Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Microb Biotechnol ; 17(6): e14480, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38858807

ABSTRACT

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.


Subject(s)
Hexosyltransferases , Membrane Proteins , Hexosyltransferases/genetics , Hexosyltransferases/metabolism , Hexosyltransferases/chemistry , Glycosylation , Membrane Proteins/metabolism , Membrane Proteins/genetics , Campylobacter/genetics , Campylobacter/enzymology , Campylobacter/metabolism , Polysaccharides/metabolism , Cell-Free System , Campylobacter jejuni/enzymology , Campylobacter jejuni/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Glycoconjugates/metabolism
2.
PLoS Pathog ; 19(9): e1011666, 2023 09.
Article in English | MEDLINE | ID: mdl-37733817

ABSTRACT

Prior infection can generate protective immunity against subsequent infection, although the efficacy of such immunity can vary considerably. Live-attenuated vaccines (LAVs) are one of the most effective methods for mimicking this natural process, and analysis of their efficacy has proven instrumental in the identification of protective immune mechanisms. Here, we address the question of what makes a LAV efficacious by characterising immune responses to a LAV, termed TAS2010, which is highly protective (80-90%) against lethal murine salmonellosis, in comparison with a moderately protective (40-50%) LAV, BRD509. Mice vaccinated with TAS2010 developed immunity systemically and were protected against gut-associated virulent infection in a CD4+ T cell-dependent manner. TAS2010-vaccinated mice showed increased activation of Th1 responses compared with their BRD509-vaccinated counterparts, leading to increased Th1 memory populations in both lymphoid and non-lymphoid organs. The optimal development of Th1-driven immunity was closely correlated with the activation of CD11b+Ly6GnegLy6Chi inflammatory monocytes (IMs), the activation of which can be modulated proportionally by bacterial load in vivo. Upon vaccination with the LAV, IMs expressed T cell chemoattractant CXCL9 that attracted CD4+ T cells to the foci of infection, where IMs also served as a potent source of antigen presentation and Th1-promoting cytokine IL-12. The expression of MHC-II in IMs was rapidly upregulated following vaccination and then maintained at an elevated level in immune mice, suggesting IMs may have a role in sustained antigen stimulation. Our findings present a longitudinal analysis of CD4+ T cell development post-vaccination with an intracellular bacterial LAV, and highlight the benefit of inflammation in the development of Th1 immunity. Future studies focusing on the induction of IMs may reveal key strategies for improving vaccine-induced T cell immunity.


Subject(s)
CD4-Positive T-Lymphocytes , Salmonella Infections , Mice , Animals , Monocytes , Vaccines, Attenuated , Inflammation
4.
Microb Cell Fact ; 21(1): 66, 2022 Apr 21.
Article in English | MEDLINE | ID: mdl-35449016

ABSTRACT

BACKGROUND: Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse. RESULTS: In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences. CONCLUSIONS: Amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community.


Subject(s)
Escherichia coli , Glycoconjugates , Bacterial Vaccines/genetics , Escherichia coli/metabolism , Glycoconjugates/metabolism , Polysaccharides/metabolism , Polysaccharides, Bacterial/metabolism , Racemases and Epimerases/metabolism , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/metabolism , Vaccines, Conjugate
5.
Nature ; 597(7877): 533-538, 2021 09.
Article in English | MEDLINE | ID: mdl-34497420

ABSTRACT

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.


Subject(s)
Bacteria/metabolism , Bioaccumulation , Duloxetine Hydrochloride/metabolism , Gastrointestinal Microbiome/physiology , Animals , Antidepressive Agents/metabolism , Antidepressive Agents/pharmacokinetics , Caenorhabditis elegans/metabolism , Cells/metabolism , Click Chemistry , Duloxetine Hydrochloride/adverse effects , Duloxetine Hydrochloride/pharmacokinetics , Humans , Metabolomics , Models, Animal , Proteomics , Reproducibility of Results
6.
Cell ; 178(6): 1299-1312.e29, 2019 09 05.
Article in English | MEDLINE | ID: mdl-31474368

ABSTRACT

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Gastrointestinal Microbiome/drug effects , Host Microbial Interactions/drug effects , Hypoglycemic Agents/therapeutic use , Metformin/therapeutic use , Agmatine/metabolism , Animals , Caenorhabditis elegans/microbiology , Cyclic AMP Receptor Protein , Escherichia coli/drug effects , Escherichia coli/genetics , Humans , Hypoglycemic Agents/pharmacology , Lipid Metabolism/drug effects , Longevity/drug effects , Metformin/pharmacology , Nutrients/metabolism
7.
J Biol Chem ; 293(24): 9506-9519, 2018 06 15.
Article in English | MEDLINE | ID: mdl-29720401

ABSTRACT

Methionine (Met) is an amino acid essential for many important cellular and biosynthetic functions, including the initiation of protein synthesis and S-adenosylmethionine-mediated methylation of proteins, RNA, and DNA. The de novo biosynthetic pathway of Met is well conserved across prokaryotes but absent from vertebrates, making it a plausible antimicrobial target. Using a systematic approach, we examined the essentiality of de novo methionine biosynthesis in Salmonella enterica serovar Typhimurium, a bacterial pathogen causing significant gastrointestinal and systemic diseases in humans and agricultural animals. Our data demonstrate that Met biosynthesis is essential for S. Typhimurium to grow in synthetic medium and within cultured epithelial cells where Met is depleted in the environment. During systemic infection of mice, the virulence of S. Typhimurium was not affected when either de novo Met biosynthesis or high-affinity Met transport was disrupted alone, but combined disruption in both led to severe in vivo growth attenuation, demonstrating a functional redundancy between de novo biosynthesis and acquisition as a mechanism of sourcing Met to support growth and virulence for S. Typhimurium during infection. In addition, our LC-MS analysis revealed global changes in the metabolome of S. Typhimurium mutants lacking Met biosynthesis and also uncovered unexpected interactions between Met and peptidoglycan biosynthesis. Together, this study highlights the complexity of the interactions between a single amino acid, Met, and other bacterial processes leading to virulence in the host and indicates that disrupting the de novo biosynthetic pathway alone is likely to be ineffective as an antimicrobial therapy against S. Typhimurium.


