High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.
BACKGROUND
With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive.


RESULTS
We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA.


CONCLUSIONS
This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.
Greub
Gilbert
G
Center for Research on Intracellular Bacteria (CRIB), Institute of Microbiology, University Hospital Center, University of Lausanne, Lausanne, Switzerland. gilbert.greub@chuv.ch
Kebbi-Beghdadi
Carole
C
Bertelli
Claire
C
Collyn
François
F
Riederer
Beat M
BM
Yersin
Camille
C
Croxatto
Antony
A
Raoult
Didier
D
D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D002695:Chlamydiales / Q000235:genetics* / Q000472:pathogenicity*; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins