1、,Proteomics of Anthrax Infections,Phil Hanna & John Yates U. of Michigan & The Scripps Research Institute pchannaumich.edu,1st Programmatic Meeting NIAID Biodefense Proteomics Research Program Silver Spring, Maryland October 25, 2004,Objectives,Document global protein & gene expression patterns at v
2、ital stages of the anthrax infection. Document global host cell protein & gene expression patterns during infection. Validate potential “choke-points”. Genetic mutants approaches for B. anthracis RNAi for host cells Structural determination of leads Crystallography cores at the SERCEB & GLRCE,Criter
3、ia,Target protein is present during infection. Target proteins mRNA is also expressed. Target protein has close homology to known essential factors, or virulence factors, from other organisms. Target protein maintains critical activities, regions or structures that are non-mammalian.,Progression of
4、Systemic Anthrax0 2 3 4 5 6 Days,B. anthracis morphologies,Endospore Stable for decades Resistant to uv, heat, desiccation &, antibiotics Metabolically dormant, yet very sensitive to germinants in environment Infectious particle,Vegetative bacilli Rapid growth (doubling time 15-20 minutes) Non-infec
5、tious,Bacillus Life Cycle,Sporulation,Vegetative Growth,Vegetative cell,B. anthracis genome,Chromosome 5.23 MB 5508 genespXO1 toxin plasmid182 KB 217 genes pXO2 capsule plasmid95 KB 113 genes,T. D. Read et al, Nature 2003,Which of these encodes the next vaccine or drug target?,B. anthracis genomenew
6、 possibilities,DNA microarray construction for RNA expression studiesMass Spectrometry-based shotgun proteomics,T. D. Read et al, Nature 2003,Sporulation in B. anthracis,Cluster analysis of growth phase-regulated B. anthracis genes,0:00,5:00,2090 genes,K-median cluster analysis -24 clusters of co-ex
7、pressed genes -4719 genes scored -3518 with 2-fold change -2090 2-fold 2consecutive time pointsArranged in order by time of activation,Growth phase dependent expression occurs in five waves,0:00,5:00,B. anthracis genomenew possibilities,DNA microarray construction for RNA expression studiesMass Spec
8、trometry-based shotgun proteomics,T. D. Read et al, Nature 2003,SEQUEST DTASelect & Contrast, 700 Proteins Identified,Tandem Mass Spectrometer,Sample: The Anthrax Spore,Digestion,Lysis,High-Throughput Proteomics: MuDPIT,MS/MS Spectrum,Good non-bias with Multi-Dimensional Protein Identification MuDPI
9、T,Theoretical proteome, B. anthracis spore,Actual proteome, B. anthracis spore,Expression of the B. anthracis spore proteome,1,2,3,4,5,Spore Proteome,873 genes;,765 proteins,Expression of the B. anthracis spore proteome,1,2,3,4,5,Spore Proteome,765 proteins,Including the ger operons,B. anthracis Ger
10、minant 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,gerLA,gerLB,gerLC,P G,* Frameshift,B. anthracis Germinant Sensors,0.03,0.1,1,3,OD600,Hours,Growth and sporulat
11、ion In mG medium,Bacillus Life Cycle,Sporulation,Target Validation #1,Determine the contributions made by each putative germinant sensor to the germination profile of B. anthracis endospores.,gerLA,gerLB,gerLC,P G,Approach: Disrupt individual sensors Assess pathway defects in resulting null endospor
12、es Assign pathway functions to each sensor,Disruption of Germinant Sensors,P G,Disruption of Germinant Sensors,Germination Analysis,Germination Assays,D-gerL,D-gerK,D-gerS,D-gerX,D-gerH,Germinant Recognition,Germination,Macrophage Associated Germination,Target Validation #2,dltA, is the first of fou
13、r 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 be
14、en 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 place
15、d 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
16、 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 result
17、s in cell shape deformities ( ) and irregular cell size. Additionally, dlt cells become autolytic during stationary phase resulting in many ghost cells ( ) in the culture.,A,B,Figure 5. Resistance to the Cationic Anti-microbial Peptide Nisin depends on the expression level of the dlt locus. 34F2 dlt
18、A: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 a
19、t 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 multi
20、plicity 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 sin
21、gle 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 defi
22、nition 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 points”Atomic structures Animal studies,Acknowledgements,Hanna Lab: Drs. Nick Bergman & Amy Herring, Erica Johnson + 1 John Yates Lab Scripps, MuDPIT proteomics Crystallography: SERCEB & GLRCE Bob Liddington (Burnham Inst.) SRI & NERCE drug screening GeneWorks Inc.,
