Project description:Proteomic investigation on the glycosylation substrates and proteome effects of altering neisserial OTases within the proteome of N. gonorrhoeae MS11
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:Within the Burkholderia genus O-linked protein glycosylation is now known to be highly conserved at the pathway and glycosylation substrate levels. While inhibition of glycosylation has been shown to be detrimental to virulence in B. cenocepacia, little is known about the role of glycosylation in Burkholderia glycoproteins. Within this study we have sought to improve our understanding of the breadth and dynamics of the B. cenocepacia O-glycoproteome to identify glycoproteins which require glycosylation for functionality. Assessing the glycoproteome across multiple common culturing media (LB, TSB, and artificial sputum medium to simulate cystic fibrosis sputum-like conditions) we demonstrate at least 141 glycoproteins are subjected to glycosylation within B. cenocepacia K56-2. Leveraging this insight, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) across culturing media and growth phases revealing most B. cenocepacia glycoproteins are express under all conditions. Examination of how the absence of glycosylation impacts the glycoproteome reveals only a subset of the glycoproteome (BCAL1086, BCAL2974, BCAL0525, BCAM0505 and BCAL0127) appear impacted by the loss of glycosylation. Assessing the proteomic and phenotypic impacts of the loss of these glycoproteins compared to glycosylation null strains revealing the loss of BCAL0525, and to a lesser extend BCAL0127, mirror the proteomic effects observed in the absence of glycosylation. Finally, we demonstrate the loss of glycosylation within BCAL0525 at Serine-358 results in both loss of motility and proteomic impacts on flagellar apparatus consistent with the loss of apparatus stability. Combined this work demonstrates that O-linked glycosylation of BCAL0525 is functionally important within B. cenocepacia.
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:The trypanosomatid protozoan parasite Leishmania has a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes in Leishmania; the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporter LPG2, and the mitochondrial fucosyltransferase FUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally observed Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both lpg2- and fut1s mutants with FUT1-dependent changes linked to marked alterations within mitochondrial associated proteins while LPG2-dependent changes impacted multiple aspects of the secretory pathway. While FUT1 has been shown to fucosylate peptides in vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans within Leishmania glycopeptides isolated using HILIC enrichment. Combined this work provides a critical proteomic resource for the community on the observable Leishmania proteome as well as highlights phenotypic changes associated with LPG2/FUT1 which may guide the development of future therapeutics.
Project description:Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins directly into infected host cells. A subset of these injected protein ‘effectors’ are enzymes that modify the structure and inhibit the function of human proteins by catalyzing the addition of unusual post-translational modifications. T3SS effectors play essential roles in bacterial virulence and their modes of action have provided great insight into the functions and the components of the innate immune system. 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. Arginine glycosylation is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are chaperoned in the bacterium to keep the effectors partially unfolded and competent for secretion, as well as for targeting the effectors to the T3SS sorting platform. The chaperones are then stripped from their effector substrates at the sorting platform and the effectors are secreted in an unfolded conformation. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected into host cells. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the E. coli glutathione synthetase (GshB) is glycosylated on an arginine residue (R256) by NleB. NleB glycosyltransferase activity is essential to C. rodentium survival in oxidative stress conditions because glycosylation of GshB results in enhanced glutathione production. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that effector activities thought to be restricted to host cell compartments additionally play important roles in regulating bacterial physiology.
Project description:Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling these diarrheal diseases, which are a significant cause of infant morbidity in the developing world. The only approved vaccine against an apicomplexan parasite targets conserved adhesins possessing a thrombospondin repeat (TSR) domains. Orthologous TSR domain-containing proteins are commonplace in the apicomplexa and C. parvum possess 12 such proteins. Here, we explore the molecular evolution and conservation of these proteins and examine their abundance in C. parvum oocysts to assess the likelihood that they may be useful as vaccine candidates. We go onto examine the glycosylation states of these proteins using antibody-enabled and ZIC-HILIC enrichment techniques, which revealed that these proteins are modified with C-linked Hex and N-linked Hex5-6HexNAc2 glycans.
Project description:Strains of Salmonella utilise two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, while ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggests that Salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.