Synthetic cell-cell communication systems

Cell-cell communication is critical for the physiological functions of diverse organisms. Even in bacteria, populations can monitor their own density and achieve coordinated expression of target genes. From the engineering perspective, cell-cell communication serves as a versatile regulatory module that enables coordination among cells in and between populations. Synthetic communication systems are required for the engineering of microbial consortia, which is an important new frontier in synthetic biology. Without synthetic communication systems, members of the microbial consortia can not communicate with each other, thus the division of labor in the microbial consortia can not be achieved. Synthetic communication systems can be used to enable dynamic gene regulation and facilitates the generation of reliable dynamics at the community level. Challenges in the area of synthetic communication systems remain the limited number of independent communication modules, crosstalk between signals, and interspecies communication. The development of new communication modules is needed to address each of these challenges.

Figure 1. Synthetic communication systems in microbial consortia, cited from reference [1]

Synthetic communication systems using AHL-based Quorum sensing systems from Gram negative bacteria

The most popular tools for engineering communication systems are based on quorum-sensing (QS) systems used by bacteria to sense and respond to changes in their local population density. Quorum sensing allows bacteria to monitor their population density for the purpose of controlling various group activities such as virulence or luminescence. By coordinating group behaviors, bacterial communities can enhance their ability to adapt to nutrient-limited conditions, defend against assaults from competing microorganisms or host immune systems, and improve their ability to acquire new genetic material that could potentially lead to antibiotic resistance.

The acyl-homoserine lactone (AHL)-based QS systems from Gram-negative organisms have been engineered extensively. Take the LuxI-LuxR system as an example, the AHL molecules are synthesized by the LuxI protein. The extracellular AHL molecules are imported into the cytoplasm to activate the transcription factor LuxR, which controls the expression of genes. The Natural LuxI-LuxR system can be split into signal Sender cell and signal Receiver cell. Synthetic AHL-based communication systems have been used for a variety of applications including triggering biofilm formation, constructing synchronized oscillators, generating patterns, sensing pathogens, developing synthetic ecosystems. M.Omar Din, et al. engineered the LuxI-LuxR AHL QS system in a clinically relevant bacterium to lyse synchronously at a threshold population density and to release drugs to treat cancer. The 2016 Imperial College team used the synthetic LuxI-LuxR AHL QS system to maintain the balance of the co-culture bacteria. The 2016 Arizona State University Team continued to engineer several new AHL-based communication systems to enrich the diversity.

Figure 2. Natural AHL-based quorum sensing system (A) and synthetic communication system (B), cited from reference [2].

The Peptide-based QS systems in Gram positive bacteria

Gram-positive bacteria use peptides, called autoinducing peptides (AIPs), as signaling molecules in the QS system. Once produced in the cell, AIPs are processed and secreted. When the extracellular concentration of the AIP is high, which occurs at high cell density, it can be either sensed by a cognate membrane-bound two-component histidine kinase receptor or it can be imported into the cytoplasm to be sensed by a cognate transcription factor. In the two component system type, AIP binding activates the receptor’s kinase activity, it autophosphorylates, and passes phosphate to a cognate cytoplasmic response regulator. The phosphorylated response regulator activates transcription of the genes in the QS regulon. The Agr system from S.aureus belongs to the two component system type. In the cytoplasmic sensing type, AIPs are transported back into the cell cytoplasm where they interact with transcription factors to modulate the transcription factor’s activity and, in turn, modulate gene expression changes. The AimR-AimP system from B.subtilis bacteriophage phi3T and the PlcR-PapR system from B.cereus belong to the cytoplasmic transcription factor type.

Fig 3. Two types of peptide-based quorum sensing systems in Gram-positive bacteria. (A) two-component signaling pathway, (B) cytoplasmic transcription factor. Cited from reference [3].

The characteristics of peptide-based quorum system from Gram positive bacteria

Compared to the AHL-based QS systems, the signal peptide encoding capacity is far more expanded. Even as short as seven amino acids, the encoded information capacity can reach to 207 different types. The peptide configuration is far more diverse than the AHL molecules, which will limit the cross talk of the signal peptide. Further more, the peptide molecules can be commercially synthesized at very low price. The peptide can also be degraded by the proteinase K or inactivated using physical or chemical methods. Thus, we envision that the development of communication systems using peptide-based QS system will facilitate the synthetic biologists to construct more sophisticated systems.

The characteristics of peptide-based quorum system from Gram positive bacteria

Currently, almost all of the synthetic communication systems that have been developed to date are functional in and between Gram-negative organisms based on the AHL QS systems, and most of the synthetic networks have been implemented in Escherichia coli. However, no communication systems using peptide-based QS systems have been developed. In this project, we want to develop synthetic communication system using the two types of peptide-based QS systems.

Gram positive bacteria comprise diverse species. Some species can cause severe human infections, such as Staphylococcus aureus and Mycobacterium tuberculosis. Some species are important industrial producers, such as L.lactis and B.subtilis. What is more, the balance between Gram-positive and Gram-negative bacteria in the microbiota is crucial for human health. However, no synthetic communication systems have been developed for the Gram positive bacteria. In this project, we want to use the L.lactis and B.subtilis as our Gram positive bacteria chassis to implement them with the synthetic communication systems.

References:

[1] Shong, J., Jimenez Diaz, M.R., and Collins, C.H. (2012). Towards synthetic microbial consortia for bioprocessing. Curr Opin Biotechnol 23, 798-802.

[2] Davis, R.M., Muller, R.Y., and Haynes, K.A. (2015). Can the natural diversity of quorum-sensing advance synthetic biology? Front Bioeng Biotechnol 3, 30.

[3] Rutherford, S.T., and Bassler, B.L. (2012). Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2.