towards host cells. Proteomics encompasses the global analysis of proteins at the organism level. The technologies included under this term have now started to be utilized for understanding how C ylobacter species respond to changes in the environment, with an emphasis on the human host, as well as to map subcellular locations of proteins, in particular those that are surface-associated. C. jejuni is also of great significance as, unlike most other bacteria, it is able to post-translationally modify its proteins. The analysis of such proteins represents a major challenge in understanding this organism at the proteomic and cellular levels. This review will examine the state-of-the-art in C ylobacter proteomics, as well as provide insights into strategies that need to be undertaken to provide a comprehensive understanding of this organism at the molecular and functional level.
Publisher: Cold Spring Harbor Laboratory
Date: 29-01-2023
DOI: 10.1101/2023.01.28.526070
Abstract: 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 β-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 ersity within the LGDH pathway, through which bacteria utilize levoglucosan as a nutrient source.
Publisher: Proceedings of the National Academy of Sciences
Date: 17-08-2022
Abstract: Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical “proline-rich” motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal “DY” segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.
Publisher: Elsevier BV
Date: 09-2014
Publisher: Public Library of Science (PLoS)
Date: 07-06-2012
Publisher: Elsevier BV
Date: 11-2012
Publisher: Cold Spring Harbor Laboratory
Date: 15-03-2021
DOI: 10.1101/2021.03.14.435332
Abstract: 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 erse 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.
Publisher: Wiley
Date: 11-02-2019
DOI: 10.1111/MMI.14182
Abstract: Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking in idual Cpx-regulated genes showed that the chaperone rotease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.
Publisher: American Association for Cancer Research (AACR)
Date: 16-06-2022
DOI: 10.1158/1055-9965.EPI-22-0096
Abstract: Over 20 susceptibility single-nucleotide polymorphisms (SNP) have been identified for esophageal adenocarcinoma (EAC) and its precursor, Barrett esophagus (BE), explaining a small portion of heritability. Using genetic data from 4,323 BE and 4,116 EAC patients aggregated by international consortia including the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON), we conducted a comprehensive transcriptome-wide association study (TWAS) for BE/EAC, leveraging Genotype Tissue Expression (GTEx) gene-expression data from six tissue types of plausible relevance to EAC etiology: mucosa and muscularis from the esophagus, gastroesophageal (GE) junction, stomach, whole blood, and visceral adipose. Two analytical approaches were taken: standard TWAS using the predicted gene expression from local expression quantitative trait loci (eQTL), and set-based SKAT association using selected eQTLs that predict the gene expression. Although the standard approach did not identify significant signals, the eQTL set–based approach identified eight novel associations, three of which were validated in independent external data (eQTL SNP sets for EXOC3, ZNF641, and HSP90AA1). This study identified novel genetic susceptibility loci for EAC and BE using an eQTL set–based genetic association approach. This study expanded the pool of genetic susceptibility loci for EAC and BE, suggesting the potential of the eQTL set–based genetic association approach as an alternative method for TWAS analysis.
Publisher: American Society for Microbiology
Date: 23-02-2022
DOI: 10.1128/SPECTRUM.01502-21
Abstract: Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability.
Publisher: Elsevier BV
Date: 10-2017
Publisher: American Chemical Society (ACS)
Date: 11-10-2021
Publisher: EMBO
Date: 2017
Abstract: Protein–protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling ( PCP ), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas‐mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome‐wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates ( TAILS ) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.
Publisher: Elsevier BV
Date: 06-2019
Publisher: Wiley
Date: 2008
Abstract: C ylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.
Publisher: American Society for Microbiology
Date: 31-08-2021
Abstract: The pathogen Acinetobacter baumannii is considered an urgent threat to human health. A. baumannii is highly resistant to treatment with antibiotics, in part due to its protective cell envelope. This bacterium is only distantly related to other bacterial pathogens, so its cell envelope has distinct properties and contains components distinct from those of other bacteria that support its function.
