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Functional Microbial Genomics…HIV

Functional Microbial Genomics…HIV. Shainn-Wei Wang, Ph.D. NCKU, College of Medicine Institute of Molecular Medicine. HIV exhibits tremendous genetic diversity. Garber D. A., et al., Lancet Infec. Dis., 2004. Functional genomics of HIV infection. Host gene re-programming to viral infection

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Functional Microbial Genomics…HIV

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  1. Functional Microbial Genomics…HIV Shainn-Wei Wang, Ph.D. NCKU, College of Medicine Institute of Molecular Medicine

  2. HIV exhibits tremendous genetic diversity Garber D. A., et al., Lancet Infec. Dis., 2004

  3. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to a viral protein • Cellular protein networks in response to assembly, replication, and latency

  4. Mock control PBMC Target cells Non-Infection: gene sets Infection: signature gene sets comparison Change in disease or functional Status

  5. The Journal of Infectious Diseases    2004;189:572-582

  6. Microarray assayand functional categories

  7. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to aviral protein • Cellular protein networks in response to assembly, replication, and latency

  8. Reprogramming the iDC • As the major activator of HIV transcription, Tat drives viral gene expression. • Tat regulates the expression of chemokines that promote lymphocyte and monocyte migration. • By recruiting susceptible host cells to infected dendritic cells,Tat may facilitate HIV dissemination

  9. Study approach • Gene chip assay • iDC mRNA • Under different treatments and different time • Which genes be increased expression • Which genes be decreased expression • RT-PCR • Up-regulated gene • Gene expression analysis • Colocalization • Maturation markers expression

  10. Expression analysis of primary iDC infected with HIV-1BAL and adeno-Tat • Expression profiles of iDC genes whose RNA levels were affected similarly by adeno-Tat and • HIV-1 infection. • Genes are divided into functional groups; the fold change in expression levels relative to the • 0 time point is displayed in red (increased expression) or green (decreased expression). • - Asterisk (*) marks IFN-inducible genes.

  11. RT-PCR analysis of selected immature dendritic cell genes whose expression is affected byTat. • Identical amounts of total RNA from iDC infected with adeno-LacZ and adeno-Tat were used. • Control β-actin mRNA was not affected by HIV-1 or adeno-Tat, confirming the microarray analysis.

  12. MCP-2 expression and SIV infection in axillary lymph nodes a, SIV Nef–expressing cells (red) in the paracortex. b, Digital overlay of images from the same field labeled for MCP-2 (green) and SIV Nef (red). Arrows indicate double-labeled cells (yellow) that are positive for both markers in the digital overlay of images. c–e, High-powered fields of lymph nodes showing SIV Nef (c),MCP-2 (d) and DC-SIGN (e, blue) expression by a single cell (indicated by arrows). f, Digital overlay of c–e shows a single SIV-infected dendritic cell (indicated by arrow) expressing all three markers (original magnification: a,b, ×200, c–f, ×400).

  13. Chemotaxis of Monocytes and activated T cells

  14. None of the typical dendritic cell maturation markers (such as CD40, CD80, CD83, CD86 and CD25) were expressed at increased levels during the time course of adeno-Tat, adeno-LacZ or HIV-1 infection

  15. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to a viral protein • Cellular protein networks in response to assembly, replication, and latency

  16. Proteome analysis • Protein-Protein interaction • Two Hybrid Sysytem • (Tandem) Affinity Tag purification method • Protein complex purification requires expression of the target protein at, or close to, its natural expression level. • Among all kinds of protein complex purification method, protein A and CBP tags allowed efficient recovery of proteins • Proteome analysis, in particular using mass spectrometry (MS), requires fast and reliable methods of protein purification.

  17. Tandem Affinity Purification Method Guillaume Rigaut, 1999 TEV cleavage site calmodulin-binding peptide protein A TAP tag

  18. Protein identification and functional analysis MALDI-TOF Protein samples from GE - Online Data mining - Functional assay

  19. Nature. 2002 Jan 10;415(6868):141-7.

  20. Synopsis of the screen a. Schematic representation of the gene targeting procedureThe TAP cassette is inserted at the C terminus of a given yeast ORF by homologous recombination, generating the TAP-tagged fusion protein. b. Examples of TAP complexes purified from different subcellular compartments separated on denaturing protein gels and stained with Coomassie. Tagged proteins are indicated at the bottom. ER, endoplasmic reticulum. c, Schematic representation of the sequential steps used for the purification and identification of TAP complexes (left), and the number of experiments and success rate at each step of the procedure (right).

