Neisserial OTases have different protein specificies
Ontology highlight
ABSTRACT: Proteomic investigation on the glycosylation substrates and proteome effects of altering neisserial OTases within the proteome of N. gonorrhoeae MS11
Project description:Burkholderia species are associated with several life-threatening human infections, often resulting in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies targeting conserved, yet unique, Burkholderia pathways are needed. One such pathway is the Burkholderia O-linked protein glycosylation system, essential for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (OGC), a five-gene cluster sufficient and required for the generation of a trisaccharide β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for ligating glycans to glycoproteins. Previous work has shown that the OGC cluster can be removed, but individual mutations associated with late-stage glycan biosynthesis are essential. Here, we explore the essentiality of late-stage O-linked glycan biosynthesis in B. cenocepacia, revealing that the completion and translocation of the O-linked trisaccharide is necessary for viability and bacterial fitness. Using inducible systems, we demonstrate toxicity dependent on multiple OGC genes and the initiation of O-linked glycan biosynthesis. Upon loss of late-stage biosynthesis, mutants exhibit notable growth defects and profound sensitivity to stresses. Proteomics and glycoproteomic analysis show that blocking late-stage glycan biosynthesis inhibits protein glycosylation and drives large membrane proteomic changes. Finally, we demonstrate that OGC mediated toxicity is not limited to blockages but can also occur via the overexpression of steps within O-linked glycan biosynthesis. Combined, these findings suggest that the O-linked glycan biosynthesis pathway of B. cenocepacia is extremely sensitive to dysregulation and may be an ideal target for the development of antimicrobial therapies.
Project description:Burkholderia species are associated with several life-threatening human infections, often resulting in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies targeting conserved, yet unique, Burkholderia pathways are needed. One such pathway is the Burkholderia O-linked protein glycosylation system, essential for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (OGC), a five-gene cluster sufficient and required for the generation of a trisaccharide Gal-GalNAc-GalNAc used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for ligating glycans to glycoproteins. Previous work has shown that the OGC cluster can be removed, but individual mutations associated with late-stage glycan biosynthesis are essential. Here, we explore the essentiality of late-stage O-linked glycan biosynthesis in B. cenocepacia, revealing that the completion and translocation of the O-linked trisaccharide is necessary for viability and bacterial fitness. Using inducible systems, we demonstrate toxicity dependent on multiple OGC genes and the initiation of O-linked glycan biosynthesis. Upon loss of late-stage biosynthesis, mutants exhibit notable growth defects and profound sensitivity to stresses. Proteomics and glycoproteomic analysis show that blocking late-stage glycan biosynthesis inhibits protein glycosylation and drives large membrane proteomic changes. Finally, we demonstrate that OGC mediated toxicity is not limited to blockages but can also occur via the overexpression of steps within O-linked glycan biosynthesis. Combined, these findings suggest that the O-linked glycan biosynthesis pathway of B. cenocepacia is extremely sensitive to dysregulation and may be an ideal target for the development of antimicrobial therapies.
Project description:Burkholderia species are associated with several life-threatening human infections, often resulting in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies targeting conserved, yet unique, Burkholderia pathways are needed. One such pathway is the Burkholderia O-linked protein glycosylation system, essential for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (OGC), a five-gene cluster sufficient and required for the generation of a trisaccharide β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for ligating glycans to glycoproteins. Previous work has shown that the OGC cluster can be removed, but individual mutations associated with late-stage glycan biosynthesis are essential. Here, we explore the essentiality of late-stage O-linked glycan biosynthesis in B. cenocepacia, revealing that the completion and translocation of the O-linked trisaccharide is necessary for viability and bacterial fitness. Using inducible systems, we demonstrate toxicity dependent on multiple OGC genes and the initiation of O-linked glycan biosynthesis. Upon loss of late-stage biosynthesis, mutants exhibit notable growth defects and profound sensitivity to stresses. Proteomics and glycoproteomic analysis show that blocking late-stage glycan biosynthesis inhibits protein glycosylation and drives large membrane proteomic changes. Finally, we demonstrate that OGC mediated toxicity is not limited to blockages but can also occur via the overexpression of steps within O-linked glycan biosynthesis. Combined, these findings suggest that the O-linked glycan biosynthesis pathway of B. cenocepacia is extremely sensitive to dysregulation and may be an ideal target for the development of antimicrobial therapies.
