Publications by authors named "Nichollas E Scott"

85 Publications

Development and Immunogenicity of a Prototype Multivalent Group B Bioconjugate Vaccine.

ACS Infect Dis 2021 Oct 11. Epub 2021 Oct 11.

VaxNewMo, St. Louis, Missouri 63110, United States.

Group B (GBS) is a leading cause of neonatal infections and invasive diseases in nonpregnant adults worldwide. Developing a protective conjugate vaccine targeting the capsule of GBS has been pursued for more than 30 years; however, it has yet to yield a licensed product. In this study, we present a novel bioconjugation platform for producing a prototype multivalent GBS conjugate vaccine and its subsequent analytical and immunological characterizations. Using a glycoengineering strategy, we generated strains of that recombinantly express the type Ia, type Ib, and type III GBS capsular polysaccharides. We then combined the type Ia-, Ib-, and III-capsule-expressing strains with an engineered exotoxin A (EPA) carrier protein and the PglS oligosaccharyltransferase. Coexpression of a GBS capsule, the engineered EPA protein, and PglS enabled the covalent attachment of the target GBS capsule to an engineered serine residue on EPA, all within the periplasm of . GBS bioconjugates were purified, analytically characterized, and evaluated for immunogenicity and functional antibody responses. This proof-of-concept study signifies the first step in the development of a next-generation multivalent GBS bioconjugate vaccine, which was validated by the production of conjugates that are able to elicit functional antibodies directed against the GBS capsule.
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http://dx.doi.org/10.1021/acsinfecdis.1c00415DOI Listing
October 2021

Characterization of the O-Glycoproteome of Tannerella forsythia.

mSphere 2021 Sep 15:e0064921. Epub 2021 Sep 15.

Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbournegrid.1008.9, Victoria, Australia.

Tannerella forsythia is a Gram-negative oral pathogen known to possess an O-glycosylation system responsible for targeting multiple proteins associated with virulence at the three-residue motif (D)(S/T)(A/I/L/V/M/T). Multiple proteins have been identified to be decorated with a decasaccharide glycan composed of a poorly defined core plus a partially characterized species-specific section. To date, glycosylation studies have focused mainly on the two S-layer glycoproteins, TfsA and TfsB, so the true extent of glycosylation within this species has not been fully explored. In the present study, we characterize the glycoproteome of by employing FAIMS-based glycopeptide enrichment of a cell membrane fraction. We demonstrate that at least 13 glycans are utilized within the glycoproteome, varying with respect to the presence of the three terminal sugars and the presence of fucose and digitoxose residues at the reducing end. To improve the localization of glycosylation events and enhance the detection of glycopeptides, we utilized trifluoromethanesulfonic acid treatment to allow the selective chemical cleavage of glycans. Reducing the chemical complexity of glycopeptides dramatically improved the number of glycopeptides identified and our ability to localize glycosylation sites by ETD fragmentation, leading to the identification of 312 putative glycosylation sites in 145 glycoproteins. Glycosylation site analysis revealed that glycosylation occurs on a much broader motif than initially reported, with glycosylation found at (D)(S/T)(A/I/L/V/M/T/S/C/G/F). The prevalence of this broader glycosylation motif in the genome suggests the existence of hundreds of potential O-glycoproteins in this organism. 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. There is evidence that these proteins are modified by glycosylation, and this modification is essential for virulence in producing disease. We have utilized novel techniques coupled with mass spectrometry to identify over 13 glycans and 312 putative glycosylation sites in 145 glycoproteins within . Glycosylation site analysis revealed that this modification occurs on a much broader motif than initially reported such that there is a high prevalence of potential glycoproteins in this organism that may help to explain its role in periodontal disease.
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http://dx.doi.org/10.1128/mSphere.00649-21DOI Listing
September 2021

Burkholderia PglL enzymes are Serine preferring oligosaccharyltransferases which target conserved proteins across the Burkholderia genus.