Publisher: Cold Spring Harbor Laboratory
Date: 07-03-2020
DOI: 10.1101/2020.03.05.976167
Abstract: Fundamental aspects of SARS-CoV-2 biology remain to be described, having the potential to provide insight to the response effort for this high-priority pathogen. Here we describe the first native RNA sequence of SARS-CoV-2, detailing the coronaviral transcriptome and epitranscriptome, and share these data publicly. A data-driven inference of viral genetic features and evolutionary rate is also made. The rapid sharing of sequence information throughout the SARS-CoV-2 pandemic represents an inflection point for public health and genomic epidemiology, providing early insights into the biology and evolution of this emerging pathogen.
Publisher: Springer Science and Business Media LLC
Date: 04-07-2019
DOI: 10.1007/S10719-019-09875-1
Abstract: Protein glycosylation is increasingly recognised as an essential requirement for effective microbial infections. Within microbial pathogen's protein glycosylation is used for both defensive and offensive purposes enabling pathogens to fortify themselves against the host immune response or to disarm the host's ability to resist infection. Although microbial protein glycosylation systems have been recognised for nearly two decades only recently has the true extend of protein glycosylation within microbes begun to be appreciated. A key enabler for this conceptual shift has been the development and application of modern approaches for the characterisation of glycosylation. Over the last decade my research has focused on the development of proteomic tools to probe microbial glycosylation. By developing workflows for glycopeptide enrichment and identification we have demostrated that it is now possible to characterise the glycoproteomes of microbial species in a truely high-throughput manner. Using these high-throughput approaches we have shown a number of bacterial species modify multiple proteins including members of the C ylobacter genus and the pathogens A. baumannii, R. solanacearum and B. cenocepacia. These studies have established that bacterial glycosylation is widespread, that glycan microheterogeneity is common place and that an extensive array of glycans are used to decorate protein compared to Eukaryotic glycosylation systems. Excitingly these approaches developed to characterise O- and N-linked bacterial glycosylation systems are equally amenable to studying newly discovered forms of microbial glycosylation such as Arginine glycosylation as well as glycosylation within the parasitic eukaryotic organisms T. gondii and P. falciparum. This work demonstrates that MS approaches can now be considered an indispensable tool for the elucidation and tracking of microbial glycosylation events.
Publisher: Elsevier BV
Date: 07-2015
Publisher: Cold Spring Harbor Laboratory
Date: 03-02-2022
DOI: 10.1101/2022.02.01.478765
Abstract: 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 t ype II s ecretion s ystem (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in u rinary t ract i nfection (UTI) remains unknown. Herein, we used a c atheter- a ssociated 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 t ype V s ecretion s ystem (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the β-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. While 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 Acinetobacter ’s urinary tract virulence, and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.
Publisher: Public Library of Science (PLoS)
Date: 16-06-2021
DOI: 10.1371/JOURNAL.PPAT.1009658
Abstract: During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E . coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N -acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.
Publisher: Elsevier BV
Date: 02-2011
Publisher: Proceedings of the National Academy of Sciences
Date: 27-08-2019
Abstract: Klebsiella pneumoniae is considered a nosocomial pathogen, usually infecting immunocompromised patients. However, a pathotype of K. pneumoniae , termed hypervirulent K. pneumoniae (hv Kp ), has emerged and is spreading throughout the community, causing severe, often fatal, disease in healthy in iduals. Moreover, reports on multidrug-resistant hv Kp isolates are increasing in frequency. It is imperative that strategies to combat hv Kp begin immediately to prevent further dissemination of this new class of “superbugs.” Here, we show that bioconjugate vaccines targeting the capsule of hv Kp can provide immunity and protection against extremely lethal hv Kp strains. Further, we demonstrate that bioconjugation is a promising technology for rapid development of efficacious vaccines against emerging bacterial threats.
Publisher: Springer Science and Business Media LLC
Date: 26-11-2018
DOI: 10.1038/S41598-018-35717-5
Abstract: Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped viruses (e.g. hepatitis A virus) can acquire an envelope during infection by hijacking host cellular pathways. In this study, we examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket paralysis virus (CrPV). Utilizing quantitative proteomics, we demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the erse strategies of nonenveloped viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.
