CheA/CheY signaling systemresponsible for chemotaxis in vitro and colonization in vivo of Campylobacter jejuni

Abstract

Objective To determine the effects of Che A and CheY proteins ofCampylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization invivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector andexpression strain, respectively, prokaryotic expression systems of cheA and cheY genes ofC. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the targetrecombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG andrCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript-II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-outmutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotacticability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting thebacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY afterDOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. Thedifference of colonization ability between cheA- mutant and wild-type of C. jejuni in micewere checked and then compared. Results The constructed prokaryotic expression systemscould efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed thecheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotacticability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis ofwild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levelsof CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). Thecolonization ability in murine jejunum of cheA- mutant was also lower than that of thewild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system(Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activatedby dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro andcolonization in vivo of C. jejuni, and this protein can be used as a target for developingnovel anti- C. jejuni drugs.