Functional microbial genomics hiv
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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


Functional microbial genomics hiv

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


Functional microbial genomics hiv

Mock control

PBMC

Target cells

Non-Infection: gene sets

Infection: signature gene sets

comparison

Change in disease or functional Status


Functional microbial genomics hiv

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


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


Functional microbial genomics hiv

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.


Functional microbial genomics hiv

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.


Functional microbial genomics hiv

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).


Functional microbial genomics hiv

Chemotaxis of Monocytes and activated T cells


Functional microbial genomics hiv

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

  • 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

  • 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.


Functional microbial genomics hiv

Tandem Affinity Purification Method

Guillaume Rigaut, 1999

TEV cleavage site

calmodulin-binding peptide

protein A

TAP tag


Protein identification and functional analysis

Protein identification and functional analysis

MALDI-TOF

Protein samples from GE

- Online Data mining

- Functional assay


Functional microbial genomics hiv

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


Functional microbial genomics hiv

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).


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.


Functional microbial genomics hiv

Genomic studies on

bacterial pathogens

何漣漪

國立成功大學

微生物及免疫學研究所


Functional microbial genomics hiv

Approaches to bacterial pathogenesis

  • Biochemical studies

  • Genetic studies

  • Genomic studies

    • Functional genomics

    • Comparative genomics

    • Proteomics

  • Bacterium-host interactions


  • Functional microbial genomics hiv

    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.


    Functional microbial genomics hiv

    FEMS Immunol Med Microbiol 50 (2007) 165–176


    Functional microbial genomics hiv

    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


    Functional microbial genomics hiv

    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


    Functional microbial genomics hiv

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


    Functional microbial genomics hiv

    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.


    Functional microbial genomics hiv

    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


    Study procedure

    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


    Functional microbial genomics hiv

    Features of the BT2-specific DNA sequences


    Functional microbial genomics hiv

    Specificity of the BT2-specific DNA sequences


    Plasmid study

    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.


    Functional microbial genomics hiv

    ORF prediction and annotation

    ORF prediction by Glimmer and GenMark

    Annotation by AutoFACT

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


    Functional microbial genomics hiv

    Plasmid Curing

    Association of BT2 plasmids with bacterial virulence in eels and mice


    Functional microbial genomics hiv

    Association of vep07 with virulence for eels


    Functional microbial genomics hiv

    • 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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