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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

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
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


Mock control

PBMC

Target cells

Non-Infection: gene sets

Infection: signature gene sets

comparison

Change in disease or functional Status



Microarray assay and functional categories
Microarray assayand functional categories


Functional genomics of hiv infection1
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


Reprogramming the idc
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


Study approach
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


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.


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.


MCP-2 expression and SIV infection in axillary lymph nodes whose expression is affected byTat.

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).


Chemotaxis of Monocytes and activated T cells whose expression is affected byTat.


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


Functional genomics of hiv infection2
Functional genomics of HIV infection 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

  • 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


Proteome analysis
Proteome analysis 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

  • 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.


Tandem Affinity Purification Method 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

Guillaume Rigaut, 1999

TEV cleavage site

calmodulin-binding peptide

protein A

TAP tag


Protein identification and functional analysis
Protein identification and functional analysis 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

MALDI-TOF

Protein samples from GE

- Online Data mining

- Functional assay


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


Synopsis of the screen 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

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).


The protein complex network and grouping of connected complexes
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.


Genomic studies on complexes

bacterial pathogens

何漣漪

國立成功大學

微生物及免疫學研究所


Approaches to bacterial pathogenesis complexes

  • Biochemical studies

  • Genetic studies

  • Genomic studies

    • Functional genomics

    • Comparative genomics

    • Proteomics

  • Bacterium-host interactions


  • Applications of microbial genomes complexes

    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.



    Comparative genomics complexes

    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


    Functional genomics complexes

    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


    What makes the biotype 2 complexesVibrio vulnificus strains virulent for the eel?


    V. vulnificus complexes 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.


    Hypothesis complexes

    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


    Study procedure
    Study procedure complexes

    • 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




    Plasmid study
    Plasmid study complexes

    • 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.


    ORF prediction and annotation complexes

    ORF prediction by Glimmer and GenMark

    Annotation by AutoFACT

    BLAST from UniRef90, UniRef100, NCBI’snrdb, COG, KEGG, PFAM, and SMART


    Plasmid Curing complexes

    Association of BT2 plasmids with bacterial virulence in eels and mice


    Association of complexesvep07 with virulence for eels


    • Summary complexes

    • 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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