Project description:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within members of the Neisseria genus O-linked protein glycosylation plays important roles in virulence and antigenic variation yet our understanding of the substrates of glycosylation are limited. Recently it was identified that even closely related Neisserial species can possess O-oligosaccharyltransferases, pglOs, that possess varying glycosylation specificities suggesting that distinct targeting activities may impact both the glycoprotome as well as the proteome of Neisserial species. Within this work we explore this concept using of Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of differences in the glycoproteomes and proteomes within N. gonorrhoeae strains expressing differing pglO alleles. We demonstrate the utility of FAIMS to expand the known glycoproteome of N. gonorrhoeae and enable comparative glycoproteomics of a recently reported panel of N. gonorrhoeae strains expressing different pglO allelic chimeras (15 pglO enzymes) with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae yet lead to minimal effects on the abundance of glycoproteins. Additionally, while DIA analysis can allow occupancy to be inferred by the absence or presence of peptides known to be modified, we observe a poor correlation between DIA measurements of non-modified versions of glycopeptides and glycoproteomic analysis. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and supports that the expression of different pglO alleles appears to drive proteomic changes independent of the glycoproteins targeted for glycosylation.
Project description:The haematopoietic cytokine thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for maintenance of the haematopoetic stem cell compartment. Tpo is a heavily glycosylated, hepatocyte-derived cytokine which functions by binding to its receptor (TpoR) on target cells and thereby activating intracellular signalling cascades that induce their proliferation and/or differentiation. In addition to its role in signal propagation, TpoR is expressed on the surface of platelets, where it contributes to regulation of Tpo levels by sequestering circulating cytokine. TpoR belongs to the homodimeric Class I cytokine receptor family but is unusual due to a duplication of the Cytokine binding Homology Region (CHR). Almost thirty years after initial discovery of TpoR, the structure of the human Tpo:TpoR interaction was recently reported. Here we determine the structure of extracellular portion of the murine Tpo:TpoR signalling complex using single particle cryo-EM. The structure reveals that Tpo:TpoR forms a largely symmetrical 1:2 complex. The cytokine cross-links the same site on the membrane-distal CHR of both receptor chains using opposing surfaces and with significantly different affinities. This orients the two membrane-proximal CHRs such that they contact one another adjacent to the plasma membrane. The potential cytokine-binding site in CHR2 is glycosylated and does not interact with Tpo. A large insertion in CHR1 that is unique to Tpo forms a partially structured loop that is disulphide bonded to CHR2 and, in one receptor chain, contacts cytokine. Biochemical analyses indicate that the glycosylated C-terminal domain of Tpo does not influence receptor binding. We demonstrate that the therapeutic TpoR agonist Romiplostim binds to the same site on the receptor as does cytokine. Our study characterises the Tpo/TpoR interaction structurally and biochemically to allow for the future development of potent TpoR agonists for therapeutic use.
Project description:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within members of the Neisseria genus O-linked protein glycosylation plays important roles in virulence and antigenic variation yet our understanding of the substrates of glycosylation are limited. Recently it was identified that even closely related Neisserial species can possess O-oligosaccharyltransferases, pglOs, that possess varying glycosylation specificities suggesting that distinct targeting activities may impact both the glycoprotome as well as the proteome of Neisserial species. Within this work we explore this concept using of Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of differences in the glycoproteomes and proteomes within N. gonorrhoeae strains expressing differing pglO alleles. We demonstrate the utility of FAIMS to expand the known glycoproteome of N. gonorrhoeae and enable comparative glycoproteomics of a recently reported panel of N. gonorrhoeae strains expressing different pglO allelic chimeras (15 pglO enzymes) with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae yet lead to minimal effects on the abundance of glycoproteins. Additionally, while DIA analysis can allow occupancy to be inferred by the absence or presence of peptides known to be modified, we observe a poor correlation between DIA measurements of non-modified versions of glycopeptides and glycoproteomic analysis. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and supports that the expression of different pglO alleles appears to drive proteomic changes independent of the glycoproteins targeted for glycosylation.