Commun Biol 2021 09 7;4(1):1045. Epub 2021 Sep 7.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.

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.
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http://dx.doi.org/10.1038/s42003-021-02588-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423747PMC
September 2021

Modern Acinetobacter baumannii clinical isolates replicate inside spacious vacuoles and egress from macrophages.

PLoS Pathog 2021 Aug 9;17(8):e1009802. Epub 2021 Aug 9.

Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376066PMC
August 2021

Synthesis of C-Mannosylated Glycopeptides Enabled by Ni-Catalyzed Photoreductive Cross-Coupling Reactions.

J Am Chem Soc 2021 Aug 4;143(32):12699-12707. Epub 2021 Aug 4.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

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 -HrTH-I, a C-mannosylated insect hormone. To complement this approach, we also performed late-stage tryptophan C-mannosylation on a diverse array of peptides, demonstrating the broad scope and utility of this methodology for preparing glycopeptides.
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http://dx.doi.org/10.1021/jacs.1c05567DOI Listing
August 2021

BonA from Forms a Divisome-Localized Decamer That Supports Outer Envelope Function.

mBio 2021 Jul 27:e0148021. Epub 2021 Jul 27.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash Universitygrid.1002.3, Clayton, Australia.

Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multidrug-resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here, we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON (acterial smY and odulation) domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division. 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. Here, we report the discovery of BonA, a protein that supports A. baumannii outer envelope function and is required for cell motility. We determine the atomic structure of BonA and show that it forms part of the cell division machinery and functions by forming a complex, features that mirror those of distantly related homologs from other bacteria. By improving our understanding of the A. baumannii cell envelope this work will assist in treating this pathogen.
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http://dx.doi.org/10.1128/mBio.01480-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406262PMC
July 2021

An atlas of protein-protein interactions across mouse tissues.

Cell 2021 Jul 1;184(15):4073-4089.e17. Epub 2021 Jul 1.

Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.
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http://dx.doi.org/10.1016/j.cell.2021.06.003DOI Listing
July 2021

NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector.

PLoS Pathog 2021 06 16;17(6):e1009658. Epub 2021 Jun 16.

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238200PMC
June 2021

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Proc Natl Acad Sci U S A 2021 05;118(19)

Infectious Diseases and Immune Defences Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 10-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.
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http://dx.doi.org/10.1073/pnas.2101918118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126837PMC
May 2021

Editorial overview: Systems biology and the rise and rise of omics approaches.

Curr Opin Chem Biol 2021 02;60:A1-A3

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia; Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia; Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, Bluestone Center for Clinical Research, New York, NY, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cbpa.2021.02.006DOI Listing
February 2021

SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells.

Sci Adv 2021 Feb 19;7(8). Epub 2021 Feb 19.

Translational Haematology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.
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http://dx.doi.org/10.1126/sciadv.abe3610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895430PMC
February 2021

Arginine glycosylation enhances methylglyoxal detoxification.

Sci Rep 2021 Feb 15;11(1):3834. Epub 2021 Feb 15.

College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA.

Type III secretion system effector proteins have primarily been characterized for their interactions with host cell proteins and their ability to disrupt host signaling pathways. We are testing the hypothesis that some effectors are active within the bacterium, where they modulate bacterial signal transduction and physiology. We previously determined that the Citrobacter rodentium effector NleB possesses an intra-bacterial glycosyltransferase activity that increases glutathione synthetase activity to protect the bacterium from oxidative stress. Here we investigated the potential intra-bacterial activities of NleB orthologs in Salmonella enterica and found that SseK1 and SseK3 mediate resistance to methylglyoxal. SseK1 glycosylates specific arginine residues on four proteins involved in methylglyoxal detoxification, namely GloA (R9), GloB (R190), GloC (R160), and YajL (R149). SseK1-mediated Arg-glycosylation of these four proteins significantly enhances their catalytic activity, thus providing another important example of the intra-bacterial activities of type three secretion system effector proteins. These data are also the first demonstration that a Salmonella T3SS effector is active within the bacterium.
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http://dx.doi.org/10.1038/s41598-021-83437-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884692PMC
February 2021

TLR2-mediated activation of innate responses in the upper airways confers antiviral protection of the lungs.

