A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Institut Pasteur

Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunosorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.

Isolation and characterisation of local strains of Chlamydophila abortus (Chlamydia psittaci serotype 1) from Tunisia.

Q fever and chlamydiosis often affect ovine and caprine flocks simultaneously or successively. Combination vaccines effective against these 2 diseases would be of great value in veterinary medicine. Unfortunately, the current effective vaccines are a live vaccine for chlamydiosis and killed vaccine for Q fever. Vaccination of mice with live chlamydiosis vaccine 1B and killed phase I vaccine against Q fever at 2 points on the back at the same time produced good protection against chlamydial abortion. This suggests that it may be possible to vaccinate ewes and goats against chlamydiosis and Q fever simultaneously.

Combined vaccination of live 1B Chlamydophila abortus and killed phase I Coxiella burnetii vaccine does not destroy protection against chlamydiosis in a mouse model.

Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.

Identification and characterisation of coding tandem repeat variants in incA gene of Chlamydophila pecorum.

Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals.

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR.

Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection.

Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum.

The lux operon derived from Photorhabdus luminescens incorporated into bacterial genomes, elicits the production of biological chemiluminescence typically centered on 490 nm. The light-producing bacteria are widely used for in vivo bioluminescence imaging. However, in living samples, a common difficulty is the presence of blue-green absorbers such as hemoglobin. Here we report a characterization of fluorescence by unbound excitation from luminescence, a phenomenon that exploits radiating luminescence to excite nearby fluorophores by epifluorescence. We show that photons from bioluminescent bacteria radiate over mesoscopic distances and induce a red-shifted fluorescent emission from appropriate fluorophores in a manner distinct from bioluminescence resonance energy transfer. Our results characterizing fluorescence by unbound excitation from luminescence, both in vitro and in vivo, demonstrate how the resulting blue-to-red wavelength shift is both necessary and sufficient to yield contrast enhancement revealing mesoscopic proximity of luminescent and fluorescent probes in the context of living biological tissues.

In vivo excitation of nanoparticles using luminescent bacteria.

Energy transfer mechanisms represent the basis for an array of valuable tools to infer interactions in vitro and in vivo, enhance detection or resolve interspecies distances such as with resonance. Based upon our own previously published studies and new results shown here we present a novel framework describing for the first time a model giving a view of the biophysical relationship between Fluorescence by Unbound Excitation from Luminescence (FUEL), a conventional radiative excitation-emission process, and bioluminescence resonance energy transfer. We show here that in homogeneous solutions and in fluorophore-targeted bacteria, FUEL is the dominant mechanism responsible for the production of red-shifted photons. The minor resonance contribution was ascertained by comparing the intensity of the experimental signal to its theoretical resonance counterpart. Distinctive features of the in vitro FUEL signal include a macroscopic depth dependency, a lack of enhancement upon targeting at a constant fluorophore concentration cf and a non-square dependency on cf. Significantly, FUEL is an important, so far overlooked, component of all resonance phenomena which should guide the design of appropriate controls when elucidating interactions. Last, our results highlight the potential for FUEL as a means to enhance in vivo and in vitro detection through complex media while alleviating the need for targeting.

In vitro characterization of Fluorescence by Unbound Excitation from Luminescence: broadening the scope of energy transfer.

Bioluminescence imaging is a powerful technique that allows for deep-tissue analysis in living, intact organisms. However, in vivo optical imaging is compounded by difficulties due to light scattering and absorption. While light scattering is relatively difficult to overcome and compensate, light absorption by biological tissue is strongly dependent upon wavelength. For example, light absorption by mammalian tissue is highest in the blue-yellow part of the visible energy spectrum. Many natural bioluminescent molecules emit photonic energy in this range, thus in vivo optical detection of these molecules is primarily limited by absorption. This has driven efforts for probe development aimed to enhance photonic emission of red light that is absorbed much less by mammalian tissue using either direct genetic manipulation, and/or resonance energy transfer methods. Here we describe a recently identified alternative approach termed Fluorescence by Unbound Excitation from Luminescence (FUEL), where bioluminescent molecules are able to induce a fluorescent response from fluorescent nanoparticles through an epifluorescence mechanism, thereby significantly increasing both the total number of detectable photons as well as the number of red photons produced.

Abdessalem Rekiki

Plate-Forme d'Imagerie Dynamique,
Imagopole,
Plate-Forme d'Imagerie Dynamique, Imagopole

Institut Pasteur

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Abdessalem Rekiki

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Institut Pasteur

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Plate-Forme d'Imagerie Dynamique,
Imagopole,
Plate-Forme d'Imagerie Dynamique, Imagopole