ABSTRACT
BACKGROUND & OBJECTIVES: The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito. METHODS: Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10 th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis. RESULTS: The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV. INTERPRETATION & CONCLUSIONS: The results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane.
Subject(s)
Aedes , Chikungunya Fever/transmission , Chikungunya virus/pathogenicity , Insect Vectors , Serratia/pathogenicity , Aedes/microbiology , Aedes/virology , Animals , Chaperonin 60/metabolism , Chikungunya Fever/pathology , Chikungunya Fever/virology , Chikungunya virus/growth & development , Female , Gastrointestinal Tract/microbiology , Gastrointestinal Tract/virology , Humans , Insect Vectors/microbiology , Insect Vectors/virology , Mice , Serratia/growth & developmentABSTRACT
A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.
