Project description:DPANN are a widespread and highly diverse group of archaea characterised by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. DPANNs are predominantly obligate ectosymbionts that depend on their host for their survival and proliferation. Despite the recent expansion in this clade, the structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we used electron cryotomography (cryo-ET) to reveal that the Candidatus Micrarchaeota” (ARM-1) interacts with its host through intercellular proteinaceous nanotubes. These tubes (~4.5 nm wide) originate in the host, extend all the way to the DPANN cytoplasm and act like tunnels for intercellular exchange. Combining cryo-ET and sub-tomogram averaging, we revealed the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states, providing mechanistic insights into substrate exchange. Additionally, we performed comparative proteomics and genomic analyses to identify host proteomic changes in response to the DPANN attachment. Our results showed striking alterations in host-proteome during symbiosis and upregulation/downregulation of key cellular pathways. Collectively, these results provided unprecedented insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.
Project description:Inducible gene expression systems are pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate-inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (PCymRC/CymRGV) which allows the tight and tunable control of protein expression within B. cenocepacia as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allows precise and tuneable gene expression in Burkholderia even within a host-pathogen context.
Project description:Inducible gene expression systems are pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate-inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (PCymRC/CymRGV) which allows the tight and tunable control of protein expression within B. cenocepacia as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allows precise and tuneable gene expression in Burkholderia even within a host-pathogen context.
Project description:Inducible gene expression systems are pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate-inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (PCymRC/CymRGV) which allows the tight and tunable control of protein expression within B. cenocepacia as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allows precise and tuneable gene expression in Burkholderia even within a host-pathogen context.
Project description:Inducible gene expression systems are pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate-inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (PCymRC/CymRGV) which allows the tight and tunable control of protein expression within B. cenocepacia as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allows precise and tuneable gene expression in Burkholderia even within a host-pathogen context.
Project description:Inducible gene expression systems are pivotal for dissecting bacterial physiology and virulence mechanisms. Across the Burkholderia genera, a limited range of inducible systems currently exist that show minimal impacts on the proteome and allow tight regulation. In this study, we engineer a set of cumate-inducible vectors for use in Burkholderia cenocepacia that offer minimal basal expression and the ability to control B. cenocepacia gene expression within Eukaryotic cells. Through mutagenesis-based studies of cumate circuits and the cumate regulator (CymR), we generate an optimized cumate circuit (PCymRC/CymRGV) which allows the tight and tunable control of protein expression within B. cenocepacia as assessed by fluorescent and protein O-linked glycosylation analysis. Using comparative proteomics, we demonstrate cumate induction leads to both reduced and orthogonal effects on B. cenocepacia compared to widely used rhamnose based induction systems. Leveraging the cell permeability of cumate and the generation of a CTX-based chromosomal integration vector, we show inducible control of protein expression is achievable during intracellular replication of B. cenocepacia. Finally, using the ability to control intracellular expression, we demonstrate the requirement of O-linked protein glycosylation for optimal B. cenocepacia intracellular replication. Combined, this work demonstrates that cumate inducible systems allows precise and tuneable gene expression in Burkholderia even within a host-pathogen context.
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:Proteomic analysis of Bacteroides thetaiotaomicron Dma2 and its impact on both the proteome and localisation of proteins across fractions