1995 was a hallmark in the history of microbiology, with the first complete bacterial genome sequencing (Fleischmann et al., 1995). Subsequently, bacterial genome sequencing experienced a considerable development, with 1,000 genomes sequenced within only 15 years. Initially, genome sequences were mostly used to address fundamental question in research, notably for studying bacterial evolution. However, the development of high throughput sequencing technologies (Hall, 2007), computational assembly of sequences, and functional inferences, gave access to a tremendous source of information and revolutionized basic aspects of microbiology. Genome analysis, also known as genome mining or in silico analysis, now constitutes an irreplaceable research tool for various aspects of microbiology. In particular, the availability of genomes from many bacterial human pathogens has opened perspectives in the fields of diagnosis, epidemiology, pathophysiology and treatment (Woodford & Johnson, 2004).
Genome sequencing of bacteria, especially those recently isolated, has several interests (Fournier et al., 2007):
1°) To obtain information on the ability of bacteria to resist antibiotics. We discovered in Acinetobacter baumannii a large resistance island that contained most genes encoding resistance markers, including mechanisms unidentified phenotypically (Fournier et al., 2006).
2°) To identify sequences specific to a given clone. We applied this technique based on genomic analysis to the genotyping of Rickettsia species and developed a tool named multi-spacer typing for these bacteria (Fournier et al., 2004). In particular, this enables tracing epidemic strains and demonstrated to be valid for other bacterial taxa, including Tropheryma whipplei (Li et al., 2008).
3°) To develop diagnostic tools. By screening genome sequences for repeats, we identified multi-copy sequences that were suitable PCR targets for Tropheryma whipplei and enabled a significantly increased detection sensitivity (Fenollar et al., 2004).
4°) To understand the evolutionary mechanisms of a given bacterial clade. By comparing the genomes of Rickettsia species, we determined that genome reduction by progressive gene degradation is an ongoing phenomenon in these bacteria (Ogata et al., 2001), and is linked to increase in virulence (Fournier et al., 2009). We also demonstrated that these strictly intracellular bacteria have conjugative plasmids and may be able to exchange genetic material (Fournier et al., 2008).
5°) To understand the adaptation of intra-amoebal bacteria to their hostile environment. The partial study of the Legionella drancourtii genome enabled us to identify genes horizontally acquired from plants and amoebae (Moliner et al., 2009b;Moliner et al., 2009a) and to speculate that these bacteria evolve in a sympatric lifestyle that leads to a larger genome due to genetic exchange (Moliner et al., 2009b).
We plan to sequence and study additional bacterial genomes to complete our understanding of genomics of intracellular bacteria. An objective is to study the genomic substratum of the observed differences between the clinical forms of the diseases caused by Rickettsia species. A second objective is to sequence rapidly all Rickettsia subspecies and compare their genomic contents in order to develop specific diagnostic tools.
We intend to sequence 12 rickettsial genomes, including R. conorii subsp. Indica, R. conorii subsp. Caspia, R. conorii subsp. Israelensis, R. sibirica subsp. Mongolitimonae, R. sibirica subsp. Sibirica, R. Helvetica, R. heilongjiangensis, R. aeschlimannii, R. parkeri, R. rhipicephali and R. montanensis.
Rickettsia conorii subsp. Indica, R. conorii subsp. caspia, and R. conorii subsp. israelensis are subspecies of the R. conorii species and are agents of Mediterranean spotted fever in India, Astrakhan and Israel, respectively. These subspecies exhibit both geographic and pathogenic specificities although they are genetically very close.
R. sibirica subsp. mongolitimonae, the agent of “lymphangitis-associated rickettsiosis” and R. sibirica subsp. sibirica, agent of Siberian tick typhus, are subspecies of R. sibirica and also exhibit phenotypic differences R. heilongjiangensis is the agent of far-eastern rickettsiosis, R. helvetica is the agent of an unnamed aneruptive rickettsiosis, and R. aeschlimannii, R. parkeri and R. rhipicephali, agents of unnamed spotted fevers. We will compare their sequences to those of all other Rickettsia sp. and identify their specificities.
Legionella drancourtii, initially named legionella-like amoebal pathogen 12 (La Scola et al., 2004), is an intra-amoebal Legionella species that possesses a bigger genome than other species within this genus. Preliminary analyses of its genome found evidence of horizontal gene transfer with plants and amoebae. We intend to compare this genomic sequence to that of Parachlamydia amoebophila, another intra-amoebal bacterium that lives in a sympatric lifestyle with other intra-amoebal microorganisms, in order to identify genes that may have been exchanged and those that are associated to this specific environment.
The sequences specific from each of the studied genomes will enable design of various types of diagnostic tools. These tools will be designed in silico at the species level or strain level. Sensitivity and specificity will be confirmed on a collection of bacteria and viruses. These tools will allow the detection of these pathogens in clinical specimens and also to estimate their clonality. For Rickettsia sp., these species-specific sequences will be incorporated into an identification microarray that will allow to by-pass systematic sequencing of PCR products.
In silico identification of the most antigenic proteins of Legionella drancourtii will also be performed. Proteins can be identified by MALDI-TOF based on sequences genome in the databanks after spotting of the antigens best reacting with antibodies in bidimensional electrophoresis. The best candidates will then be expressed through several techniques among which the expression into Escherichia coli and the in-vitro expression by the RTS system from Roche.
All selected bacteria will be sequenced by a combination of two complementary methods by a dedicated engineer and a technician: the GS-FLX Titanium system (Roche) that we already have, which produces up to 600 million bases per run, and the Solexa system (Illumina) which results in up to 2 billion bases per run. The latest system was ordered recently and will be available in our laboratory in2010. Onceobtained, the sequences will be treated through a home-made pipeline by a bioinformatic engineer, and the evaluation of the genomic repertoires and diversity by comparison with reference strains will be performed. Further immuno-proteomic studies will be conducted by one specialized engineer. For this work, we intend to recruit two bioinformatics students (one PhD students, one Post-doctorate fellow):
After culture, purification and DNA extraction, bacterial genomes will be sequenced and annotated. Proteins will be resolved on bi-dimensional gels and checked for reaction with animal sera. Reactive proteins will be spotted and identified by MALDI-TOF. Selected genes will be express to generate proteins that will be tested serologically. Finally, studies on metabolic pathways will results in attempts to design axenic media allowing the culture of these intracellular bacteria.
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