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Hello;
I'm Christine Van Duyn, Ph.D.
[Creativity Meets Science]
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Summary of Pseudomonas aeruginosa virulence factors. Pseudomonas aeruginosa has a variety of cell surface-associated factors and secreted effectors that contributes to its virulence. The protective exopolysaccharide, alginate, as well as biosurfactants called rhamnolipids that aid in swarming motility are secreted by P. aeruginosa. Immunogenic lipopolysaccharides (LPS) causes inflammation and results in immune system dysregulation. Type IV pili are involved in attachment to epithelial cells, initiates swarming motility, and facilitates communication between cells within a biofilm. Quorum sensing (QS) involves cell-to-cell communication by means of diffusible signaling molecules that mediate synchronization of coordinated responses to environmental stimuli. Surface-associated bacterial communities called biofilms provide resistant to antimicrobial reagents and offer protection from elimination by host immune cells. Multiple efflux pumps provide resistance to several classes of antibiotics including aminoglycosides, polymyxins, fluoroquinolones, and ß-lactam antibiotics. Secretion of alkaline protease by the Type I Secretion System (T1SS) results in heme acquisition via the destruction of red blood cells (RBCs) and blocking of complement-mediated lysis by neutrophils and RBCs. The Type II Secretion System (T2SS) releases elastases that break down host tissue and impair healing, especially in patients with burn wounds. The Type V Secretion System (T5SS) secretes lipolytic enzymes, lipases, and proteases that cause tissue damage. Additionally, the T5SS secretes several adhesins involved in initial attachment and development of biofilms. The Type III Secretion System (T3SS) secretes pore-forming effectors directly into cell membranes of eukaryotic cells, inhibiting phagocytosis and inducing apoptosis of immune cells. The Type VI Secretion System (T6SS) secretes toxic effectors directly into the membranes of other bacteria using a needle-like apparatus that deploys from a contractile outer sheath, causing them to lyse. T6SS are important for interbacterial competition and affect both Gram-positive and Gram-negative bacteria.
Model of the Gac/Rsm Signaling Network.
The Gac/Rsm signal transduction system in P. aeruginosa controls the reversible transition between acute and chronic infections. The RsmA regulatory protein binds to the promoters of genes relevant for acute infection while repressing genes associated with chronic infections. GacA phosphorylation by GacS stimulates the production of the small RNAs RsmY and RsmZ, which bind to RsmA, repressing its activation of virulence factors associated with acute infections and allowing for the activation of virulence factors associated with chronic infections. The sensor kinase LadS works in concert with GacS in activating RsmY and RsmZ production while the sensor kinase RetS forms a protein-protein complex with GacS and blocks production of RsmY and RsmZ. Created using Biorender.
The Gac/Rsm signal transduction system in P. aeruginosa controls the reversible transition between acute and chronic infections. The RsmA regulatory protein binds to the promoters of genes relevant for acute infection while repressing genes associated with chronic infections. GacA phosphorylation by GacS stimulates the production of the small RNAs RsmY and RsmZ, which bind to RsmA, repressing its activation of virulence factors associated with acute infections and allowing for the activation of virulence factors associated with chronic infections. The sensor kinase LadS works in concert with GacS in activating RsmY and RsmZ production while the sensor kinase RetS forms a protein-protein complex with GacS and blocks production of RsmY and RsmZ. Created using Biorender.
Schematic of Experimental-Computational Methodology of RIL-seq
RIL-seq (RNA Interaction by Ligation and Sequencing) relies on the binding of sRNAs to the hexameric RNA binding protein (RBP), Hfq, which facilitates base pairing with its target mRNA. Bacteria carrying a Flag- tagged Hfq protein are grown under desired conditions and are UV-irradiated to cross-link Hfq to bound RNA molecules. Cells are then lysed and Hfq is immunoprecipitated with bound RNA-RNA complexes. The unbound regions are trimmed with RNases and the bound RNAs are ligated together following 5' and 3' phosphorylation of RNA with T4 Polynucleotide Kinase (T4 PNK). Hfq is degraded using Proteinase K and RNA-RNA fragments are isolated, reverse transcribed into cDNA, and analyzed by Illumina sequencing. Fragments are classified as "chimeric" if the two fragments map to different genomic locations and classified as "S-chimeras" if they are overrepresented and statistically-significant. S-chimeras are then analyzed to expand knowledge of regulatory networks. Hfq shown as a pink hexamer. The flag tag shown as an orange diamond Lock icons represent ligated RNAs.
Created using Biorender.
