Diphtheria is a disease caused by toxigenic strains of Corynebacterium diphtheriae, a gram positive rod bacteria that produces diphtheria toxin. This toxin is an AB toxin which has two components, subunit A and B. The subunit B has a function in facilitating the process of toxin internalization via endocytosis by binding to a receptor called heparin-binding epidermal growth factor-like growth factor (HB-EGF). On the other hand, subunit A is the active part of the toxin halts protein synthesis inside the cell by ribosylating elongation factor 2 (EF2).1-2
Beside working as receptor for diphtheria toxin, HB-EGF belongs to epidermal growth factor (EGF) family that functions in growth and differentiation. There are two forms of HB-EGF, mature soluble form that has function as mitogen and chemotactic factor and transmembrane form which functions in juxtacrine growth and adhesion factor. HB-EGF is known to be involved in normal physiological processes such as blastocyst implantation, wound healing, pathological processes such as tumor growth, SMC hyperplasia, and atherosclerosis.3
EnvZ/OmpR Two Component System in Escherichia coli
Two component system (TCS) is a basic stimulus-response mechanism that allows microorganisms to sense and respond to environmental changes. It usually consists of two fundamental units which are histidine kinase (HK) and response regulator (RR).4 EnvZ/OmpR system is one of the most studied TCS in E. coli. This system regulates the expression of outer membrane porin (OmpC and OmpF) in response to osmolarity changes.5 The system naturally works as follows.
Figure 1. EnvZ/OmpR two component system in E. coli.
HB-EGF/EnvZ as diphtheria diagnostic tool system
Due to HB-EGF capacity to bind diphtheria toxin, we decided to make a chimeric protein that consists of HB-EGF receptor domain and EnvZ signalling domain in our project. This chimeric protein later will be referred as HE. Our project also used GFP gene that is regulated under OmpC promoter that can be activated by binding with phosphorylated OmpR. Once phosphorylated OmpR regulates the OmpC promoter, the downstream gene of the OmpC promoter, which will later be substituted with green fluorescent protein (GFP), will be activated. The expression of GFP is positively regulated by the binding of DT to HE. The schematic mechanism of how our project system work is shown in Figure 2.
In our project’s genetic circuit, HE is constitutively expressed under t5 promoter. Naturally, EnvZ responds to the environment’s osmolarity changes regulating the phosphorylation of OmpR transcription factor. Phosphorylated OmpR level decreases when the medium’s osmolarity is low, and increases when the medium’s osmolarity is high.
In our project, our EnvZ - HB-EGF - chimeric protein will be activated upon binding of diphtheria toxin (DT). Once activated, HE will be phosphorylated, which will cause OmpR to be phosphorylated as well. Phosphorylated OmpR will bind to OmpC promoter and initiates the transcription of GFP gene downstream to the promoter. GFP emits green light (520nm) which makes our bacteria to glow in green color.
Considering the presence of native EnvZ in E. coli, the expression of GFP can also be activated by the presence of high concentration of salt in the medium. In order to prevent a false positive result and this genetic circuit can work as a diagnostic tool to detect DT, we modify our medium’s salt concentration medium to be low enough so that the phosphorylated OmpR is kept low. Therefore, once the diphtheria toxin binds with the HE, prominent expression of green fluorescence can be observed
Using modified diphtheria toxin to test the HB-EGF/EnvZ diagnostic tool system
Concerning the toxicity of diphtheria toxin, we did not use any native diphtheria toxin or C. diphtheria at all in our project. We used a modified version of diphtheria toxin, which will be later called diphtox, which does not contain the toxic domain presence in diphtheria toxin wild type. Diphtox only consists fragment that binds to HB-EGF receptor. We expressed diphtox using biobrick that was created by Team UI Indonesia 2018 (BBa_K2607000), later will be called as DT18. This part consists of rbs and diphtox gene. The schematic structure of this part is shown below.
To make some improvements in this project as well as fulfilling gold medal criteria, we made some improvements to part DT18. We changed the RBS sequence that can boost the expression of diphtox and added red fluorescence protein (RFP) to make bacterial colony selection easier after transformation. This improved part later will be referred as DT19. The schematic structure of this improved part is shown below.
Both part DT18 and DT19 will be used as diphtox that serves as a substitute of diphtheria toxin that will activate the HB-EGF/EnvZ-based diphtheria diagnostic tool system.
BioBrick used for making HB-EGF/EnvZ diagnostic tool system
To make this system, we used two biobricks which are part HE and part OG. Part HE consists of t5 promoter, RBS, HE coding sequence, and terminator. The coding sequence of HE was generated by amino acid sequences from structural modelling of HB-EGF/EnvZ chimeric protein whereas the other components were obtained from iGEM registries. The schematic structure of Part HE is shown below.
Part OG consists of OmpC promoter, rbs, GFP coding sequence, and terminator. The whole part was already present in the iGEM registry and provided in iGEM Distribution Kit 2019. The schematic structure of part OG is shown below.
References
Baron S. Medical Microbiology. University of Texas Medical Branch at Galveston; 1996.
Naglich JG, Metherall JE, Russell DW, Eidels L. Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor. Cell. 1992 Jun 12;69(6):1051-61.
Raab G, Klagsbrun M. Heparin-binding EGF-like growth factor. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer. 1997 Dec 9;1333(3):F179-99.
Selvamani V, Ganesh I, Maruthamuthu M, Eom GT, Hong SH. Engineering chimeric two-component system into Escherichia coli from Paracoccus denitrificans to sense methanol. Biotechnol and Bioprocess Engineering. 2017 Jun 1;22(3):225-30.
Ganesh I, Ravikumar S, Lee SH, Park SJ, Hong SH. Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system. J Biotechnol. 2013 Dec 1;168(4):560-6