Publisher: Springer Science and Business Media LLC
Date: 10-12-2021
Publisher: Springer New York
Date: 25-11-2016
DOI: 10.1007/978-1-4939-6536-6_18
Abstract: The glycosylation systems of C ylobacter jejuni (C. jejuni) are considered archetypal ex les of both N- and O-linked glycosylations in the field of bacterial glycosylation. The discovery and characterization of these systems both have revealed important biological insight into C. jejuni and have led to the refinement and enhancement of methodologies to characterize bacterial glycosylation. In general, mass spectrometry-based characterization has become the preferred methodology for the study of C. jejuni glycosylation because of its speed, sensitivity, and ability to enable both qualitative and quantitative assessments of glycosylation events. In these experiments the generation of insightful data requires the careful selection of experimental approaches and mass spectrometry (MS) instrumentation. As such, it is essential to have a deep understanding of the technologies and approaches used for characterization of glycosylation events. Here we describe protocols for the initial characterization of C. jejuni glycoproteins using protein- eptide-centric approaches and discuss considerations that can enhance the generation of insightful data.
Publisher: American Society for Clinical Investigation
Date: 08-03-2021
Publisher: Cold Spring Harbor Laboratory
Date: 28-08-2020
DOI: 10.1101/2020.08.27.271338
Abstract: 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 s les 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 s les 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. Glanders 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.
Publisher: Elsevier BV
Date: 03-2017
Publisher: Cold Spring Harbor Laboratory
Date: 26-10-2023
Publisher: Springer Science and Business Media LLC
Date: 04-02-2021
DOI: 10.1038/S41589-020-00727-W
Abstract: Tryptophan C-mannosylation is an unusual co-translational protein modification performed by metazoans and apicomplexan protists. The prevalence and biological functions of this modification are poorly understood, with progress in the field h ered by a dearth of convenient tools for installing and detecting the modification. Here, we engineer a yeast system to produce a erse array of proteins with and without tryptophan C-mannosylation and interrogate the modification's influence on protein stability and function. This system also enabled mutagenesis studies to identify residues of the glycosyltransferase and its protein substrates that are crucial for catalysis. The collection of modified proteins accrued during this work facilitated the generation and thorough characterization of monoclonal antibodies against tryptophan C-mannosylation. These antibodies empowered proteomic analyses of the brain C-glycome by enriching for peptides possessing tryptophan C-mannosylation. This study revealed many new modification sites on proteins throughout the secretory pathway with both conventional and non-canonical consensus sequences.
Publisher: American Chemical Society (ACS)
Date: 04-08-2021
DOI: 10.1021/JACS.1C05567
Abstract: The biological functions of tryptophan C-mannosylation are poorly understood, in part, due to a dearth of methods for preparing pure glycopeptides and glycoproteins with this modification. To address this issue, efficient and scalable methods are required for installing this protein modification. Here, we describe unique Ni-catalyzed cross-coupling conditions that utilize photocatalysis or a Hantzsch ester photoreductant to couple glycosyl halides with (hetero)aryl bromides, thereby enabling the α-C-mannosylation of 2-bromo-tryptophan, peptides thereof, and (hetero)aryl bromides more generally. We also report that 2-(α-d-mannopyranosyl)-L-tryptophan undergoes facile anomerization in the presence of acid: something that must be considered when preparing and handling peptides with this modification. These developments enabled the first automated solid-phase peptide syntheses of C-mannosylated glycopeptides, which we used to map the epitope of an antibody, as well as providing the first verified synthesis of
Publisher: American Chemical Society (ACS)
Date: 11-09-2009
DOI: 10.1021/PR900544X
Abstract: C ylobacter jejuni is the most common cause of bacterial gastroenteritis in the developed world. Immunoproteomics highlighted a 42-45 kDa antigen that comigrated on two-dimensional (2-DE) gels with the C. jejuni major outer membrane protein (MOMP). Predictive analysis revealed two candidates for the identity of the antigen, the most likely of which was the surface-associated lipoprotein, JlpA. Recombinant JlpA (rJlpA) reacted with patient sera, confirming that JlpA is antigenic. Polyclonal antibodies raised against rJlpA reacted against 3 JlpA mass variants from multiple C. jejuni. These variants differed by approximately 1.5 kDa, suggesting the presence of the N-linked C. jejuni glycan on two sites. Soybean agglutinin affinity and 2-DE purified 2 JlpA glycoforms (43.5 and 45 kDa). Their identities were confirmed using mass spectrometry following trypsin digest. Glycopeptides within JlpA variants were identified by proteinase-K digestion, graphite micropurification and MS-MS. Sites of glycosylation were confirmed as asparagines 107 and 146, both of which are flanked by the N-linked sequon. Sequence analysis confirmed that the N146 sequon is conserved in all C. jejuni genomes examined to date, while the N107 sequon is absent in the reference strain NCTC 11168. Western blotting confirmed the presence of only a single JlpA glycoform in both virulent (O) and avirulent (GS) isolates of NCTC 11168. MS analysis showed that JlpA exists as 3 discrete forms, unmodified, glycosylated at N146, and glycosylated at both N(146/107), suggesting glycan addition at N146 is necessary for N107 glycosylation. Glycine extracts and Western blotting revealed that doubly glycosylated JlpA was the predominant form on the C. jejuni JHH1 surface however, glycosylation is not required for antigenicity. This is the first study to identify N-linked glycosylation of a surface-exposed C. jejuni virulence factor and to show strain variation in glycosylation sites.
