Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae

Particle and Fibre Toxicology

Shirley Luckhart¹  Aaron C Brault²  Michael Anishchenko²  Kong Cheung¹  Lattha Souvannaseng¹  Ashley A Brenton¹ 

[1] Department of Medical Microbiology and Immunology, School of Medicine, University of California Davis, 95616 Davis, CA, USA;Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 3156 Rampart Rd, 80521 Fort Collins, CO, USA

关键词: Immunity;    Single nucleotide polymorphism;    Malaria;    Plasmodium;    MAPK;    Mosquito;    Anopheles;   

Others  :  803920
DOI  :  10.1186/1756-3305-7-287

 received in 2014-02-11, accepted in 2014-06-13,  发布年份 2014

【 摘 要 】

Background

Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.

Methods

We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.

Results

The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.

Conclusion

MEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.

【 授权许可】

   
2014 Brenton et al.; licensee BioMed Central Ltd.

【 图 表 】

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