  21. The protein complex network, and grouping of connected complexes Links were established between complexes sharing at least one protein. For clarity, proteins found in more than nine complexes were omitted. The graphs were generated automatically by a relaxation algorithm that finds a local minimum in the distribution of nodes by minimizing the distance of connected nodes and maximizing distance of unconnected nodes. In the upper panel, cellular roles of the individual complexes are colour coded: red, cell cycle; dark green, signalling; dark blue, transcription, DNA maintenance, chromatin structure; pink, protein and RNA transport; orange, RNA metabolism; light green, protein synthesis and turnover; brown, cell polarity and structure; violet, intermediate and energy metabolism; light blue, membrane biogenesis and traffic. The lower panel is an example of a complex (yeast TAP-C212) linked to two other complexes (yeast TAP-C77 and TAP-C110) by shared components. It illustrates the connection between the protein and complex levels of organization. Red lines indicate physical interactions as listed in YPD22.

  22. Genomic studies on bacterial pathogens 何漣漪 國立成功大學 微生物及免疫學研究所

  23. Approaches to bacterial pathogenesis • Biochemical studies • Genetic studies • Genomic studies • Functional genomics • Comparative genomics • Proteomics • Bacterium-host interactions

  24. Applications of microbial genomes 1. Design of new antimicrobial agents and vaccines. Cegelski L, Marshall GR, Eldridge GR, and Hultgren SJ. 2008. The biology and future prospects of antivirulence therapies. Nature Reviews Microbiology 6: 17-26. 2. Development of high-throughput detection or diagnosis of microbial pathogens.

  25. FEMS Immunol Med Microbiol 50 (2007) 165–176

  26. Comparative genomics Helicobacter pylori diversity at the genome level Definition of the core genome and the variable gene pool Identification of strain-, species- and genus-specific genes Worldwide coevolution and spreading with the human host (by Multi Locus Sequence Typing, MLST) Microevolution and genetic variability within single human hosts H. pylori evolution during early infection and disease progression FEMS Immunol Med Microbiol 50 (2007) 165–176

  27. Functional genomics Transcriptome analysis in conditions mimicking those encountered in the host (acidity, growth phase, iron starvation and attachment to gastric cells) Transcriptome analysis in mutants deficient in regulators (eg. Ars, Fur, s28, s54) Transcriptome analysis in mutants deficient in regulators in response to acidity and metal metabolism FEMS Immunol Med Microbiol 50 (2007) 165–176

  28. What makes the biotype 2 Vibrio vulnificus strains virulent for the eel?

  29. V. vulnificus are divided into three biotypes: biotypes 1, 2, and 3, by the differences of biochemical properties and host range. Among these biotypes, only biotype 2 can infect eels and has caused economical losses in brackish-water anguilliculture in Europe. Two major serovars of biotype 2: serovars E and A. Serovar E strains have been isolated from human cases.

  30. Hypothesis The virulence determinants for eels may be encoded from regions that are present in biotype 2, but not biotype 1, strains Suppression subtractive hybridization (SSH) Tester: one biotype 2 strain Drivers: three biotype 1 strains Identification of the biotype 2-specific DNA fragments

  31. Study procedure • Suppression subtractive hybridization (SSH) • Tester DNA (biotype 2) and driver DNA (biotype 1, three strain) • Identification of the biotype 2-specific DNA fragments • Subtraction library (PCR product, mixture) • Southern hybridization • with probes derived from the subtraction products • PCR cloning library • with probes derived from two different clones • Dot blot hybridization to identify the identical subtractive clones • Cloning and Sequencing: BT2-specific DNA sequences

  32. Features of the BT2-specific DNA sequences

  33. Specificity of the BT2-specific DNA sequences

  34. Plasmid study • Plasmid curing • Plasmid sequemcing • Isolation of plasmid DNA • Construction of shotgun library (> 10 X coverage) • DNA sequence determination by an autosequencer (ABI 3700) • DNA sequence assembly by Phred/Phrap/Consed • Closing of gaps by primer walking on linking clones and PCR products amplified with primers derived from end sequences of the contigs. • DNA sequence confirmation by restriction mapping with a variety of enzymes.

  35. ORF prediction and annotation ORF prediction by Glimmer and GenMark Annotation by AutoFACT BLAST from UniRef90, UniRef100, NCBI’snrdb, COG, KEGG, PFAM, and SMART

  36. Plasmid Curing Association of BT2 plasmids with bacterial virulence in eels and mice

  37. Association of vep07 with virulence for eels

  38. Summary • The eel-virulent V. vulnificus strains contain a virulence plasmid. Curing of this plasmid from the host results in loss of virulence for eels, but not for mice. Furthermore, acquisition of the virulence plasmid restores virulence for eels. • A gene, vep07, in the virulence plasmid pR99 that encodes a hypothetical protein is associated with virulence for eels and resistance to killing by eel serum. • 3. The virulence plasmid can be transferred between the biotype 2 strains by conjugation in the presence of a self-transmissible plasmid and maintain stably in the host cell. It can not be readily transferred to the biotype 1 strains, however.

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