Design | WHU-China

Part 1: Secretion

Overview

Lpp' OmpA secretion system

This year we genetically engineer Escherichia coli into probiotic
Intestide.Our proposed design encompasses three interconnected
modules:

Part 1:
Secretion

Part 2:
Adhesion

Part 3:
Biosafety

Part 1: Secretion

Overview

Fig. 1 The genetic circuit of secretion system

To enable the engineered bacteria to produce short peptides in the intestine, we have designed a secretion system. This system employs the Lpp'OmpA secretion system to expose and anchor the protein on the outer membrane of Escherichia coli. Subsequently, enterokinases in the small intestine cleave the short peptides at the DDDDK cleavage site near the C-terminal of the protein.

Lpp' OmpA secretion system

Lpp'OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins^[1]. As a signal peptide, Lpp facilitates the modification and maturation of the protein in the periplasm. It then enables transport from the periplasm through the inner leaflet of the outer membrane, resulting in the formation of inner surface lipoproteins. Subsequently, OmpA transports the protein through the outer leaflet and anchors it on the cell surface.

Fig. 2 The schematic view of Lpp'OmpA surface display system

Peptide synthesis

Following the Lpp'OmpA chimera, the protein contains a region for short peptide generation, including the DDDDK sequence and the target short peptide. The DDDDK sequence serves as a cleavage site for enterokinase, which cleaves the protein after the lysine residue (K). Enterokinase is an enzyme naturally secreted in the small intestine. Consequently, once the chimera has directed the protein to the outer membrane surface, the DDDDK-short peptide will be exposed outside the cell. Enterokinase in the small intestine will then cleave the peptide at the DDDDK site, releasing the target short peptide into the intestinal environment.

Fig. 3 The schematic view of peptide synthesis and secretion process

Part 2: Adhesion

We design the adhesion construct to ensure that two types of bacteria adhere to each other and the surface of small intestinal epithelial cells. It comprises two parts: adhesins and a display toolbox.

Fig. 4 The schematic view of peptide synthesis and secretion process

There are heat shock protein 60 (HSP60) on the surface of the small intestinal mucosa^[2], and it's proven that HSP60 can bind to Listeria adhesion protein (LAP) originating from non-pathogenic Listeria Innocua^[3]. In our project, we take advantage of these features and display LAP and HSP60 on the surface of our engineering bacteria, so that our bacteria can stick to each other and reside in the intestine^[2][3].

Fig. 5 A model representing the adhesion of two bacteria and intestine

Fig. 6 A detailed partitioning for Neae, among which LysM domain for peptidoglycan binding while β-barrel for transmembrane insertion

Sure, it is necessary to display the adhesins on the outer membrane of E. coli. To achieve this target, we choose the display toolbox Neae. Neae is the abbreviation of intimin N-terminus, which includes a short N-terminal signal peptide (export tag) to direct its trafficking to the periplasm, a LysM domain for peptidoglycan binding, and a β-barrel for transmembrane insertion. Adhesins are fused to the C terminus of Neae to form the Adhesion construct^[4][5].

Fig. 7 A model representing Neae can display adhesin on the outer membrane of Gram-negative bacteria(Timmis et al.,2019)

Fig. 8 The expected effect of the adhesion construct

Part 3: Biosafety

Overview

Fig. 9 The genetic circuit of Biosafety system

Upon reaching the intestines, the engineered bacteria are designed not to multiply indefinitely, thus preventing them from displacing the native gut microbiota. Additionally, they are programmed to die immediately upon exposure to the external environment. To enhance the controllability and safety of the engineered bacteria, we have designed a predator-prey quorum sensing system. This system ensures that the bacteria maintain a target quantity within the intestines and are programmed to die upon exposure to oxygen.

Basic circuit

Fig. 10 A basic circuit of quorum sensing suicide system, adapted from L. You et.al (2004)

In the LuxI/LuxR system, the LuxI protein synthesizes a small, diffusible signaling molecule known as acyl-homoserine lactone (AHL). As cell density increases, AHL accumulates both outside and inside the cells. It binds to and activates the LuxR transcriptional regulator, which in turn induces the expression of the killing gene (E) under the control of the luxI promoter (P_luxI). Elevated levels of the killing protein ultimately result in cell death.