Publisher: Cold Spring Harbor Laboratory
Date: 13-07-2023
DOI: 10.1101/2023.07.13.548920
Abstract: Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by O uter M embrane V esicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron ( Bt ). Our screening led us to the identification of a novel family of D ual M embrane-spanning A nti-sigma factors (Dma), which regulate OMV biogenesis in Bt . We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal β-barrel embedded in the OM its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.
Publisher: Wiley
Date: 12-07-2013
DOI: 10.1111/MMI.12300
Abstract: Multi-drug resistant strains of Acinetobacter baumannii are increasingly being isolated in hospitals worldwide. Among the virulence factors identified in this bacterium there is a general O-glycosylation system that appears to be important for biofilm formation and virulence, and the capsular polysaccharide, which is essential for resistance to complement killing. In this work, we identified a locus that is responsible for the synthesis of the O-pentasaccharide found on the glycoproteins. Besides the enzymes required for the assembly of the glycan, additional proteins typically involved in polymerization and transport of capsule were identified within or adjacently to the locus. Mutagenesis of PglC, the initiating glycosyltransferase prevented the synthesis of both glycoproteins and capsule, resulting in abnormal biofilm structures and attenuated virulence in mice. These results, together with the structural analysis of A. baumannii 17978 capsular polysaccharide via NMR, demonstrated that the pentasaccharides that decorate the glycoproteins are also the building blocks for capsule biosynthesis. Two linked subunits, but not longer glycan chains, were detected on proteins via MS. The discovery of a bifurcated pathway for O-glycosylation and capsule synthesis not only provides insight into the biology of A. baumannii but also identifies potential novel candidates for intervention against this emerging pathogen.
Publisher: Springer Science and Business Media LLC
Date: 07-09-2021
DOI: 10.1038/S42003-021-02588-Y
Abstract: 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 systems is far more limited. Within this work we explore the conservation of glycoproteins and glycosylation sites across the pan- Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach, we generate high-confidence glycoproteomes 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 B. cenocepacia isolates. 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. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharyltransferases that target conserved and shared protein substrates.
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.CARRES.2018.05.008
Abstract: Many monoclonal antibodies (mAbs) used in cancer immunotherapy mediate tumour cell lysis by recruiting natural killer (NK) cells a phenomenon known as antibody-dependent cellular cytotoxicity (ADCC). Eliminating core-fucose from the N-glycans of a mAb enhances its capacity to induce ADCC. As such, inhibitors of fucosylation are highly desirable for the production of mAbs for research and therapeutic use. Herein, we describe a simple synthesis of 6,6,6-trifluoro-l-fucose (F3Fuc), a metabolic inhibitor of fucosylation, and demonstrate the utility of this molecule in the production of low-fucose mAbs from murine hybridoma cell lines.
Publisher: Cold Spring Harbor Laboratory
Date: 09-04-2021
DOI: 10.1101/2021.04.08.439081
Abstract: Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A. baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium’s pathobiology, which remains poorly understood. A. baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A. baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A. baumannii . We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A. baumannii .
