炭疽感染的蛋白质组学课件

Proteomics of Anthrax Infections Phil Hanna 765 proteins 173 genes in common Expression of the B. anthracis spore proteome 1 2 3 4 5 Spore Proteome 765 proteins 400 expressed constitutively 130 58 23 25 125Including the ger operons B. anthracis Germinant Sensors Tri-cistronic operons Expressed late during sporulation Disruption leads to a germinant specific defect in germination Chromosomal: gerH gerS gerL gerK gerY* gerA* pXO1: gerX gerLAgerLBgerLC P G * Frameshift B. anthracis Germinant Sensors 1567234 0.03 0.1 1 3 OD600 gerH gerS gerL gerK gerY* gerX gerA* Red = Repressed Green = Expressed Hours Growth and sporulation In mG medium Bacillus Life Cycle Vegetative Growth gerE Sporulation H2O Ca+ Na+ H+ DPA K+ K+ Germination Stage IStage II Proteases CLE Outgrowth Target Validation #1 Determine the contributions made by each putative germinant sensor to the germination profile of B. anthracis endospores. gerLAgerLBgerLC P G Approach: Disrupt individual sensors Assess pathway defects in resulting null endospores Assign pathway functions to each sensor Disruption of Germinant Sensors gerLAgerLBgerLC P G ALacZ oriTs LacIq Pspac pNFd13 KanR 30 C LacIq Pspac gerLA:pNFd13 ALacZABC KanR oriTs 39 C P G Disruption of Germinant Sensors LacIq Pspac gerLA:pNFd13 ALacZABC KanR oriTs P G IPTG Isogenic Complementation -gal Assayed for Expression Analysis Sensor Null Endospore X Sporulation Sensor Null Endospore X Germination Analysis Sensor Null Endospores 39C sporulation Single isolate (ger_:pNFd13) Sensor Complemented Endospores 39C sporulation - IPTG 39C sporulation + IPTG Diluted Endospores in Germinant Solution Null or Complemented Endospores In Aqueous suspension Germination Assays H2O Ca+ Na+ H+ DPA K+ K+ in density = in OD600 Loss of heat-resistance (plating) Ca-release assays DPA release assay Dye uptake assays -gerL-gerK -gerS-gerX -gerH Germinant Recognition O OHHO HOCH2NN N NH2 Adenosine (Purine nucleosides) (Aromatic Amino Acids) gerL gerK gerS gerX gerH Germination Macrophage Associated Germination Target Validation #2 dltA, is the first of four genes in the dlt operon, which controls the incorporation of D-alanine into lipoteichoic acids on the cell surface. Essential for: maintenance of normal cell shape, control of autolytic enzymes, and resistance to cationic anti-microbial peptides in Gram positive organisms. Loss of dlt operon has been shown to decrease the virulence of S. aureus and L. monocytogenes. It has many characteristics of an potential candidate for drug development targeting in B. anthracis. Figure 2. Expression analysis. Integration of pNFd13 into the targeted locus results in the B- galactosidase reporter being placed downstream of the native promoter for expression analysis. Panel A shows a typical growth curve of B. anthracis in modified G medium. Panel B shows the expression patters of dlt and gerH during this growth cycle. Panel C shows the expression patters of these loci based on microarray analysis under identical growth conditions (2). The pNFd13 analysis verifies and quantitates the microarray data. A B C gerH dltA Figure 4. Loss of dlt results in cell shape defects. Wild type (A) or the dlt disruption strain (B) were grown overnight on BHI plates and analyzed by Gram staining. Loss of dlt results in cell shape deformities ( ) and irregular cell size. Additionally, dlt cells become autolytic during stationary phase resulting in many ghost cells ( ) in the culture. AB Figure 5. Resistance to the Cationic Anti-microbial Peptide Nisin depends on the expression level of the dlt locus. 34F2 dltA:pNFd13 or 34F2 gerA:pNFd13 (a control strain with pNFd13 inserted at a silent locus). Cells were grown to early stationary phase then back diluted into fresh media containing the indicated concentration of IPTG and serial dilutions of Nisin. After 16 hours, growth was assessed by optical density at 600nm. The minimum inhibitory concentration (MIC) was recorded and varied by concentration of IPTG in the growth medium. Figure 6. The dlt system contributes to B. anthracis survival in cultured RAW macrophage. 34F2 dltA:pNFd13 or 34F2 gerA:pNFd13 endospores were added to RAW macrophage at a multiplicity of infection of 10. The infection was allowed to proceed for the indicated time, after which glass coverslips were removed from each infection well, vortexed in water, and bacterial colony forming units were counted by dilution plating. Results shown are triplicate dilution titers from a single representative of three individual experiments. Research Plan for 05 Phase 1 0-18 months Protein & Gene analyses of B. anthracis during lab growth. Validation of potential essential & surface genes. Phase 2 12-36 months Protein & gene analyses during B. anthracis infection stages. Molecular definition of B. anthracis infection kinetics. Phase 3 24-48 months Analysis of infection by other B.a. strains, other Bacillus species and purified virulence factors. Phase 4 24-60 months Detailed validation of critical factors “choke poi