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  Director : Jean-François Charles depuis le 27 Octobre 2000 (antérieurement, Bernard Papierok) (jcharles@pasteur.fr)


  abstract

 

Our research activities in 2000 have been focused on the following points :
Mode of action of Bacillus sphaericus toxins : structure / function relationship; channel formation in artificial membranes.
Receptors to B. sphaericus toxins in mosquito larvae Culex pipiens : identification, characterisation, cloning and functional expression in heterologous hosts.
Mechanisms of resistance of C. pipiens to B. sphaericus toxins.
Research of new mosquitocidal toxins and improvement of B. sphaericus potency by co-expression of other mosquitocidal toxins.
Molecular basis of mosquito-arbovirus interactions (Aedes sp. / dengue and yellow fever).
Entomopathogenic fungi : bio-diversity / molecular relationship in fonction of host origin.



  report

cale


A. Research

1. Receptors of Bacillus sphaericus toxins in mosquito larvae (Jean-François Charles, Christina Nielsen-LeRoux)

A specific receptor to the Bacillus sphaericus binary toxin (Bin) has been identified on mosquito larvae. This receptor (a 60-kDa alpha-glucosidase) has been cloned and expressed in insect culture cells (Sf9), in collaboration with a team from the INRA (Antibes). On another hand, the cloning and the expression in Sf9 cells of the homologous receptor from a resistant colony of C. pipiens has been done. The study of their respective functionalities showed the necessity of the GPI anchoring in the cell membrane.


2. Channels formed by the B. sphaericus Bin toxin (Jean-François Charles)

In collaboration with a team from the National Research Council of Montreal, we showed that the BinA component of the Bin toxin was able to insert and make pores in artificial phospholipidic membranes. This confirmed the active role of the BinA component, the BinB part being responsible for the specific binding to the receptor.


3. Structure/function relationships of the B. sphaericus Bin toxin (Jean-François Charles, Christina Nielsen-LeRoux)

The reasons for the non toxicity to mosquito larvae of a natural variant of the Bin toxin have been investigated. This absence of larvicidal activity is not due to an absence of binding to the receptor, but to a mutation in the BinA componant at the position 93, which, when reverted, restores the larvicidal activity on C. pipiens.


4. Mosquito larval resistance to Bacillus sphaericus and identification of new toxic compounds (Christina Nielsen-LeRoux)

Several Culex pipiens populations have developed resistance against the binary toxin of B. sphaericus. The mechanisms involve either a modification of the binding step (toxin—receptor) or a mechanism (unknown) probably located after the initial binding event. There is evidences that at least two mechanisms may co-exist in Culex populations from the Montpellier region (South of France). Resistance to B. sphaericus has been determined as recessive in several cases, but is seems to be dominant in an Indian colony.
Two natural strains of B. sphaericus expressed a larvicidal activity towards larvae resistant to commercial B. sphaericus strains. Their potential in management of resistance and identification of new toxic compounds are under study. The field application of one of these strains may delay the appearance of resistance.


5. Characterisation of novel mosquitocidal toxins (A. Delécluse)

A screening program had been previously initiated to identify novel mosquitocidal toxins from various Bacillus thuringiensis strains, that could replace those presently used. Several interesting toxins were thus identified and three, namely Cry29A, Cry30A, and Cyt2Bc from B. thuringiensis medellin, remained to be biologically characterised. Cry29A and Cry30A were bioassayed against Aedes aegypti, Culex pipiens and Anopheles stephensi mosquito larvae. Both were found inactive when tested alone but synergised the activity of the other native toxins from Bt medellin. Cyt2Bc was also bioassayed in the same conditions and found active against all the species tested.


6. Mosquito resistance to novel toxins (A. Delécluse and M. Wirth)

In collaboration with M. Wirth (Riverside University, California), we studied the cross-resistance to novel toxins, Cyt1Ab, Cyt2Ba et Cry19A, in Culex quinquefasciatus colonies resistant to either B. sphaericus ou Bt israelensis toxins. All three polypeptides were found toxic to resistant larvae.


7. Improvement of Bacillus sphaericus entomopathogenic properties (V. Juárez-Pérez, M. Porcar, and A. Delécluse)

One way to improve the entomopathogenic properties of B. sphaericus is to construct recombinant bacteria expressing high levels of different combinations of toxins. We therefore selected B. sphaericus sequences corresponding to strong promoters: 10 sequences have been identified and further characterised. The construction of a recombinant B. sphaericus containing one of these sequences and a toxin gene previously identified in the screening program above mentioned is in progress.


8. Molecular basis of mosquito-arbovirus interactions (A. Delécluse and V. Juárez-Pérez)

A new research program has recently been initiated, that aims at 1) understanding the molecular mechanisms underlying the arbovirus (dengue and yellow fever) transmission by mosquitoes and 2) studying the dengue genome evolution during its replication in the mosquito.


9. Natural occurrence of B. thuringiensis in Camargue (Christina Nielsen-LeRoux, Isabelle Thiéry)

In collaboration with the "Entente Interdépartementale de Démoustication" (EID Mediterranée, Montpellier), we are involved in the evaluation of the natural occurrence of B. thuringiensis in the aim to determine in which way, after treatment with a mosquito larvicidal product based on Bt israelensis, viable spores may accumulate and persist in the environment. This project belongs to the LIFE programme of the European Union.


10. Entomopathogenic fungi (Bernard Papierok)

Studies on entomopathogenic fungi were focused on the bio-diversity, either in the field (associations between Entomophthoral and Homoptera in Senegal and New Guinea) or in the laboratory (by using molecular tools to precise, for the Erynia genus, the inter- and intra-specific relationships in function of the host origin.


B. WHO Collaborative Centre / IEBC Collection (Jean-François Charles)
(see also :
www.pasteur.fr/sante/clre/cadrecnr/bentomo-index.html)

The Entomopathogenic Bacteria laboratory is a WHO Collaborative Centre for entomopathogenic bacteria used in the biological control of vectors of tropical diseases agents. We identified in 2000, by using serotyping (based on flagellar H-antigens), biochemical characters and polypeptide patterns of proteins from parasporal inclusions, 160 strains mainly from South America, North Africa, Corea and India. More than 2/3 of these strains belonged to B. thuringiensis.
The International Entomopathogenic Bacillus Collection (IEBC Collection) has more than 6000 strains : 3600 B. thuringiensis, 600 B. sphaericus and 1800 other Bacillus. The Center also evaluated the entomopathological activity, against 3 mosquito species, of strains and products addressed for expertise.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Marie-France Blanc

Jean-François Charles (IP)

Bernard Papierok (IP)

Armelle Delécluse (IP)

Christine Nielsen-Le Roux (IP)

Philippe Laurent (CNRS)

Victor Juarez, Postdoc

Manel Porcar, Postdoc

Patricia Vary, Professeur, Northern Illinois University, USA

Chantal Francis, étudiante en DESS Biotechnologie, Université Victor Segalen, Bordeaux

Myriam Hajaij, étudiante en thèse, Faculté des Sciences de Tunis

Jérôme Prêtre, étudiant en licence, Faculté de Franche-Comté, Besançon

Isabelle Thiéry

Sylviane Hamon

Sylvie Brémont

Huguette Ripouteau

Nelly Delattre

Vidalia Patricio

Anne-Marie Fillodeau

Christine Viley


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