Team:UI Indonesia/Project
OVERVIEW
Diphtheria is becoming a prominent issue in Indonesia as its incidence is increasing recently. It also causes various complications, leading to morbidity and mortality. We realized the urgency of fast, reliable, and cheap early detection method for diphtheria infection as one of means necessary for its eradication. Therefore, we created a chimeric between native Escherichia coli Tar chemotaxis receptor and human HB-EGF receptor so the bacterium may recognize diphtheria toxin. Moreover, we combined CheA and CheY in E. coli chemotaxis system with LuxAB and eYFP, respectively. When in contact, LuxAB and eYFP create resonance energy transfer system in which LuxAB gives its emission to eYFP. Without diphtheria toxin, CheA will be in phosphorylated state, allowing interaction with CheY and energy transfer, resulting in yellow color. Toxin binding into chimeric receptor will inhibit CheA phosphorylation, hindering interaction with CheY and energy transfer, resulting in blue color (i.e. LuxAB native color).
Pathogenesis of Diphtheria: How Does Corynebacterium diphtheriae Cause the Disease?
Corynebacterium diphtheriae is a Gram-positive rod bacterium that causes diphtheria. It produces exotoxin with two fragments (AB toxin). Fragment B facilitates toxin internalization within host cell via endocytosis upon binding HB-EGF receptor,while fragment A catalyzes modification of ribosomal elongation factor-2; therefore, it suppresses protein synthesis
Tar-mediated Chemotaxis System in Escherichia coli
In E. coli, chemotaxis mediated by methyl-accepting chemotaxis proteins (MCPs) has been widely studied. MCPs are transmembrane chemoreceptors with periplasmic ligand binding domain and cytoplasmic signaling domain. To date, four different E. coli MCPs have been identified: Tar, Tsr, Trg and Tap chemoreceptors.
Tar chemoreceptor mediates E. coli movement towards aspartate and maltose (attractant molecules). It undergoes conformational change upon attractant molecule binding, leading to inhibition of CheA protein autophosphorylation and subsequent CheY activity. This causes the flagellum facilitates the bacterium to swin towards attractant substance via opposite motor protein FliM rotation. On the other hand, CheA autophosphorylation would cause prominent rotation of motor protein to make tumbling effect.
LuxAB-eYFP Fluorescence Resonance Energy Transfer (FRET) System
Basically, a molecule is excited to higher energy state when it absorbs a photon energy. This molecule relaxes back to ground state when the energy is emitted back to the environment or transferred into another molecule. FRET is a phenomenon in which non-radioactive energy is transferred from excited donor molecule to acceptor molecule via dipole-dipole interactions. Molecules involved in this phenomenon are called fluorophores as they emit fluorescence according to their respective emission spectrum after absorbing higher photon energy. LuxAB and eYFP are one of the most widely studied paired fluorophores. In this case, LuxAB is the donor fluorophore as it emits cyan colored light with relatively high energy (peak emission at 490 nm). eYFP serves as the acceptor fluorophore when in close contact with LuxAB, as it absorbs high energy from LuxAB that is overlapped with its own absorption spectrum and emits yellow colored light with lower energy (peak emission at 530 nm). To be utilized in macromolecules interaction studies, LuxAB and eYFP should be incorporated with the molecules of interest. When the molecules of interest are in contact, energy transfer between LuxAB and eYFP will happen and its efficiency can be measured with fluorescence-lifetime imaging microscopy method.
OUR PROJECT
Our Protocols can be accessed here
Our Lab Notes can be accessed here
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RESULTS AND DISCUSSIONS
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RESULTS AND DISCUSSIONS