Introduction
Edwardsiella piscicida is an intracellular parasitic pathogenic bacterium belonging to the genus Edwardsiella. It is a primary causative agent of edwardsiellosis in marine farmed fish worldwide, capable of causing large-scale mortality in economically important fish species such as turbot (Scophthalmus maximus). This bacterium inflicts significant economic losses on the aquaculture industry.
Figure 1 Schematic diagram of Edwardsiella piscicida invading Scophthalmus maximus
Fish vaccines share a similar mechanism with vaccines administered to humans. They involve introducing specially treated pathogenic microorganisms (such as bacteria, viruses, etc.) or their metabolites into fish bodies via methods like injection and immersion. This process stimulates the fish's own immune system to produce antibodies, thereby protecting the fish against infections caused by the corresponding diseases.
Table 1 comparison of three vaccination routes for fish vaccines
In the immunization operations of fish vaccines, immersion immunization is the simplest immunization method. It can meet the efficiency requirements of large-scale farming, reduce the stress and mortality risks during the fry stage, and cover the immunization needs across the entire developmental stages of fish. However, the immersion method still has limitations, including relatively low immune protection rate and short duration of immunity.
In the preliminary study, we subjected zebrafish to immersion infection using fluorescent protein-labeled Edwardsiella piscicida, and acquired imaging data via a fluorescence microscope. Image analysis results showed that distinct fluorescent signals were observable in the zebrafish body, confirming that the fluorescent protein-labeled Edwardsiella piscicida had successfully invaded the zebrafish host, colonized relevant tissues, and achieved effective dissemination. This provides direct visual evidence for the infectivity of the bacterium under immersion conditions.
Figure 2 Fluorescence Microscopic Images of Zebrafish Infected with Fluorescent Protein-Labeled Edwardsiella piscicida
Based on the aforementioned research, we plan to replace Edwardsiella piscicida with Escherichia coli to construct an engineered strain capable of linear movement under blue light and tumbling under green light. Furthermore, we will integrate a light irradiation device to achieve precise regulation of the engineered strain's movement states, thereby enhancing the invasive capacity of the vaccine strain. Additionally, we will verify the efficacy of this light-driven micro-nanorobot using equipment such as fluorescence microscopes.
Figure 3 Schematic Diagram of Tumbling and Linear Motion of the Engineered Strain
Strategy 1:The Flagellar Movement Control System in Escherichia Coli
The flagellum of Escherichia coli consists of several key components: Flagellar Motor: Converts the chemical energy from ion gradients (H⁺/Na⁺) into mechanical rotational force to drive flagellar rotation. It includes: MS Ring: The starting point of torque transmission, connecting the C Ring (direction switch) and the Rod.
- C Ring: Controls the rotation direction (clockwise/counterclockwise) and responds to chemical signals.
- Stator (e.g., MotA/B): A transmembrane ion channel that generates torque using proton flow to push the MS Ring to rotate.
- Rod: Transmits the motor's torque to the Hook and requires high rigidity to resist torsional deformation.
- LP Ring: Supports the rotation of the Rod and reduces friction loss.
- Hook: A flexible "universal joint" that connects the Rod and the Filament, transmitting torque and buffering deflection stress.
- Export Apparatus: Assembles flagellar components (similar to Type III secretion system, T3SS) and serves as the assembly base for the Rod.
Like numerous other biological signaling systems, the E.coli chemotaxis pathway relies on reversible protein phosphorylation as its signaling mechanism.That said, the core signaling biochemistry differs somewhat between prokaryotes and eukaryotes.CheA functions as a kinase that utilizes ATP to undergo autophosphorylation at a particular histidine residue. Subsequently, phospho-CheA molecules act as donors in autokinase reactions, transferring phosphoryl groups to specific aspartate residues within CheY.
Phosphorylated CheY promotes clockwise (CW) rotation of the flagellar motors, while phosphorylated CheB exhibits high methylesterase activity toward methyl-accepting chemotaxis proteins (MCPs). These activated response regulators have a brief lifespan due to their rapid loss of phosphoryl groups through spontaneous self-hydrolysis. Additionally, CheZ accelerates the dephosphorylation of phospho-CheY, ensuring that locomotor responses to fluctuations in the availability of signaling phosphoryl groups to CheY occur rapidly.
In terms of the molecular mechanism underlying the direction switching of the flagellar motor, when there is no CheY protein binding, the stator units that rotate clockwise are located outside the FliGCC subring of the C ring, driving the flagellar motor to rotate counterclockwise. When CheY binds to the C ring, it promotes a conformational change in the C ring, which contracts toward the center of the flagellar motor and shifts upward as a whole. This further induces a conformational change in the FliGCC domain, ultimately leading to a 180° flip of the αtorque on FliGCC. Consequently, the stator units move from the outside to the inside of FliG, undergoing spatial relocation on the inner membrane, which prompts the C ring to rotate clockwise, thereby achieving the direction switching of the flagellar motor.