Prey-predator system

Fig. 11 The genetic circuit of prey and predator system, adapted from F.K. Balagaddé et.al(2008)

In this system, we employ two fundamental circuits: the LuxI/LuxR system and the LasI/LasR system. Our design designates the peptide producer as the predator and the controller as the prey.

When the prey density is low, predator cells undergo apoptosis due to the constitutive expression of the suicide gene (ccdB). In the prey cells, LuxI synthesizes an acyl-homoserine lactone (AHL) known as 3OC₆HSL. As prey density increases, 3OC₆HSL accumulates in the extracellular environment and, at sufficiently high concentrations, binds to the LuxR transcriptional regulator in predator cells. This binding leads to the increased expression of the antidote gene (ccdA), rescuing the predator cells from apoptosis. Conversely, LasI in the predator cells produces another AHL, 3OC₁₂HSL, which diffuses into the prey cells and binds with LasR, activating the expression of the ccdB gene to initiate "hunting".

Therefore, when the controller density is low, the peptide producers will die. Only when the controller reaches a certain density can the peptide producers survive. If the density of the peptide producers becomes too high, the controllers are "hunted," reducing their density and causing the death of the peptide producers once again. This dynamic system ensures that our bacteria maintain a specific range of densities.

Oxygen-killing

Fig. 12 The genetic circuit of oxygen-killing system

To ensure that our engineered bacteria die immediately after being excreted from the body, without impacting the prey-predator system through additional ccdA activity, we have designed an Oxygen-killing module within the controller.

This module utilizes P_nar, an oxygen-inducible promoter, which activates only in low-oxygen environments and is suppressed in high-oxygen conditions. The P_R promoter, which is lytic, contains binding sites that are recognized by the repressor protein Cl. When Cl is bound to these sites, p_R is inhibited.

Within the gut, where oxygen levels are low, P_nar activates the expression of Cl, which in turn inhibits P_R and reduces ccdB expression, allowing the controller cells to survive. However, once the bacteria exit the gut and encounter high-oxygen environments, Cl expression is suppressed. This removal of Cl inhibition allows P_R to promote ccdB expression, leading to the self-destruction of the controller cells.

References

[1]

S. Nicchi, M. Giuliani, F. Giusti, L. Pancotto, D. Maione, I. Delany, C.L. Galeotti, C. Brettoni, Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems, Microbial Cell Factories 20(1) (2021) 33.

[2]

Kim KP, Jagadeesan B, Burkholder KM, Jaradat ZW, Wampler JL, Lathrop AA, Morgan MT, Bhunia AK. Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor. FEMS Microbiol Lett. 2006 Mar;256(2):324-32. doi: 10.1111/j.1574-6968.2006.00140.x.

[3]

Drolia, R., Amalaradjou, M. A. R., Ryan, V., Tenguria, S., Liu, D., Bai, X., Bhunia, A. K. (2020). Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nature Communications, 11(1), 6344. doi:10.1038/s41467-020-20200-5

[4]

Glass, D. S., & Riedel-Kruse, I. H. (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell, 174(3), 649-658.e616. doi:https://doi.org/10.1016/j.cell.2018.06.041

[5]

Timmis, K., Timmis, J. K., Brüssow, H., & Fernández, L. Á. (2019). Synthetic consortia of nanobody-coupled and formatted bacteria for prophylaxis and therapy interventions targeting microbiome dysbiosis-associated diseases and co-morbidities. 12(1), 58-65. doi:https://doi.org/10.1111/1751-7915.13355

[6]

L. You, R.S. Cox, R. Weiss, F.H. Arnold, Programmed population control by cell-cell communication and regulated killing, Nature 428(6985) (2004) 868-871.

[7]

F.K. Balagaddé, H. Song, J. Ozaki, C.H. Collins, M. Barnet, F.H. Arnold, S.R. Quake, L. You, A synthetic Escherichia coli predator–prey ecosystem, Molecular Systems Biology 4(1) (2008) 187.