Publisher: Elsevier BV
Date: 03-2023
Publisher: Wiley
Date: 05-03-2014
DOI: 10.1111/MMI.12540
Abstract: Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O-linked protein glycosylation system in B. cenocepacia K56-2. The PglLBc O-oligosaccharyltransferase (O-OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the C ylobacter jejuni N-glycosylation system to a Neisseria meningitides-derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56-2, PglLBc is responsible for the glycosylation of 23 proteins involved in erse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc-HexNAc-Hex, which is unrelated to the O-antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first ex le of post-translational modification in Bcc with implications for pathogenesis.
Publisher: Elsevier BV
Date: 09-2020
Publisher: American Society for Microbiology
Date: 30-10-2015
Abstract: Phenotypic variation is prevalent in the zoonotic pathogen C ylobacter jejuni , the leading agent of enterocolitis in the developed world. Heterogeneity enhances the survival and adaptive malleability of bacterial populations because variable phenotypes may allow some cells to be protected against future stress. Exposure to hyperosmotic stress previously revealed prevalent differences in growth between C. jejuni strain 81-176 colonies due to resistant or sensitive phenotypes, and these isolated colonies continued to produce progeny with differential phenotypes. In this study, whole-genome sequencing of isolated colonies identified allelic variants of two purine biosynthesis genes, purF and apt , encoding phosphoribosyltransferases that utilize a shared substrate. Genetic analyses determined that purF was essential for fitness, while apt was critical. Traditional and high-depth licon-sequencing analyses confirmed extensive intrapopulation genetic variation of purF and apt that resulted in viable strains bearing alleles with in-frame insertion duplications, deletions, or missense polymorphisms. Different purF and apt alleles were associated with various stress survival capabilities under several niche-relevant conditions and contributed to differential intracellular survival in an epithelial cell infection model. Amplicon sequencing revealed that intracellular survival selected for stress-fit purF and apt alleles, as did exposure to oxygen and hyperosmotic stress. Putative protein recognition direct repeat sequences were identified in purF and apt , and a DNA-protein affinity screen captured a predicted exonuclease that promoted the global spontaneous mutation rate. This work illustrates the adaptive properties of high-frequency genetic variation in two housekeeping genes, which influences C. jejuni survival under stress and promotes its success as a pathogen. IMPORTANCE C. jejuni is an important cause of bacterial diarrheal illness. Bacterial populations have many strategies for stress survival, but phenotypic variation due to genetic ersity has a powerful advantage: no matter how swift the change in environment, a fraction of the population already expresses the survival trait. Nonclonality is thus increasingly viewed as a mechanism of population success. Our previous work identified prominent resistant/sensitive colonial variation in C. jejuni bacteria in response to hyperosmotic stress in the work presented here, we attribute that to high-frequency genetic variation in two purine biosynthesis genes, purF and apt . We demonstrated selective pressure for nonlethal mutant alleles of both genes, showed that single-cell variants had the capacity to give rise to erse purF and apt populations, and determined that stress exposure selected for desirable alleles. Thus, a novel C. jejuni adaptive strategy was identified, which was, unusually, reliant on prevalent genetic variation in two housekeeping genes.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-06-2023
Abstract: Extracellular vesicles are produced in all three domains of life, and their biogenesis has 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. Bacteroides thetaiotaomicron ( Bt ), a prominent member of the human intestinal microbiota, produces significant amounts of outer membrane vesicles (OMVs) which have been proposed to play key physiological roles. Here, we employed a dual marker system, consisting of outer membrane- and OMV-specific markers fused to fluorescent proteins to visualize OMV biogenesis by time-lapse microscopy. Furthermore, we performed comparative proteomic analyses to show that, in Bt , the OMV cargo is adapted for the optimal utilization of different polysaccharides. We also show that a negatively charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. Our results demonstrate that OMV production is the result of a highly regulated process in Bt .
Publisher: American Society for Microbiology
Date: 20-02-2019
DOI: 10.1128/IAI.00534-19
Abstract: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella -containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes.
