Capturing prokaryotic dark matter genomes.

Prokaryotes are the most diverse and abundant cellular life forms on Earth. Most of them, identified by indirect molecular approaches, belong to microbial dark matter. The advent of metagenomic and single-cell genomic approaches has highlighted the metabolic capabilities of numerous members of this dark matter through genome reconstruction. Thus, linking functions back to the species has revolutionized our understanding of how ecosystem function is sustained by the microbial world. This review will present discoveries acquired through the illumination of prokaryotic dark matter genomes by these innovative approaches.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

PhylArray: phylogenetic probe design algorithm for microarray.

MOTIVATION: Microbial diversity is still largely unknown in most environments, such as soils. In order to get access to this microbial 'black-box', the development of powerful tools such as microarrays are necessary. However, the reliability of this approach relies on probe efficiency, in particular sensitivity, specificity and explorative power, in order to obtain an image of the microbial communities that is close to reality. RESULTS: We propose a new probe design algorithm that is able to select microarray probes targeting SSU rRNA at any phylogenetic level. This original approach, implemented in a program called 'PhylArray', designs a combination of degenerate and non-degenerate probes for each target taxon. Comparative experimental evaluations indicate that probes designed with PhylArray yield a higher sensitivity and specificity than those designed by conventional approaches. Applying the combined PhyArray/GoArrays strategy helps to optimize the hybridization performance of short probes. Finally, hybridizations with environmental targets have shown that the use of the PhylArray strategy can draw attention to even previously unknown bacteria.

Serodiagnostic studies in an immunocompetent individual infected with Encephalitozoon cuniculi.

Little is known about the prevalence and clinical significance of infection with Encephalitozoon species in immunocompetent individuals. In the present study, by using indirect immunofluorescence technique (IFAT), Western blot, and recombinant antigens of the spore wall (SWP1) and polar tube (PTP1, PTP2, and PTP3 ), we analyzed the IgG antibody response of a laboratory worker who was infected with Encephalitozoon cuniculi. Serum samples were analyzed 1, 20, 32, and 38 months after infection. After 1 month, by use of IFAT, only spore-wall antigens were recognized, an antibody reaction that changed toward both the spore wall and polar tube in the following months. By use of Western blot analysis, a characteristic pattern that recognized multiple bands was noticed. Reaction against SWP1 was present in all 4 serum samples. The IgG response against PTP1, PTP2, and PTP3 was not detectable 1 month after infection, but became evident in the follow-up serum samples. Serum samples showed cross-reactivity with the spore wall of Encephalitozoon hellem and Encephalitozoon intestinalis, but only little cross-reactivity with the polar tube of these parasites. This is the first study to our knowledge that provides full details about the antibody response against a specified Encephalitozoon species in an immunocompetent person. The results strongly encourage the development and use of reliable serodiagnostic methods, which will provide information about the prevalence and clinical significance of Encephalitozoon species infection in humans.

Homology modeling and calculation of the cobalt cluster charges of the Encephazlitozoon cuniculi methionine aminopeptidase, a potential target for drug design.

Fumagillin is a potent anti-angiogenic drug used in cancer treatments. It is also one of the few molecules active against the Enterocytozoon and Encephalitozoon parasites responsible for various clinical syndromes in HIV-infected or immunosuppressive treated patients. Its toxicity, however, makes desirable the design of more specific molecules. The fumagillin target, as anti-angiogenic agent, is the methionine aminopeptidase, an ubiquitous metallo-enzyme responsible for the removing of the N-terminal methionine in nascent proteins. By analogy, it has been proposed that this enzyme could also be the target in the parasites. As a first approach to verify this and to determine if it would be possible to design a more specific derivative, we have built a homology model of the E. cuniculi aminopeptidase. The charges of the two cobalt ions present in the active site and of the side-chains involved in their binding were computed using ab-initio methods. A preliminary comparison of the interactions of the fumagillin and of a related compound, the TNP-470, with both the human and the parasitic enzymes strongly support the hypothesis that the parasitic aminopeptidase is indeed the target of the fumagillin. It also suggests that the TNP-470 interact identically with both enzymes while there could be small differences in case of the fumagillin.