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: Parasitology and Mycology

Title of the PhD project: Molecular basis of host cell invasion by the malaria parasite

Name of the lab: Malaria Biology and Genetics

Head of the lab: Robert Ménard

PhD advisor: Robert Ménard

Email address: rmenard@pasteur.fr

Web site address of the lab:

Doctoral school affiliation and University: B3MI

 

 

 

Presentation of the laboratory and its research topics:

Our laboratory focuses on the pre-erythrocytic phase of malaria. This phase extends from injection of the parasite in the skin by the mosquito to the onset of red blood cell infection, which causes the clinical symptoms of the disease. Our major aim is a functional understanding of the important host-parasite interactions that occur during the parasite journey to the liver, as well as inside hepatocytes. For this, we use a combination of molecular genetics, cell biology and in vivo imaging approaches. We are also addressing how the host can mount a protective immune response against this phase of Plasmodium infection. We use rodent Plasmodium models of infection in our studies.

 

Description of the project:

Malaria remains the most deadly parasitic disease worldwide, still killing about a million people each year. The disease is due to a eukaryotic parasite, called Plasmodium. Like other apicomplexan parasites (including Toxoplasma and Cryptosporidium spp. among other human pathogens), Plasmodium spp. multiplies only inside host cells and generates motile extracellular parasite forms called zoites. Plasmodium zoites come in two forms: the sporozoite, the parasite form injected by the mosquito that invades hepatocytes in the liver, and the merozoite, which invades red blood cells (RBCs) and is associated with pathology.

 

One central question in basic malaria research is how zoites invade host cells. Zoite invasion takes only a few seconds, and is thought to depend on a parasite actin-myosin motor located underneath the parasite plasma membrane. A defining feature of host cell invasion by apicomplexan zoites is the presence of a tight junction (TJ) between the parasite apex and the host cell membranes. The TJ is thought to act as a stable anchor on which the zoite motor exerts traction to move the zoite in a vacuole inside the host cell.

 

The exact composition of this TJ, however, is still unknown. One of the reasons for this gap in knowledge is that, until recently, the nature and function of the TJ could not be addressed genetically since inactivation of any essential gene is lethal in Plasmodium. We have established conditional mutagenesis techniques in rodent Plasmodium parasites making use of the Flp-FRT and Cre-loxP site-specific recombination systems. They allow for the first time to investigate the function of proteins that are essential to zoite invasion, via the construction of recombinant parasites in which a gene of interest has been deleted specifically at the sporozoite or merozoite stages. 

 

Recently, these technical advances have allowed us to invalidate the previously established model of TJ composition, which consisted of two interacting parasite proteins, AMA1 and RON2, supposed to link the parasite motor to the host cell cytoskeleton. Therefore, the proteins that are involved in shaping the TJ remain unknown. We have selected a small set of possible candidate TJ proteins based on evolutionary, gene expression and protein localization criteria. The project we propose will consist in testing the role of selected candidates using a variety of experimental tools, including not only powerful molecular genetic approaches to construct conditional mutants but also state-of-the-art imaging technologies to characterize their phenotype and thus understand protein function. The experimental system we use is Plasmodium berghei, a plasmodial species that infects rodents, can be easily cycled through mosquitoes and thus offers the possibility to follow the entire parasite life cycle in vivo and to analyze both the merozoite and sporozoite stages.

 

Proteins of interest will be targeted genetically by conditional mutagenesis techniques at both the sporozoite and merozoite stages. This includes the construction of recombinant DNA constructs, parasite transfection, selection of recombinant parasites in mice, and parasite cloning by limiting dilution. After verification of the expected locus in recombinant parasites by PCR/Southern blot analysis, parasites will be fed to mosquitoes in a dedicated on-campus mosquito production and infection facility. Sporozoites will be dissected out from mosquito salivary glands, and used to infect cultured hepatocytes, inside which merozoites are produced. Merozoite-RBC interactions will be investigated using imaging flow cytometry. In parallel, selected proteins of interest will be characterized further by biochemical methods (including co-immunopreciptation techniques to identify binding partners) as well as localization techniques (including correlative structural (three-dimensional) imaging).

 

In conclusion, this project aims at using state of the art tools to characterize the TJ made by malaria parasites to invade their host cells. In turn, identification of new TJ components might be useful to identify new vaccine targets for preventing Plasmodium zoite invasion of host cells.

 

 

References:

 

Lacroix C, Giovannini D, Combe A, Bargieri DY, Späth S, Panchal D, Tawk L, Thiberge S, Carvalho TG, Barale JC, Bhanot P, Ménard R. 2011. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nature Protocols 6, 1412-1428.

Giovannini D, Spaeth S, Lacroix C, Perazzi A, Bargieri D, Lagal V, Lebugle C, Combe A, Thiberge S, Baldacci P, Tardieux I, Ménard R. 2011. Independent roles of AMA1 and RON4 in distinct steps of host cell invasion by Apicomplexa. Cell Host Microbe 10, 591-602.

Bargieri D, Lagal V, Tardieux I, Ménard R. 2012. Host cell invasion by apicomplexans: what do we know? Trends in Parasitology 28, 131-135.

Bargieri D, Andenmatten N, Lagal V, Thiberge S, Whitelaw JA, Tardieux I, Meissner M, Ménard R. 2013. Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nature Communications, In Press.

 

 

Keywords:

 

Malaria – Invasion – Mutagenesis

 

Expected profile of the candidate (optional):

 

Contact:

 

Robert Ménard: rmenard@pasteur.fr

 

Mis à jour le 16/09/2013