JCI Insight 2021 03 8;6(5). Epub 2021 Mar 8.

Department of Microbiology and Immunology, the University of Melbourne, the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

The impact of respiratory virus infections on global health is felt not just during a pandemic, but endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen-independent innate immunity in the upper airways can prevent the spread of respiratory virus infection to the vulnerable lower airways. Activation of TLR2, when restricted to the nasal turbinates, resulted in prompt induction of innate immune-driven antiviral responses through action of cytokines, chemokines, and cellular activity in the upper but not the lower airways. We have defined how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to 7 days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.
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http://dx.doi.org/10.1172/jci.insight.140267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021123PMC
March 2021

Yeast- and antibody-based tools for studying tryptophan C-mannosylation.

Nat Chem Biol 2021 04 4;17(4):428-437. Epub 2021 Feb 4.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

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 hampered by a dearth of convenient tools for installing and detecting the modification. Here, we engineer a yeast system to produce a diverse 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.
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http://dx.doi.org/10.1038/s41589-020-00727-wDOI Listing
April 2021

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

Virulence 2021 12;12(1):493-506

Wellcome-Wolfson Institute of Experimental Medicine, Queen's University Belfast , Belfast, UK.

We recently described a protein -glycosylation pathway conserved in all species of the genus that results in the 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. , the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of . We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with and . Our results indicate that protein -glycosylation in can be exploited as a biomarker for diagnosis of -associated infections.
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http://dx.doi.org/10.1080/21505594.2021.1876440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849680PMC
December 2021

Editorial overview: Systems biology and the rise and rise of omics approaches.

Curr Opin Chem Biol 2021 Jan 5. Epub 2021 Jan 5.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia; Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia; Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, Bluestone Center for Clinical Research, New York, NY, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cbpa.2020.12.001DOI Listing
January 2021

Glycoproteomics: growing up fast.

Curr Opin Struct Biol 2021 06 2;68:18-25. Epub 2020 Dec 2.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Melbourne 3000, Australia. Electronic address:

Glycoproteomics is a rapidly growing field which seeks to identify and characterise glycosylation events at a proteome scale. Over the last few years considerable effort has been made in developing new technologies, enrichment systems, and analysis strategies to enhance the quality of glycoproteomic studies. Within this review we discuss the recent developments in glycoproteomics and the current state of the art approaches for analysing glycosylated substrates. We highlight key improvements in mass spectrometry instrumentation coupled with the advancements in enrichment approaches for key classes of glycosylation including mucin-O-glycosylation, O-GlcNAc glycosylation and N-linked glycosylation which now allow the identification/quantification of hundreds to thousands of glycosylation sites within individual experiments. Finally, we summarise the emerging trends within glycoproteomics to illustrate how the field is moving away from studies simply focused on identifying glycosylated substrates to studying specific mechanisms and disease states.
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http://dx.doi.org/10.1016/j.sbi.2020.10.028DOI Listing
June 2021

Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.

Mol Cell Proteomics 2020 Dec 3;20:100005. Epub 2020 Dec 3.

Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia. Electronic address:

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, 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 developed 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 four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization 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.
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http://dx.doi.org/10.1074/mcp.RA120.002370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7950127PMC
December 2020

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses.

J Proteome Res 2021 01 30;20(1):599-612. Epub 2020 Oct 30.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.

Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three species (, and ), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of subsp. also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.
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http://dx.doi.org/10.1021/acs.jproteome.0c00565DOI Listing
January 2021

2020 FASEB Science Research Conference on Microbial Glycobiology, July 13-14, 2020.

FASEB J 2020 11 8;34(11):14069-14072. Epub 2020 Oct 8.

Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, UK.

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http://dx.doi.org/10.1096/fj.202002191DOI Listing
November 2020

The Glycoprotease CpaA Secreted by Medically Relevant Acinetobacter Species Targets Multiple -Linked Host Glycoproteins.

mBio 2020 10 6;11(5). Epub 2020 Oct 6.

Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA

Glycans decorate proteins and affect their biological function, including protection against proteolytic degradation. However, pathogenic, and commensal bacteria have evolved specific glycoproteases that overcome the steric impediment posed by carbohydrates, cleaving glycoproteins precisely at their glycosylation site(s). Medically relevant strains employ their type II secretion system (T2SS) to secrete the glycoprotease CpaA, which contributes to virulence. Previously, CpaA was shown to cleave two -linked glycoproteins, factors V and XII, leading to reduced blood coagulation. In this work, we show that CpaA cleaves a broader range of -linked human glycoproteins, including several glycoproteins involved in complement activation, such as CD55 and CD46. However, only CD55 was removed from the cell surface, while CD46 remained unaltered during the infection assay. We show that CpaA has a unique consensus target sequence that consists of a glycosylated serine or threonine residue after a proline residue (P-S/T), and its activity is not affected by sialic acids. Molecular modeling and mutagenesis analysis of CpaA suggest that the indole ring of Trp493 and the ring of the Pro residue in the substrate form a key interaction that contributes to CpaA sequence selectivity. Similar bacterial glycoproteases have recently gained attention as tools for proteomic analysis of human glycoproteins, and CpaA appears to be a robust and attractive new component of the glycoproteomics toolbox. Combined, our work provides insight into the function and possible application of CpaA, a member of a widespread class of broad-spectrum bacterial glycoproteases involved in host-pathogen interactions. CpaA is a glycoprotease expressed by members of the complex, and it is the first bona fide secreted virulence factor identified in these species. Here, we show that CpaA cleaves multiple targets precisely at -glycosylation sites preceded by a Pro residue. This feature, together with the observation that sialic acid does not impact CpaA activity, makes this enzyme an attractive tool for the analysis of -linked human protein for biotechnical and diagnostic purposes. Previous work identified proteins involved in blood coagulation as targets of CpaA. Our work broadens the set of targets of CpaA, pointing toward additional roles in bacterium-host interactions. We propose that CpaA belongs to an expanding class of functionally defined glycoproteases that targets multiple -linked host glycoproteins.
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http://dx.doi.org/10.1128/mBio.02033-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542363PMC
October 2020

The Effector SseK3 Targets Small Rab GTPases.

Front Cell Infect Microbiol 2020 19;10:419. Epub 2020 Aug 19.

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.

During infection, species inject multiple type III secretion system (T3SS) effector proteins into host cells that mediate invasion and subsequent intracellular replication. At early stages of infection, exploits key regulators of host intracellular vesicle transport, including the small GTPases Rab5 and Rab7, to subvert host endocytic vesicle trafficking and establish the containing vacuole (SCV). At later stages of intracellular replication, interactions of the SCV with Rab GTPases are less well defined. Here we report that Rab1, Rab5, and Rab11 are modified at later stages of infection by SseK3, an arginine acetylglucosamine (GlcNAc) transferase effector translocated via the pathogenicity island 2 (SPI-2) type III secretion system. SseK3 modified arginines at positions 74, 82, and 111 within Rab1 and this modification occurred independently of Rab1 nucleotide binding. SseK3 exhibited Golgi localization that was independent of its glycosyltransferase activity but Arg-GlcNAc transferase activity was required for inhibition of alkaline phosphatase secretion in transfected cells. While SseK3 had a modest effect on SEAP secretion during infection of HeLa229 cells, inhibition of IL-1 and GM-CSF cytokine secretion was only observed upon over-expression of SseK3 during infection of RAW264.7 cells. Our results suggest that, in addition to targeting death receptor signaling, SseK3 may contribute to infection by interfering with the activity of key Rab GTPases.
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http://dx.doi.org/10.3389/fcimb.2020.00419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466453PMC
June 2021

CDK13 cooperates with CDK12 to control global RNA polymerase II processivity.

