Project description:Sulforaphane is a naturally occurring, potent antioxidant and anti-inflammatory compound, found in cruciferous plants such as broccoli. Recently there have been a large number of clinical trials assessing broccoli sprout extracts as sulforaphane-based therapies for conditions including fibrosis, cancer and preeclampsia. As sulforaphane is orally administered, there is also the potential for impact on the gut microbiome. Here, we have determined the effect of sulforaphane on the growth of 43 common human gastrointestinal bacterial commensals and pathogens, which represented the four main phyla found in the human gastrointestinal microbiome. The pathogenic Escherichia coli strain ECE2348/69 showed the most significant increases in growth in the presence of sulforaphane compared to control conditions. Proteomic analysis of this isolate showed that sulforaphane increased anaerobic respiration, whilst metabolomic profiling identified differentially produced metabolites involved in amino acid biosynthesis and known to decrease inflammation in human cells. Therefore, sulforaphane can increase growth of specific gastrointestinal bacterial isolates, correlating with increased production of anti-inflammatory metabolites, that may provide a novel mechanism for modulating inflammatory states in patients.
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:Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyze the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria but to date its role in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. Pipecolic acid derived compounds, SF235 and AN296 demonstrate inhibitory activities against CbMip and these compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain RSA439 in axenic culture with comparative proteomic demonstrating selective alterations in response to these agents. This study also showed that exposure of C. burnetii to Mip inhibitors resulted in increased sensitivity to oxidative stress. Furthermore, compounds SF235 and AN296 demonstrated protective activity in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for growth and replication and that inhibitors against CbMip offer potential as novel therapeutics against C. burnetii.
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:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within pathogenic members of the Neisseria genus O-linked protein glycosylation is associated with virulence yet the depth of the glycoproteome, or if glycosylation plays additional roles in Neisserial physiology are largely unknown. Recently it was identified that even closely related members of the Neisseria genus can possess O-Oligosaccharyltransferases, pglOs, that possess distinct targeting activities suggesting extensive glycoproteome diversity in terms of the substrates capable of being glycosylated across Neisserial species. Within this work we explore this concept using 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 enabling the characterization of the glycoproteomes of wild type N. gonorrhoeae MS11 as well as a recently reported panel of 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. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy independent of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and the impact of glycosylation on bacterial species.