In vivo deciphering of the early steps of Bacillus anthracis interactions with the vascular, lymphatic and resident immune cells during infection. – Deciphering In Vivo Host Anthrax Interac

Constructions of monofluorescente strains: We use genetic techniques adapted to Gram positive bacteria (construction in Escherichia coli and transfer by electroporation or conjugation hétérogramique).
Imaging ex vivo infections in two-photon microscopy: This is intravital microscopy techniques adapted to the analysis of moving organs such as the lung.

Constructions of monofluorescentes strains: Difficulties were encountered. Mutants under control of genomic integration sspB promoter were obtained, but these constructions interfered negatively with sporulation. The promoter scrA was used, as it is expressed in spores and bacilli and is functional with GFP fluorescence is low in spores and intense in bacilli. This promoter has been cloned into a suitable vector, and the genes of fluorescent proteins CFP, dnTomato, mCherry and Crimson were incorporated downstream .
Imaging ex vivo infections in two-photon microscopy : Analysis of ex vivo lung infections required to develop techniques allowing analysis of the dynamics of tissues after infection under the microscope over periods on the order of several hours to discriminate repercussions physiological infection on major cellular functions (viability , migration). We have then developed tools for image analysis by post- treatment in a Matlab code. The parasitic movements are eliminated by trimming images by analysis of the cross - correlation signal of second harmonic generation (SHG ) of the collagen foam . This system allows a stable analysis cropping over a long term .

Preliminary results allow the implementation of effective phase of the project with the implementation of the study models of infection.

Publication:
D. Fiole, C. Touvrey, A. Quesnel-Hellmann, J. Douady, J.-N.
Tournier. Shape-based tracking allows functional discrimination of two immune cell subsets expressing the same fluorescent tag in mouse lung explant.
PLoS ONE, 2012;7(6):e39831. Epub 2012 Jun 22.

Oral communication:
D. Fiole, P. Deman, J. Mathieu, P.L. Goossens, J. Douady, J.-N. Tournier. Two-photon microscopy of inhalational anthrax infection reveals novel immunological synapses.
Bacillus ACT, Septembre 2013, Vancouver, Canada.

J.-N. Tournier. Two-photon imaging of pulmonary anthrax infection reveals novel immunological synapses.
ASM biodefense, Février 2013, Washington DC, USA.

This project addresses the problematic raised in Thematic axis 7, concerning the analysis and evaluation of the risk arising from a NRBC threat, more specifically the biological threat due the consequences of anthrax contamination. It presents a strong dual outcome of the research, both civilian and with specificity for answering defense needs.
The bioterrorist anthrax US attack in 2001 has exemplified the life threatening consequences on the health of the civil and military personnel exposed to anthrax spores, and the ensuing economy and societal disruptions. A better understanding of the disease could thus be a means to decrease the health consequences of exposure to anthrax spores and thus impact the costs generated by such bioterrist acts for the society.
The initial steps of anthrax infection are still insufficiently understood. A gap of knowledge exist on the precise mechanisms used by B. anthracis to interact, successfully invade and disseminate into the host.
A wealth of data has been accumulated in in vitro systems, but their relevance to in vivo conditions of infection is regularly questionned. Translation to in vivo models are severely hampered by a lack of appropriate methods.
The proposed project aims at imaging in vivo and in real time the initial steps of anthrax, both cutaneous and inhalational, by combining powerful complementary fluorescence methodologies, biphotonic microscopy and ex vivo 3-D reconstruction on BSL3 B. anthracis infected tissues of mice displaying fluorescent lymphatic and blood vessels, or fluorescent immune cells (such as dendritic cells). Through these imaging technologies combined to histological and ultrastructural analysis, we will visualize and characterize in real time the entry of B. anthracis, the local dynamics of the bacterial differentiation steps (germination and capsule and toxin production) and dynamically define the local subversive effects of the toxins on the local host control mechanisms during an infection; more specifically on the integrity of lymphatic and blood endothelial cells and the dynamics of innate immune cell recruitment. Overall, the combination of these imaging approaches will provide a unique and novel picture of B. anthracis infection.
The outcomes of this project include: - a better understanding of the critical initial steps of anthrax, both cutaneous and inhalational - the development of new molecular approaches to the early treatment of B. anthracis infection and vaccination strategies. - the development of novel visualization technologies to follow in real time an infection that could be applied to other pathogens of interest This work should also make it possible to improve the societal and economic countermeasures to be taken in cases of biological terrorist threats.