Publisher: Springer Science and Business Media LLC
Date: 23-06-2022
Publisher: Cold Spring Harbor Laboratory
Date: 23-04-2020
DOI: 10.1101/2020.04.21.052845
Abstract: 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 erse 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 C ylobacter 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 ersity within bacterial proteomes.
Publisher: Cold Spring Harbor Laboratory
Date: 04-09-2019
DOI: 10.1101/757575
Abstract: O -linked protein glycosylation is a conserved feature of the Burkholderia genus. For Burkholderia cenocepacia , the addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-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. Protein 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 C ylobacter 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.
Publisher: Elsevier BV
Date: 04-2015
DOI: 10.1016/J.JPROT.2014.10.024
Abstract: Standard approaches to studying an interactome do not easily allow conditional experiments but in recent years numerous groups have demonstrated the potential for co-fractionation/co-migration based approaches to assess an interactome at a similar sensitivity and specificity yet significantly lower cost and higher speed than traditional approaches. Unfortunately, there is as yet no implementation of the bioinformatics tools required to robustly analyze co-fractionation data in a way that can also integrate the valuable information contained in biological replicates. Here we have developed a freely available, integrated bioinformatics solution for the analysis of protein correlation profiling SILAC data. This modular solution allows the deconvolution of protein chromatograms into in idual Gaussian curves enabling the use of these chromatography features to align replicates and assemble a consensus map of features observed across replicates the chromatograms and in idual curves are then used to quantify changes in protein interactions and construct the interactome. We have applied this workflow to the analysis of HeLa cells infected with a Salmonella enterica serovar Typhimurium infection model where we can identify specific interactions that are affected by the infection. These bioinformatics tools simplify the analysis of co-fractionation/co-migration data to the point where there is no specialized knowledge required to measure an interactome in this way. We describe a set of software tools for the bioinformatics analysis of co-migration/co-fractionation data that integrates the results from multiple replicates to generate an interactome, including the impact on in idual interactions of any external perturbation. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.
Publisher: Elsevier BV
Date: 2021
Publisher: Springer Science and Business Media LLC
Date: 15-09-2017
DOI: 10.1038/S41467-017-00571-Y
Abstract: O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.
Publisher: American Society for Microbiology
Date: 10-2019
DOI: 10.1128/IAI.00528-19
Abstract: Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, and there are few options for effective therapy. Therefore, there is a need to identify antibacterial targets for the development of novel treatments. Cyclophilins are a family of highly conserved enzymes important in multiple cellular processes.
Publisher: American Chemical Society (ACS)
Date: 30-03-2023
Publisher: Springer Science and Business Media LLC
Date: 05-01-2023
DOI: 10.1038/S41589-022-01219-9
Abstract: C -linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C -mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C -mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.
Publisher: Cold Spring Harbor Laboratory
Date: 19-12-2019
DOI: 10.1101/2019.12.17.868638
Abstract: Rhizobia are nitrogen fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria with the soil and rhizosphere. Here we show that free living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway resulting in production of sulfolactate (SL) as the major metabolic end-product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter cassette, sulfo-ED enzymes and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates (data are available via ProteomeXchange with identifier PXD015822). Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a non-specific aldehyde reductase, respectively. This work shows that rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.
Publisher: American Chemical Society (ACS)
Date: 11-2013
DOI: 10.1021/PR400783J
Abstract: A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
Publisher: Cold Spring Harbor Laboratory
Date: 11-2022
DOI: 10.1101/2022.11.01.514596
Abstract: Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (c Kp ) and hypervirulent K. pneumoniae (hv Kp ). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hv Kp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hv Kp and c Kp , 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 c Kp and hv Kp 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 c Kp and hv Kp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hv Kp and some c Kp strains, due to capsule blocking the O-antigen. Currently 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 c Kp and hv Kp in murine models of pneumonia and bacteremia, suggesting that capsule-based vaccines offer superior protection from some K. pneumoniae infections.
Publisher: Springer Science and Business Media LLC
Date: 24-06-2019
DOI: 10.1038/S41467-019-10706-Y
Abstract: Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.
Publisher: Springer Science and Business Media LLC
Date: 23-08-2022
Publisher: American Society for Microbiology
Date: 02-11-2016
Abstract: The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi . By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l , d -endopeptidase activity. TagX shows homology to known bacteriophage l , d -endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium. IMPORTANCE Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter , and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery.