Figure 4 Schematic Diagram of Flagellar Rotation Direction Regulated by Partial Bacterial Chemotaxis Networks
Strategy 2: Blue Light-Controlled Linear Movement of Escherichia Coli
The pDawn system functions as a reliable gene expression platform regulated by light activation. Specifically, blue light triggers the expression of target genes, with this effect being significantly amplified by either higher light intensities or prolonged exposure durations. The underlying molecular mechanism of this system is explained as follows.
A key component of the system is the histidine kinase YF1, which contains a light-oxygen-voltage (LOV) domain sensitive to blue light. In the dark (without blue light exposure), YF1 undergoes autophosphorylation and transfers the phosphate group to its paired response regulator FixJ. Phosphorylated FixJ then activates substantial transcription from the FixK2 promoter, a process that is inhibited when YF1 absorbs blue light.
All elements of the YF1/FixJ two-component system (TCS) are integrated into the medium-copy plasmid pDawn. Here, YF1 and FixJ are constitutively expressed as a bicistronic operon under the control of the LacIq promoter. Target genes can be inserted in a single cloning step through a multiple-cloning site (MCS) located downstream of the pFixK2 promoter. Additionally, the λ phage repressor cI, whose expression is driven by FixK2, inhibits the λ promoter pR within pDawn. This configuration reverses the signal polarity, resulting in light-dependent activation of gene expression: when blue light is present, FixK2 activity is suppressed, leading to reduced cI production. Consequently, the λ promoter pR is derepressed, initiating transcription of the target gene.
Thus, we utilize the pDawn system for the expression of the CheZ gene. Under blue light induction, the CheZ protein is expressed, which induces ccw rotation of the flagella and promotes the linear movement of bacteria.
Figure 5 Linear Motion Induced by Blue Light
Strategy 3: Green Light-Controlled Tumbling Motion of Escherichia Coli
The second light sensor was engineered by cloning the Synechocystis PCC 6803 ccaS-ccaR-cpcG2 genomic cluster into a ColE1-origin plasmid, and replacing the native cpcG2 output gene with CheA or CheY.CcaS is a SK with a N-terminal PCB-binding cyanobacteriochrome GAF domain and a C-terminal histidine kinase domain.CcaS holoprotein is produced in a green absorbing, phosphatase active ground state (Pg). Green light switches CcaS Pg to a kinase active Pr conformation, where it phosphorylates the CcaR.CcaR∼P binds to a G-box operator within PcpcG2,activating transcription.
In this project, the CcaS/CcaR dual-plasmid green light system was used to express CheA and CheY proteins respectively, thereby achieving the goal of regulating the tumbling movement of engineered strains via green light.
Figure 6 Tumbling Motion Induced by Green Light
Pathway Construction
GROUP 1: pBBR1MCS-5-CheA、pBBR1MCS-5-CheY、pBBR1MCS-5-CheZ
Group 1 comprises three plasmids.The pBBR1MCS-5 vector was used to express the CheA, CheY, and CheZ genes respectively, which served to familiarize with the experimental procedure and conduct a preliminary exploration of bacterial motility.
Figure 7 schematic diagram of pBBR1-MCS-CheA
Figure 8 schematic diagram of pBBR1-MCS-CheY
Figure 9 schematic diagram of pBBR1-MCS-CheZ
GROUP 2: pBBR1MCS-5-CheA-Flag、pBBR1MCS-5-CheY-Flag、pBBR1MCS-5-CheZ-Flag
Group 2 comprises three plasmids.Building upon Group 1, Flag tags were respectively added to the three target genes, which were used in Western blot to verify the protein expression status.
Figure 10 schematic diagram of pBBR1-MCS-CheA-Flag
Figure 11 schematic diagram of pBBR1-MCS-CheY-Flag
Figure 12 schematic diagram of pBBR1-MCS-CheZ-Flag
GROUP 3:pDawn-CheZ、pDawn-CheZ-Flag、pDawn-sfGFP
Group 3 comprises three plasmids, which express CheZ, CheZ-Flag, and sfGFP respectively under blue light induction. Among them, sfGFP is used for fluorescence detection with a multi-functional microplate reader to confirm the successful construction of the expression system and conduct semi-quantitative evaluation of expression intensity, thereby providing a reference for the optimization of induction conditions.