Subject(s)
Methionine/metabolism , Salmonella Infections/metabolism , Salmonella typhimurium/growth & development , Salmonella typhimurium/pathogenicity , Animals , Biological Transport , Biosynthetic Pathways , Female , HeLa Cells , Humans , Male , Metabolome , Mice , Mice, Inbred C57BL , Salmonella typhimurium/metabolism , Virulence
8.
Oncotarget ; 8(55): 93303-93304, 2017 Nov 07.
Article in English | MEDLINE | ID: mdl-29212144
9.
Cell ; 169(3): 442-456.e18, 2017 04 20.
Article in English | MEDLINE | ID: mdl-28431245

ABSTRACT

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.


Subject(s)
Antineoplastic Agents/metabolism , Escherichia coli/metabolism , Fluorouracil/metabolism , Gastrointestinal Microbiome , Animals , Autophagy , Caenorhabditis elegans , Cell Death , Colorectal Neoplasms/drug therapy , Diet , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , Models, Animal , Pentosyltransferases/genetics
10.
Curr Opin Microbiol ; 17: 99-105, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24440968

ABSTRACT

Salmonella enterica subsp. enterica includes several very important human serovars including Typhi, Paratyphi, Typhimurium and Enteritidis. These bacteria cause a significant global burden of disease, typically classified into enteric fever, gastroenteritis and, more recently, invasive non-typhoidal salmonellosis (iNTS). Vaccines have been developed for one of these serovars, S. Typhi and the recent increase in iNTS cases has resulted in a push to develop new vaccines that will inhibit disease by S. Typhimurium and S. Enteritidis, the most common iNTS S. enterica serovars. The development of new human vaccines has been informed by studies in the murine model of typhoid fever based on S. Typhimurium infections of very 'sensitive' (Nramp-1(S)) mice, which has some obvious deficiencies, not the least that antibodies protect humans against S. Typhi infection but are only weakly protective in 'sensitive' mice infected with S. Typhimurium. S. Typhimurium also lacks Vi, the target of protective antibodies in typhoid fever. Notwithstanding these deficiencies, the murine model has identified a very complex series of innate and adaptive immune responses to infection that might be exploited to develop new vaccines. Equally, advances in understanding the pathogenesis of infection, through pathogenomics and more sophisticated animal models will likely contribute to the development of novel immunogens.


Subject(s)
Salmonella Vaccines , Animals , Disease Models, Animal , Humans , Mice , Salmonella Infections/prevention & control
11.
Proc Natl Acad Sci U S A ; 110(6): 2252-7, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23345426

ABSTRACT

IFN-γ is critical for immunity against infections with intracellular pathogens, such as Salmonella enterica. However, which of the many cell types capable of producing IFN-γ controls Salmonella infections remains unclear. Using a mouse model of systemic Salmonella infection, we observed that only a lack of all lymphocytes or CD90 (Thy1)(+) cells, but not the absence of T cells, Retinoic acid-related orphan receptor (ROR)-γt-dependent lymphocytes, (NK)1.1(+) cells, natural killer T (NKT), and/or B cells alone, replicated the highly susceptible phenotype of IFN-γ-deficient mice to Salmonella infection. A combination of antibody depletions and adoptive transfer experiments revealed that early protective IFN-γ was provided by Thy1-expressing natural killer (NK) cells and that these cells improved antibacterial immunity through the provision of IFN-γ. Further analysis of NK cells producing IFN-γ in response to Salmonella indicated that less mature NK cells were more efficient at mediating antibacterial effector function than terminally differentiated NK cells. Inspired by recent reports of Thy1(+) NK cells contributing to immune memory, we analyzed their role in secondary protection against otherwise lethal WT Salmonella infections. Notably, we observed that a newly generated Salmonella vaccine strain not only conferred superior protection compared with conventional regimens but that this enhanced efficiency of recall immunity was afforded by incorporating CD4(-)CD8(-)Thy1(+) cells into the secondary response. Taken together, these findings demonstrate that Thy1-expressing NK cells play an important role in antibacterial immunity.


Subject(s)
Interferon-gamma/biosynthesis , Killer Cells, Natural/immunology , Killer Cells, Natural/microbiology , Salmonella Infections, Animal/immunology , Salmonella typhimurium , Adoptive Transfer , Animals , Cell Differentiation/immunology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Interferon-gamma/deficiency , Interferon-gamma/genetics , Killer Cells, Natural/classification , Killer Cells, Natural/pathology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/microbiology , Lymphocyte Subsets/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Salmonella Infections, Animal/microbiology , Salmonella typhimurium/genetics , Salmonella typhimurium/immunology , Thy-1 Antigens/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...