Publisher: Cold Spring Harbor Laboratory
Date: 02-08-2023
DOI: 10.1101/2023.08.01.551581
Abstract: We report the isolation of Paracoccus onubensis strain Merri, which can grow on the rare sulfosugar sulfofucose (6-deoxy-6-sulfo-D-galactose) as sole carbon source, leading to its breakdown and release of sulfite. Genome sequencing and comparative proteomics of s les grown on glucose versus sulfofucose identified a genetic locus containing most of the genes for a sulfoglycolytic pathway for breakdown of sulfofucose to the 3-carbon organosulfonate sulfolactaldehyde, and a biomineralization pathway for catabolism of sulfolactaldehyde to pyruvate and sulfite. A candidate sulfotagatose-1-phosphate aldolase was expressed and was active on the epimeric substrate sulfofructose-1-phosphate. Its 3D X-ray structure revealed a close homology to sulfofructose-1-phosphate aldolase from the sulfoglycolytic Embden-Meyerhof-Parnas pathway. A patchwork sulfofucose catabolism pathway is proposed involving a sulfofucolytic Embden-Meyerhof-Parnas and short-chain organosulfonate biomineralization pathway. Sulfosugars are important compounds in the biogeochemical sulfur cycle and are produced by a wide range of photosynthetic plants, algae and cyanobacteria. While the pathways for the biosynthesis and catabolism of sulfoquinovose are well studied, much less is known about the rarer sulfosugar sulfofucose. We report a bacterium that can use sulfofucose as sole carbon source and achieve its complete breakdown to produce sulfite. We propose that sulfofucose catabolism is achieved using enzymes of the sulfoglycolytic Embden-Meyerhof-Parnas pathway that tolerate a change in stereochemistry at C4 of the sulfosugar. The sulfolactaldehyde output of this pathway is broken down to sulfite through a biomineralization pathway via sulfolactate. Microbial metabolism of this rare sulfosugar can be achieved through repurposing enzymes within known sulfosugar and short-chain organosulfonate catabolism catabolism.
Publisher: Elsevier BV
Date: 11-2016
Publisher: Oxford University Press (OUP)
Date: 03-11-2015
Publisher: Elsevier BV
Date: 08-2012
Publisher: Elsevier BV
Date: 09-2019
Publisher: Informa UK Limited
Date: 28-01-2021
Publisher: Elsevier BV
Date: 08-2011
Publisher: Proceedings of the National Academy of Sciences
Date: 23-04-2021
Abstract: Neutralizing antibodies are important for immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and as therapeutics for the prevention and treatment of COVID-19. We identified high-affinity nanobodies against SARS-CoV-2 receptor-binding domain and found that nanobody cocktails consisting of two noncompeting nanobodies were able to block ACE2 engagement with RBD variants present in human populations and potently neutralize both wild-type SARS-CoV-2 and the N501Y D614G variant at low concentrations. Prophylactic administration of nanobody cocktails reduced viral loads in mice infected with the N501Y D614G SARS-CoV-2 virus, showing that nanobody cocktails are useful as prophylactic agents against SARS-CoV-2.
Publisher: Elsevier BV
Date: 02-2018
Publisher: Elsevier BV
Date: 09-2019
Publisher: Informa UK Limited
Date: 11-12-2014
DOI: 10.1586/14789450.2015.987756
Abstract: The opportunistic human pathogen Acinetobacter baumannii persists in the healthcare setting because of its ability to survive exposure to various antimicrobial and sterilization agents. A. baumannii's ability to cause multiple infection types complicates diagnosis and treatment. Rapid detection of A. baumannii infections would likely improve treatment outcomes. Recently published Acinetobacter glycoproteomic data show the prevalence of O-linked glycoproteins, suggesting the possibility for an O-glycan-based detection technology. O-glycan biosynthesis is required for protein glycosylation and capsular polysaccharide production in A. baumannii. Recent publications demonstrate key roles for protein glycosylation and capsular polysaccharide in the pathogenicity of A. baumannii. Targeted antimicrobial development against O-glycan biosynthesis may produce new effective treatment options for A. baumannii infections. Here, we discuss how the data gathered through Acinetobacter glycoproteomics can be used to develop technologies for rapid diagnosis and reveal potential antimicrobial targets. In addition, we consider the efficacy of glycoconjugate vaccine development against A. baumannii.
