In Vivo Programmed Gene Expression Based on Artificial Quorum Networks

Abstract

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.

FIG 1

Construction and in vitro performance of QS. (A) Structures of the cell density-regulated expression systems QS1 to QS3. P_luxI, promoter of the luxI gene; P_luxR, promoter of the luxR gene; TT, transcription terminator; RBS, ribosome binding site. Katushka is a reporter gene encoding red fluorescent protein. (B) Protein expression levels of E. coli BL21 strains containing QS1 to QS3 were evaluated by determining the relative fluorescence values (RFV) of red fluorescent protein. The BL21(DE3) strain containing a traditional T7 expression system was configured as a control. (C) Cell density-regulated expression of the QS3 system. E. coli BL21 harboring the QS3 system was cultivated in a fed-batch model, and the OD₆₀₀ was maintained below 0.3 by feeding fresh medium continuously. Autoinducers of the QS system were exogenously added to the medium or not.

FIG 2

Construction and in vitro performance of araQS. (A) Linear sketch of araQS. aiiA is quorum quenching gene from B. thuringiensis; P_araBAD is the arabinose promoter. (B) Protein expression of the araQS system regulated by an arabinose signal. Arabinose was added to the medium or not. Cell cultures were harvested and adjusted to an OD₆₀₀ of 1 for fluorescence detection at the indicated time points. (C) Cell density-regulated expression of araQS. E. coli BL21 harboring araQS was cultivated via fed-batch culture, and the OD₆₀₀ was maintained below 0.3 by feeding fresh medium continuously. Autoinducers of QS were exogenously added to the medium or not.

FIG 3

In vivo performance of araQS. (A and B) Protein expression of E. coli(araQS) in zebrafish larvae. Fish larvae, immersed in a bacterial suspension for 2 h, were observed by using an inverted fluorescence microscope at different time intervals, and the corresponding number of bacteria in larvae was counted by coating LB agar plates with homogenized larvae at each time point. (C and D) Protein expression of E. coli(araQS) in adult zebrafish. The internal organs of 20 fish from each group were surgically extracted at different time intervals. The tissue samples were further analyzed by Western blotting using an antibody specific to Katushka, and the corresponding number of bacteria in adult zebrafish was counted by coating LB agar plates with the tissue samples.

FIG 4

In vitro performance of araQS in different bacterial hosts. The araQS plasmid was transformed into Edwardsiella tarda, Vibrio anguillarum, Salmonella Typhimurium, and Staphylococcus aureus, and the resultant recombinant bacterial hosts were cultivated in LB medium with or without the addition of arabinose. Simultaneously, the recombinant bacteria were cultivated in a fed-batch model to maintain the OD₆₀₀ below 0.3 by feeding fresh medium continuously. Each of the cultures was harvested and adjusted to an OD₆₀₀ of 1 for fluorescence detection at the indicated time points.

FIG 5

In vitro growth and in vivo colonization of QS3 and araQS. (A) Growth of E. tarda WED loaded with the QS3 or araQS plasmid in arabinose-rich medium. The blank plasmid pUTat was used as the control. (B) Counts of viable bacteria in zebrafish injected with plasmid-loaded WED strains (5 × 10⁴ CFU per tail) at 12, 24, and 48 h. Ten fish were set as a pool, and three parallel experiments were done. At the indicated time points, 10 fish from each group were randomly picked and homogenized, and the corresponding number of bacteria was counted by coating LB agar plates with homogenized fish.

FIG 6

Construction of the recombinant vector vaccine WED(araQS-G). (A) Plasmid construction of araQS-G. (B) Expression analysis of GAPDH in WED(araQS-G) by SDS-PAGE. Recombinant bacteria were cultivated under different conditions for 9 h. HCD, high cell density; LCD, low cell density; ara +, medium with arabinose added; ara −, arabinose-free medium; M, protein molecular marker.