Figure 13 schematic diagram of pDawn-CheZ
Figure 14 schematic diagram of pDawn-CheZ-Flag
Figure 15 schematic diagram of pDawn-CheZ-sfGFP
GROUP 4: pSR43.6r&pSR58.6、pSR43.6r&pSR58.6-CheA、pSR43.6r&pSR58.6-CheA-Flag、pSR43.6r&pSR58.6-CheY、pSR43.6r&pSR58.6-CheY-Flag
Figure 16 schematic diagram of pSR43.6r
Figure 17 schematic diagram of pSR58.6-CheY
Figure 18 schematic diagram of pSR58.6-CheA
Figure 19 schematic diagram of pSR58.6-CheY-Flag
Figure 20 schematic diagram of pSR58.6-CheA-Flag
GROUP 5: pDawn-CheZ-sfGFP、pSR43.6r&pSR58.6-CheA-sfGFP、pSR43.6r&pSR58.6-CheY-sfGFP
Figure 21 schematic diagram of pDawn-CheZ-sfGFP
Figure 22 schematic diagram of pSR58.6-CheA-sfGFP
Figure 23 schematic diagram of pSR58.6-CheY-sfGFP
References
[1] 王启要. 中国鱼类疫苗技术研发及应用研究进展[J]. 大连海洋大学学报,2022,37(1):1-9.
[2] Kojima S, Blair DF. The bacterial flagellar motor: structure and function of a complex molecular machine. Int Rev Cytol. 2004;233:93-134.
[3] Tan J, Zhang X, Wang X, et al. Structural basis of assembly and torque transmission of the bacterial flagellar motor. Cell. 2021 May 13;184(10):2665-2679.e19.
[4] Zhang J, Luo Y, Poh CL. Blue Light-Directed Cell Migration, Aggregation, and Patterning. J Mol Biol. 2020 May 1;432(10):3137-3148.
[5] Tan J, Zhang L, Zhou X, et al. Structural basis of the bacterial flagellar motor rotational switching. Cell Res. 2024 Nov;34(11):788-801.
[6] Perlova T, Gruebele M, Chemla YR. Blue Light Is a Universal Signal for Escherichia coli Chemoreceptors. J Bacteriol. 2019 May 8;201(11):e00762-18.
[7] Schmidl S R, Sheth R U, Wu A, et al. Refactoring and optimization of light-switchable Escherichia coli two-component systems[J]. ACS synthetic biology, 2014, 3(11): 820-831.
[8] Kurzthaler C, Zhao Y, Zhou N, et al. Characterization and control of the run-and-tumble dynamics of Escherichia coli[J]. Physical Review Letters, 2024, 132(3): 038302.
[9] Soffer G, Perry J M, Shih S C C. Real-time optogenetics system for controlling gene expression using a model-based design[J]. Analytical Chemistry, 2021, 93(6): 3181-3188.
[10] Clavier T. Contrôle génétique de la croissance et des conditions de culture pour maximiser la production biotechnologique chez E. coli[D]. Université Grenoble Alpes, 2024.
[11] Kumar S, Khammash M. Platforms for optogenetic stimulation and feedback control[J]. Frontiers in bioengineering and biotechnology, 2022, 10: 918917.
[12] Zhang Y, Xue X, Fang M, et al. Upconversion optogenetic engineered bacteria system for time-resolved imaging diagnosis and light-controlled cancer therapy[J]. ACS Applied Materials & Interfaces, 2022, 14(41): 46351-46361.
[13] Zhao R, Collins E J, Bourret R B, et al. Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ[J]. Nature structural biology, 2002, 9(8): 570-575.
[14] Kennedy E N, Barr S A, Liu X, et al. Azorhizobium caulinodans chemotaxis is controlled by an unusual phosphorelay network[J]. Journal of bacteriology, 2022, 204(2): e00527-21.
[15] Ou B, Lv H, Ge H, et al. Deletion of the cheZ gene results in the loss of swimming ability and the decrease of adhesion ability to Caco-2 cells in Escherichia coli Nissle 1917[J]. Folia Microbiologica, 2023, 68(3): 395-402.
[16] Karmakar R. State of the art of bacterial chemotaxis[J]. Journal of Basic Microbiology, 2021, 61(5): 366-379.
[17] Zhang X, Feng Z, Li H, et al. Ingestible optoelectronic capsules enable bidirectional communication with engineered microbes for controllable therapeutic interventions[J]. Nature Microbiology, 2025: 1-13.
[18] Wadhwa N, Berg H C. Bacterial motility: machinery and mechanisms[J]. Nature reviews microbiology, 2022, 20(3): 161-173.
[19] Zhulin I B, Berry M A, Andrianova E P. Diverse domain architectures of CheA histidine kinase, a central component of bacterial and archaeal chemosensory systems[J]. 2023.
[20] Li X, Zhang C, Xu X, et al. A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells. Nucleic Acids Res. 2020 Apr 6;48(6):e33.