Publisher: Cold Spring Harbor Laboratory
Date: 14-08-2018
DOI: 10.1101/391623
Abstract: Toxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii , has highly glycosylated thrombospondin repeat domains (TSR). At least seven C- linked and three O -linked glycosylation sites exist within MIC2, with % occupancy at O -glycosylation sites. We demonstrate that the addition of O -glycans to MIC2 is mediated by a protein O -fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 O -glycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.
Publisher: Elsevier BV
Date: 04-2013
Publisher: Elsevier BV
Date: 08-2023
Publisher: Springer Science and Business Media LLC
Date: 23-10-2017
Publisher: Springer Science and Business Media LLC
Date: 20-07-2015
DOI: 10.1038/NSMB.3060
Abstract: Sialyltransferases of the mammalian ST8Sia family catalyze oligo- and polysialylation of surface-localized glycoproteins and glycolipids through transfer of sialic acids from CMP-sialic acid to the nonreducing ends of sialic acid acceptors. The crystal structure of human ST8SiaIII at 1.85-Å resolution presented here is, to our knowledge, the first solved structure of a polysialyltransferase from any species, and it reveals a cluster of polysialyltransferase-specific structural motifs that collectively provide an extended electropositive surface groove for binding of oligo-polysialic acid chain products. The ternary complex of ST8SiaIII with a donor sugar analog and a sulfated glycan acceptor identified with a sialyltransferase glycan array provides insight into the residues involved in substrate binding, specificity and sialyl transfer.
Publisher: Wiley
Date: 14-06-2011
Abstract: Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10 mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel antioxidant proteins that contribute to its increased resistance against oxidative stress.
Publisher: American Society for Microbiology
Date: 28-06-2022
Abstract: Although general protein glycosylation systems have been well recognized in prokaryotes, the processes governing their distribution, function, and evolution remain poorly understood. Here, we have begun to address these gaps in knowledge by comparative analyses of broad-spectrum O -linked protein glycosylation manifest in species within the genus Neisseria that strictly colonize humans.
Publisher: American Society for Microbiology
Date: 02-2017
DOI: 10.1128/IAI.00620-16
Abstract: Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to d en interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD.
Publisher: American Chemical Society (ACS)
Date: 25-08-2014
DOI: 10.1021/PR5005554
Abstract: C ylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.
Publisher: eLife Sciences Publications, Ltd
Date: 27-06-2019
Publisher: American Society for Microbiology
Date: 28-06-2022
Abstract: While 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.
Publisher: American Society for Microbiology
Date: 18-12-2019
Abstract: Protein glycosylation is increasingly recognized as a common posttranslational 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 C ylobacter jejuni , O -linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR ( BCAM1868 ) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic 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.
Publisher: Cold Spring Harbor Laboratory
Date: 23-07-2020
DOI: 10.1101/2020.07.22.216903
Abstract: 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-s ling 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 s les. Using Field Asymmetric Waveform IMS (FAIMS) fractionation we show that at low compensation voltages (CVs) short aliphatic glycopeptides can be enriched from complex s les 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 C ylobacter 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.
Publisher: eLife Sciences Publications, Ltd
Date: 31-07-2019
DOI: 10.7554/ELIFE.46615
Abstract: Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a ‘one-size fits all’ membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of ,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For ex le, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.
Publisher: Cold Spring Harbor Laboratory
Date: 29-11-2019
DOI: 10.1101/859785
Abstract: 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 alent lectins that recognise the GlcNAc-α-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.
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2012
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2018
End Date: 2020
Funder: Australian Research Council
View Funded ActivityStart Date: 2016
End Date: 2018
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 03-2021
End Date: 04-2024
Amount: $534,500.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2021
End Date: 12-2024
Amount: $850,770.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2018
End Date: 12-2020
Amount: $269,734.00
Funder: Australian Research Council
View Funded Activity