RIL-seq (RNA Interaction by Ligation and Sequencing) relies on the binding of sRNAs to the hexameric RNA binding protein (RBP), Hfq, which facilitates base pairing with its target mRNA. Bacteria carrying a Flag- tagged Hfq protein are grown under desired conditions and are UV-irradiated to cross-link Hfq to bound RNA molecules. Cells are then lysed and Hfq is immunoprecipitated with bound RNA-RNA complexes. The unbound regions are trimmed with RNases and the bound RNAs are ligated together following 5' and 3' phosphorylation of RNA with T4 Polynucleotide Kinase (T4 PNK). Hfq is degraded using Proteinase K and RNA-RNA fragments are isolated, reverse transcribed into cDNA, and analyzed by Illumina sequencing. Fragments are classified as "chimeric" if the two fragments map to different genomic locations and classified as "S-chimeras" if they are overrepresented and statistically-significant. S-chimeras are then analyzed to expand knowledge of regulatory networks. Hfq shown as a pink hexamer. The flag tag shown as an orange diamond Lock icons represent ligated RNAs.
Created using Biorender.
Model of σ22-mediated activation of the alginate biosynthetic operon in response to cell wall stress. (A) Genetic map of the algT/U operon of Pseudomonas aeruginosa. Black arrows represent promoter regions and the blue arrow indicates autoregulation by the gene product of algT, σ22 (B) Model of σ22 under normal conditions where it is sequestered by the anti-sigma factor MucA. MucB, another anti-sigma factor is predicted to interact with the periplasmic domain of MucA and MucD encodes a periplasmic serine protease. Under normal conditions the major housekeeping sigma factor, σ70 directs the RNA polymerase to the promoter sequence at -10 and -35 (C) Model of σ22 activation under conditions of cell wall stress. The alternative sigma factor, σ22 is released from sequestration by MucA. σ22 replaces the housekeeping sigma factor and directs the RNA polymerase to the algD operon, which is responsible for alginate overproduction. The most common mutation in P. aeruginosa CF isolates is a mucA22 mutation, which leads to a truncated MucA that cannot sequester MucB appears to interact with the periplasmic domain of MucA as part of signal transduction mechanism. Periplasmic MucD may play a role in signal transduction. P. aeruginosa strains in CF patients frequently undergo mucA22 mutation, which truncates MucA, resulting in constitutive σ22 expression and alginate overproduction. Figure created using Biorender.
Schematic of the alginate biosynthetic complex and its encoding algD operon.
(A) Predicted structure of the alginate biosynthetic complex of P. aeruginosa (B) Genetic map of the algD operon.
Proteins and genes are color-coded according to their predicted function. Created using Biorender.
(A) Predicted structure of the alginate biosynthetic complex of P. aeruginosa (B) Genetic map of the algD operon.
Proteins and genes are color-coded according to their predicted function. Created using Biorender.
Summary of P. aeruginosa infections
(A) Summary of predisposing conditions for P. aeruginosa infections (B) Schematic of a healthy lung compared to a CF lung. In a healthy lung (Left), a functional CFTR (CF transmembrane conductance regulator) moved water and chloride ions across epithelial cells, thinning the airway mucus and allowing for clearance of bacteria. In a CF lung (Right), aberrant chloride transport from a defective or absent CFTR leads to a buildup of dehydrated mucus which leads airway obstruction, polymicrobial infection, and persistent inflammation. In addition, neutrophils release tissue-damaging NETs (neutrophil extracellular traps) and Pseudomonas aeruginosa secretes an exopolysaccharide called alginate, further exacerbating pulmonary complications. Created using Biorender.
(A) Summary of predisposing conditions for P. aeruginosa infections (B) Schematic of a healthy lung compared to a CF lung. In a healthy lung (Left), a functional CFTR (CF transmembrane conductance regulator) moved water and chloride ions across epithelial cells, thinning the airway mucus and allowing for clearance of bacteria. In a CF lung (Right), aberrant chloride transport from a defective or absent CFTR leads to a buildup of dehydrated mucus which leads airway obstruction, polymicrobial infection, and persistent inflammation. In addition, neutrophils release tissue-damaging NETs (neutrophil extracellular traps) and Pseudomonas aeruginosa secretes an exopolysaccharide called alginate, further exacerbating pulmonary complications. Created using Biorender.
Summary of Regulation by Bacterial sRNAs.
Christine Van Duyn, Ph.D.
I received my Ph.D. in Microbiology and Immunology from
Virginia Commonwealth University in Richmond, VA in
November 2020. My hope is to combine my scientific knowledge and passion for research with my creativity to produce interactive images and engaging presentations to make science accessible to everyone.
Let's work together.
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