Tài liệu Báo cáo khoa học: A second independent resistance mechanism to Bacillus sphaericus binary

A second independent resistance mechanism to Bacillus

sphaericus binary toxin targets its a-glucosidase receptor

in Culex quinquefasciatus

Tatiany Patrı´cia Roma˜o1

, Karlos Diogo de Melo Chalegre1

, Shana Key1

, Constaˆncia

Fla´via Junqueira Ayres1

, Cla´udia Maria Fontes de Oliveira1

, Osvaldo Pompı´lio de-Melo-Neto2

and Maria Helena Neves Lobo Silva-Filha1

1 Department of Entomology, Centro de Pesquisas Aggeu Magalha˜es ⁄ Fundac¸a˜o Oswaldo Cruz, Recife-PE, Brazil

2 Department of Microbiology, Centro de Pesquisas Aggeu Magalha˜es ⁄ Fundac¸a˜o Oswaldo Cruz, Recife-PE, Brazil

Culex quinquefasciatus has an important role in the

spread of diseases world wide, and, in Brazil, this

species is the major vector of lymphatic filariasis

which remains an endemic disease in some urban

areas. The status of Culex sp. as a disease vector

has greatly increased in recent years vis a vis the

spread of the West Nile virus in the Americas. Ade￾quate strategies of vector control are essential to

interrupt disease transmission, and the search for

effective control agents has shown that the use of

bacterial larvicides is an alternative for overcoming

the negative effects of synthetic insecticides com￾monly used in mosquito control programs. Bacillus

sphaericus is the most successful biological larvicide

commercially available to control Culex. Field trials

have proved its effectiveness for reducing population

density in areas where Culex is a source of nuisance

or vector of diseases [1–3]. The most important

B. sphaericus features are its selective spectrum of

action, extended persistence in the breeding sites and

the facilities for its large-scale production, storage

and spraying.

Keywords

Bacillus sphaericus; binding site; Culex

quinquefasciatus; a-glucosidase; resistance

Correspondence

M. H. N. L. Silva-Filha, Centro de Pesquisas

Aggeu Magalha˜es-Fiocruz, Avenue Moraes

Reˆgo s ⁄ n Cidade Universita´ria, Recife-PE,

Brazil 50670-420

Tel: +55 81 21012553

Fax: +55 81 34532449

E-mail: [email protected]

Note

Nucleotide sequence data has been

submitted to the GenBank database under

the accession number DQ333335.

(Received 15 December 2005, revised 27

January 2006, accepted 13 February 2006)

doi:10.1111/j.1742-4658.2006.05177.x

The entomopathogen Bacillus sphaericus is an important tool for the vector

control of Culex sp., and its effectiveness has been validated in field trials.

The appearance of resistance to this bacterium, however, remains a threat

to its use, and attempts have been made to understand the resistance mech￾anisms. Previous work showed that the resistance to B. sphaericus in a

Culex quinquefasciatus colony is associated with the absence of the

60-kDa binary toxin receptor in larvae midgut microvilli. Here, the gene

encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and

sequenced from a susceptible colony. The deduced amino-acid sequence

confirmed its identity as an a-glucosidase, and analysis of the correspond￾ing gene sequence from resistant larvae implicated a 19-nucleotide deletion

as the basis for resistance. This deletion changes the ORF and originates a

premature stop codon, which prevents the synthesis of the full-length

Cqm1. Expression of the truncated protein, however, was not detected

when whole larvae extracts were probed with antibodies raised against an

N-terminal 45-kDa recombinant fragment of Cqm1. It seems that the pre￾mature stop codon directs the mutated cqm1 to the nonsense-mediated

decay pathway of mRNA degradation. In-gel assays confirmed that a

single a-glucosidase protein is missing from the resistant colony. Further

in vitro affinity assays showed that the recombinant fragment binds to the

toxin, and mapped the binding site to the N-terminus of the receptor.

Abbreviations

BBMF, brush border membrane fraction; Bin, binary; GPI, glycosylphosphatidylinositol; NMD, nonsense-mediated decay.

1556 FEBS Journal 273 (2006) 1556–1568 ª 2006 The Authors Journal compilation ª 2006 FEBS