Sci Adv 2020 May 29;6(18). Epub 2020 Apr 29.

The Peter MacCallum Cancer Centre, Melbourne, 3000 VIC, Australia.

The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.
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http://dx.doi.org/10.1126/sciadv.aaz5041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190357PMC
May 2020

Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19.

Nat Med 2020 09 13;26(9):1428-1434. Epub 2020 Jul 13.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts, most targeting the viral 'spike' glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2). Eliciting neutralizing antibodies that block S-ACE2 interaction, or indirectly prevent membrane fusion, constitute an attractive modality for vaccine-elicited protection. However, although prototypic S-based vaccines show promise in animal models, the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.
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http://dx.doi.org/10.1038/s41591-020-0995-0DOI Listing
September 2020

Influence of Protein Glycosylation on Physiology.

Front Microbiol 2020 17;11:1191. Epub 2020 Jun 17.

Department of Microbiology, University of Georgia, Athens, GA, United States.

is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that infection can result from the consumption or handling of contaminated food products, but is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, , the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two subsp. protein glycosylation () mutants ( and ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H and increased hydrogenase activity, suggesting that N-glycosylation in is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the pathway and its enzymes, heterologous expression in followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in . In addition, the mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of , but also expands our knowledge on the influence of protein N-glycosylation on cell physiology.
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http://dx.doi.org/10.3389/fmicb.2020.01191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313396PMC
June 2020

Open Database Searching Enables the Identification and Comparison of Bacterial Glycoproteomes without Defining Glycan Compositions Prior to Searching.

Mol Cell Proteomics 2020 09 23;19(9):1561-1574. Epub 2020 Jun 23.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia. Electronic address:

Mass spectrometry has become an indispensable tool for the characterization 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. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using -linked glycopeptides of as well as -linked glycopeptides of and revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species (). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.
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http://dx.doi.org/10.1074/mcp.TIR120.002100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143609PMC
September 2020

A Sulfoglycolytic Entner-Doudoroff Pathway in Rhizobium leguminosarum bv. trifolii SRDI565.

Appl Environ Microbiol 2020 07 20;86(15). Epub 2020 Jul 20.

School of Chemistry, University of Melbourne, Parkville, Victoria, Australia

Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol 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, 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. 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 nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil. 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, 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.
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http://dx.doi.org/10.1128/AEM.00750-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376563PMC
July 2020

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

Nat Commun 2020 05 13;11(1):2265. Epub 2020 May 13.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

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 divalent 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.
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http://dx.doi.org/10.1038/s41467-020-16223-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221086PMC
May 2020

An intra-bacterial activity for a T3SS effector.

Sci Rep 2020 01 23;10(1):1073. Epub 2020 Jan 23.

College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA.

Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins into host cells. A subset of these injected protein 'effectors' are enzymes that inhibit the function of host proteins by catalyzing the addition of unusual post-translational modifications. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected, due to the activity of chaperones that keep the effectors in a structural state permissive for secretion. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the bacterial glutathione synthetase (GshB) is glycosylated by NleB on arginine residue R256. NleB-mediated glycosylation of GshB resulted in enhanced GshB activity, leading to an increase in glutathione production, and promoted C. rodentium survival in oxidative stress conditions. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that arginine-GlcNAcylation, once thought to be restricted to host cell compartments, also plays an important role in regulating bacterial physiology.
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http://dx.doi.org/10.1038/s41598-020-58062-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978387PMC
January 2020
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