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
Shainn-Wei Wang, Ph.D.
NCKU, College of Medicine
Institute of Molecular Medicine
Garber D. A., et al., Lancet Infec. Dis., 2004
Non-Infection: gene sets
Infection: signature gene sets
Change in disease or functional Status
RT-PCR analysis of selected immature dendritic cell genes whose expression is affected byTat.
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
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
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).
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
Approaches to bacterial pathogenesis complexes
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.
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
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