PglL enzymes of Burkholderia species are serine preferring oligosaccharidetransferases which targets conserved proteins and sites across the genus -DsbA1 analysis
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ABSTRACT: Glycosylation analysis of DsbA1 proteins expressed in Burkholderia cenocepacia strains
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: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:Enterobacter bugandensis is one of species from the E. cloacae complex (ECC) that has been predominantly associated to neonatal sepsis. A major concern with E. bugandensis and ECC bacteria in general is the frequent appearance of multidrug resistant isolates including those resistant to last-resort antibiotics, such as polymyxins, for which these microbes are in the ESKAPE list of global threat pathogens. Here, we investigated polymyxin B (PmB) resistance and heteroresistance in E. bugandensis by transcriptomics and a gene deletion approach using two clinical isolates. Genes encoded in the CrrAB-regulated operon including crrC and kexD were highly upregulated in both strains in the presence of PmB. We show in one of these isolates that ∆crrC and ∆kexD mutants exhibited lower levels of PmB resistance and heteroresistance than the parental strain. Moreover, the heterologous expression of CrrC and KexD proteins increased PmB resistance in a sensitive E. ludwigii clinical isolate and in the Escherichia coli K12 strain W3110. We also showed that the efflux pump AcrAB and TolC contribute to PmB resistance and heteroresistance. Deletion of the regulatory genes phoPQ and crrAB cause reduced PmB resistance and heteroresistance, while deletion of pmrAB did not have any effect. Our results also reveal that the addition of L-Ara4N into the lipid A, mediated by the arnBCADTEF operon, is critical to determine PmB resistance, while the deletion of eptA, encoding a PEtN transferase had no effect. Finally, PmB resistance did not correlate with pathogenicity in the Galleria mellonella infection model.
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: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: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:This project examines the impact of different growth media on the protein compositions of membranes of Bacteroides thetaiotaomicron.
Project description: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.