PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

 

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

 

Department: Infection and Epidemiology

Title of the PhD project: Exploring Leishmania donovani-rodent host interactions : an approach in real time at the cellular and tissue level

Name of the lab: Trypanosomatids Infectious Processes Lab

Head of the lab: Dr Paola MINOPRIO

PhD advisor: Dr Thierry LANG

Email address: thierry.lang@pasteur.fr

Web site address of the lab: http://www.pasteur.fr/recherche/unites/tcruzi/minoprio/minoprio.html

Doctoral school affiliation and University:

Compléxité du Vivant-“ at the Paris 6 University

 

 

 

Presentation of the laboratory and its research topics:

 

The  laboratory’s  primary  goal  is  to  identify  molecules,  which  are  both  implicated  in  parasite  evasion  of  the host’s immune system and could ultimately be used as targets in the development of new therapies. The laboratory  has been studying experimental models of human Chagas disease and animal trypanosomosis, two of the “most neglected  diseases”  that  afflict  the  poor  and  powerless  in  developing  regions  of  sub-Saharan  Africa,  Asia  and the Americas. Chagas’  disease, the third largest disease burden in Latin America, is caused by Trypanosoma cruzi. The  team  has  recently  drawn  its  attention  to  other  protozoan  parasites of  the  trypanosomatid family which are equally considered as “neglected”: Leishmania (L) donovani and L. major, responsible respectively for  visceral  and  cutaneous Leishmaniasis. The pathogenesis differs among  those trypanosomatid  infectious  processes,  reflecting  the  different  interactions  of  these  parasites  with their  hosts,  but  important  immunological  dysfunctions  are  involved  in  the  development  of  these  seriously disabling illnesses. Aiming at better exploring the well-established experimental models of infections we  have  further developed  our  strategies  of  study  in  order  to  decipher  the  more  precisely  as  possible  the  interaction  of  these microorganisms  with  their  hosts. To circumvent the major constraints inherent to studying parasite /host interactions in the field, we had both developed in vivo animal models of trypanosomosis (1-4) and leishmaniasis (5-7). Our team uses a highly interdisciplinary approach to generate and validate innovative tools for real time imaging of these Trypanosomatidae in their mammalian hosts and insect vectors. We showed that the rodent experimental model reproduce most features of the infection in human. More than reflecting only the main parasitological parameters of  the  animal  infection,  the  rodent model  can  be  used  to  elucidate  the immunopathological  mechanisms involved in parasite evasion and persistence, and the tissue damage seen during infection and disease.

 

 

 

Description of the project:

(1 page, Arial font size 11 : 600 words in total with at least 50% dedicated specifically to the proposed PhD project(s))

 

During the infectious cycle, trypanosomatid pathogens of the genus Leishmania alternate between the insect promastigote stage and the vertebrate amastigote stage that proliferates inside infected host tissues provoking the pathology of the disease (5). The major scope of our work is to accurately delineate and characterize Trypanosomatidae-driven processes in rodent tissues following parasite inoculation. The small size of these eukaryotic parasites and the spatial resolution limits of conventional imaging technologies have hampered analysis of host-parasite interactions at the single cell level. We have previously established and validated innovative imaging technologies to visualize tissue localization dynamics of Leishmania and Trypanosoma in vertebrate hosts (5). We built upon our expertise in cutting edge imaging and cellular host interactions to develop and use bioluminescence/fluorescence quantitative imaging with 2D and 3D spatial resolution (8, 9). By using bioluminescent and/or far red fluorescent parasites, we investigated the possibility to delineate the various phases of the infectious process by monitoring the Leishmania populations and analyzing the cellular mechanisms at the host and tissue (7, 9).

 

PhD project

Monitoring of Leishmania donovani in laboratory rodents

The objective of the proposed project is primarily to monitor disease progression by examining host-parasite interactions over time and in the three-dimensional space of living mice and analyse the early steps of the infection. Secondly, the goal is to test the hypothesis that a correlation may exist between the virulence of L. donovani and the B- cell function thus favoring the mechanisms of parasite escape of T cell responses implemented by the host. In order to monitor the early development and dissemination of L. donovani at the inoculation site in mouse dermis after inoculation, a panel of intravital imaging technologies will be used to visualize the infection process. Animals infected by bioluminescent/fluorescent parasites will be alternatively observed by epifluorescence microscopy, optical fiber, two-photon and spinning-disk laser scanning microscopy, widefield Apotome confocal microscopy and IVIS Spectrum imaging system. The next exciting step will be to monitor the infection in vivo and especially to understand the early steps of the infection (parasite migration, proliferation, differentiation, interaction with host cells …).

Parameters that account for the establishment of parasites in mice will be determined in real time during the phase of parasite implantation and expansion. We will use an innovative approach for monitoring and measuring simultaneously parasite load and immune responses (9) after inoculation of L. donovani. A main limitation for in vivo studies of experimental visceral leishmaniasis is the loss of L. donovani virulence in culture. In order to correlate expression of virulence with infectivity and pathogenicity “virulent” and “attenuated” bioluminescent L. donovani will be used to generate new transgenic parasites over-expressing factors previously demonstrated to be important for the survival of L. donovani amastigotes in the liver and the spleen (10). Several candidates will be chosen as candidate virulence/survival factors that will be over-expressed in virulent and attenuated parasites to establish transgenic lines with distinct virulence phenotypes. Evaluation of the survival and expansion of these parasites will be performed by monitoring bioluminescent parasite burden in macrophages and in hamsters. By using a hamster model as part of a proof-of-principle study we will  be able testing the ability of luciferase-expressing L. donovani to cause visceral leishmaniases (VL) following various clinical signs of the disease, including fever, pallor, wasting, and hepato-splenomegaly. These quantitative imaging studies will open up possibilities to analyze infectious processes in mice after inoculation of “virulent” and “attenuated” L. donovani.   The parasite size populations, humoral and T cell responses will be studied in vivo and ex-vivo during the different phases of the infection. The investigation of the cellular mechanisms at the host and tissue levels with high temporal and/or spatial resolution will contribute to the elucidation of the dynamics of intracellular trafficking events The resulting data obtained in this work will greatly enhance our understanding of leishmaniasis pathogenic process, and provide a major breakthrough in anti-parasitic research.

 

References:

1.         N. Chamond et al., PLoS Negl Trop Dis 4, e792 (2010).

2.         S. D'Archivio et al., PLoS Negl Trop Dis 5, e1461 (Dec, 2011).

3.         S. D'Archivio et al., PLoS Negl Trop Dis 7, e1976 (Jan, 2013).

4.         S. Goyard et al., Parasitol Int,  (Jul 25, 2013).

5.         T. Lang, H. Lecoeur, E. Prina, Trends Parasitol 25, 464 (Oct, 2009).

6.         E. Giraud et al., PLoS Negl Trop Dis 6, e1980 (Dec, 2012).

7.         E. Giraud et al., Parasitol Int,  (Aug 31, 2013).

8.         H. Lecoeur et al., Microbes Infect 12, 46 (Jan, 2010).

9.         E. de La Llave et al., Cell Microbiol 13, 81 (Jan, 2011).

10.       P. Pescher, T. Blisnick, P. Bastin, G. F. Spath, Cell Microbiol,  (Mar 23, 2011).

 

 

 

Keywords: Leishmania donovani, real time  imaging, RT-qPCR, virulence, rodents, experimental animal model,

 

 

 

 

Contact:

Dr Thierry LANG

thierry.lang@pasteur.fr

Tel +33 1 45 68 86 73

 

 

 

 

Mis à jour le 16/09/2013