Project description:Burkholderia species are associated with several life-threatening human infections, often resulting in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies targeting conserved, yet unique, Burkholderia pathways are needed. One such pathway is the Burkholderia O-linked protein glycosylation system, essential for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (OGC), a five-gene cluster sufficient and required for the generation of a trisaccharide β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for ligating glycans to glycoproteins. Previous work has shown that the OGC cluster can be removed, but individual mutations associated with late-stage glycan biosynthesis are essential. Here, we explore the essentiality of late-stage O-linked glycan biosynthesis in B. cenocepacia, revealing that the completion and translocation of the O-linked trisaccharide is necessary for viability and bacterial fitness. Using inducible systems, we demonstrate toxicity dependent on multiple OGC genes and the initiation of O-linked glycan biosynthesis. Upon loss of late-stage biosynthesis, mutants exhibit notable growth defects and profound sensitivity to stresses. Proteomics and glycoproteomic analysis show that blocking late-stage glycan biosynthesis inhibits protein glycosylation and drives large membrane proteomic changes. Finally, we demonstrate that OGC mediated toxicity is not limited to blockages but can also occur via the overexpression of steps within O-linked glycan biosynthesis. Combined, these findings suggest that the O-linked glycan biosynthesis pathway of B. cenocepacia is extremely sensitive to dysregulation and may be an ideal target for the development of antimicrobial therapies.
Project description:Burkholderia species are associated with several life-threatening human infections, often resulting in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies targeting conserved, yet unique, Burkholderia pathways are needed. One such pathway is the Burkholderia O-linked protein glycosylation system, essential for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (OGC), a five-gene cluster sufficient and required for the generation of a trisaccharide β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for ligating glycans to glycoproteins. Previous work has shown that the OGC cluster can be removed, but individual mutations associated with late-stage glycan biosynthesis are essential. Here, we explore the essentiality of late-stage O-linked glycan biosynthesis in B. cenocepacia, revealing that the completion and translocation of the O-linked trisaccharide is necessary for viability and bacterial fitness. Using inducible systems, we demonstrate toxicity dependent on multiple OGC genes and the initiation of O-linked glycan biosynthesis. Upon loss of late-stage biosynthesis, mutants exhibit notable growth defects and profound sensitivity to stresses. Proteomics and glycoproteomic analysis show that blocking late-stage glycan biosynthesis inhibits protein glycosylation and drives large membrane proteomic changes. Finally, we demonstrate that OGC mediated toxicity is not limited to blockages but can also occur via the overexpression of steps within O-linked glycan biosynthesis. Combined, these findings suggest that the O-linked glycan biosynthesis pathway of B. cenocepacia is extremely sensitive to dysregulation and may be an ideal target for the development of antimicrobial therapies.
Project description:Burkholderia species are associated with several life-threatening human infections, often resulting in high morbidity and mortality rates due to their innate resistance to antibiotics. To improve clinical outcomes, new therapies targeting conserved, yet unique, Burkholderia pathways are needed. One such pathway is the Burkholderia O-linked protein glycosylation system, essential for virulence in Burkholderia cenocepacia and Burkholderia pseudomallei. This system relies on the O-Glycosylation gene Cluster (OGC), a five-gene cluster sufficient and required for the generation of a trisaccharide β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc used for protein glycosylation, and the distally encoded oligosaccharyltransferase, pglL, responsible for ligating glycans to glycoproteins. Previous work has shown that the OGC cluster can be removed, but individual mutations associated with late-stage glycan biosynthesis are essential. Here, we explore the essentiality of late-stage O-linked glycan biosynthesis in B. cenocepacia, revealing that the completion and translocation of the O-linked trisaccharide is necessary for viability and bacterial fitness. Using inducible systems, we demonstrate toxicity dependent on multiple OGC genes and the initiation of O-linked glycan biosynthesis. Upon loss of late-stage biosynthesis, mutants exhibit notable growth defects and profound sensitivity to stresses. Proteomics and glycoproteomic analysis show that blocking late-stage glycan biosynthesis inhibits protein glycosylation and drives large membrane proteomic changes. Finally, we demonstrate that OGC mediated toxicity is not limited to blockages but can also occur via the overexpression of steps within O-linked glycan biosynthesis. Combined, these findings suggest that the O-linked glycan biosynthesis pathway of B. cenocepacia is extremely sensitive to dysregulation and may be an ideal target for the development of antimicrobial therapies.